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Type I Interferons Modulate the Expression of Thyroid Peroxidase, Sodium/Iodide Symporter, and Thyroglobulin Genes in Primary Human Thyrocyte Cultures

Departments of Internal Medicine (N.C., S.C., A.D., E.F., F.M.), Oncology (R.G., F.B.), and Surgery (P.F., P.B., D.G.), University of Pisa, 56126 Pisa, Italy

Address all correspondence and requests for reprints to: Nadia Caraccio, M.D., Metabolism and Endocrinology Unit, Department of Internal Medicine, University of Pisa, Via Roma 67, 56126 Pisa, Italy. E-mail: n.caraccio{at}int.med.unipi.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We evaluated in primary human thyrocyte cultures the effect of interferon (IFN)- and -ß on the expression of thyroid peroxidase (TPO), sodium/iodide symporter (NIS), and thyroglobulin (Tg) as well as T₄ release. Human thyrocyte cultures were carried out with fresh normal thyroid tissue. Gene and protein expression of Tg, TPO, and NIS were assessed by RT-PCR and Western blot analysis after 24, 48, and 72 h of treatment with TSH alone (10 mIU/ml) and in combination with IFN or -ß (10⁴ U/ml). IFN inhibited the TSH-stimulated gene expression of Tg, TPO, and NIS in a time-dependent manner without significant differences between IFN and -ß. Moreover, the addition of both type I IFNs clearly reduced the TSH-stimulated protein expression of Tg, TPO, and NIS after 72 h of exposure. Finally, this down-regulation was associated with a reduction of T₄ release by almost 50%. In conclusion, our study shows that both IFN and -ß down-regulate the TSH-stimulated expression of Tg, TPO, and NIS as well as T₄ release. Indeed, the development of hypothyroidism during type I IFN therapy may be related, at least in part, to an abnormal expression and function of key proteins involved in iodine uptake and organification.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
INTERFERONS (IFNs) ARE a family of distinct proteins that share a high degree of homology with respect to amino acid sequence and three-dimensional structure (1). IFNs have been divided into two major subgroups according to their immunogenicity and plasma membrane binding receptor (2). Type I IFNs (IFN and IFNß) have been widely used to treat patients with chronic viral hepatitis, malignant disorders, and multiple sclerosis (3, 4, 5). Several intervention studies described an association between IFN therapy and transient or permanent thyroid dysfunction, particularly in patients with preexisting thyroid autoimmunity (6, 7, 8, 9, 10, 11). Hypothyroidism was initially described in patients being treated with IFN for breast cancer (6); since then, the association between IFN therapy and subclinical or overt thyroid dysfunction has been described in both malignancies and chronic viral hepatitis (B and C), with a frequency ranging from 2.5 to 31% (9, 11). More recently, IFNß therapy has also been associated with a relatively high risk (1–13%) of developing thyroid dysfunction (10, 12, 13). Although most side effects of IFN therapy appear to be a consequence of immune system dysregulation (3, 8, 9), the mechanisms by which these cytokines induce thyroid dysfunction are still not well elucidated. Yamazaki et al. (14) demonstrated, in vitro, that IFN and -ß added to human thyroid follicle cultures inhibited iodine uptake and organification as well as thyroxine release. Moreover, Roti et al. (15) described a subtle defect in the organification of iodine, determined with the iodide-perchlorate discharge test, in patients receiving IFN therapy.

These data demonstrate a direct inhibitory effect on thyrocyte function of both IFN and -ß; so far, however, the molecular mechanisms by which hypothyroidism may develop during type I IFN therapy have not been clearly understood. In particular, whether thyroid function impairment results from direct toxicity of IFNs on thyrocytes or an abnormal expression and function of key enzymes involved in iodine uptake and organification remains to be elucidated. Highly specialized enzymes regulate iodine metabolism and hormonogenesis in thyroid cells, among which sodium iodide symporter (NIS) and thyroid peroxidase (TPO) are the most important. NIS is a plasma membrane protein that catalyzes the active accumulation of iodide in the thyroid gland, a major step in the biosynthesis of thyroid hormones. NIS couples the inward translocation of Na⁺ down its electrochemical gradient to the simultaneous inward translocation of I^– against its electrochemical gradient (16, 17), using the energy source of Na⁺/K⁺ ATPase (18, 19). TPO is an integral apical membrane glycoprotein of thyroid follicular cells, which bears a catalytic activity for two substrates and is responsible for both the iodination and the coupling of tyrosine residues in thyroglobulin (Tg), leading to thyroid hormone generation (20).

