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. 2010 Oct;91(5):420–425. doi: 10.1111/j.1365-2613.2010.00715.x

Thyrocyte interleukin-18 expression is up-regulated by interferon-γ and may contribute to thyroid destruction in Hashimoto's thyroiditis

Zhe Liu 1,1, Haining Wang 1,1, Wenhua Xiao 1, Chen Wang 1, Guoqiang Liu 1, Tianpei Hong 1
PMCID: PMC3003839  PMID: 20586818

Abstract

Interferon-γ (IFN-γ) has a direct role in thyroid destruction in autoimmune thyroiditis. Interleukin-18 (IL-18), a pro-inflammatory cytokine with potent IFN-γ inducing activities, may play an important role in Th1-mediated autoimmune diseases. The purpose of this study was to characterize the expression and localization of IL-18 in the thyroid tissues of Hashimoto's thyroiditis (HT) and to investigate the effect of IFN-γ on IL-18 expression in isolated human thyroid follicular cells (TFCs). Thyroid tissues obtained from six euthyroid patients with HT and six control subjects were used to detect IL-18 expression by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. Human TFCs were isolated and incubated for 48 h with or without IFN-γ, tumour necrosis factor-α (TNF-α) or IL-1β. IL-18 expression was analysed by RT-PCR, immunofluorescent double staining and western blot. We found that IL-18 expression was increased in the thyroid tissues of HT compared with control thyroid tissues. TFCs were major cell types expressing IL-18 in the thyroid tissues of HT. IL-18 was constitutively expressed in isolated human TFCs, and the expression was significantly up-regulated by IFN-γ rather than TNF-α or IL-1β. Western bolt revealed that a 24-kDa band corresponding to pro-IL-18 was broadened in the lysates of IFN-γ-treated TFCs. Our results demonstrated that IL-18 expression is up-regulated in the TFCs of HT patients and in primary human TFCs exposed to IFN-γ. Therefore, intrathyroidal interaction between IL-18 and IFN-γ may have a role in promoting the local immune response, which contributes to the thyroid destruction seen in HT.

Keywords: autoimmune thyroiditis, cytokine, thyroid gland

Introduction

Interleukin-18 (IL-18), previously called interferon-γ (IFN-γ) inducing factor, was identified originally as a pro-inflammatory cytokine derived from Kupffer cells of mice conditioned with Propionibacterium acnes and challenged with lipopolysaccharide (LPS) (Okamura et al. 1995). In synergy with IL-12, IL-18 stimulates IFN-γ production in T cells, potentiates type 1 T helper (Th1) cell development, enhances production of Th1-type cytokines and induces NK cell cytotoxicity (Boraschi & Dinarello 2006). Moreover, it has been shown that IL-18 is produced by a variety of cell types originated from both immune and non-immune systems, suggesting that IL-18 may have a wide range of cellular sources and functions apart from being a macrophage-derived inducer of IFN-γ production from Th1 cells (Boraschi & Dinarello 2006). IL-18 is synthesized as a non-functional pro-IL-18, and the precursor protein is then processed via either caspase-1-dependent or caspase-1-independent pathway into a bioactive mature form (Ghayur et al. 1997; Sugawara et al. 2001).

Hashimoto's thyroiditis (HT) is an autoimmune disorder in which Th1-mediated immune response contributes to the destruction of thyroid follicular cells (TFCs) (Heuer et al. 1996; Phenekos et al. 2004). Th1 cytokine IFN-γ is produced intrathyroidally by infiltrating inflammatory cells, acting in a paracrine manner (Weetman 1994,Heuer et al. 1996). IFN-γ potentiates the expression of major histocompatibility complex class II molecules and adhesion molecules in TFCs (Weetman 1994). Importantly, a direct role of IFN-γ in autoimmune thyroid destruction has been documented in the thyroid-specific IFN-γ transgenic mice (Caturegli et al. 2000). Furthermore, it has been reported that thyrocytes responding to IFN-γ are essential for the development of lymphocytic spontaneous autoimmune thyroiditis and inhibition of thyrocyte proliferation in NOD.H-2h4 mice (Yu et al. 2006).

