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The recent cloning and molecular characterization of the sodium iodide-symporter (NIS) has inspired novel approaches to the diagnosis and treatment of thyroidal and nonthyroidal malignancies. This article briefly reviews the physiologic regulation of NIS expression by cytokines, the expression in benign and malignant thyroidal diseases, and the expression in extrathyroidal tissues. Current concepts for NIS-based cancer therapy in thyroidal and extrathyroidal tumors are presented. The recent discovery of NIS expression in a majority of breast cancers as well as its promising application for prostate cancer imply potential applications in diagnostic imaging and radioiodine anticancer therapy for these highly common and lethal malignancies.

The sodium iodide symporter (NIS), first identified in FRTL-5 cells, plays a critical role in iodide transport in the thyroid gland and in the production of the iodine-containing thyroid hormones. The aim of our study was to examine the regulation of NIS RNA steady-state levels and protein expression as well as functional activity in FRTL-5 cells. FRTL-5 cells cycling in media containing thyrotropin (TSH) were incubated for 48 hours with dexamethasone (10(-8)-10(-5) M), triiodothyronine (T3; 10(-9)-10(-6) M), methimazole (100 microM), propylthiouracil (PTU; 100 microM), perchlorate (10 microM) and potassium iodide (40 microM). In other experiments, cells were treated for 48 hours with various cytokines including interleukin-6 (IL-6) (100 U/mL), interferon-gamma (IFN-gamma) (100 U/mL), tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml), IL-1alpha (100 U/mL), and IL-1beta (100 U/mL). Northern blot analysis using a 32P-labeled rat NIS-specific cDNA probe (nucleotides 1397-1937) revealed NIS mRNA as a single species of approximately 3 kb. When normalized for beta-actin mRNA signal intensities, NIS RNA steady-state levels in viable FRTL-5 cells were suppressed by approximately 80% after incubation with dexamethasone and T3 in a concentration-dependent manner. Iodide accumulation was decreased by up to 40% after incubation with dexamethasone and T3, respectively, in a concentration-dependent manner. Using a rabbit polyclonal rNIS-specific antibody, Western blot analysis of FRTL-5 cell membranes revealed a 60% and 70% suppression of NIS protein expression after treatment with T3 (0.1 microM) and dexamethasone (1 microM), respectively. In additon, NIS RNA steady-state levels were decreased by approximately 50% after treatment of monolayers with methimazole, PTU, and potassium iodide, respectively. Incubation with methimazole and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. Treatment of FRTL-5 cells with IL-6 and IL-1beta resulted in a 30% decrease of NIS RNA steady-state levels. IL-6 did not alter NIS functional activity, but IL-1beta suppressed iodide accumulation by approximately 25%. IFN-gamma and perchlorate failed to alter NIS RNA steady-state levels. In contrast to IFN-gamma that had no effect on iodide accumulation, perchlorate almost completely suppressed iodide accumulation. TNF-alpha and IL-1alpha failed to alter NIS RNA steady-state levels in higher passage numbers of FRTL-5 cells, whereas treatment with TNF-alpha and IL-1alpha of early passages of FRTL-5 cells (20 cell passages) resulted in a 70% and 40% decrease of NIS RNA steady-state levels, respectively, and in a 20% suppression of NIS functional activity. In conclusion, our data suggest that various agents known to affect iodide transport are capable of differentially altering NIS gene expression and function in cultured thyroid cells. Suppression of NIS gene expression and function by certain cytokines may be responsible, at least in part, for the impaired radioiodine uptake by thyroid tissue in certain forms of thyroiditis.

There are several thyroid antigens including human sodium iodide symporter (hNIS), thyrotropin receptor (TSH-R), thyroid peroxidase (TPO), and thyroglobulin (Tg) that have been considered to be thyroid-specific proteins involved in the pathogenesis of autoimmune thyroid diseases. We examined the expression of these thyroid-tolerance related genes in normal human thymus, the lymphoid organ responsible for the induction of central T-cell self. Reverse transcription-polymerase chain reaction (RT-PCR) amplifications were performed with 4 pairs of oligonucleotide primers specific for the hNIS, TSH-R, TPO, and Tg genes, respectively. Gene-specific transcripts were confirmed by Southern hybridization using digoxigenin-labeled internal oligonucleotide probes. To monitor cDNA integrity and quantity, all samples were coamplified with a pair of intron-spanning human beta-actin-specific oligonucleotide primers. Furthermore, using a highly sensitive immunostaining technique and antibodies specific for these 4 antigens, we examined whether NIS-, TSH-R-, TPO-, and Tg-specific immunoreactivity can be detected and localized in normal human thymus. RT-PCR and Southern hybridization revealed expression of each of these 4 thyroid-related genes in normal human thymus. In addition, immunohistochemical analysis of frozen tissue sections derived from normal human thymus showed marked immunoreactivity for NIS, TSH-R, and Tg as well as weaker staining for TPO. Control reactions using isotype matched nonimmune immunoglobulins were consistently negative. Taken together, our results suggest that NIS-, TSH-R-, TPO-, and Tg-RNA are present and actively processed to immunoreactive NIS-, TSH-R-, TPO-, and Tg-like protein in human thymus. These data support the concept that pre-T lymphocytes may be educated to recognize thyroid-related epitopes expressed in thymus, and, thus, to generate self-tolerance against these thyroid-related antigens.

