β (CCL2) and α (CXCL10) chemokine modulations by cytokines and peroxisome proliferator-activated receptor-α agonists in Graves' ophthalmopathy

in Journal of Endocrinology
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  • 1 Department of Internal Medicine, Endocrinology Unit, Department of Otorhinolaryngology, Clinical Biochemistry Unit, School of Medicine, University of Pisa, Via Roma 67, I-56100 Pisa, Italy

No data are present in the literature about the effect of cytokines on the prototype β chemokine (C-C motif) ligand 2 (CCL2) or of peroxisome proliferator-activated receptor α (PPARα (PPARA)) activation on CCL2 and CXCL10 chemokines secretion in fibroblasts or preadipocytes in Graves' ophthalmopathy (GO). We have tested the effect of interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα) on CCL2, and for comparison on the prototype α chemokine (C-X-C motif) ligand 10 (CXCL10), and the possible modulatory role of PPARα activation on secretion of these chemokines in normal and GO fibroblasts or preadipocytes in primary cell cultures. This study shows that IFNγ alone, or in combination with TNFα, stimulates the secretion of CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO at levels similar to those observed in controls. IFNγ and TNFα also stimulated CXCL10 chemokine secretion as expected. The presence of PPARα and PPARγ (PPARG) in primary fibroblasts or preadipocytes of patients with GO has been confirmed. PPARα activators were able to inhibit the secretion of CXCL10 and CCL2, while PPARγ activators were confirmed to be able to inhibit CXCL10 but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists. In conclusion, CCL2 and CXCL10 are modulated by IFNγ and TNFα in GO. PPARα activators inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.

Abstract

No data are present in the literature about the effect of cytokines on the prototype β chemokine (C-C motif) ligand 2 (CCL2) or of peroxisome proliferator-activated receptor α (PPARα (PPARA)) activation on CCL2 and CXCL10 chemokines secretion in fibroblasts or preadipocytes in Graves' ophthalmopathy (GO). We have tested the effect of interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα) on CCL2, and for comparison on the prototype α chemokine (C-X-C motif) ligand 10 (CXCL10), and the possible modulatory role of PPARα activation on secretion of these chemokines in normal and GO fibroblasts or preadipocytes in primary cell cultures. This study shows that IFNγ alone, or in combination with TNFα, stimulates the secretion of CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO at levels similar to those observed in controls. IFNγ and TNFα also stimulated CXCL10 chemokine secretion as expected. The presence of PPARα and PPARγ (PPARG) in primary fibroblasts or preadipocytes of patients with GO has been confirmed. PPARα activators were able to inhibit the secretion of CXCL10 and CCL2, while PPARγ activators were confirmed to be able to inhibit CXCL10 but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists. In conclusion, CCL2 and CXCL10 are modulated by IFNγ and TNFα in GO. PPARα activators inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.

Introduction

Recent evidences have shown that CXC α-chemokines (Th1), especially CXCL9, CXCL10, and CXCL11, play an important role in the initial phases of autoimmune thyroid dysfunction (Garcià-Lòpez et al. 2001, Antonelli et al. 2004, 2006a,b, 2007).

We have recently demonstrated that the secretion of CXCL9, CXCL10, and CXCL11 in primary cultures of Graves' ophthalmopathy (GO) fibroblasts and preadipocytes can be stimulated by interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα; Antonelli et al. 2006c, 2009a), suggesting that GO retro-orbital cells participate in the self-perpetuation of inflammation by releasing chemokines (under the influence of cytokines) and inducing the recruitment of activated T cells in the orbit.

Furthermore, other studies have demonstrated recently a possible role for chemokine (C-C motif) ligand 2 (CCL2; also known as monocyte chemoattractant protein-1 (MCP1)) in GO. Hwang et al. (2009) showed that orbital fibroblasts from patients with GO overexpress CD40 and that CD154 (the ligand for CD40) upregulated the expression of CCL2. Furthermore, Chen et al. (2008) found that the expression of CCL2 was higher in the orbital fat of GO patients than in controls.

The peroxisome proliferator-activated receptors α (PPARα (PPARA)), PPARγ (PPARG), and PPARδ (PPARD) are ligand-activated nuclear receptors with a wide range of effects on metabolism, cell proliferation, and differentiation (Michalik et al. 2006). In addition, abundant evidence suggests that the PPARs are also important regulators of the immune system. Of considerable interest, ligands for PPARγ and PPARα have therapeutic activity in several rodent models of inflammatory and autoimmune diseases (Spencer et al. 1997, Delerive et al. 2000, Lovett-Racke et al. 2004, Okamoto et al. 2005, Lee et al. 2007, Oliveira et al. 2007), suggesting that they might have similar activity in human disease as well.