The aim of the present study was to evaluate in primary human thyroid cell cultures the possible effect of IFN and -ß on TPO, NIS, and Tg gene and protein expression as well as on thyroid hormone synthesis and release.

	Materials and Methods

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Culture of thyroid cells

Human thyrocytes were obtained from normal thyroid tissue of five euthyroid patients operated on for benign follicular nodules. All the patients gave their written informed consent and the study protocol was approved by the local ethical committee. In detail, tissue samples were carefully dissected and minced with scissors into small pieces and incubated with type II collagenase (200 U/ml, Gibco-BRL, Grand Island, NY) in a shaking water bath at 37 C for 4–6 h. Thyroid cells were suspended in DMEM culture medium containing 10% fetal calf serum (Sigma Chemical, Milan, Italy) and penicillin-streptomycin (Sigma). Cells were plated in Falcon 25-cm² tissue culture flasks for primary culture (Becton Dickinson, Franklin Lakes, NJ) with DMEM medium and incubated at 37 C in humidified air atmosphere containing 5% CO₂; the medium was changed every 3 d. After 24 h the supernatant containing no adherent cells was removed. The primary cultures of thyroid cells formed a confluent monolayer within 7–9 d; the cells were treated when they had reached 70–80% confluence.

Thyroid cell culture treatments

Cells were treated with TSH alone (10 mIU/ml) and in combination with 10⁴ U/ml IFN (Intron A, Schering, Kenilworth, NJ) or -ß (ß Feron, Pharmades, Florence, Italy) for 24, 48, and 72 h. For the 48- and 72-h experiments, the culture medium with TSH and IFNs was replaced daily. At the end of each time period, cells were removed from the plate by trypsinization (Gibco) and centrifuged at 1000 x g for 5 min. Cells were resuspended with PBS (pH 7.4) and centrifuged. PBS was removed and the pellet was immediately frozen in liquid nitrogen and stored at –80 C until use for RNA extraction.

RNA extraction

Total RNA was extracted using a RNeasy minikit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). The RNeasy procedure represents a novel technology that combines the selective binding properties of a silica gel-based membrane with the speed of a microspin principle. High-quality RNA was then eluted in 30 µl water. Purified RNA was digested with RNAase-free DNase to guarantee that RNA was completely free of DNA contamination.

RT-PCR analysis

A constant amount of total RNA (5 µg) was reverse transcribed at 42 C for 60 min in a total 20 µl reaction volume using first-strand cDNA synthesis kit (Roche Diagnostics Corp., Indianapolis, IN). The cDNA was incubated at 95 C for 5 min to inactivate the reverse transcriptase, and served as a template DNA for 30 rounds of amplification using the Cycler thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA).

PCR was performed in a standard 25 µl reaction mixture consisting of 10 mM Tris-HCL, 50 mM KCL, 1.5 mM MgCl₂ (pH 8.3), 0.2 mM deoxynucleotide triphosphates, 20 pmol of each sense and antisense primer, and 2.5 U AmpliTaq DNA polymerase (Laboratoires Eurobio, Les Ulis Cedex, France). PCR primers for Tg, TPO, NIS, and ß-actin, used as internal control, were as follows: Tg, 5'-primer 5'-GTTGGCAACCTCATCGT-3' and 3'-primer 5'-AATTCTGCAGTGCCTGGT-3', the amplification yielding a 663-bp DNA product corresponding to fragment 7657–7674, according to the published sequence of the gene (21); TPO, 5'-primer, 5'-ACTGCACACGCTGTGGCTGC-3' and 3'-primer, 5'-TGCAGTTTGGCTGGTCTTGC-3', the amplification yielding a 434-bp DNA product corresponding to fragment 1299–1733, according to the published sequence of the gene (22); NIS, 5'-primer, 5'-TCTCTCAGTCAACGCCTCT-3' and 3'-primer, 5'-ATCCAGGATGGCCACTTCTT-3', the amplification yielding a 299-bp DNA product corresponding to a fragment 152–560, according to the published sequence of the gene (23); and ß-actin, 5'-primer, 5'-CACGGCATTGTAACCAACTG-3' and 3'-primer, 5'-TCTCAGCTGTGGTGCTGAAG-3', the amplification yielding a 470-bp DNA product.

