Human fetal thyroid cell growth in vitro: system characterization and cytokine inhibition.

We have studied primary human fetal thyroid cell monolayers as an in vitro model for thyroid cell growth and function. Fetal thyroid cells grew slowly in a serum-free medium under basal conditions (i.e. without insulin and TSH), with a doubling time of 112 +/- 7 h (mean +/- SEM), indicating autonomous growth capacity. Addition of insulin (10 micrograms/ml) lead to increased growth, with a doubling time of 43.0 +/- 2.5 h, and TSH further reduced the doubling time in a dose-dependent manner, with the highest growth rate at 1-10 mU/ml (doubling time, 27 +/- 0.5 h). These growth rates were only observed when cells were subjected to a short culture time (12 h) before investigation, whereas after 96 h of culture the fetal thyroid cell growth rate was reduced by up to 50%. Addition of more than 5% serum completely inhibited the growth stimulation initiated by insulin and TSH. The accumulation of extracellular cAMP by the fetal cell monolayer was induced by TSH in a dose-dependent manner and reached a maximum effect at 10 mU/ml (4.8 +/- 0.6 pmol cAMP). Basal thyroglobulin (Tg) release was 26.9 +/- 0.5 ng/10(5) cells.day. Insulin decreased Tg release to 16.7 +/- 0.5 ng/10(5) cells,day, whereas TSH increased it up to 52.5 +/- 1.0 ng/10(5) cells.day. The T cell cytokine gamma-interferon, a product of the lymphocytic infiltrate in autoimmune thyroid disease, significantly reduced both insulin and TSH-stimulated cellular growth as well as accumulation of Tg. In conclusion, human fetal thyroid cell monolayers grew at high velocity under defined experimental conditions in vitro. These conditions included a low serum concentration, short culture time before investigation, and insulin/TSH supplementation. Furthermore, this human thyroid model confirms our earlier observations on the influence of cytokines in thyroid cell growth regulation.

Induction of human thyroid cell ICAM-1 (CD54) antigen expression and ICAM-1-mediated lymphocyte binding.

To further understand the mechanism of lymphocyte accumulation within the thyroid gland in autoimmune thyroid disease we have examined the expression, regulation, and functional significance of the intercellular adhesion molecule-1 (ICAM-1, CD54) in human thyroid monolayer cells and immortalized thyroid cell clones. Human thyroid monolayer cells derived from both normal and abnormal human thyroid tissue showed low basal expression of the ICAM-1 antigen by flow cytometric assessment (mean % +/- SD positive cells = 13.7 +/- 6.1) compatible with the presence of ICAM-1 positive nonthyroid cells within the monolayer cultures. However, thyroid cell ICAM-1 antigen expression was further induced by exposure to recombinant human interferon-gamma (IF-gamma). At 100 U/ml, IF-gamma induced ICAM-1 expression in 56.0 +/- 19.0% of thyroid monolayer cells. Even greater expression of ICAM-1 antigen was induced by IF-gamma in human fetal thyroid cell monolayers of high purity (up to 80% of ICAM-1 positivity) thyroid monolayers established from a patient with Graves' disease (up to 84%), and in two immortalized human thyroid cell clones, 12S and TAD-2 (up to 61%). Furthermore, dose-response curves for ICAM-1 and HLA-DR antigen induction by increasing concentrations of IF-gamma showed that ICAM-1 antigen gene induction was 10-fold more responsive to IF-gamma than the HLA-DR antigen gene. In order to explore the functional consequence of ICAM-1 antigen expression by thyroid epithelial cells we examined the binding of peripheral blood mononuclear cells to thyroid monolayer cells and immortalized thyroid cells. These studies revealed a preferential adhesion of human PBMC to IF-gamma-treated thyroid monolayers compared to untreated control monolayer cells. Furthermore, this IF-gamma-induced cell adhesion was specifically inhibited by monoclonal anti-ICAM-1. These experiments demonstrate not only the capacity of human thyroid epithelial cells to express ICAM-1 antigen in the presence of a cytokine but, in addition, identify ICAM-1 antigen as responsible for enhanced T cell binding to thyroid epithelial cells. ICAM-1 antigen may, therefore, play an important role in T cell targeting and accumulation within the thyroid gland in autoimmune thyroid disease.

