Interleukin-6 stimulates thyrotropin receptor expression in human orbital preadipocyte fibroblasts from patients with Graves' ophthalmopathy.

The thyrotropin receptor (TSHR) is the thyroid autoantigen against which stimulating autoantibodies are directed in Graves' hyperthyroidism. Recent evidence suggests that TSHR may also serve as an orbital autoantigen in Graves' ophthalmopathy (GO) and that expression of this protein is increased in the fatty connective tissues of the orbit in this condition. It has been shown that orbital fibroblasts from patients with GO increase thyrotropin (TSH)-dependent cyclic adenosine monophosphate (cAMP) production and TSHR gene expression when cultured under conditions known to stimulate adipocyte differentiation. In the current study, we wanted to determine whether treatment of these cells with particular cytokines (each 1 ng/mL) during differentiation might further augment TSHR expression. We found that exposure to interleukin (IL)-6 increased TSHR expression above control levels in cells from patients with GO. In contrast, this cytokine did not affect TSHR expression in normal orbital cells. Neither IL-4 nor IL-1alpha had a significant stimulatory effect in either normal or Graves' cultures. These findings suggest that IL-6 may play a role in the pathogenesis of GO by increasing expression of the putative autoantigen within the adipose/connective tissues of the orbit.

Cloning of the mouse sodium iodide symporter.

The iodide-concentrating ability of the thyroid gland is essential to the production of thyroid hormone. We report the nucleotide and amino acid sequence of the mouse sodium iodide symporter (mNIS), which mediates this activity within the thyroid gland. An open reading frame of 1,857 nucleotides codes for a protein of 618 amino acids with 95% identity to rat NIS and 84% identity to human NIS. Transient expression of the mNIS cDNA in Chinese hamster ovary (CHO) cells, a nonthyroid cell line, resulted in sodium-dependent, perchlorate-sensitive iodide uptake. Western blot analysis of membrane preparations of CHO cells transiently transfected with mNIS cDNA showed a band of 90 kd when probed with an antibody directed against rat NIS. mNIS will serve as an important reagent in determining the role of NIS in experimental thyroid diseases and for monitoring the immune response to in animal models of NIS-mediated gene therapy.

Induction of leptin expression in orbital preadipocyte fibroblasts.

Graves' ophthalmopathy (GO) is an autoimmune disease characterized by an increase in the volume of the orbital fatty/connective tissues and extraocular muscles. This volume change is due to expansion of the adipose tissues and to accumulation of glycosaminoglycans and edema within the connective tissues of the orbit. We have shown previously that a subpopulation of confluent human orbital preadipocyte fibroblasts can be induced in vitro to differentiate into cells with morphological features of adipocytes and that these cultures express functional thyrotropin receptor (TSHR). In order to identify and study these cells further, we examined the expression of leptin protein and TSHR and leptin mRNA in these cultures. Using immunocytochemistry with objective measurement of immunofluorescent staining intensity on digitized microscopic images, we determined leptin protein expression to be 6 to 37 times greater in differentiated cultures than in control cultures. In addition, we showed that the expression of both genes is enhanced in differentiated cultures. We suggest that an unknown humoral stimulus, present in Graves' disease, might act to induce the differentiation of normal orbital fibroblasts into TSHR-bearing adipocytes. This process would be expected to result in expansion of the orbital adipose tissues and increased TSHR expression within the orbit.

Expression of the sodium iodide symporter in human kidney.