About chemokines, it has been recently shown that fenofibrate represses interleukin 17 (IL17 (IL17a)) and IFNγ expression and improves colitis in Il10-deficient mice by inhibiting the expression of the gene-encoding chemokine (C-X-C motif) ligand 10 (CXCL10) and by repressing CXCL10 gene promoter activity in TNFα-treated HT-29 cells (Lee et al. 2007).

Treatment of fibroblasts and preadipocytes with PPARγ activators (rosiglitazone and pioglitazone) at near-therapeutic doses significantly inhibited IFNγ-stimulated CXC chemokine secretion, strongly suggesting that PPARγ might be involved in the regulation of IFNγ-induced chemokine expression in human thyroid autoimmunity and GO (Antonelli et al. 2006c, 2009a). More recently, we have shown that CXCL9 and CXCL11 chemokines are modulated by PPARα agonist secretion in Graves' and normal thyrocytes (Antonelli et al. 2010). Furthermore, PPARα has been found to be expressed in fibroblasts (Pasquali et al. 2004) and preadipocytes (Antonelli et al. 2012) in GO patients.

However, until now, to our knowledge, no data are present in the literature about the effect of cytokines on CCL2 in GO or of PPARα activation on CCL2 and CXCL10 chemokine secretions in fibroblasts and preadipocytes in GO. Here, we have tested the effects of IFNγ and TNFα on CCL2 (the prototype Th2 chemokine) and the possible modulatory role of PPARα activation on CCL2, and for comparison on the prototype Th1 CXCL10 chemokine, secretion in GO fibroblasts or preadipocytes in primary culture.

Materials and Methods

Fibroblast and preadipocyte cell cultures

Orbital adipose/connective tissue samples were obtained from five euthyroid patients (two on l-thyroxine after thyroidectomy) undergoing orbital decompression for severe GO during the inactive phase of the disease (all previously treated with antithyroid medication and systemic corticosteroids; none treated with orbital radiotherapy). All subjects gave their informed consent to the study, which was approved by the local Ethics Committee.

GO tissue explants were minced and placed directly in plastic culture dishes, to let preadipocyte fibroblasts proliferate, as described previously (Bahn et al. 1989). Cells were propagated in medium 199 with 20% v/v fetal bovine serum (FBS; Gibco; Invitrogen Ltd.), penicillin (100 IU/ml), and gentamicin (20 μg/ml) in a humidified 5% CO2 incubator at 37 °C and maintained with medium 199 containing 10% FBS and antibiotics. Orbital cells were grown to confluence in six-well plates in medium 199 with 10% FBS in order to initiate adipocyte differentiation, which was carried out as reported previously (Antonelli et al. 2006c, 2010). The differentiation protocol was continued for 10 days, and the medium was replaced every 3–4 days. Separately, fibroblasts obtained from the same patients' orbital tissues were maintained for the same period in the medium lacking various components necessary for complete adipocyte differentiation (i.e. cPGI2, dexamethasone, and IBMX, 3-isobuty1-1-methylxanthine; Antonelli et al. 2010). Control fibroblasts and preadipocytes were obtained from dermal tissues (not affected by any pathology) of the same patients.

Orbital preadipocyte fibroblast cultures were plated in medium 199 with 10% FBS, grown to confluence, and subjected to either the differentiation protocol or the nondifferentiation conditions. Cells were washed, stained with filtered 0.21% Oil Red O and subsequently with Mayer's hematoxylin solution (MHS-32; Sigma), and visualized using an Olympus IX50 light microscope and photographed at ×20 as previously reported (Antonelli et al. 2009a, 2012).

CXCL10 and CCL2 secretion assays

For CXCL10 and CCL2 secretion assays, cells were seeded in 96-well plates at a concentration of 30 000 cells/ml as previously reported (Antonelli et al. 2009a) and incubated (24 h) with IFNγ (500, 1000, 5000, and 10 000 IU/ml; R&D Systems, Minneapolis, MN, USA) and 10 ng/ml TNFα (R&D Systems), alone or in combination (Antonelli et al. 2009a, 2012). After 24 h, the supernatant was collected and kept frozen at −20 °C until CXCL10 assay.