In view of the potential importance of IL-18 in the Th1-type immune response, IL-18 may have a key role in the autoimmune pathology of HT. Indeed, IL-18 expression in TFCs which is generally observed in close relation to a lymphocytic infiltrate has been shown in thyroid tissue from a patient with HT, suggesting that IL-18 might be a secreted immunomodulator in HT (Takiyama et al. 2002). However, intrathyroidal inflammatory cascade via interaction between IFN-γ and IL-18 remains to be elucidated. Therefore, the aims of this study were to investigate whether IL-18 was expressed in the TFCs of patients with HT and whether IFN-γ affected IL-18 expression in isolated human TFCs.

Materials and methods

Regents

Recombinant human (rh) IFN-γ and rhIL-1β were purchased from R & D Systems (Minneapolis, MN, USA). rhTNF-α and rh-pro-IL-18 were from PeproTech (Rocky Hill, NJ, USA). DMEM, foetal calf serum (FCS), collagenase IV and trypsin were purchased from Gibco Biotechnology (Carlsbad, CA, USA). DMEM which contains 11 mM glucose was supplemented with 20 mM HEPES buffer, 2 mM l-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin. Rabbit anti-human IL-18 polyclonal antibody was purchased from MBL International (Woburn, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody was from Zymed Laboratories (San Francisco, CA, USA). The primers for IL-18 and β-actin were from Beijing Aoke Biotechnology (Beijing, China). All other chemicals were purchased from Sigma (St Louis, MO, USA) unless otherwise indicated.

Specimens

For ex vivo studies, thyroid tissues were obtained from six euthyroid patients with HT (five females and one male, with an average age of 42.3 ± 10.4 years) by using automatic biopsy device for diagnostic purposes. Normal thyroid tissues collected from six laryngocarcinoma patients undergoing total laryngectomy (four females and two males, with an average age of 43.8 ± 5.7 years) were included as controls. Freshly sampled thyroid tissues were used for both immunohistochemical staining and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. For in vitro studies, normal thyroid tissues adjacent to tumour were taken from thyroid tumour patients who received thyroid resection. The thyroid tissues were used as a source for TFC isolation and culture. Informed consent was obtained from each patient. The project was approved by the Ethical Committee of Peking University Health Science Center.

Isolation and culture of TFCs

Fresh thyroid tissues were washed with ice-cold HBSS, and fat and connective tissues were carefully removed. After minced mechanically, the tissues were digested with 3 U/ml collagenase IV for 120 min at 37 °C, followed by digestion with 0.5 U/ml trypsin for another 30 min. The suspension was passed through a 200-μm filter to remove undigested material and centrifuged for 10 min at 300 g at 4 °C. Following several washes with HBSS with centrifugation for 2 min at 300 g, the cellular pellet was resuspended in DMEM supplemented with 20% FCS, and incubated at 37 °C in humidified CO2 incubator. After incubation for 48 h, the medium containing 10% FCS was changed, and the cells were further preincubated for another 48 h. Eligible TFC preparations used in this study were identified as more than 90% of cells expressing thyroglobulin as determined by immunofluorescent staining. The TFCs were treated with 250 U/ml (or 500 U/ml) IFN-γ, 500 U/ml TNF-α or 50 U/ml IL-1β for 48 h. After treatment, the cells were collected for RT-PCR, immunofluorescence and western blot analyses.

RT-PCR analysis

Total RNA was extracted from both the thyroid tissues and the TFCs using TRIzol reagent (Gibco) according to the manufacture's instructions. cDNA was prepared with the SuperScription First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) as described previously (Hong et al. 2000). The cDNAs were amplified by polymerase chain reaction (PCR). The sequence of primers used for PCR was 5′-ATG GCT GCT GAA CCA GTA G-3′ and 5′-TCT ACA GTC AGA ATC AGT CAT-3′ (225 bp) for IL-18, and 5′-GGC ATG GGT CAG AAG GAT TCC-3′ and 5′-ATG TCA CGC ACG ATT TCC CGC-3′ (500 bp) for β-actin. Each semiquantitative RT-PCR analysis was performed with a set of IL-18 primers in combination with a set of primer for house-keeping gene β-actin as internal standard. The conditions of RT-PCR reaction were as follows: 1 cycle of 94 °C, 5 min; 30 cycles of 94 °C, 30 s; 55 °C, 30 s; 72 °C, 45 s and 1 cycle of 72 °C, 5 min. The RT-PCR products were separated on a 1.5% agarose gel. The gel was stained with ethidium bromide and scanned using an ultraviolet gel imaging system (Bio-Rad, Hercules, CA, USA). Gene expression levels were represented as ratios of target gene to co-amplified internal standard.

