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Differential Interactions between Th1/Th2, Th1/Th3, and Th2/Th3 Cytokines in the Regulation of Thyroperoxidase and Dual Oxidase Expression, and of Thyroglobulin Secretion in Thyrocytes in Vitro

Unité de Morphologie Expérimentale, Université catholique de Louvain, B-1200, Brussels, Belgium

Address all correspondence and requests for reprints to: A.-C. Gérard, Ph.D., Unité de Morphologie Expérimentale, Université catholique de Louvain, Université catholique de Louvain-5251, 52 Avenue East Mounier, B-1200 Brussels, Belgium. E-mail: anne-catherine.gerard{at}uclouvain.be.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypothyroidism, together with glandular atrophy, is the usual outcome of destructive autoimmune thyroiditis. The impairment in the thyroid function results either from cell destruction or from Th1 cytokine-induced alteration in hormonogenesis. Here, we investigated the impact of the local immune context on the thyroid function. We used two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes incubated with IL-1/interferon (IFN) together with IL-4, a Th2 cytokine, or with TGF-β, or IL-10, two Th3 cytokines. We first observed that IL-4 totally blocked IL-1/interferon -induced alteration in dual oxidase and thyroperoxidase expression, and in thyroglobulin secretion. By contrast, TGF-β and IL-10 had no such effect. They rather repressed thyrocyte function as do Th1 cytokines. In addition, IL-4 blocked IL-10-induced repression of thyrocyte function, but not that induced by TGF-β. In conclusion, Th1 cytokine- and IL-10-induced local inhibitory actions on thyroid function can be totally overturned by Th2 cytokines. These data provide new clues about the influence of the immune context on thyrocyte function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
HASHIMOTO’S THYROIDITIS (HT) is a destructive autoimmune disease characterized by the infiltration of the parenchyma by inflammatory cells. The immune response is mostly cell mediated and is accompanied by a gradual loss of function, eventually leading to permanent hypothyroidism (1).

Cytokines that act as mediators of immune responses are classified into different groups. Th1 cytokines, usually termed pro-inflammatory, are involved in the regulation of cell-mediated immune responses and play a leading role in HT (2, 3, 4). Th2 cytokines are involved in the control of antibody production, in disease remission, and the suppression of immune responses. Actions resulting from Th1 and Th2 cells are mutually suppressive (5, 6, 7, 8). More recently, a third group composed of Th3 cytokines was described, including IL-10 and TGF-βs (8, 9). Both TGF-βs and IL-10 are immunosuppressive and play a role in the resolution of inflammatory responses in a large variety of organ-specific autoimmune diseases (9, 10). They are also involved in various biological processes, including tumorigenesis and fibrosis (10, 11, 12, 13, 14, 15).

Th1 cytokines exert direct local effects in the thyroid through receptors located on thyrocytes (16, 17, 18, 19, 20, 21). They affect the expression of thyroid-specific proteins, including thyroglobulin (Tg), thyroperoxidase (TPO), Na⁺/I ^– symporter, and the H₂O₂ generating enzymes [dual activity oxidase (DUOX)], which are all down-regulated (22, 23, 24, 25, 26). Thus, hypothyroidism may accompany HT not only because of irreversible apoptotic cell death (27, 28), but also because of impairment of the thyroid function without cellular destruction (26). This may explain the observation made by clinicians in their daily medical practice about the highly variable clinical outcome of HT patients. Some of them have a glandular atrophy along with pronounced hypothyroidism, whereas others remain euthyroid or subclinical hypothyroid for a while. Some patients can even sometimes recover from hypothyroidism toward euthyroidism, suggesting a possible reversibility of the immunological injury (29, 30, 31). Mechanisms responsible for changes in the thyroid function are not yet quite explained. Our previous paper (26) provided some clues on how IL-1/interferon (IFN) may alter TPO and DUOX protein expression without affecting cell survival. For instance, their effects are partially mediated by nitric oxide (NO) and can be overshadowed by increased concentrations of TSH, suggesting a mutual antagonism between Th1 cytokines and TSH-activated intracellular pathways. In the present paper, we propose that the local immune context and interactions between cytokines of different nature could also deeply influence the functional outcome of inflammatory thyroid glands. We looked at whether IL-4, a Th2 cytokine, and TGF-β or IL-10, two Th3 cytokines, may reverse IL-1/IFN (Th1 cytokines)-induced inhibitory effects on thyroid function. To answer this question, we used three different in vitro models of thyrocytes [two rat thyroid cell lines (PCCL3 and FRTL-5) and one in the human (thyrocytes in primary cultures)], in which, in the presence of IL-1/IFN, thyroid function is hampered without cell death (26).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell cultures
FRTL-5 cells (32) were a gift from Dr. P. Kopp (Northwestern University, Chicago, IL), and PCCL3 cells (a continuous line of nontransformed rat thyroid follicular cells) (33) were from Dr. F. Miot (Université Libre de Bruxelles, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Brussels, Belgium). FRTL-5 cells were grown to 80–90% confluence in Coon’s modified Ham’s F12 medium (BRL-Life Technologies, Inc., Paisley, Strathclyde, UK) supplemented with 5% newborn calf serum (NCS), 5 mU/ml TSH, 10 µg/ml insulin, 5 µg/ml transferrin, 10 ng/ml somatostatin, 10 ng/ml glycyl-L-histidyl-L-lysine-acetate, and 3.2 ng/ml hydrocortisone in a humidified atmosphere (5% CO₂). Cells were then incubated with insulin (1 µg/ml) and TSH (1 mU/ml) for 3 d (all reagents were from Sigma, Bornem, Belgium). For PCCL3, Coon’s medium was supplemented with 5% NCS, 1 mU/ml TSH, 1 µg/ml insulin, and 5 µg/ml transferrin. Recombinant rat IL-1 (2 ng/ml; CHEMICON Intl., Inc., Temecula, CA) and recombinant rat IFN (100 U/ml, CHEMICON) were added for 3 additional days, in combination or not with IL-4 (0.2, 2, or 20 ng/ml; a gift from Dr. Vanderbruggen, Université catholique de Louvain, Institute of Cellular Pathology, Brussels, Belgium), TGF-β (0.2, 2, or 20 ng/ml; CHEMICON), or IL-10 (0.2, 2, or 20 ng/ml; R&D Systems, Abingdon, UK) in the same medium containing 0.5% NCS and 1 mU/ml TSH. Finally, IL-10 (2 ng/ml) or TGF-β (2 ng/ml) was added alone or in combination with IL-4 (2 ng/ml).