To establish how the PPARα or PPARγ activators affect IFNγ-induced chemokine secretion, cells were treated (24 h) with IFNγ (1000 IU/ml) and TNFα (10 ng/ml) in the presence or absence of increasing concentrations of PPARα agonists (5, 10, 50, and 100 μmol/l fenofibrate; 50, 100, 200, and 400 μmol/l gemfibrozil or ciprofibrate; Sigma–Aldrich Corp.) or PPARγ agonists (0, 1, 5, 10, and 20 μmol/l rosiglitazone; Glaxo SmithKline, or pioglitazone; Alexis Biochemicals, Lausen, Switzerland; Antonelli et al. 2012).

Supernatants were assayed by ELISA for CXCL10 and CCL2 concentrations. The experiments were repeated three times with the different cell preparations.

Cell cultures and PPARα or PPARγ agonist treatment

Cell cultures were treated (24 h) with PPARγ agonists (0, 1, 5, 10, or 20 μmol/l rosiglitazone or pioglitazone) or PPARα agonists (5, 10, 50, and 100 μmol/l fenofibrate; 50, 100, 200, and 400 μmol/l gemfibrozil or ciprofibrate). Control cultures were grown (24 h) in the same medium containing vehicle (absolute ethanol, 0.47% v/v) without PPARα or PPARγ agonists. Some cultures were examined by phase contrast microscopy using an Olympus IX50 light microscope.

Lysis and homogenization of cell preparations were performed, and the sample was immediately assayed for its protein concentration by conventional methods (Antonelli et al. 2006c).

ELISA for CXCL10 and CCL2

Culture supernatants CXCL10 and CCL2 levels were assayed by a quantitative sandwich immunoassay using a commercially available kit (R&D Systems). The mean minimum detectable dose for CXCL10 was 1.35 pg/ml; the intra- and inter-assay coefficients of variation (CV) were 3.1 and 6.8% respectively. The mean minimum detectable dose for CCL2 was 4.6 pg/ml; the intra- and interassay CV were 4.6 and 5.7% respectively.

RT-PCR for PPARα and PPARγ

Total RNA (400 ng) was reverse transcribed using TaqMan RT reagents kit as previously reported (Antonelli et al. 2010). Primers and probes for PPARα and PPARγ were from Applied Biosystems (TaqMan Gene Expression Assay; Hs00231882_m1 and Hs00234592_m1 respectively).

The amount of target, normalized to the endogenous reference glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Pre-Developed TaqMan Assay Reagents; Applied Biosystems) and relative to a calibrator (quantitative PCR human reference total RNA), was given by calculation (Antonelli et al. 2010).

Data analysis

Values are given as mean±s.d. for normally distributed variables (in text), or mean±s.e.m. (in figures), otherwise as median and (interquartile range). Mean group values were compared using ANOVA for normally distributed variables, otherwise by the Mann–Whitney U (two groups) or Kruskal–Wallis (three groups or more) tests. Proportions were compared by the χ2 test. Post hoc comparisons on normally distributed variables were carried out using the Bonferroni–Dunn test.

Results

PPARα and PPARγ expression in GO fibroblasts and preadipocytes

PPARα and PPARγ mRNAs were detectable in all primary orbital fibroblast cells. According to RT-PCR results, the relative level of PPARα expression vs the reference gene (GAPDH) ranges from 0.4 to 0.9 and the expression of PPARγ from 0.1 to 0.2. The results obtained in control fibroblasts are summarized in Table 1. PPARα and PPARγ were expressed by RT-PCR more in GO fibroblasts than in controls. Similar results were present in preadipocytes (data not shown).

Table 1

Relative level of peroxisome proliferator-activated receptor α (PPARα) or PPARγ mRNA in fibroblasts from Graves' ophthalmopathy (GO) or control fibroblasts by RT-PCR. The amount of target was normalized to the endogenous reference GAPDH mRNA (PPARα/GAPDH mRNA or PPARγ/GAPDH mRNA) and expressed as range of values

PPARα/GAPDHPPARγ/GAPDH
GO fibroblasts0.41–0.870.10–0.21
Control fibroblasts0.12–0.340.04–0.08

PPAR agonists and chemokines in GO fibroblasts and preadipocytes

CXCL10 was undetectable in the supernatants collected from primary GO fibroblast cultures. IFNγ dose dependently induced the CXCL10 release (ANOVA, P<0.001; Table 2); similar results were observed in preadipocytes (ANOVA, P<0.001). TNFα alone had no effect (chemokines remaining undetectable), while the combination of IFNγ and TNFα had a significant synergistic effect on the CXCL10 secretion both in fibroblasts (ANOVA, P<0.0001) and in preadipocytes (ANOVA, P<0.0001).