The effect of androgen, estrogen, and growth factors on the proliferation of cultured fibroblasts derived from human fetal and adult prostates.

Stromal enlargement plays a key role in the development of benign prostatic hypertrophy in humans. Human prostatic fibroblasts were obtained from fetal and adult prostates and characterized as to their androgen and estrogen receptor status and growth in response to dihydrotestosterone (DHT), estradiol (E2), hydroxyflutamide (OH-FLU), hydrocortisone, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). In addition, the ability of hormones and growth factors to induce the messenger RNA (mRNA) for the c-fos protooncogene was assessed as a measure of the early, direct effects of these compounds on cellular proliferation. Nuclear androgen receptors were demonstrable by immunocytochemistry in both fetal and adult cells. Nuclear estrogen receptor staining was negative. Neither E2 nor hydrocortisone increased cellular proliferation. Both EGF and bFGF did increase cellular growth. DHT (10(-8)-10(-7) M) had a significant stimulatory effect on cell growth only in serum-free media. OH-FLU addition enhanced DHT induced proliferation. Changing the media during the course of the experiment obliterated the stimulatory effect of DHT. Both EGF (10 ng/ml) and bFGF (20 ng/ml) increased the mRNA for the c-fos protooncogene. DHT (10(-7) M) did not induce the mRNA for c-fos. We conclude that EGF, bFGF, and DHT (especially in combination with OH-FLU) increase the proliferation of human prostatic fetal and adult fibroblasts in vitro. E2 has no effect on fibroblast proliferation. The stimulatory effects of EGF and bFGF are direct, whereas the effect of DHT appears to be indirect, possibly mediated via the increased production and/or secretion of growth factors.

The positive regulation of human thyrotropin (TSH) receptor messenger ribonucleic acid by recombinant human TSH is at the intranuclear level.

We have assessed the regulatory influence of human recombinant TSH (rec-hTSH) on its homologous receptor (TSHR) using a well characterized human fetal thyroid monolayer cell culture technique. Under the culture conditions employed, fetal human thyroid cells showed basal expression of TSHR-specific mRNA transcripts, and the addition of rec-hTSH (1 U/L) induced up to an 8-fold increase in specific mRNA over a 48-h observation period. This induction was simulated by bromo-cAMP in a dose-dependent manner, indicating that the stimulatory effect of rec-hTSH was active at the postreceptor level. Furthermore, there was no detectable increase in the transcription rate of the TSHR gene after stimulation with rec-hTSH for 12-36 h, although a marked increase in thyroglobulin-specific mRNA was observed. Rec-hTSH also had no influence on the half-life of TSHR-specific mRNA, which remained at approximately 16 h in the presence or absence of rec-hTSH. These data indicate that rec-hTSH induced up-regulation in human thyroid cell TSHR-specific mRNA and that the mechanism of this regulation was likely to be secondary to a posttranscriptional nuclear event involving changes in the regulation of primary unspliced mRNA for the TSHR.

Human chorionic gonadotropin (hCG) interacts directly with recombinant human TSH receptors.

We have previously shown that highly purified urinary hCG has the potential to both stimulate the intracellular accumulation of cyclic AMP and induce growth of immortalized rat thyroid cells. We have now compared the ability of recombinant human TSH and purified urinary hCG preparations to stimulate Chinese hamster ovary (CHO) cells which have been transfected with the human TSH receptor. Only transfected CHO cells expressing recombinant TSH receptors, but not control CHO cells, were stimulated by hCG to release cyclic AMP in a dose-related manner and the effect of 100 IU of HCG was equivalent to approximately 9.2 uU of rec-hTSH. These data demonstrate that hCG interacts directly with the human TSH receptor.

Thyrotropin receptor autoantibodies induce human thyroid cell growth and c-fos activation.