BACKGROUND: The human sodium iodide symporter (hNIS) is a transmembrane protein that mediates the active transport of iodide in the thyroid gland. Following cloning of NIS, NIS expression has been detected in a broad range of nonthyroidal tissues, suggesting that iodide transport in these tissues is conferred by the expression of functional NIS protein. METHODS: The aim of this study was to examine functional hNIS expression in kidney by reverse transcription-polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), immunohistochemistry, and Western blot analysis accompanied by iodide accumulation studies in kidney cells. RESULTS: Using a pair of full-length hNIS-specific oligonucleotide primers, RT-PCR followed by Southern hybridization revealed hNIS mRNA expression in normal human kidney tissue. The PCR products were subjected to automated sequencing and revealed full identity with the published human thyroid-derived NIS cDNA sequence. Furthermore, positive protected bands indicating the presence of hNIS mRNA were apparent in RPA gel lanes corresponding to human kidney cells as well as Chinese hamster ovary (CHO) cells stably transfected with hNIS cDNA and Graves' thyroid tissue. Immunohistochemical analysis of normal human kidney tissue using a mouse monoclonal hNIS-specific antibody showed marked hNIS-specific immunoreactivity confined to tubular cells, while no hNIS-specific immunoreactivity was detected in the glomeruli. NIS protein expression in human kidney cells was further confirmed by Western blot analysis. In addition, accumulation of (125)I was detected in human kidney cells in vitro and was shown to be sodium dependent and sensitive to perchlorate. CONCLUSIONS: Functional hNIS expression was demonstrated in the renal tubular system, suggesting that renal iodide transport may be, at least in part, an active process driven by NIS.

Effect of tumor necrosis factor-alpha, interferon-gamma, and transforming growth factor-beta on adipogenesis and expression of thyrotropin receptor in human orbital preadipocyte fibroblasts.

Graves' ophthalmopathy (GO) is an orbital autoimmune disease that is closely associated with Graves' hyperthyroidism. Examination of retroorbital tissues in GO reveals an accumulation of glycosaminoglycans, increased fat volume, lymphocytic infiltration, and the presence of several inflammatory cytokines. A subpopulation of human orbital fibroblasts can be differentiated in vitro into cells with the morphologic features of adipocytes. We demonstrated recently that these differentiated cultures show increased expression of functional TSH receptor (TSHr). To determine whether the presence of inflammatory cytokines might impact adipogenesis or TSHr expression in these cultures, we treated orbital fibroblasts from normal individuals or GO patients with tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta. We found that each of these cytokines inhibits TSH-dependent cAMP production and TSHr gene expression, and that TNF-alpha and IFN-gamma also inhibit morphological adipocyte differentiation. When cytokines were added after differentiation, the inhibition was less pronounced. Our results suggest that TNF-alpha, IFN-gamma, and transforming growth factor-beta may act within the orbit in GO to modulate expression of the putative orbital autoantigen, TSHr. In addition, the former two cytokines may play a role in determining the extent to which the volume of the orbital adipose tissue increases in this condition.

Differentiation of human orbital preadipocyte fibroblasts induces expression of functional thyrotropin receptor.

Although the autoantigen involved in Graves' hyperthyroidism is known to be the TSH receptor (TSHr), whether this antigen plays a primary role in the pathogenesis of Graves' ophthalmopathy (GO) is unclear. We sought to determine whether fibroblasts derived from orbital adipose/connective tissue are capable of differentiating into adipocytes that bear immunoreactive and functional TSHr. In addition, we assessed relative levels of TSHr gene expression in normal and GO orbital adipose/connective tissue specimens. GO and normal orbital preadipocyte fibroblasts, cultured under conditions known to stimulate adipocyte differentiation, showed evidence of adipogenesis and positive immunostaining for TSHr protein. In addition, significantly more cAMP was produced in response to TSH stimulation in the differentiated cultures than in undifferentiated cultures derived from the same individuals' cells. Other studies demonstrated relatively greater TSHr gene expression in GO than in normal orbital tissue specimens. These results indicate that orbital preadipocyte fibroblasts increase their TSHr expression with differentiation and suggest that these cells play an important role in the pathogenesis of GO. Furthermore, our studies support the concept that TSHr may be an important target antigen in this condition. Factors that stimulate adipocyte differentiation and TSHr expression in the orbit in GO have yet to be defined.

Thyrotropin receptor expression in cultured Graves' orbital preadipocyte fibroblasts is stimulated by thyrotropin.