Human thyroids from multinodular goiters were obtained at surgery after patients gave their informed consent. Thyrocytes were isolated according to Nilsson et al. (34) and suspended in modified Earle’s medium without phenol red containing 5% NCS, penicillin (50 U/ml), streptomycin (50 µg/ml), and Fungizone (2.5 µg/ml) (BRL-Life Technologies). They were plated in six-well plates (50 µg DNA/well) and cultured in a humidified atmosphere (5% CO₂) with 1 mU/ml TSH. After 1 wk with TSH, cells were incubated for 3 additional days with recombinant human IL-1 (2 ng/ml; R&D Systems) and recombinant human IFN (100 U/ml; R&D Systems) in the same medium but containing 0.5% NCS, in combination or not with IL-4 (2 ng/ml) or IL-10 (2 ng/ml). To investigate the role of NO, N°-nitroso-L-arginine methyl ester (L-NAME) (2.5 mM; Sigma) was added together with Th1 cytokines. The experiment was repeated twice.

For each experimental condition, the media of six wells were used for nitrite and Tg ELISA assays. Thereafter, cells from the first three wells were allocated to Western blotting and those from the last three to RT-PCR.

Nitrite assay
Nitrite accumulation in the medium of human thyrocytes was measured by the Griess reaction using a commercially available kit (Promega, Madison, WI).

Viability assay
Cell viability was assessed using the Alamar blueTM assay (Biosource Intl., Camarillo, CA), as previously described (26).

ELISA
The secretion of Tg by human thyrocytes was assessed by ELISA. Microplates were coated overnight at 4 C with a first monoclonal antihuman Tg antibody [A3 clone; a gift from J. J. M. DeVijlder, Amsterdam, The Netherlands; 10 µg/ml in carbonate buffer (0.05 M) (pH 9.6)]. After washing with PBS containing 0.1% Tween 20, wells were saturated with 5% nonfat dry milk in PBS (pH 7.4). After washing, 100 µl of each supernatant was incubated for 1 h at 37 C, thereafter washed and incubated for 1 h at 37 C with 100 µl of a secondary polyclonal anti-Tg antibody (DakoCytomation, Carpinteria, CA). One hundred microliters of EnVision antirabbit (1/20; DakoCytomation) were incubated 30 min at 37 C, and the reaction was revealed with OPD (o-Phenylenediamine; Sigma). Results were read at 492 nm and reported to a human Tg standard curve. To determine the optimal antibody concentration, dilutions of primary and secondary antibodies were performed in preliminary titrations.

Western blotting
Thyrocytes from three individual wells were suspended in Laemmli buffer [50 mM Tris HCl (pH 6.8), 2% sodium dodecyl sulfate, and 10% glycerol], containing a protease inhibitor cocktail (Sigma), and were sonicated for 30 sec. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL). DUOX (antibody provided by F. Miot) and TPO (antibody provided by J. Ruf, Université de la Méditerranée, Marseille, France) Western blottings were performed as previously described (26). Briefly, proteins (30 µg/lane) were heated at 95 C for 5 min in the loading buffer (Laemmli buffer containing 100 mM dithiothreitol and 0.1% bromophenol blue), separated by 8% SDS-PAGE, and transferred onto a nitrocellulose membrane (Hybond ECL; Amersham Biosciences, Rosendaal, The Netherlands). Membranes were blocked for 1 h at room temperature in PBS (pH 7.4), 5% non fat dry milk, 0.1% Tween, and incubated overnight at 4 C with the primary antibody at a dilution of 1:4000. Membranes were incubated for 1 h at room temperature with EnVision (1:200) peroxidase-labeled secondary antibody, and visualized with enhanced chemiluminescence (SuperSignal West Pico; Pierce) on CL-Xposure TM films (Pierce). Only DUOX expression was analyzed in FRTL-5 and PCCL3 cells.