Samples were subjected to amplification, and PCR conditions were as follows: TPO gene, denaturation at 94 C (1 min), annealing 58 C (1 min), and extension at 72 C (1 min) for 32 cycles; Tg gene, denaturation at 94 C (1 min), annealing at 58 C (1 min), and extension at 72 C (1 min) for 32 cycles; NIS gene, denaturation at 94 C (1 min), annealing at 58 C (1 min), and extension 72 C (1 min) for 35 cycles; and ß-actin gene, denaturation at 94 C (1 min), annealing at 58 C (1 min) and extension 72 C (1 min) for 32 cycles.

Amplified PCR products were run on a 2% agarose gel containing 0.5 µg/ml ethidium bromide. As the negative control, DNA template was omitted in each reaction. Each experiment was carried out at least three times with different batches of cells on different days to verify reproducibility.

Acquisition of gel images and semiquantitative analysis

We tested a number of cycles ranging from 24 to 40 to select the appropriate number of cycles so that the amplification products were clearly visible on agarose gel and in the exponential range. The optimal number of cycles and the annealing temperatures were in the same range for the specific RNA of interest and the internal control (ß-actin) so that both can be measured on the same gel.

Images of the RT-PCR ethidium bromide-stained agarose gels were acquired with a high-performance charge-coupled device camera (Cohu Inc., San Diego, CA), and quantification of the bands were performed by Kodak Digital Science 1.0 (Kodak Italy, Cinisello Balsamo). Band intensity was expressed as relative absorbance units. The ratio between the sample RNA to be determined and ß-actin was calculated to normalize for initial variations in sample concentration and as a control for reaction efficiency (24).

Western blot analysis

Cellular proteins were isolated from lysate of thyrocytes. Cells were collected and added to lysis buffer [50 mM Tris buffer (pH 8.0), 150 mM NaCl, 0.03% sodium azide, 0.1% sodium dodecyl sulfate, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate] for 20 min at 4 C. The supernatants were collected and the protein concentration determined spectrophotometrically (Bio-Rad, Melville, NY) according to the method of Bradford (25). The total protein content was determined by staining duplicate lanes with Coomassie blue. An identical amount of protein for each lysate (15 µg/ml) was resolved to 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis at a constant voltage (110 V). Proteins were transferred to a nitrocellulose membrane (Bio-Rad). Blocking was carried out by using 5% nonfat dried milk for 2 h at room temperature. The membrane was then incubated overnight at 4 C with 1:20 dilution of mouse antihuman TPO mAb (Alexis Biochemicals, Lausen, Switzerland), 1:200 dilution of mouse antihuman NIS mAb (Chemicon International, Hofheim/Ts, Germany) directed against an epitope in the region of amino acids 625–643 of human NIS, and 1:500 dilution of mouse antihuman Tg mAb (Chemicon International).

The membrane was washed three times for 15 min with Tris-buffered saline and Tween 20 and incubated with an antimouse IgG and biotinylated antibody and subsequently coupled with streptavidin-biotinylated horseradish peroxidase complex. The enhanced chemiluminescence system (ECLplus, Amersham, Little Chalfont, Buckinghamshire, UK) was used for the detection. ß-Actin goat polyclonal IgG antibody (1:500 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) was used as the internal control protein. Standard scanning densitometry was performed on the immunoreactive bands, with normalization of densitometry measured to ß-actin.