Our laboratory has shown recently that the thyrotropin receptor (TSHr) is expressed in orbital adipose/connective tissues from patients with Graves' ophthalmopathy (GO), and that this receptor is not demonstrable in orbital tissues from normal individuals. In order to study the regulation of TSHr expression in the orbit in GO, we treated cultures of Graves' orbital preadipocyte fibroblasts with the TSHr ligand thyrotropin (TSH; 100 mU/L). We found increased expression of both intact (2.4 kb) and variant (1.3 kb) TSHr mRNA and of TSHr protein in orbital fibroblasts following 72 hr treatment with TSH. These studies suggest that the expression of this receptor in the orbit in vivo may be stimulated by TSH or other TSHr ligands, and that this stimulation may be important in the development of GO.

Thyrotropin receptor expression in Graves' orbital adipose/connective tissues: potential autoantigen in Graves' ophthalmopathy.

It is acknowledged that the TSH receptor (TSHr) on thyroid follicular cells is the autoantigen involved in the hyperthyroidism of Graves' disease. However, whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of Graves' ophthalmopathy (GO) are unclear. We sought to detect the expression of TSHr messenger ribonucleic acid (mRNA) and protein in orbital adipose/connective tissue specimens and in human orbital preadipocyte fibroblast cultures using liquid hybridization analysis and immunohistochemical methods. We demonstrated intact and variant TSHr mRNA transcripts and TSHr-like immunoreactivity in orbital adipose/connective tissue specimens from patients with GO. In addition, TSHr-like immunoreactivity was detected in early passage GO preadipocyte fibroblast cultures that were shown to include some adipose cells. In contrast, neither TSHr mRNA nor protein was detected in normal orbital adipose/connective tissue specimens or in late passage GO orbital fibroblast cultures containing no lipid-laden adipose cells. In conclusion, we showed that TSHr is expressed in the adipose/connective tissue of the diseased orbit in GO. In addition, TSHr is demonstrable in early passage GO preadipocyte orbital fibroblast cultures that contain a subpopulation of adipocytes. Subsequent passaging of these cells results in the loss of both TSHr expression and adipocyte-specific staining. These results suggest that both the expression of this receptor and the accumulation of adipose tissue in the orbit in GO may be induced in vivo by a humoral factor(s) not present in the cell culture environment.

Thyrotropin receptor expression in adrenal, kidney, and thymus.

Because the thyrotropin receptor (TSHR) has long been considered a thyroid-specific protein, its presence in extrathyroidal tissues has been controversial. In this study, we sought to detect and quantify this potentially low abundance mRNA in various extrathyroidal tissues using liquid hybridization analysis (LHA) and to detect protein with immunohistochemical studies. Strongly positive protected bands, indicating the presence of both intact (2.4 kb) and variant (1.3 kb) TSHr mRNA, were apparent in LHA gel lanes corresponding to normal thyroid, Graves' thyroid, and thymus. Less abundant protected bands of the same sizes were present in lanes corresponding to normal adrenal, and samples from normal kidney were faintly positive. The full-length transcript:variant transcript ratio was approximately 1:1 in all positive tissues. Immunohistochemical analysis of TSHR-like reactivity in paraffin-embedded thymus, adrenal, and kidney revealed specific staining in each of these tissues. No TSHR mRNA or TSHR-like immunoreactivity was detected in samples from several other normal human tissues. We conclude that measurable TSHR mRNA and protein expression is not restricted to the thyroid gland. Further study is warranted to determine whether these extrathyroidal receptors play a role in normal physiology or in disease.

Interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptor inhibit IL-1-induced glycosaminoglycan production in cultured human orbital fibroblasts from patients with Graves' ophthalmopathy.