RT-PCR
Cells were suspended in TriPure isolation reagent (Roche Diagnostics GmbH, Mannheim, Germany), and total RNA was purified according to the manufacturer’s protocol. RT was performed by incubating 2 µg RNA with 200 U Moloney murine leukemia virus reverse transcriptase (Invitrogen, Merelbeke, Belgium) in the recommended buffer containing 1 µl Rnasin (Promega), 0.5 mM each deoxynucleotide triphosphate (Promega), 2 µM oligo-deoxythymidine (Sigma), and 10 mM dithiothreitol (20 µl final volume) overnight at 42 C. H₂0 (80 µl) was then added, and the end products were used for PCR amplifications. cDNA (2.5 µl) was mixed with 0.65 U TaKaRa Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), 0.2 mM deoxynucleotide triphosphate, and 0.5 µM specific primers. Amplification cycles were as follows: 94 C for 3 min, cycles of 94 C/1 min, annealing temperature/2 min, and 72 C/1 min, followed by 10 min/72 C. Specific primers used were: 5'-GTGGCTGGCTGACATCAT-3' (sense human DUOX-1/2), 5'-TGCAGGGAGTTGAAGAA-3' (antisense human DUOX-1/2); 5'-CACGATGCAGAGAAACCTCAA-3' (sense human TPO), 5'-ATAGACTGGAGGGAGCCAT-3' (antisense human TPO), 5'-GTGGCTGGCTGACATCAT-3' (sense rat DUOX1/2), 5'-CCGTGAACAGACTCCTGT-3 (antisense rat DUOX1/2), 5'-CAGGTTTTGGTGGGAGAA-3' (sense rat TPO), 5'-CTGCACACTCATTAACATCTT-3' (antisense rat TPO), 5'-CATCCTGCGTCTGGACCT-3' (sense human and rat β-actin), and 5'-AGGAGGAGCAATGATCTTGAT-3' (antisense human and rat β-actin). All primers were intron spanning to avoid genomic DNA amplification. The annealing temperatures were 62 C for β-actin, 60 C for human TPO and rat DUOX, 56 C for human DUOX, and 58 C for rat TPO. PCR products were separated by agarose gel (1 or 1.5%) electrophoresis.

Data analysis and statistics
Data were expressed as mean ± SEM (n = 6 for nitrite and Tg ELISA). Statistical analyses were performed using ANOVA, followed by the Tukey-Kramer Multiple Comparison Test (GraphPad InStat, San Diego, CA), and P < 0.05 was considered statistically significant.

Western blots were scanned and quantified by densitometry using the National Institutes of Health (NIH) Scion Image Analysis Software (NIH, Bethesda, MD). Values were expressed as mean ± SEM of one representative experiment (n = 5 or 3 for human thyrocytes, and n = 6 for FRTL-5 and PCCL3 cells). Digital images of ethidium bromide-stained agarose gels were quantified by densitometry using the NIH Scion Image Analysis Software. Values were normalized by reporting the signal intensity to β-actin expression. Values were expressed as mean ± SEM of one representative experiment performed in triplicate for both FRTL-5 cells and human thyrocytes, and n = 6 for PCCL3 cells. The statistical analysis was performed using the unpaired t test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-4 counters IL-1/IFN-induced effects in PCCL3, FRTL-5, and human thyrocytes
DUOX protein was detected in cells incubated with TSH (1 mU/ml) as a single band of 179 kDa in control FRTL-5 and PCCL3 cells. Its expression was down-regulated by IL-1/IFN. Coincubation with IL-4 blocked the Th1-induced inhibitory effect on DUOX protein expression in PCCL3 cells and in a dose-dependent manner (Fig. 1A). Cells treated with IL-4 alone showed no alteration in DUOX protein expression (data not shown). No effect on cell viability was observed whatever the dose used (data not shown). IL-4 at a dose of 2 ng/ml was then used for the following experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 1. IL-4 counteracts the inhibitory effects of IL-1/IFN in PCCL3 and FRTL-5 cells. A, Total protein extracts of PCCL3 cells were analyzed by Western blotting using a DUOX antibody. IL-4 was incubated along with IL-1/IFN at increasing concentrations of 0.2, 2, and 20 ng/ml. B, Quantification of DUOX protein expression (upper panel) and of DUOX (middle panel) and TPO (lower panel) mRNA in PCCL3 and FRTL-5 cells. Densitometric values of Western blots are expressed as mean ± SEM of one representative experiment (n = 6). Densitometric values of RT-PCR detection of DUOX and TPO mRNAs, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment (n = 6 and 3 for PCCL3 and FRTL-5 cells, respectively). IL-4 was added at a concentration of 2 ng/ml. *, P < 0.05 vs. control cells. +, P < 0.05 vs. IL-1/IFN-treated cells.