T₄ evaluation in supernatants

T₄ concentration in the supernatants of cultured thyrocytes, treated with TSH alone and TSH plus IFN or -ß, was evaluated by specific RIA (ICN Pharmaceuticals Inc., Costa Mesa, CA). T₄ values were normalized to a fixed number of cells (2.5 x 10⁴). To avoid interassay variations, all samples were measured in the same run; in our laboratory the intraassay variation was 4%.

Statistical analysis

Data are expressed as the mean ± SE. Statistical analysis was performed using Student’s t test for paired data and one-way ANOVA as appropriate. Statistical significance was assigned when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tg, TPO, and NIS mRNA expression

The mRNA expression of Tg, TPO, and NIS was low in TSH-deprived cultures (Figs. 1A, 2A, and 3A, respectively). The exposure of thyrocytes to TSH significantly increased the expression of all the three tested genes in a time-dependent fashion (Figs. 1B, 2B, and 3B). The addition of both IFN and -ß induced a significant down-regulation of TSH-stimulated Tg, TPO, and NIS mRNA expression after 48 (Figs. 1C, 2C, and 3C, respectively) and 72 h of exposure (Figs. 1D, 2D, and 3D, respectively). IFN inhibited the TSH-stimulated Tg, TPO, and NIS expression in a time-dependent manner without significant differences between IFN and -ß. The maximal (72 h) down-regulation of TSH-stimulated gene expression ranged from 48 to 70% for Tg, 52 to 61% for TPO, and from 47 to 67% for NIS.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Semiquantitative multiplex RT-PCR in the same tube comparing the PCR-amplified DNA of the Tg gene with ß-actin gene products in monolayer primary cultures of thyrocytes deprived of TSH (TSH–) after 24, 48, and 72 h of exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) (A). Lane M, DNA molecular-weight marker (100 bp, Ladder Eurobio). RT-PCR products were demonstrated on 2% agarose gel stained by 0.5 µg/ml ethidium bromide. Bars represent band intensity, expressed as relative absorbance units of the ratio between Tg cDNA and the internal control ß-actin, in TSH-deprived cells and plus TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). *, P = 0.001 vs. TSH-; **, P 0.05 vs. TSH 24 h; ***, P < 0.02 vs. TSH 48 h; °, P = 0.03; °°, P < 0.02 vs. TSH 48 h; #, P = 0.002 vs. TSH 72 h.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Semiquantitative multiplex RT-PCR in the same tube comparing the PCR-amplified DNA of the TPO gene with ß-actin gene products in monolayer primary cultures of thyrocytes deprived of TSH (TSH–) after 24, 48, and 72 h of exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) (A). Lane M, DNA molecular-weight marker (100 bp, Ladder Eurobio). RT-PCR products were demonstrated on 2% agarose gel stained by 0.5 µg/ml ethidium bromide. Bars represent band intensity, expressed as relative absorbance units of the ratio between TPO cDNA and the internal control ß-actin, in TSH-deprived cells and plus TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). *, P < 0.01 vs. TSH-; **, P = 0.004 vs. TSH 24 h; ***, P < 0.05 vs. TSH 48 h; °, P = 0.001, and °°, P = 0.0001 vs. TSH 48 h; #, P = 0.0001 vs. TSH 72 h.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Semiquantitative multiplex RT-PCR in the same tube comparing the PCR-amplified DNA of the NIS gene with ß-actin gene products in monolayer primary cultures of thyrocytes deprived of TSH (TSH–) after 24, 48, and 72 h of exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) (A). Lane M, DNA molecular-weight marker (100 bp, Ladder Eurobio). RT-PCR products were demonstrated on 2% agarose gel stained by 0.5 µg/ml ethidium bromide. Bars represent band intensity, expressed as relative absorbance units of the ratio between NIS cDNA and the internal control ß-actin, in TSH-deprived cells and plus TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). *, P < 0.0001 vs. TSH-; **, P < 0.002 vs. TSH 24 h; ***, P < 0.001 vs. TSH 48 h; °, P = 0.03, and °°, P = 0.02 vs. TSH 48 h; #, P < 0.001 vs. TSH 72 h.

