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Division of Nephrology, Endocrinology and Vascular Medicine and
¹ Division of Rheumatology and Hematology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan

(Requests for offprints should be addressed to K Mori; Email: kokimori{at}mail.tains.tohoku.ac.jp)

	Abstract

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Tumor necrosis factor- (TNF) may play a role in the development of autoimmune thyroiditis such as Hashimoto’s thyroiditis. In the present study, we examined whether TNF induced its own expression in FRTL-5 rat thyroid cells. Lipopolysaccharide (LPS) markedly increased TNF mRNA levels in FRTL-5 cells as assessed by semiquantitative RT-PCR. In addition, LPS-stimulated cells released TNF protein into the culture medium. Similarly, TNF induced its own gene and protein expression in FRTL-5 cells as assessed by RT-PCR and metabolic labeling and immunoprecipitation of TNF. The autoinduction of TNF gene was also observed in TNF-stimulated human thyroid epithelial cells. TNF induction was specific to LPS and TNF since interferon- or amiodarone failed to increase TNF mRNA levels in FRTL-5 cells. Human TNF induced rat TNF gene expression, indicating that type 1 TNF receptor (TNF-R) is involved in the autoinduction. TNF did not increase either type 1 or type 2 TNF-R mRNA levels, suggesting that upregulation of TNF receptors is not involved in the autoinduction of TNF. Although the biological significance of autoinduction of TNF remains unclear, our results suggest that thyroid epithelial cells may participate in the development of autoimmune thyroiditis through production of TNF. Furthermore, inhibition of TNF production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor necrosis factor- (TNF) is a pleiotropic cytokine that acts as a central regulator of inflammation and immunity. This cytokine is detectable in thyroid tissues obtained from patients with Hashimoto’s thyroiditis (Ajjan et al. 1996, Aust et al. 1996). In accordance, we and others have demonstrated enhanced TNF gene expression in the inflamed thyroid of BioBreeding/Worcester (BB/W) rats (Mori et al. 1998, Bluher et al. 1999). Furthermore, TNF augments interferon- (IFN)-induced class II major histocompatibility complex (MHC) antigen expression (Weetman & Rees 1988, Zakarija et al. 1988). In FRTL-5 rat thyroid cells, TNF induces interferon regulatory factor-1 (Mori et al. 1999), which plays a role in immune responses (Taniguchi et al. 1997). Taken together, these results suggest that TNF may be involved in the development of autoimmune thyroiditis such as Hashimoto’s thyroiditis.

While the majority of TNF detected in inflamed thyroid tissues is produced by infiltrating inflammatory cells (Aust et al. 1996), studies demonstrate the production of TNF by thyroid epithelial cells (Zheng et al. 1992, Mori et al. 1998). It may be possible that TNF produced by infiltrating inflammatory cells induces its own expression in thyrocytes in the area of infiltration, since autoinduction of TNF has been demonstrated in HL-60 human promyelocytic leukemia cells and in rat tracheal epithelial cells (Spriggs et al. 1990, Bader & Nettesheim 1996). If so, thyrocyte-produced TNF may stimulate infiltrating cells to aggravate inflammation in autoimmune thyroiditis. However, autoinduction of TNF in thyroid epithelial cells has never been reported. In the present study, we examined whether TNF induced its own expression in FRTL-5 cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Recombinant rat IFN and recombinant mouse TNF were obtained from Genzyme (Cambridge, MA, USA). Recombinant human TNF was purchased from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide (LPS; Escherichia coli O55:B5) and amiodarone (AMD) were purchased from Sigma Chemical Co. (St Louis, MO, USA). -³²P-dATP was purchased from New England Nuclear Corporation (Boston, MA, USA). ³⁵S-methionine (TRAN³⁵S-LABEL) was obtained from ICN Biomedicals (Irvine, CA, USA). Anti-p65 subunit of nuclear factor kappa B (NF-B) and preimmune rabbit serum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Antibodies to IB and phosphorylated IB (Ser 32) were obtained from New England Biolabs (Beverly, MA, USA). The chemiluminescence detection system (ECL) was purchased from Amersham Biosciences (Piscataway, NJ, USA). The Superscript preamplification system containing Superscript II reverse transcriptase was obtained from Gibco/BRL (Grand Island, NY, USA). All other chemicals and reagents were obtained from commercial sources and were of reagent or molecular biology grade.