Identical results were obtained in primary cultures of human thyrocytes. DUOX and TPO proteins were detected as a single band of 179 and 110 kDa, respectively, in control thyrocytes incubated with TSH (1 mU/ml). Without TSH, TPO and, to a lesser extent, DUOX expression was low, as expected (data not shown) (26). IL-1/IFN significantly decreased DUOX and TPO protein expression (40 and 38% decrease, respectively; P < 0.05), an effect prevented by IL-4 cotreatment. Similarly, DUOX and TPO mRNA expression was significantly reduced after IL-1/IFN treatment (47 and 63% decrease, respectively; P < 0.05). In the presence of IL-4, DUOX and TPO mRNA expression was restored (Fig. 2, A and B). IL-1/IFN significantly decreased Tg production (51% decrease; P < 0.05; Fig. 2C), as previously reported (22, 24, 35). In cells coincubated with IL-4, Tg release was restored.

View larger version (21K): [in this window] [in a new window]   FIG. 2. IL-4 counteracts the inhibitory effects of IL-1/IFN in human thyrocytes. A, DUOX protein (left panel) and mRNA (right panel) expression in human normal thyrocytes. B, TPO protein (left panel) and mRNA (right panel) expression in human normal thyrocytes. Densitometric values of Western blot results are expressed as mean ± SEM of one representative experiment performed in triplicate. Densitometric values of RT-PCR, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment performed in triplicate. IL-4 was added at a concentration of 2 ng/ml. C, Tg production by human thyrocytes. Tg accumulation in the supernatant was assayed by ELISA. Results are expressed as means ± SEM of six individual wells of one representative experiment. IL-4 was added at a concentration of 2 ng/ml. D, Nitrite accumulation in the culture medium of human thyrocytes. Results are expressed as means ± SEM of six individual wells of one representative experiment. IL-4 was added at a concentration of 2 ng/ml. *, P < 0.05 vs. control cells. +, P < 0.05 vs. IL-1/IFN-treated cells.

IL-1/IFN-induced DUOX and TPO down-regulation is not affected by Th3 cytokines in PCCL3 and FRTL-5 cells
In PCCL3 cells, TGF-β alone induced a dose-dependent down-regulation of DUOX protein expression (data not shown). In contrast with IL-4, the inhibitory effects of IL-1/IFN on DUOX protein expression were not counteracted when cells were coincubated with TGF-β (Fig. 3A). No effect on cell viability was observed (data not shown). The dose of 2 ng/ml was used for the following experiments.

View larger version (12K): [in this window] [in a new window]   FIG. 3. TGF-β does not reverse the inhibitory effects of IL-1/IFN in PCCL3 and FRTL-5 cells. A, Total protein extracts of PCCL3 cells were analyzed by Western blotting using a DUOX antibody. TGF-β was incubated along with IL-1/IFN at increasing concentrations of 0.2, 2, and 20 ng/ml. B, Quantification of DUOX protein expression (upper panel) and of DUOX (middle panel) and TPO (lower panel) mRNA in PCCL3 and FRTL-5 cells. Densitometric values of Western blots are expressed as mean ± SEM of one representative experiment (n = 6). Densitometric values of RT-PCR detection of DUOX and TPO mRNAs, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment (n = 6 and 3 for PCCL3 and FRTL-5 cells, respectively). TGF-β was added at a concentration of 2 ng/ml. *, P < 0.05 vs. control cells.

To verify if the well-known inhibitory action of TGF-β (37) was specific or inherent to all Th3 cytokines, we tested a second Th3 cytokine, IL-10. In PCCL3 cells, IL-10 alone also inhibited DUOX protein expression in a dose-dependent manner (data not shown). In contrast with IL-4, the inhibitory effects of IL-1/IFN on DUOX protein expression were not counteracted when cells were coincubated with IL-10 (Fig. 4A). No effect on cell viability was observed (data not shown). The concentration of 2 ng/ml was used for the following experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 4. IL-10 does not reverse the inhibitory effects of IL-1/IFN in PCCL3 cells. A, Total protein extracts of PCCL3 cells were analyzed by Western blotting using a DUOX antibody. IL-10 was incubated along with IL-1/IFN at increasing concentrations of 0.2, 2, or 20 ng/ml. B, Quantification of DUOX protein expression (upper panel) and of DUOX (middle panel) and TPO (lower panel) mRNA in PCCL3. Densitometric values of Western blots are expressed as mean ± SEM of one representative experiment (n = 6). Densitometric values of RT-PCR detection of DUOX and TPO mRNAs, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment (n = 6). IL-10 was added at a concentration of 2 ng/ml. *, P < 0.05 vs control cells.

IL-1/IFN significantly decreased DUOX and TPO protein expression (45 and 51% decrease, respectively; P < 0.05) in primary cultures of human thyrocytes. IL-10 cotreatment did not prevent this effect. Similar results were obtained when analyzing DUOX and TPO mRNA expression by RT-PCR. The expression of the two mRNAs was significantly reduced after IL-1/IFN treatment (46 and 73% decrease, respectively; P < 0.05), but not restored by IL-10 (Fig. 5, A and B). The inhibition of Tg release induced by IL-1/IFN was not prevented by IL-10 (Fig. 5C).