The protein expression of Tg, TPO, and NIS was evaluated after treatment of thyrocytes with either TSH alone or TSH plus IFN or -ß and is shown in Figs. 4 through 6, respectively. ß-Actin, used as the control protein, was equally expressed at any time under all conditions. Like mRNA, the exposure of thyrocytes to TSH increased the protein expression of Tg, TPO, and NIS in a time-dependent fashion (Figs. 4B, 5B, and 6B, respectively). The addition of both IFN and -ß significantly reduced the TSH-stimulated expression of all three tested proteins after 72 h of exposure (P = 0.002, P = 0.01, and P < 0.005, respectively). However, IFNß showed a significant inhibition of the expression of Tg, TPO, and NIS also at 48 h (P = 0.02, P < 0.05, and P = 0.01, respectively).

View larger version (24K): [in this window] [in a new window]   FIG. 4. Effect of 24-, 48-, and 72-h exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) on protein expression of Tg and the internal control ß-actin in monolayer primary thyrocyte cultures (A). Bars represent band intensity, expressed as relative absorbance units of the ratio between Tg and ß-actin protein expression, induced by TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). Western blotting analysis was carried out as described in Materials and Methods. *, P < 0.001 vs. TSH 24 h; **, P < 0.05 vs. TSH 48 h; °, P = 0.02 vs. TSH 48 h; °°, P = 0.002 vs. TSH 72 h.

View larger version (25K): [in this window] [in a new window]   FIG. 5. Effect of 24-, 48-, and 72-h exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) on protein expression of TPO and the internal control ß-actin in monolayer primary thyrocyte cultures (A). Bars represent band intensity, expressed as relative absorbance units of the ratio between TPO and ß-actin protein expression, induced by TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). Western blotting analysis was carried out as described in Materials and Methods. *, P = 0.01 vs. TSH 24 h; **, P < 0.05 vs. TSH 48 h; °, P < 0.05 vs. TSH 48 h; ##, P = 0.01 vs. TSH 72 h.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Effect of 24-, 48-, and 72-h exposure to bovine TSH alone (10 mIU/ml) and in combination with 104 U/ml IFN (+IFN) or -ß (+IFNß) on protein expression of NIS and the internal control ß-actin in monolayer primary thyrocyte cultures (A). Bars represent band intensity, expressed as relative absorbance units of the ratio between NIS and ß-actin protein expression, induced by TSH alone at 24, 48, and 72 h (B); TSH plus IFN or -ß at 48 h (C); and TSH plus IFN or -ß at 72 h (D). Western blotting analysis was carried out as described in Materials and Methods. *, P = 0.05 vs. TSH 24 h; **, P < 0.05 vs. TSH 48 h; °, P = 0.01 vs. TSH 48 h; °°, P < 0.005 vs. TSH 72 h.

TSH induced a time-dependent significant increase of T₄ release by thyrocytes. In detail, T₄ level was 0.07 ± 0.01 ng/dl (0.9 ± 0.1 pmol/liter) in the absence of TSH (TSH–), 0.17 ± 0.03 (2.1 ± 0.4) after 24 h of treatment with TSH (P = 0.001 vs. TSH–), 0.23 ± 0.06 (3.2 ± 0.9) after 48 h (P = 0.006 vs. TSH– and P = 0.04 vs. TSH 24 h), and 0.27 ± 0.05 (3.7 ± 0.6) after 72 h (P = 0.002 vs. TSH– and P = 0.01 vs. TSH 48 h) (Fig. 7A). The concomitant exposure of thyrocytes to IFN or -ß did not significantly affect the TSH-induced T₄ release at 24 h (Fig. 7B), whereas both IFNs induced a significant decrease of T₄ release at either 48 h (P = 0.001 and P = 0.02 vs. TSH alone; corresponding to 47 and 30% reduction, respectively) (Fig. 7C) or 72 h (P < 0.001 vs. TSH alone; corresponding to 52 and 48% reduction, respectively) (Fig. 7D).