Cell culture

FRTL-5 cells were obtained from ATCC (Rockville, MD, USA), proliferated in a thyrotropin (TSH)-dependent manner and possessed all the properties previously described (Mori et al. 2001). Cells were grown in Coon’s modified Ham’s F12 medium supplemented with 5% calf serum containing bovine TSH (1 mU/ml), bovine insulin (10 µg/ml), human transferrin (5 mg/ml), glycyl-L-histidyl-L-lysine (2 ng/ml), somatostatin (10 ng/ml) and hydrocortisone (0.36 ng/ml; 6H medium). After cells approached confluence, cells were cultured for 7 days in media devoid of TSH (5H medium). This treatment allowed cells to be quiescent as shown previously (Mori et al. 2001). Human thyroid epithelial cells were obtained and cultured as previously described (Trieb et al. 1992) with modification. Normal thyroid tissues were removed from two patients who underwent thyroidectomy for papillary carcinoma. Informed consent was obtained from each patient and the study protocol was approved by the local ethics committee. The tissues were washed with PBS and minced into small pieces. They were digested with 1 mg/ml collagenase I (Sigma Chemical Co.) and 2.4 U/ml dispase II (Roche Diagnostics GmBH, Penzbeg, Germany) in PBS at 37 °C for 45 min. This procedure was repeated for 75 min with fresh enzyme mixture. The resulting cell suspension was filtered through 100 µm mesh and washed in 6H medium. Cells were cultured in 6H medium for 15 h followed by vigorous washing to remove non-adherent and loosely adherent cells. The remaining adherent cells were detached by treatment with trypsin EDTA solution (Gibco/BRL) and plated in culture flasks and 96-well flat-bottomed plates. More than 95% cells were stained with serum containing a high titer of anti-thyroglobulin and anti-thyroid peroxidase antibodies from a patient with Hashimoto’s thyroiditis (data not shown). The culture medium was changed twice a week. On the day of experiment, cells were incubated with test reagents for the indicated time and harvested for analyses. Treatment of cells did not result in cell detachment and had no effect on cell viability, as assessed by trypan blue exclusion.

Extraction of soluble nuclear proteins

Soluble nuclear proteins were obtained as previously described (Mori et al. 1999). In brief, FRTL-5 cells were washed twice with ice-cold PBS, and harvested in 1 ml hypotonic buffer containing 10 mM N-(2-hydroxyethyl) piperazine-N’-(2-ethane sulfonic acid; HEPES)-KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM NaF and 1 mM Na₃VO₄. After incubation on ice for 15 min, cells were vortexed for 10 s in hypotonic buffer containing 0.1% NP-40, followed by incubation on ice for 10 min. Nuclei were pelleted by centrifugation, washed twice with hypotonic buffer and then the nuclear pellets were incubated at 4 °C for 30 min in hypertonic buffer containing 20 mM HEPES-KOH, pH 7.9, 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM NaF, 1 mM Na₃VO₄ and 20% glycerol. Supernatants were collected after centrifugation and stored at – 80 °C until use. Protein concentrations were measured by the Bradford method (Bradford 1976).

Electrophoretic mobility shift assay (EMSA)

The synthetic oligonucleotides, 5'-CAAACAGGGGGCT TTCCCTCCTC-3' and 5'-GAGGAGGGAAAGCCCC CTGTTTG-3', containing the NFB (B3) site in rat TNF gene promoter (Nathens et al. 1997), were used for the detection of the protein–DNA complex by EMSA. The double-stranded probes were end-labeled using Klenow-DNA polymerase and -³²P-dATP. Nuclear proteins (5 µg) were incubated with 40 fmol ³²P-labeled probe at 22 °C for 30 min in buffer containing 10 mM HEPES-KOH, pH 7.9, 100 mM KCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 0.5 mM PMSF, 10% glycerol, 0.05% NP-40 and 2 µg poly(dI-dC)-poly (dI-dC). The nucleoprotein complexes were resolved by nondenaturing electrophoresis in a 5% polyacrylamide gel at 4 °C in buffer containing 45 mM Tris–HCl, pH 8.0, 45 mM boric acid and 1 mM EDTA. Gels were dried and exposed to BioMax MS film (Eastman Kodak, Rochester, NY, USA) at –80 °C. For competition experiments, a 100-fold molar excess of the unlabeled oligonucleotides was added 15 min before incubation of nuclear extracts with radiolabeled probes. In supershift assays, nuclear proteins were incubated for 1 h at 22 °C with 1 µl anti-p65 subunit of NFB or preimmune rabbit serum followed by addition of radiolabeled probes.