View larger version (19K): [in this window] [in a new window]   FIG. 5. IL-10 does not reverse the inhibitory effects of IL-1/IFN in human thyrocytes. A, DUOX protein (left panel) and mRNA (right panel) expression in human normal thyrocytes. B, TPO protein (left panel) and mRNA (right panel) expression in human normal thyrocytes. Densitometric values of Western blots are expressed as mean ± SEM of one representative experiment performed in triplicate. Densitometric values of RT-PCR, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment performed in triplicate. C, Tg production by human thyrocytes. Tg accumulation in the supernatant was assayed by ELISA. Results are expressed as means ± SEM of six individual wells of one representative experiment. D, Nitrite accumulation in the culture medium of human thyrocytes. Results are expressed as means ± SEM of six individual wells of one representative experiment. *, P < 0.05 vs. control cells. +, P < 0.05 vs. IL-1/IFN-treated cells.

IL-4 counters IL-10- but not TGF-β-induced DUOX and TPO down-regulation in PCCL3 cells
In PCCL3 cells, the inhibition of DUOX protein expression induced by IL-10, but not that of TGF-β (another Th3 cytokine), was reversed after coincubation with IL-4 (Fig. 6A). Identical results were obtained when DUOX and TPO mRNA expression was analyzed (Fig. 6, B and C).

View larger version (15K): [in this window] [in a new window]   FIG. 6. IL-4 counteracts the inhibitory effects of IL-10, but not those of TGF-β in PCCL3 cells. A, Densitometric values of DUOX protein expression are expressed as mean ± SEM of one representative experiment (n = 6). IL-4 (2 ng/ml) cotreatment with IL-10 (2 ng/ml) restored DUOX protein expression but had no influence on TGF-β (2 ng/ml)-induced down-regulation. B and C, RT-PCR detection of DUOX (B) and TPO (C) mRNAs. Densitometric values, adjusted to the β-actin signal, are expressed as mean ± SEM of one representative experiment (n = 6). IL-4 reversed the decrease in the expression of both mRNA induced by IL-10, but not that induced by TGF-β. *, P < 0.05 vs. control cells. +, P < 0.05 vs. IL-10-treated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-4 reversed IL-1/IFN effects in the two rat thyroid cell lines, as well as in human thyrocytes. It blocked the decrease in DUOX and TPO expression, both at transcriptional and translational levels, and provoked a complete recovery of Tg secretion. These effects are evocative of those reported in pancreatic islet cells. Therefore, after a 12-h treatment with Th1 cytokines, whereas cell survival is not affected, IL-4 is able to reverse the decrease in insulin secretion. After a treatment of 72 h, IL-4 has no more effect on insulin secretion, but blocks cell apoptosis (5). In our model there was no cell death. Thus, changes in protein expression or secretion resulted from a direct effect of IL-4 on thyroid cell function, instead of an eventual recovery from IL-1/IFN-induced cell death (26). This obviously does not exclude the involvement of other mechanisms of protection activated in vivo, such as the likelihood for IL-4 to interact directly with inflammatory cells.

IL-4 effects were not mediated by NO, even though its production was partially blunted. Thus, whereas IL-4 fully restored DUOX and TPO expression, L-NAME, a potent NO synthase inhibitor, was unable of such an effect (26). Likewise, Tg secretion was restored by IL-4, but not L-NAME (35). Of note, IL-4 effects were observed in FRTL-5 and PCCL3 cells, two cell models that are known for their inability to release NO. Thus, IL-4 may directly interfere with IL-1/IFN transduction pathways in thyrocytes.

TGF-β did not influence IL-1/IFN-induced down-regulation of DUOX and TPO expression in PCCL3 and FRTL-5 cells. These results are in contrast with those observed in pancreatic β-cells, in which TGF-β can prevent IL-1β-induced inhibition of insulin secretion. In addition, TGF-β is able to suppress the immune reaction in experimental autoimmune encephalomyelitis and during allergy (10, 38, 39, 40). These discrepancies are not really surprising. Our results should actually be interpreted having in mind previously reported TGF-β effects in the thyroid gland. Therefore, accumulated evidence indicates that TGF-β alters several important steps of hormonogenesis as well as the thyroid cell proliferation (37, 41, 42, 43). We also found that DUOX protein expression was reduced in cells incubated with TGF-β alone and that pretreatment with TGF-β made Th1 cytokine-induced effects even worse instead of switching them off (data not shown). This suggests that in contrast with IL-4, TGF-β is a potent inhibitor of thyroid function and cannot reverse Th1 effects. Nevertheless, cytokine-induced effects should be interpreted differently because they are investigated in vivo or in vitro. An in vivo paper (44) lately reported a favorable role for TGF-β in the outcome of thyroiditis. Thus, TGF-β may overturn Th1 cytokine-induced inhibitory effects by acting directly on Th1 lymphocytes. By contrast, when it acts directly on thyrocytes, its inhibitory effects become predominant.