Graves' disease encompasses hyperthyroidism and a diffuse goiter associated with autoantibodies to the TSH receptor (TRAb). Although the cause of the goiter formation has been attributed to TRAb, the limited growth pattern of human adult thyroid cells in vitro has caused such a conclusion to be based on studies of nonhuman thyroid cell growth. We have recently characterized a predictable and precise technique for the measurement of human thyroid cell proliferation and function using fetal thyroid cells and have used this system to examine the influence of TRAb on human thyroid cell growth. Highly purified human immunoglobulin G (hIgG) preparations from normal individuals (n = 5) had no significant influence on human thyroid cell growth. However, hIgG from patients with detectable TRAb (TRAb-hIgG) (n = 13) induced a dose-related increase in extracellular cAMP (maximum effect at 0.1 mg/ml) and a 3-fold increase in human thyroid cell growth over a 4-day period (maximum effect at 1.5 mg/ml). Under basal thyroid cell culture conditions there were detectable, but low, levels of mRNA specific for the protooncogene c-fos, and this was markedly, and rapidly, induced by the addition of TRAb-hIgG but not normal hIgG. These data demonstrate induction of cellular growth by TRAb-hIgG in an homologous human thyroid cell culture system. Such observations support the hypothesis that goiter formation in patients with Graves' disease is, at least in part, secondary to the growth stimulating activity of TRAb-hIgG.

Recombinant human thyroid-stimulating hormone: initial bioactivity assessment using human fetal thyroid cells.

We have assessed the bioactivity of newly available recombinant human TSH (rec-hTSH) using human fetal thyroid cells, with the longer term aim of assessing its use for clinical applications. Rec-hTSH caused a consistent and dose-related increase in thyroid monolayer cell cAMP release and human thyroglobulin (hTg) secretion, confirming its bioactivity. Repetitive studies (n = 5) allowed us to derive an estimated biopotency for the rec-hTSH preparation examined of 5.6 IU/mg compared to 10 IU/mg for commercially available bovine TSH for human use. The rec-hTSH had a bioimmune ratio of 0.55, similar to that of purified pituitary hTSH standards, Furthermore, rec-hTSH induced thyroid epithelial cell growth, as evidenced by a decrease in thyroid cell doubling time from 54 +/- 2.1 to 31 +/- 1.7 h (P less than 0.005). Hence, rec-hTSH is a potent glycoprotein hormone preparation when measured in a homologous human thyroid cell culture system. Rec-hTSH could serve as a future definitive International Standard and has the potential for a useful diagnostic and therapeutic reagent.

Positive regulation of human thyrotropin receptor mRNA by thyrotropin.

We have assessed the influence of natural and recombinant thyrotropin (TSH) on mRNA specific for the human TSH receptor (TSHR) in normal and abnormal adult human thyroid monolayer cells. Using physiological concentrations of TSH (less than 100 mU/L), a marked increase in the level of TSHR mRNA was observed within 12 hours and reached a greater than 1000% increase after 24 hours exposure. At high TSH concentrations (greater than 1000 mU/L), this increase in TSHR-specific mRNA was markedly reduced. However, at no time were basal TSHR mRNA levels suppressed even with 100 U/L of TSH for 48 hours. These observations demonstrate ligand-induced up-regulation of the human TSHR mRNA by physiologically relevant concentrations of TSH.

[Reparative processes of the iris after irradiation with the argon-ion laser (author's transl)]. / Reparative Vorgänge an der Iris des pigmentierten Kaninchenauges nach Bestrahlung mit dem Argon-Laser.

The reparative processes of the pigmented iris of the rabbit were analysed with ultrastructural methods. 1. Clearing of the damaged area by macrophages is the first step in the reparative processes. Clump cells are macrophages which are observed from the first day of the injury until the ninth week. 2. Repair of the anterior surface of the iris is largely finished after 32 days. 3. The repair of collagenous fibres reaches its maximum activity 32 days after irradiation. 4. The pigment epithelium has only an insignificant regeneration potential. 5. Irradiation of the iris by the argon-ion laser results in an atrophic, hyperpigmented scar. The rapid regeneration of a lesion induced by the argon-ion laser in the rabbit iris casts doubt as to whether this method could be applied to the human eye with equal success.