Western blot analysis

Cells were washed with ice-cold washing buffer containing 10 mM sodium phosphate, pH 7.4, 137 mM NaCl and 1 mM Na₃VO₄ and solubilized for 30 min at 4 °C in the lysis buffer containing 50 mM Tris–HCl, pH 7.5, 137 mM NaCl, 2 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na₃VO₄, 1 mM NaF and 0.1% Triton X-100. Supernatants were collected after centrifugation and aliquots containing 50 µg protein were mixed with Laemmli sample buffer and separated by SDS-PAGE (Laemmli 1970). Proteins were transferred to a nitrocellulose membrane by electroblotting. After blocking with 1% BSA in Tris–buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with rabbit polyclonal antibody to serine-phosphorylated IB (1:1000) overnight at 22 °C. The membranes were washed and incubated with anti-rabbit IgG conjugated with horseradish peroxidase (1:2000) for 1 h at 22 °C. Blots were visualized using the ECL detection system. After stripping, the membranes were reprobed with rabbit polyclonal anti-IB (1:1000).

RT-PCR

Cells were washed twice with ice-cold PBS and total cellular RNA was isolated by the acid guanidinium–thiocyanate–phenol–chloroform extraction method of Chomczynski and Sacchi (1987), using Trizol (Gibco/BRL). Four micrograms total RNA were reverse transcribed with Superscript II reverse transcriptase according to the manufacturer’s instructions. All PCRs were performed in a 50 µl reaction volume containing 1 µl aliquots from each cDNA reaction, 10 pmol of each upstream and downstream primer, 1.25 units Taq polymerase (Takara, Otsu, Japan), 0.2 mM of each dNTP, and 2 mM MgCl₂. Amplification was performed at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s followed by a 5-min extension at 72 °C. The primer sequences, product length and PCR cycles shown in Table 1 were used for the detection of TNF, two types of TNF receptors, TNF-R1 and TNF-R2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in FRTL-5 cells. Primer pairs for rat TNF span introns, and extra bands suggesting genomic DNA contamination were not seen. Human TNF and human ß-actin gene expression was determined using a human TNF primer pair (BioSource International, Camarillo, CA, USA) and a human ß-actin primer pair (Takara). Control reactions without reverse transcription were carried out in parallel. Cycle numbers were chosen to lie in the linear range of amplification. Rat GAPDH and human ß-actin were used as an internal standard to verify that equal amounts of RNA had been subjected to RT-PCR. PCR products were separated on 1.5% agarose gel and visualized with ethidium bromide. All PCR products were cloned into pGEM-T vector (Promega, Madison, IL, USA) and sequenced by the dideoxy chain termination method.

TNF protein production in TNF-stimulated cells was assessed by immunoprecipitation of ³⁵S-labeled TNF (Zhou et al. 2000). Briefly, cells were washed with PBS and incubated in methionine-free RPMI (ICN Biomedicals) supplemented with L-glutamine, penicillin-streptomycin, 1% calf serum and ³⁵S-methionine (TRAN³⁵S-LABEL; 100 µCi/ml) for 24 h in the presence or absence of 50 ng/ml TNF. Cells were washed and lyzed in the lysis buffer. Samples were clarified by centrifugation and standardized to protein concentration. Cell extracts were precleared twice by incubating with protein A/G agarose (Santa Cruz Biotechnology). The samples were incubated with 5 µg antibody to TNF (Genzyme) or normal goat IgG overnight and protein A/G agarose was added for an additional 2 h. The beads were washed 5 times and eluted in Laemmli sample buffer. The proteins were separated by SDS-PAGE and dried gels were subjected to autoradiography.