To verify whether findings with TGF-β were specific for this molecule, but not necessarily for other Th3 cytokines, we looked at the effects of IL-10. They were similar. Thus, IL-10 inhibited DUOX and TPO expression, without affecting Th1 inhibitory effects. It is the first time that a direct inhibitory effect of IL-10 on thyrocyte function is reported. In fact, little is known about how IL-10 is affecting the outcome of autoimmune thyroiditis. In vivo reports indicate a rather beneficial role in experimental autoimmune thyroiditis (45, 46, 47). Additional investigations are required to figure out what IL-10 really does in the thyroid, and should be performed in vitro and in vivo to sort out direct (on thyrocytes) and indirect (on infiltrating cells) effects of this cytokine. At this point, it seems reasonable to propose that, as for TGF-β, IL-10 inhibits the thyroid cell function when it acts directly on thyrocytes, but plays instead a rather favorable role in reducing autoimmunity when it is systemically administered.

In addition to its ability to reverse inhibitory effects of Th1 cytokines, IL-4 was also able to switch off inhibitory effects of IL-10, but not those of TGF-β. These results suggest that both Th3 cytokines actually act through different pathways and that IL-4 may interfere with transduction pathways activated by ILs but not by TGF-β. Although appropriate tools were not used in this study to sort out this question, literature data suggest that, indeed, intracellular signaling pathways are most likely different. Therefore, ILs act mainly through the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) signal pathway, involving various STAT molecules (STAT6 for IL-4, STAT1 for IFN, and STAT1, 3, and 5 for IL-10), and nuclear factor B for IL-1 (12, 48, 49), whereas TGF-β acts through Smads (50, 51). Unfortunately, nothing is known yet about the signaling pathways activated by IL-10 and IL-4 in the thyroid cells.

Despite the obvious limitation of in vitro models, our data bring new insights on how cytokines functionally influence the thyrocyte, and why the thyroid function of HT patients may vary between hypothyroidism and euthyroidism. Although thyroiditis is usually considered as predominantly of Th1 type (4), different cytokines may actually coexist (2, 3, 27). Depending on their nature and/or local content, Th1 cytokines may either be lethal or impair thyroid function by altering hormone synthesis. In the presence of Th2 cytokines (e.g. IL-4), the intraglandular conditions change because they can block thyrocyte-specific Th1 cytokine-induced effects, notwithstanding parallel effects on immune cells. These results are in line with those previously reported in which Th1 cytokine effects faded away in the presence of TSH. This could explain why hashitoxicosis patients with TSH receptor autoantibodies may switch from hypothyroidism to euthyroidism and even hyperthyroidism.

In conclusion, when thyrocytes are treated with IL-1/IFN, their viability is not affected, but DUOX, TPO, and Tg expression or production is hindered. This can be overturned by IL-4, a Th2 cytokine. Therefore, the ability of Th1 cytokines to affect the thyroid hormonal status is influenced by the local immune context. By contrast, Th3 cytokines, TGF-β and IL-10, failed to switch off Th1 effects but, rather, exerted inhibitory effects as Th1 cytokines. IL-10- but not TGF-β-induced inhibition was reversed by IL-4, indicating that various intracellular signaling pathways are differentially regulated according to the cytokine predominant in the scene. Further experiments are required to sort out what intracellular signaling pathways are activated by these cytokines.

	Acknowledgments

 
We thank Dr Y. Nizet (Unité d’Immunologie Expérimentale, Université catholique de Louvain) for his technical support.

	Footnotes

 
The work is supported by a Fonds spécial de recherche grant from Université catholique de Louvain, Belgium. A.-C.G. is the recipient of a grant from the Belgian National Fund for Scientific Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 10, 2008

Abbreviations: DUOX, Dual oxidase; FRTL-5, Fischer Rat Thyroid Low Serum 5%; HT, Hashimoto’s thyroiditis; IFN, interferon; L-NAME, N°-nitroso-L-arginine methyl ester; NCS, newborn calf serum; NO, nitric oxide; PCCL3, pancreatic cancer cell line 3; STAT, signal transducer and activator of transcription; Tg, thyroglobulin; TPO, thyroperoxidase.

Received September 24, 2007.