Immunohistochemistry

The thyroid tissues were fixed in 10% buffered formaldehyde, processed by standard procedure and embedded in paraffin. Immunohistochemical staining was performed on 4 μm-thick paraffin-embedded thyroid tissue sections. Briefly, sections were deparaffinized in xylene and rehydrated in graded alcohol. The tissues were incubated for 20 min with 3% hydrogen peroxide in methanol to block endogenous peroxidase activity. After washed in phosphate-buffered saline (PBS), the sections were incubated with rabbit anti-human IL-18 antibody overnight at 4 °C. On negative control sections, the step with the primary antibody was omitted. Then, the slides were rinsed with PBS again and incubated with HRP-conjugated goat anti-rabbit IgG antibody for 30 min at 37 °C. Finally, the reaction was visualized with 3, 3′-diaminobenzidine tetrahydrochloride for 2–3 min, which resulted in a brown colour.

Immunofluorescence

The TFCs were grown on coverslips and then fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were rinsed with PBST (PBS with 0.5% Triton X-100) thrice and incubated in PBS containing 10% goat serum and 0.01% Triton X-100 for blocking, and then incubated overnight at 4 °C with rabbit anti-human IL-18 antibody or mouse anti-human thyroglobulin monoclonal antibody. After three washes with PBST, the cells were incubated with TRITC-conjugated anti-rabbit or FITC-conjugated anti-mouse IgG for 1 h at room temperature. Following three washes with PBS, the samples were mounted on slides and examined with a fluorescence microscope.

Western blot

The cell lysates were mixed with SDS loading buffer and the protein in the lysates was separated by 15% SDS–PAGE. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. After blocked with blocking buffer, the membrane was incubated overnight at 4 °C with rabbit anti-human IL-18 antibody. Protein bands were made visible using HRP-conjugated goat anti-rabbit IgG antibody and the enhanced chemiluminescence reagent (Amersham Bioscience, Arlington Heights, IL, USA).

Statistical analysis

Data are expressed as mean ± SD. Wilcoxon's matched-pair test was used for statistical analysis. P < 0.05 was considered statistically significant.

Results

IL-18 expression was increased in the TFCs of HT patients

The thyroid tissues were obtained freshly from six patients with HT and six control subjects. In histological studies, the sections of thyroid tissues were stained with haematoxylin-eosin. In contrast to the control, histopathological changes including diffuse lymphocytic infiltration were observed in HT. Moreover, oxyphilic change was evident in the cytoplasm of the remaining epithelial cells, whereas hyperplasia was prominent in some epithelial cells (Figure 1a,b). First, we examined IL-18 mRNA expression in human thyroid glands using RT-PCR analysis. As shown in Figure 1c, IL-18 mRNA was constitutively expressed in the normal thyroid tissues of control subjects. The relative levels of IL-18 mRNA transcripts in the thyroid tissues were significantly increased in HT compared with control (0.92 ± 0.18 vs. 0.16 ± 0.03, P < 0.05). However, RT-PCR approach could not allow us to discriminate which cells actually produce IL-18. Therefore, we secondly used immunohistochemical staining to verify the above finding and to detect the precise localization of IL-18 expression in thyroid tissues. Positive staining for IL-18 was barely detectable in the normal thyroid tissues of control subjects, whereas more diffuse and qualitatively increased IL-18 staining in the cytoplasm was observed in the thyroid tissues of HT patients (Figure 1d,e). Moreover, TFCs near infiltrating lymphocytes were the major cell types expressing IL-18 in the thyroid tissues from patients with HT (Figure 1e).

Figure 1.