[Morphology of the primary damage caused by the argon-ion laser to the iris of the pigmented rabbit (author's transl)]. / Die Morphologie der Primärschäden des Argon-Ion-Lasers an der Iris des pigmentierten Kaninchenauges.

The effects of the argon-ion laser upon the iris of the pigmented rabbit were analysed by ultrastructural methods. 1. Apart from the physical parameters of the energy source, the damage strongly depends on the concentration, location, and distribution of the iris pigment. 2. The irradiation of the iris results in the formation of a crater. Depending on the distance of the epicentre of the impact, various degrees of ultrastructural damage are observed. a. The region immediately adjacent to the crater lumen at a revealing distance of 25 microns consists of homogenous collagenous masses, revealing a vacuolar structure. As the only residuals of destroyed cells, melanin granules are observed within the homogenous masses. b. Destruction of the collagen fibrils and their disintegration into subfibrils with elimination of all cell compartments is found in an area ranging from 25 microns to 300 microns from the wall of the crater. c. Damage to the chromatin structure is visible up to a distance of 300 microns to 375 microns from the wall of the crater. 3. Characterised by an invasion of macrophages into the damaged area, the cleaning phase starts 24 h after irradiation. 4. The results of this experiment indicate that because of the great energy dose required for man with the inherent widespread tissue damage and low probability of a lasting iridectomy, the cw argon-ion laser appears to be an unsuitable energy source for clinical iridectomy.

[Long term observations after irradiation of the iris by the argon laser (author's transl)]. / Langzeitbeobachtungen nach Bestrahlung der Iris mit dem Argonlaser.

Eleven pigmented rabbit irides were irradiated with the argon laser and were examined electron-microscopically at several intervals between 15 minutes and 256 days after exposure. It was shown that cells start to migrate into the area of the lesion already on the fourth day. After 32 days the area of the lesion is completely closed. 256 days after exposure only a slight impression remained on the anterior surface of the iris.

A 64 kDa membrane antigen is a recurrent epitope for natural autoantibodies in patients with Graves' thyroid and ophthalmic diseases.

OBJECTIVE: We have explored the recently described 64 kDa extraocular muscle antigen that is associated with autoantibodies in the serum of patients with severe Grave's ophthalmopathy. The localization of the antigen and the specificity of autoantibodies for both eye muscle antigens and ophthalmopathy patients were investigated. DESIGN: Western blotting and immunoprecipitation of metabolically labelled antigen from eye muscle and control tissues with sera from ophthalmopathy, Graves' without ophthalmopathy, and normals were used. PATIENTS: Sera from normals (n = 9), patients with recent onset Graves' ophthalmopathy (n = 23), and patients with Graves' disease without ophthalmopathy (n = 8) were utilized. MEASUREMENTS: Immunoblots using detergent phase separated (amphiphilic) antigen preparations from fetal eye muscle, skeletal muscle and control tissues were quantitated. Metabolically labelled eye muscle and skeletal muscle antigens were immunoprecipitated using patient and control IgG. RESULTS: In the eye muscle detergent phase, immunoreactivity around 64 kDa was detected in 30% of the patients with ophthalmopathy (n = 23) as well as 38% of patients with Graves' disease and no ophthalmopathy (n = 8) and in 30% of normal sera (n = 9). There was significantly more of this anti-64 kDa reactivity in sera from the ophthalmopathy patients compared with the normals (P less than 0.01). 64 kDa reactivity to detergent phase antigens prepared from human thyroid, skeletal muscle, brain, and liver was also observed with these positive sera indicating the polyreactivity of the IgG interactions to conserved antigens in this region. CONCLUSIONS: We conclude that IgG antibodies binding to a recurrent 64 kDa antigen are present in many normal human sera, with increased concentrations detectable in sera from Graves' ophthalmopathy patients. Such 'specificity-crossover' with similar molecular weight transmembrane antigens is likely to be caused by natural autoantibodies reacting with recurrent autoepitopes rather than a factor aetiological in the disease process.