TNF ELISA

TNF released into the culture medium by FRTL-5 cells treated with LPS for the indicated times was measured by a rat TNF enzyme-linked immunosorbent assay (ELISA) kit (BioSource International), according to the manufacturer’s instructions. The detection limits for rat TNF were 0.7 pg/ml. Human TNF was measured by a human TNF ELISA kit (BioSource International) with the minimum detectable level of 0.1 pg/ml. Data are the means of triplicate culture supernatants ± S.D. and are representative of two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Fisher’s protected least significant difference test. A level of P<0.05 was considered statistically significant.

	Results

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EMSA was performed to verify that TNF and LPS activate the transcription factor NF-B, which plays a central role in the TNF- and LPS-stimulated intracellular signaling pathway in FRTL-5 cells. No NF-B binding activity was detected in unstimulated cells (Fig. 1). Mouse TNF induced a rapid and marked increase in NF-B binding activity. Addition of a 100-fold molar excess of unlabeled oligonucleotides to the binding reaction completely blocked the binding activity. Furthermore, the complex formed in response to TNF was supershifted by the anti-p65 subunit of NF-B, but not by preimmune rabbit serum (Fig. 1), indicating that activated NF-B containing the p65 subunit was induced in response to TNF. Consistently, stimulation of cells with mouse TNF resulted in an increase in phosphorylated IB levels and a concomitant decrease in total IB levels at 10 and 30 mm after stimulation (Fig. 2). Similar to TNF, LPS activated NF-B (Fig. 1), consistent with previous studies (Guha & Mackman 2001). Thus, we confirmed that the major signaling molecule, NF-B, was activated in response to TNF and LPS in FRTL-5 cells.

View larger version (91K): [in this window] [in a new window]   Figure 1 Effect of TNF or LPS on nuclear NF-B binding activity in FRTL-5 cells. Nuclear proteins were incubated with 32P-labeled synthetic oligonucleotides containing the NF-B element (B3) of rat TNF gene promotor and NF-B binding activity was analyzed with EMSA. Cells were stimulated with 10 ng/ml mouse TNF or 100 µg/ml LPS for the indicated times (0, (unstimulated), 10, 30, 60 min). P, 32P-labeled probe only and C, a 100-fold molar excess of unlabeled oligonucleotides were included as competitors. The complex formed in response to TNF or LPS was supershifted by the anti-p65 subunit of NF-B (p65), but not by preimmune rabbit serum (NS). The data presented are representative of three separate experiments.

View larger version (24K): [in this window] [in a new window]   Figure 2 Effect of mouse TNF on IB phosphorylation and degradation in FRTL-5 cells. Cells were incubated with 10 ng/ml mouse TNF for the indicated times (min). Cellular proteins (50 µg/lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Phosphorylated and total IB were detected with antibodies to serine-phosphorylated and total IB and by the ECL detection system. The data presented are representative of three separate experiments.

View larger version (37K): [in this window] [in a new window]   Figure 3 (A) Induction of TNF gene expression by LPS in FRTL-5 cells. Cells were incubated with 100 µg/ml LPS for the indicated times (h). Total RNA was reverse transcribed and the resulting cDNA was used for the PCR. The relative amounts of TNF mRNA were normalized with GAPDH mRNA levels. The data presented are representative of three separate experiments. (B) Production of TNF by LPS-stimulated FRTL-5 cells. Cells were incubated with 100 µg/ml LPS for the indicated times (h). TNF levels in culture medium were determined using an ELISA kit. Data are the means of triplicate culture supernatants ± S.D. and are representative of two separate experiments. n.d., not detectable. *P<0.05 compared with unstimulated cells (0 h).