Figure 1

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Up-regulation of IL-18 expression in the thyroid follicular cells (TFCs) of patients with Hashimoto's thyroiditis (HT). Histological sections were stained with haematoxylin-eosin in the thyroid tissues of control (a) and HT (b). RT-PCR analysis showed that constitutive IL-18 mRNA expression was observed in the control thyroid tissues and dramatically increased in the thyroid tissues of HT. n = 6. P < 0.05, vs. control (c). In immunohistochemical staining, IL-18-positive cells were barely detected in the control thyroid tissues (d). More diffuse and stronger staining for IL-18 was found in the thyroid tissues of HT, and the expression was mainly localized in the cytoplasm of TFCs (e). The staining omitted the step with anti-human IL-18 antibody in those of HT was included as negative control (f). Magnification 200×.

IFN-γ up-regulated IL-18 expression in isolated human TFCs

Human TFCs were isolated from the normal thyroid tissues adjacent to thyroid tumour and incubated for 48 h with 250 U/ml (or 500 U/ml) IFN-γ, 500 U/ml TNF-α or 50 U/ml IL-1β. RT-PCR analysis showed that constitutive IL-18 mRNA expression was found in the isolated human TFCs. The expression was significantly up-regulated in a dose-dependent manner following a 48-h exposure to 250–500 U/ml IFN-γ and unchanged by a 48-h exposure to 500 U/ml TNF-α or 50 U/ml IL-1β (Figure 2a,b). To exclude the possibility that IL-18 expression derived from contamination of resident leucocytes, double staining for IL-18 and thyroglobulin was examined using immunofluorescence method. Increased IL-18 expression was co-localized with thyroglobulin in the IFN-γ-treated human TFCs (Figure 3). To identify whether IL-18 protein was precursor or mature forms, western blot analysis was further used to detect IL-18 expression in the isolated human TFCs. A 24-kDa band, found when blotting lane was loaded with 15 ng of rh-pro-IL-18 as positive control, was found in the lysates of control TFCs and broadened in the lysates of IFN-γ-treated TFCs. However, an 18-kDa band corresponding to mature IL-18 was undetected in the lysates from both groups of cells (Figure 2c).

Figure 2.

Figure 2

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Effect of IFN-γ on IL-18 expression in isolated TFCs. Human TFCs were incubated for 48 h with different cytokines. RT-PCR analysis showed that IL-18 mRNA was constitutively expressed in control cells. The expression was up-regulated by 250 U/ml IFN-γ rather than 500 U/ml TNF-α or 50 U/ml IL-1β (a). IFN-γ dose-dependently stimulated IL-18 mRNA expression in the cells (b). Western blot analysis showed that a 24-kDa protein, consistent with rh-pro-IL-18 loaded in positive control lane, was found in the lysates of control cells. The expression was increased in the lysates of IFN-γ-treated cells. However, an 18-kDa mature IL-18 was undetected in the lysates from both groups of cells (c). Data were obtained from four independent experiments in a and two in b–c. *P < 0.05, vs. control.

Figure 3.

Figure 3

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Immunofluorescent staining of IL-18 and thyroglobulin in isolated human TFCs treated with IFN-γ. Green colour showed thyroglobulin expression (a). Red colour showed IL-18 expression (b). Yellow colour indicated co-expression of IL-18 and thyroglobulin (c). Magnification 200×.

Discussion

In this study, RT-PCR analysis revealed that constitutive IL-18 mRNA expression was found in normal human thyroid tissues. A higher level of IL-18 mRNA expression was detected in the thyroid tissues of HT patients compared with those of normal controls. Furthermore, immunohistochemical study identified that increased IL-18 expression was mainly localized in the cytoplasm of TFCs adjacent to lymphocyte infiltration in the thyroid tissues from patients with HT. These findings are consistent with a previous report that IL-18 expression in the TFCs was generally observed in close relation to a lymphocytic infiltrate and distributed in the same follicles expressing Fas-L and HLA-DR in thyroid tissue from a HT patient (Takiyama et al. 2002). All these results suggest that IL-18 production from TFCs may play an important role in the pathogenesis of HT.