Accepted for publication December 28, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Reed PR, Davies TF 2002 Hypothyroidism and thyroiditis. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky WH, eds. Williams textbook of endocrinology. 10th ed. Philadelphia: Saunders; 423–455
2. Rapoport B, McLachlan SM 2001 Thyroid autoimmunity. J Clin Invest 108:1253–1259[CrossRef][Medline]
3. Stassi G, De Maria R 2002 Autoimmune thyroid disease: new models of cell death in autoimmunity. Nat Rev Immunol 2:195–204[CrossRef][Medline]
4. Colin IM, Isaac J, Dupret P, Ledant T, D’Hautcourt JL 2004 Functional lymphocyte subset assessment of the Th1/Th2 profile in patients with autoimmune thyroiditis by flowcytometric analysis of peripheral lymphocytes. J Biol Regul Homeost Agents 18:72–76[Medline]
5. Marselli L, Dotta F, Piro S, Santangelo C, Masini M, Lupi R, Realacci M, del Guerra S, Mosca F, Boggi U, Purrello F, Navalesi R, Marchetti P 2001 Th2 cytokines have a partial, direct protective effect on the function and survival of isolated human islets exposed to combined proinflammatory and Th1 cytokines. J Clin Endocrinol Metab 86:4974–4978[Abstract/Free Full Text]
6. Walz M, Overbergh L, Mathieu C, Kolb H, Martin S 2002 A murine interleukin-4-Ig fusion protein regulates the expression of Th1- and Th2-specific cytokines in the pancreas of NOD mice. Horm Metab Res 34:561–569[CrossRef][Medline]
7. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE 1999 The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 17:701–738[CrossRef][Medline]
8. Gor DO, Rose NR, Greenspan NS 2003 TH1-TH2: a procrustean paradigm. Nat Immunol 4:503–505[CrossRef][Medline]
9. Prud’homme GJ, Piccirillo CA 2000 The inhibitory effects of transforming growth factor-β-1 (TGF-β1) in autoimmune diseases. J Autoimmun 14:23–42[CrossRef][Medline]
10. Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA 2006 Mechanisms of immune suppression by interleukin-10 and transforming growth factor-β: the role of T regulatory cells. Immunology 117:433–442[CrossRef][Medline]
11. Boyman O, Purton JF, Surh CD, Sprent J 2007 Cytokines and T-cell homeostasis. Curr Opin Immunol 19:320–326[CrossRef][Medline]
12. Moore KW, de Waal MR, Coffman RL, O’Garra A 2001 Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683–765[CrossRef][Medline]
13. Louis H, Van Laethem JL, Wu W, Quertinmont E, Degraef C, Van den Berg K, Demols A, Goldman M, Le Moine O, Geerts A, Deviere J 1998 Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology 28:1607–1615[CrossRef][Medline]
14. Loser K, Apelt J, Voskort M, Mohaupt M, Balkow S, Schwarz T, Grabbe S, Beissert S 2007 IL-10 controls ultraviolet-induced carcinogenesis in mice. J Immunol 179:365–371[Abstract/Free Full Text]
15. Demols A, Van Laethem JL, Quertinmont E, Degraef C, Delhaye M, Geerts A, Deviere J 2002 Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol 282:G1105–G1112
16. Yu S, Sharp GC, Braley-Mullen H 2006 Thyrocytes responding to IFN- are essential for development of lymphocytic spontaneous autoimmune thyroiditis and inhibition of thyrocyte hyperplasia. J Immunol 176:1259–1265[Abstract/Free Full Text]
17. Svenson M, Kayser L, Hansen MB, Rasmussen AK, Bendtzen K 1991 Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5. Cytokine 3:125–130[CrossRef][Medline]
18. Kasai K, Hiraiwa M, Emoto T, Kuroda H, Hattori Y, Mochizuki Y, Nakamura T, Shimoda S 1990 Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells. Horm Metab Res 22:75–79[Medline]
19. Vella V, Mineo R, Frasca F, Mazzon E, Pandini G, Vigneri R, Belfiore A 2004 Interleukin-4 stimulates papillary thyroid cancer cell survival: implications in patients with thyroid cancer and concomitant Graves’ disease. J Clin Endocrinol Metab 89:2880–2889[Abstract/Free Full Text]
20. Franzen A, Piek E, Westermark B, ten Dijke P, Heldin NE 1999 Expression of transforming growth factor-β1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function. Endocrinology 140:4300–4310[Abstract/Free Full Text]
21. Rasmussen AK, Diamant M, Blichert-Toft M, Bendtzen K, Feldt-Rasmussen U 1997 The effects of interleukin-1β (IL-1β) on human thyrocyte functions are counteracted by the IL-1 receptor antagonist. Endocrinology 138:2043–2048[Abstract/Free Full Text]
22. Caraccio N, Giannini R, Cuccato S, Faviana P, Berti P, Galleri D, Dardano A, Basolo F, Ferrannini E, Monzani F 2005 Type I interferons modulate the expression of thyroid peroxidase, sodium/iodide symporter, and thyroglobulin genes in primary human thyrocyte cultures. J Clin Endocrinol Metab 90:1156–1162[Abstract/Free Full Text]
23. Caturegli P, Hejazi M, Suzuki K, Dohan O, Carrasco N, Kohn LD, Rose NR 2000 Hypothyroidism in transgenic mice expressing IFN- in the thyroid. Proc Natl Acad Sci USA 97:1719–1724[Abstract/Free Full Text]
24. Rasmussen AK, Bendtzen K, Feldt-Rasmussen U 2000 Thyrocyte-interleukin-1 interactions. Exp Clin Endocrinol Diabetes 108:67–71[CrossRef][Medline]
25. Vasu C, Holterman MJ, Prabhakar BS 2003 Modulation of dendritic cell function and cytokine production to prevent thyroid autoimmunity. Autoimmunity 36:389–396[CrossRef][Medline]