View larger version (24K): [in this window] [in a new window]   FIG. 7. T4 concentrations in supernatants of primary human thyrocyte cultures deprived of TSH (TSH–) and after 24, 48, and 72 h of exposure to 10 mIU/ml bovine TSH are reported (A). The effect of 24-, 48-, and 72-h concomitant exposure to TSH plus 104 U/ml IFN (+IFN) or -ß (+IFNß) on T4 release is reported (B, C, and D, respectively). Data are expressed as mean ± SE. The conversion factor for the expression of T4 concentrations as SI units (picomoles per liter) is 12.87. *, P = 0.001 vs. TSH-; **, P = 0.04 vs. TSH 24 h; ***, P = 0.01 vs. TSH 48 h; °, P = 0.001, and °°, P = 0.02 vs. TSH 48 h; #, P < 0.001 vs. TSH 72 h.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The current study shows that, similarly to type II IFN, both IFN and -ß are able to inhibit, in vitro, the TSH-stimulated expression of NIS, TPO, and Tg. To our knowledge, this is the first study demonstrating that type I IFNs directly down-regulate the transcription of key enzymes involved in iodine uptake and organification.

In accordance with previous observations (31, 32, 33), we found that the expression of Tg, TPO, and NIS mRNA is low in TSH-deprived thyrocyte cultures and significantly increased by TSH in a time-dependent fashion. The addition of both IFN and -ß to the culture medium reduced the TSH-stimulated transcription of all three tested genes by almost 50%. Semiquantitative multiplex RT-PCR analysis of the expression levels of the transcripts from the same sample showed that the inhibitory effect of type I IFNs was time dependent, reaching the statistical significance after 48 and 72 h of exposure. Furthermore, Western blotting evaluation showed that both type I IFNs also down-regulated the TSH-stimulated protein expression of Tg, TPO, and NIS.

A previous study (14) demonstrated that type I IFNs are able to inhibit both iodine uptake and T₄ release in thyroid follicle cultures, but the molecular mechanism bearing this inhibitory effect still has not been clarified. It is well known that a decrease in TPO cell content markedly affects thyroid function leading to hypothyroidism (34). In fact, thyroid peroxidase activity is responsible for both the iodination and the coupling of tyrosine residues in thyroglobulin, leading to thyroid hormone generation (20). However, in the thyroid cell, many steps are involved in the pathway of hormone synthesis and secretion, including active transport of iodide into the follicular cell by the functional activity of NIS, which couples the inward translocation of Na⁺ down its electrochemical gradient to the simultaneous inward translocation of I^– against its electrochemical gradient (16, 17). It is worth noting that NIS mRNA is not expressed in thyroid carcinoma cell lines that have lost iodide uptake activity (23). Overall these data support the hypothesis that the inhibitory effect of type I IFNs on the TSH-stimulated expression of NIS, TPO, and Tg may translate into a defect of thyroid hormone synthesis and secretion. Accordingly, in the current study, both IFN and -ß inhibited the TSH-induced T₄ release from thyrocytes by almost 50%. Indeed, a direct down-regulation of thyroid cell function may play a role in the pathogenesis of hypothyroidism in patients undergoing type I IFN therapy. The current study, however, was carried out with relatively acute experiments (up to 72 h) and may not completely reflect what happens in the thyroid gland after long-term exposure to type I IFNs, as occurs during clinical use of such cytokines. A further limitation of the present study is the use of semiquantitative methods for the analysis of gene and protein expression levels; however, our study was not designed to measure minor modifications or the exact number of molecules but the presence or not of significant changes in TPO, NIS, and Tg expression levels.

In conclusion, our data demonstrate that, in vitro, both IFN and -ß are able to down-regulate the TSH-stimulated expression of TPO, NIS, and Tg as well as T₄ release from thyrocytes. These findings suggest that the development of hypothyroidism without autoimmunity during type I IFN therapy may be related, at least in part, to an abnormal expression and function of key enzymes involved in iodine uptake and organification.

	Acknowledgments

 
We thank Dr. L. Boldrini for her advice in elaborating data.

	Footnotes

 
First Published Online November 23, 2004

Abbreviations: IFN, Interferon; NIS, sodium/iodide symporter; Tg, thyroglobulin; TPO, thyroid peroxidase.

This work was partly supported by grants from Ministero della Ricerca Scientifica e Tecnologica (Rome, Italy).

Received June 19, 2004.

Accepted October 25, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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