An accumulation of glycosaminoglycans (GAG) is a feature characteristic of orbital connective tissues from patients with Graves' ophthalmopathy (GO) that leads directly to the clinical expressions of the disease. Interleukin-1 (IL-1), produced by macrophages and fibroblasts within the diseased orbit, stimulates GAG synthesis by orbital fibroblasts. We designed the current study to determine whether particular agents might block this effect and thus be useful in the treatment of GO. Orbital fibroblast cultures were grown to confluence and incubated for 48 h with IL-1 (1-10 U/mL) alone or IL-1 (10 U/mL) in combination with IL-1 receptor antagonist (IL-1ra; 1-40 ng/mL) or soluble IL-1 receptor (sIL-1R; 0.25-10 micrograms/mL). Cells were labeled with [3H]glucosamine and processed for GAG quantitation. The addition of IL-1 alone stimulated GAG synthesis by 73-176% (mean, 104%; P < 0.05). Significant inhibition of IL-1-stimulated GAG synthesis was observed after treatment of normal fibroblasts with IL-1ra at a concentration of 5 ng/mL (12.5-fold molar excess; mean, 33%; P < 0.05); essentially complete inhibition was achieved at 40 ng/mL (100-fold molar excess; mean, 86%; P < 0.05). Significant inhibition of GAG synthesis by sIL-1R was observed at a concentration of 0.5 microgram/mL (720-fold molar excess; mean, 79%; P < 0.05), and inhibition was essentially complete at 1 microgram/mL (1440-fold molar excess; mean, 89%; P < 0.05). IL-1ra and sIL-1R are potent inhibitors of IL-1-induced GAG production by cultured human orbital fibroblasts. Our results suggest that these two compounds, shown in early trials to be safe when administered parenterally, may be useful in the prevention or treatment of GO.

A polymorphism in the extracellular domain of the thyrotropin receptor is highly associated with autoimmune thyroid disease in females.

We and others have described previously a polymorphism at the first position of codon 52 (C52 --> A52) of the human thyrotropin receptor (hTSHr) gene. To determine its potential significance, we studied female (n = 100) and male (n = 25) patients with autoimmune thyroid disease (Graves' disease, n = 91; Hashimoto's thyroiditis, n = 34) and normal individuals [n = 121, female (n = 69), male (n = 52)]. Screening was performed using AciI restriction enzyme digestions of PCR-amplified genomic DNA. All codon 52 polymorphisms were verified by direct DNA sequencing. Data were analyzed using Chi-square or Fisher exact tests and p-values were corrected for multiple comparisons. Our studies demonstrated that this polymorphism is highly associated with autoimmune thyroid disease in the female population (corrected p = 0.008). We found no such association in the male population. Within females, there was a greater association between Graves' disease and the polymorphism (corrected p = 0.017) than between Hashimoto's thyroiditis and the polymorphism (corrected p = 0.090). The polymorphism was present in a higher proportion of Graves' disease patients with Graves' ophthalmopathy and pretibial dermopathy (40%) or Graves' ophthalmopathy, pretibial dermopathy, and acropachy (60%), than in patients with Graves' disease alone (15%), or Graves' disease and Graves' ophthalmopathy alone (17%). In conclusion, a polymorphism (C52 --> A52) of the hTSHr is associated with autoimmune thyroid disease in females.

"Cryptic" dinucleotide polymorphism in the 3' region of the factor IX gene shows substantial variation among different populations.

Long tandem dinucleotide repeats composed of alternating purines and pyrimidines [RY(i)] are abundant and highly polymorphic. "Simple" RY(i) are predominantly composed of one tandem repeat of a dinucleotide sequence. In contrast, "cryptic" RY(i) [cRY(i)] are composed of multiple short dinucleotide repeats. Herein, we describe the racial distribution of alleles for a polymorphic cRY(i) in the factor IX gene. Allele I is absent in Asians, whereas allele III is rare or absent in Caucasians or blacks. A polymorphic cRY(i) analyzed previously shows even more dramatic variation among racial groups, hinting that a battery of cRY(i) might have utility in assessing the racial origin of a DNA sample.

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.

Alu repeats in the human factor IX gene: the rate of polymorphism is not substantially elevated.