It has been demonstrated that IL-18 expression is up-regulated in the target organs of several Th1-mediated autoimmune diseases, such as pancreatic islets in type 1 diabetic animal model (Rothe et al. 1997), intestinal mucosal epithelial cells in patients with Crohn's disease (Pizarro et al. 1999), kidney in patients with lupus nephritis (Tucci et al. 2008), neurons in animal model of multiple sclerosis (Wildbaum et al. 1998) salivary glands in patients with Sjögren's syndrome (Sakai et al. 2008). Systemic administration of exogenous IL-18 promotes diabetes development in 4-week-old non-obese diabetic (NOD) mice injected intramuscularly with IL-18 expression plasmid DNA (Oikawa et al. 2003). IL-18 blockade results in prevention or amelioration of the diseases process and preservation of target tissue integrity and function in several animal models of Th1-mediated disorders (Bombardieri et al. 2007; Wildbaum et al. 1998). In NOD mice exposed to cyclophosphamide, IL-18 mRNA expression in the endocrine pancreas coincided with the onset of insulitis and diabetes. IFN-γ expression in Th1 insulitis was preceded by IL-18 expression in pancreatic islets (Rothe et al. 1997). Similarly, intrathyroidal IL-18 mRNA expression was succeeded by the induction of IFN-γ mRNA expression with subsequent lymphocyte infiltration in thyroid tissues from an animal model of spontaneous autoimmune thyroiditis (Kaiser et al. 2002). Moreover, the direct toxic effect of IFN-γ on thyrocytes has been shown in animal models of autoimmune thyroiditis (Caturegli et al. 2000; Yu et al. 2006). Therefore, it is conceivable that IL-18 produced by TFCs may have a promoting role as an enhancer of intrathyroidal Th1-type immune responses early in autoimmune thyroiditis and contribute to the activation of inflammatory cascade leading to thyroid destruction in HT.

In this study, we show for the first time that IL-18 is constitutively expressed in isolated human TFCs and up-regulated by IFN-γ in a dose-dependent manner. Our data suggest that human TFCs are one of the sources of thyroid IL-18, thus adding IL-18 to the list of cytokines produced by human TFCs. In analogy to that finding reported in pancreatic β cells (Hong et al. 2000), IFN-γ stimulates IL-18 production from human TFCs. This effect might represent a mechanism of positive feedback and play a role in the exacerbation of thyroid destruction, if IL-18 is secreted or released during TFC damage. Moreover, IFN-γ induced IL-18 gene expression in TFCs may be due to the activation of inducible promoter 1 rather than constitutive promoter 2 (Kim et al. 2000). IFN consensus sequence-binding protein and activator protein-1, whose binding sites are located in the regions of IL-18 gene promoter 1, are critical elements for the maximal induction of IL-18 promoter activity by IFN-γ (Kim et al. 2000).

IL-18 is synthesized as a precursor molecule without a signal peptide and requires caspase-1 for cleavage into a mature peptide (Ghayur et al. 1997). In this study, pro-IL-18 but not mature IL-18 was found in the lysates of both control and IFN-γ-treated human TFCs by western blot analysis. This is similar to a previous report that primary human oral epithelial cells constitutively express IL-18 as a 24-kDa precursor protein rather than an 18-kDa mature protein (Sugawara et al. 2001). The secretion of bioactive IL-18 from the epithelial cells was induced by neutrophil proteinase 3 combined with LPS after IFN-γ priming via a caspase-1-independent pathway (Sugawara et al. 2001). Likewise, it is possible that proteinase 3 may be involved in pro-IL-18 processing in human TFCs. The physiological importance and pathological role of IL-18 originating from TFCs remain unclear. It is conceivable that IFN-γ secreted by thyroid-infiltrating activated T cells induces TFC IL-18 expression and that IL-18 liberated from TFCs, either actively or as a result of TFC destruction, may further stimulate T cell IFN-γ production, thereby establishing a vicious circle.

In summary, pro-IL-18 is constitutively expressed in human TFCs, and the expression is significantly up-regulated in both the TFCs of HT patients and the isolated TFCs exposed to IFN-γ. The results indicate that intrathyroidal IL-18 up-regulation is an immunological feature of HT and that interaction between IL-18 and IFN-γ may play an important role in promoting the local immune response.

Acknowledgments

We thank Yuanli Zhu, Yanlin Yang and Li Chen for their excellent technical assistance. We also thank all the patients for their great contribution to this study. This work was supported by grants from the National Natural Science Foundation of China (30771032 and 30700879), the Chinese National 973 Program (2006CB503908) and the Chinese National 863 Program (2006AA02A112).

Disclosure summary

The authors have nothing to disclose.

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