26. Gerard AC, Boucquey M, van den Hove MF, Colin IM 2006 Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1/IFN-, an effect partially mediated by nitric oxide. Am J Physiol Endocrinol Metab 291:E242–E253
27. Liblau RS, Singer SM, McDevitt HO 1995 Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 16:34–38[CrossRef][Medline]
28. Mackenzie WA, Davies TF 1987 An intrathyroidal T-cell clone specifically cytotoxic for human thyroid cells. Immunology 61:101–103[Medline]
29. Fatourechi V, McConahey WM, Woolner LB 1971 Hyperthyroidism associated with histologic Hashimoto’s thyroiditis. Mayo Clin Proc 46:682–689[Medline]
30. Kraiem Z, Baron E, Kahana L, Sadeh O, Sheinfeld M 1992 Changes in stimulating and blocking TSH receptor antibodies in a patient undergoing three cycles of transition from hypo to hyper-thyroidism and back to hypothyroidism. Clin Endocrinol (Oxf) 36:211–214[Medline]
31. Takasu N, Yamada T, Sato A, Nakagawa M, Komiya I, Nagasawa Y, Asawa T 1990 Graves’ disease following hypothyroidism due to Hashimoto’s disease: studies of eight cases. Clin Endocrinol (Oxf) 33:687–698[Medline]
32. Ambesi-Impiombato FS, Parks LA, Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA 77:3455–3459[Abstract/Free Full Text]
33. Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M, Vecchio G 1987 One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Mol Cell Biol 7:3365–3370[Abstract/Free Full Text]
34. Nilsson M, Husmark J, Nilsson B, Tisell LE, Ericson LE 1996 Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function. Eur J Endocrinol 135:469–480[Abstract/Free Full Text]
35. Rasmussen AK, Di Marco R, Diamant M, Feldt-Rasmussen U, Bendtzen K 1994 Nitric oxide production is not involved in the effects of interleukin-1 β on cAMP, thyroglobulin and interleukin-6 in TSH-stimulated human thyroid cells. Autoimmunity 19:239–245[Medline]
36. van den Hove MF, Stoenoiu MS, Croizet K, Couvreur M, Courtoy PJ, Devuyst O, Colin IM 2002 Nitric oxide is involved in interleukin-1-induced cytotoxicity in polarised human thyrocytes. J Endocrinol 173:177–185[Abstract]
37. Pekary AE, Hershman JM 1998 Tumor necrosis factor, ceramide, transforming growth factor-β1, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells. Endocrinology 139:703–712[Abstract/Free Full Text]
38. Mabley JG, Cunningham JM, John N, Di Matteo MA, Green IC 1997 Transforming growth factor β 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing β-cell line. J Endocrinol 155:567–575[Abstract/Free Full Text]
39. McGeachy MJ, Anderton SM 2005 Cytokines in the induction and resolution of experimental autoimmune encephalomyelitis. Cytokine 32:81–84[CrossRef][Medline]
40. Bach JF 2001 Non-Th2 regulatory T-cell control of Th1 autoimmunity. Scand J Immunol 54:21–29[CrossRef][Medline]
41. Tsushima T, Arai M, Saji M, Ohba Y, Murakami H, Ohmura E, Sato K, Shizume K 1988 Effects of transforming growth factor-β on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture. Endocrinology 123:1187–1194[Abstract/Free Full Text]
42. Taton M, Lamy F, Roger PP, Dumont JE 1993 General inhibition by transforming growth factor β 1 of thyrotropin and cAMP responses in human thyroid cells in primary culture. Mol Cell Endocrinol 95:13–21[CrossRef][Medline]
43. Colletta G, Cirafici AM, Di Carlo A 1989 Dual effect of transforming growth factor β on rat thyroid cells: inhibition of thyrotropin-induced proliferation and reduction of thyroid-specific differentiation markers. Cancer Res 49:3457–3462[Abstract/Free Full Text]
44. Seddon B, Mason D 1999 Regulatory T cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4(+)CD. J Exp Med 189:279–288[Abstract/Free Full Text]
45. Wang SJ, Xu HX, Liu GZ 2000 Tripterygium wilfordii saponins and interleukin-10 prevent induction of experimental autoimmune thyroiditis by dendritic cells. Acta Pharmacol Sin 21:919–923[Medline]
46. Mignon-Godefroy K, Rott O, Brazillet MP, Charreire J 1995 Curative and protective effects of IL-10 in experimental autoimmune thyroiditis (EAT). Evidence for IL-10-enhanced cell death in EAT. J Immunol 154:6634–6643[Abstract]
47. Batteux F, Trebeden H, Charreire J, Chiocchia G 1999 Curative treatment of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for interleukin-10. Eur J Immunol 29:958–963[CrossRef][Medline]
48. Haque SJ, Sharma P 2006 Interleukins and STAT signaling. Vitam Horm 74:165–206[Medline]
49. Cnop M, Welsh N, Jonas JC, Jorns A, Lenzen S, Eizirik DL 2005 Mechanisms of pancreatic β-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54(Suppl 2):S97–S107
50. Feng XH, Derynck R 2005 Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693[CrossRef][Medline]
51. Costamagna E, Garcia B, Santisteban P 2004 The functional interaction between the paired domain transcription factor Pax8 and Smad3 is involved in transforming growth factor-β repression of the sodium/iodide symporter gene. J Biol Chem 279:3439–3446[Abstract/Free Full Text]

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