Previous data suggested an elevated rate of polymorphism in Alu sequences. Direct genomic sequencing was performed on five Alu repeats in the factor IX gene in at least 20 unrelated males of European and Asian descent (40 kb total). In these Alu sequences, the estimated rate of polymorphism in Caucasians (HE = 7.1 x 10(-4); HN = 4.5 x 10(-4) is similar to previously examined nonrepetitive sequences in the factor IX gene, and about twofold lower than previous estimates of the average rate of polymorphism for the X-chromosome which utilized random genomic clones to detect RFLPs. The aggregate data on the rate of polymorphism in Alu sequences suggest that mutations due to gene conversions at these sites are infrequent.

Detection of TSH receptor RNA in cultured fibroblasts from patients with Graves' ophthalmopathy and pretibial dermopathy.

Fibroblasts are target cells for the autoimmune process in Graves' ophthalmopathy and pretibial dermopathy. Because the autoantigen involved in the hyperthyroidism of Graves' disease is the TSH receptor, we sought to determine whether RNA encoding this receptor might be present in retroocular and pretibial fibroblasts. RNA was reverse transcribed and the resulting cDNA was amplified by the polymerase chain reaction using primers spanning a region of the extracellular domain of the human TSH receptor. The predicted amplified product, verified by direct sequencing, was detected when RNA was derived from fibroblasts, but not from the nonfibroblast cells studied. The demonstration in fibroblasts of RNA encoding this important autoantigen in Graves' disease suggests that the TSH receptor might play a role in the pathogenesis of the connective tissue manifestations of this disease.

Diagnostic test for mucopolysaccharidosis. II. Rapid quantification of glycosaminoglycan in urine samples collected on a paper matrix.

The direct 1,9-dimethylmethylene blue (DMB) method for quantifying sulfated glycosaminoglycan (GAG) in urine (Clin Chem 1989; 35:374-9) has been adapted to a convenient means for sample collection and transport as a test to identify individuals with mucopolysaccharidosis (MPS) storage diseases. Results correlated moderately well (r = 0.85) with those of a commonly used, but more laborious, quantitative method. In studying factors to maximize differentiation of pathological from normal values, we found that GAG excretion (expressed as milligrams GAG per gram creatinine) fits a logarithmic function with respect to age and varies markedly below age five years. This must be considered in developing normative values and forming diagnoses. Of 112 separate urine specimens obtained from 41 MPS patients representing the major MPS diseases, glycosaminoglycan excretion by all exceeded that for age-matched normal individuals. The convenience of this method allowed us to establish the first normative values for three-week-old infants (n = 435) found to have a mean glycosaminoglycan excretion of 179 (SD 86.3) mg of GAG per gram of creatinine. This method improves the diagnostic capability for those MPS diseases that have been particularly difficult to identify (Sanfilippo's syndrome and Morquio's syndrome), and may also provide a test for other disorders with previously unrecognized abnormal excretion of glycosaminoglycan (e.g., mucolipidosis and acromesomelic dysplasia). Most importantly, this MPS diagnostic test is unique in its suitability for mass screening of newborn infants.

Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion.

This direct method for quantifying excessive urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviates cumbersome and labor-intensive procedures for separating glycosaminoglycans from other constituents of urine. Pediatric pharmaceutical formulations (except heparin), in concentrations expected in urine, do not interfere with spectrophotometry, nor does protein. Results can be expressed in terms of urinary creatinine; thus the test is applicable to very small urine specimens (0.1 mL), such as those obtainable from neonates. In a pilot study, results of the direct DMB test for 48 urine specimens agreed with the clinical diagnosis, and quantitative measurements correlated moderately (r = 0.76) with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyridinium chloride). The present method was also used to assess metabolic correction in a patient with Hurler's syndrome after treatment by bone-marrow transplantation. This quantitative method surmounts the major technical problems of developing mass screening programs for infants, thus offering the potential for earlier diagnosis and treatment of mucopolysaccharidosis diseases.