Infiltrating immunocompetent cells act to establish and perpetuate the orbital autoimmune process in thyroid eye disease (TED). Until recently, it has remained unclear whether T cells infiltrating the orbital connective/fatty tissue and extraocular muscles represent a primary immune response that is specifically directed against orbital antigens. In addition, despite a close clinical and temporal association of the thyroidal and extrathyroidal manifestations in Graves' disease (GD), it has not been proven whether T cells infiltrating thyroid, orbital, and pretibial tissue in patients with TED and pretibial dermopathy (PTD) are directed against certain antigenic determinants shared between these anatomically distinct tissues. Using polymerase chain reaction (PCR)-based molecular analysis of T-cell antigen receptor (TcR) variable (V) region genes, we have demonstrated marked restriction of orbital and pretibial TcR Valpha and Vbeta genes in patients with active TED and PTD. In addition, molecular analysis of T cells in paired samples of extraocular muscle and orbital connective/fatty tissue revealed usage of similar TcR V genes. In contrast, TcR V gene restriction was either absent or much less pronounced in patients with longstanding TED and PTD. Comparison of TcR V genes in T cells obtained from thyroid gland, orbital tissue, pretibial tissue, and peripheral blood of three individual patients with active GD, TED, and PTD also revealed marked restriction and, in addition, striking similarities of TcR V gene usage. Sequencing of complementarity determining regions 3 (CDR3) and junctional domains of TcR Vbeta genes confirmed oligoclonality of intrathyroidal, orbital, and pretibial T cells. Moreover, several conserved junctional motifs were shared by T cells infiltrating the thyroid gland and the extrathyroidal sites. Taken together, these data suggest that, in patients with GD and extrathyroidal manifestations, similar antigenic determinants may be responsible for recruitment and oligoclonal expansion of T cells both within the thyroid gland and in the involved extrathyroidal sites.

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.

Our concepts and understanding of the etiology, evolution, and propagation of Graves' ophthalmopathy have become much more sophisticated that they were 10 years ago. Given our current state of knowledge, the following scheme for the pathogenesis of Graves' ophthalmopathy can be proposed. Circulating T cells in patients with Graves' disease, directed against an antigen on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the retroorbital space. Of the cell types residing in these tissues, fibroblasts are most likely to act as both target and effector cells of the retroorbital immune process. This includes those fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from fibroblasts located in the retroorbital connective tissue. By contrast, convincing evidence implicating the human extraocular myocyte itself (rather than the tissue conglomerate of extraocular muscle) as a primary target in GO remains to be demonstrated. Together with adipocytes, fibroblasts may also serve as target and effector cells in pretibial myxedema. How autoreactive T cells escape deletion by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations is unknown. T cells are recruited to and infiltrate the orbit via certain adhesion receptors, which may also play a costimulatory role in T cell activation and facilitate antigen recognition. Analysis of variable region gene usage of the T cell antigen receptors in retroorbital T cells of patients with active GO reveals limited variability, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroocular and pretibial fibroblasts are likely important effector cells in Graves' ophthalmopathy and pretibial dermopathy. Histologic similarities exist between the tissues involved in these clinically diverse extrathyroidal manifestations of Graves' disease. Both conditions are characterized by an accumulation of glycosaminoglycans (GAGs) and an infiltration of lymphocytes. We have shown that particular cytokines, probably released by the local inflammatory cell infiltrate, are capable of stimulating GAG synthesis by retroocular and pretibial fibroblasts. In order to explain the site-selective involvement of these anatomically distinct areas in Graves' disease, we sought to identify unique characteristics shared by retroocular and pretibial fibroblasts. We have shown that affected retroocular and pretibial fibroblasts demonstrate an enhanced HLA-DR response to interferon-gamma treatment and that retroocular fibroblasts are especially sensitive to the GAG-stimulating effect of this cytokine. In addition, we have shown that only affected retroocular and pretibial fibroblasts express the 72 kDa heat shock protein. Therefore, affected retroocular and pretibial fibroblasts possess unique immunologic features that may render them more susceptible to the autoimmune process in Graves' disease. Chronic stimulation of fibroblasts by cytokines released in the local inflammatory milieu may result in excessive GAG production by these cells. The accumulation of these hydrophilic mucopolysaccharides, with attendant edema, leads to the clinical manifestations of Graves' ophthalmopathy and pretibial dermopathy.