View larger version (37K): [in this window] [in a new window]   Figure 4 (A) Induction of TNF gene expression in TNF-stimulated FRTL-5 cells. Cells were incubated with 10 ng/ml mouse TNF for the indicated times (h). The data presented are representative of three separate experiments. (B) Dose-dependent induction of TNF gene expression in mouse TNF-stimulated FRTL-5 cells. Cells were treated with various doses of TNF for 2 h. The data presented are representative of three separate experiments. (C) De novo TNF protein synthesis by TNF-treated FRTL-5 cells. Cells were stimulated with 50 ng/ml mouse TNF and metabolically labeled with 35S-methionine for 24 h. Cells were lyzed, and TNF molecules were immunoprecipitated using anti-TNF antibody or normal goat IgG. Immunoprecipitates were subjected to SDS-PAGE under reducing conditions. Gels were dried and newly synthesized TNF proteins were visualized by autoradiography. The data presented are representative of two separate experiments.

View larger version (25K): [in this window] [in a new window]   Figure 5 (A) Production of TNF by LPS-stimulated human thyroid epithelial cells. Cells were incubated with 100 µg/ml LPS for the indicated times (h). TNF levels in culture medium were determined using an ELISA kit. Data are the means of triplicate culture supernatants ± S.D. and are representative of two separate experiments. n.d., not detectable. *P<0.05 compared with unstimulated cells (0 h). (B) Induction of TNF gene expression in TNF-stimulated human thyroid cells. Cells were incubated with 10 ng/ml human TNF for the indicated times (h). The data presented are representative of three separate experiments.

View larger version (36K): [in this window] [in a new window]   Figure 6 TNF-R1 and TNF-R2 gene expression in TNF-stimulated FRTL-5 cells. Cells were incubated with 10 ng/ml mouse TNF for the indicated times (h). The data presented are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, TNF gene expression was clearly induced in FRTL-5 cells in response to LPS or TNF, as assessed by RT-PCR. In contrast, LPS- or TNF-stimulated FRTL-5 cells produced a very small amount of TNF protein. Taken together, our results suggest that the FRTL-5 cell line may be a suitable model to determine the regulatory mechanism involved in TNF gene expression, but not in TNF protein synthesis, in the thyroid. Accordingly, establishment of more sensitive methods such as the immuno-PCR (Sanna et al. 1995) is clearly required to analyze TNF biosynthesis in FRTL-5 cells. Alternatively, this issue could be addressed by the establishment of thyroid-derived cells that produce much higher amounts of TNF protein. In this regard, human thyroid epithelial cells may be suitable since LPS-stimulated cells can produce larger amounts of TNF than can FRTL-5 cells as shown in Fig. 5A.

TNF exerts its biological effects through binding to two distinct cell surface receptors, TNF-R1 and TNF-R2 (Lewis et al. 1991). Previous studies demonstrated that thyroid cells possess TNF receptors (Pang et al. 1989, 1996). However, TNF-R gene expression remained to be elucidated in TNF-stimulated thyroid cells. In the present study, we demonstrate detectable levels of TNF-R1 and TNF-R2 mRNA in unstimulated FRTL-5 cells. However, TNF failed to increase transcript levels. These results suggest that TNF induces its own expression without upregulating its receptor. The effects of TNF on its receptor gene expression seem to be tissue-specific. In rat tracheal epithelial cells, TNF downregulates TNF-R1 mRNA levels (Bader & Nettesheim 1996). In contrast, this cytokine increases TNF-R1 transcripts in rat oligodendrocytes (Dopp et al. 1997). Finally, human TNF, which binds to murine TNF-R1 but not to TNF-R2 (Lewis et al. 1991), induced TNF gene expression, indicating that TNF-R1 is involved in autoinduction of TNF in FRTL-5 cells.

In summary, we demonstrate that TNF induces its own expression in FRTL-5 cells. Although the biological significance of this phenomenon remains to be elucidated, our results suggest that thyrocyte-produced TNF may regulate immune and inflammatory reactions in the thyroid and thus may be involved in the development of autoimmune thyroiditis such as Hashimoto’s thyroiditis. Furthermore, inhibition of TNF production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.

	Acknowledgements

 
This study was presented, in part, at the 74th Annual Meeting of the American Thyroid Association, Los Angeles, CA, USA in 2002. There is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 21 July 2005
Accepted 21 July 2005
Made available online as an Accepted Preprint 5 August 2005

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Mori, K. Articles by Ito, S. Search for Related Content PubMed PubMed Citation Articles by Mori, K. Articles by Ito, S.