Table 2

In fibroblasts, from patients with Graves' ophthalmopathy (GO), interferon γ (IFNγ) dose dependently induced the CXCL10 release (ANOVA, P<0.001); similar results were observed in preadipocytes (ANOVA, P<0.001). Tumor necrosis factor α (TNFα) alone had no effect (chemokines remaining undetectable), while the combination of IFNγ and TNFα had a significant synergistic effect on the CXCL10 secretion both in fibroblasts (ANOVA, P<0.0001) and in preadipocytes (ANOVA, P<0.001)

ControlIFNγ (0 IU/ml)IFNγ (500 IU/ml)IFNγ (1000 IU/ml)IFNγ (5000 IU/ml)IFNγ (10 000 IU/ml)TNFα (10 ng/ml)IFNγ (1000 IU/ml)+TNFα (10 ng/ml)
GO fibroblasts (CXCL10 pg/ml)00167±62410±78563±94591±10701876±88
Preadipocytes (CXCL10 pg/ml)0092±32210±54361±78443±8102653±148

Treating GO fibroblasts or preadipocytes with rosiglitazone or pioglitazone, together with IFNγ and TNFα stimulation, CXCL10 release was dose dependently inhibited (ANOVA, P<0.0001) as expected (Antonelli et al. 2006c, 2009a).

By treating GO fibroblasts with fenofibrate (Fig. 1A), gemfibrozil (Fig. 1B), or ciprofibrate (Fig. 1C), together with IFNγ and TNFα stimulation, CXCL10 release was dose dependently inhibited. Similar results were observed in preadipocytes, in which fenofibrate, ciprofibrate, or gemfibrozil, dose dependently inhibited the IFNγ- and TNFα-stimulated CXCL10 release (ANOVA, P<0.0001; data not shown).

Figure 1
Figure 1

Increasing doses of fenofibrate (Feno; 5, 10, 50, and 100 μmol/l; A), gemfibrozil (Gem; 50, 100, 200, and 400 μmol/l; B), or ciprofibrate (Cipro; 50, 100, 200, and 400 μmol/l; C) inhibit CXCL10 release from orbital fibroblasts stimulated with interferon (IFNγ) (IFNg, 1000 IU/ml) and tumor necrosis factor (TNFα) (TNFa, 10 ng/ml), (IFNg+TNFa; P<0.0001 by ANOVA). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test.

Citation: Journal of Endocrinology 213, 2; 10.1530/JOE-11-0488

The inhibition of the CXCL10 chemokine secretion induced by PPARα agonists, at the highest dosage, was stronger than that induced by PPARγ activators both in fibroblasts (Fig. 2A) and in preadipocytes (Fig. 2B). However, the relative potency of the PPARα agonists on the inhibition of the CXCL10 chemokine was different for each compound.

Figure 2
Figure 2

The inhibition of the CXCL10 chemokine secretion induced by PPARα agonists at the maximum dosage in GO fibroblasts (A) or preadipocytes (B; fenofibrate (Feno; 100 μmol/l), gemfibrozil (Gem; 400 μmol/l), and ciprofibrate (Cipro; 400 μmol/l)) was stronger than that induced by PPARγ activators (pioglitazone (Pio 20; 20 μmol/l) and rosiglitazone (Rosi 20; 20 μmol/l)). Bars are mean±s.e.m. *P<0.05 or less vs IFNγ (IFNg)+TNFα (TNFa) by Bonferroni–Dunn test. °P<0.05 or less vs IFNg+TNFa+Rosi 20 or IFNg+TNFa+Pio 20 by Bonferroni–Dunn test.

Citation: Journal of Endocrinology 213, 2; 10.1530/JOE-11-0488

CCL2 was detectable (546±93 pg/ml) in the supernatants collected from primary GO fibroblast or preadipocyte cultures (751±106 pg/ml). IFNγ dose dependently induced CCL2 release in fibroblasts (CCL2: 546±93, 723±85, 970±65, 1076±131, and 1204±171 pg/ml, respectively, with IFNγ: 0, 500, 1000, 5000, and 10 000 IU/ml; ANOVA, P<0.001) and in preadipocytes (CCL2: 751±106, 937±96, 1123±125, 1411±147, and 1504±168 pg/ml, respectively, with IFNγ: 0, 500, 1000, 5000, and 10 000 IU/ml; ANOVA, P<0.001). TNFα alone had no effect on CCL2 release (with TNFα: 0, 1, and 10 ng/ml). The combination of IFNγ and TNFα had a significant synergistic effect on CCL2 secretion (ANOVA, P<0.0001; Fig. 3) in primary GO fibroblasts (Fig. 3A) and in preadipocytes (Fig. 3B).

Figure 3
Figure 3

CCL2 was detectable in the supernatants collected from orbital fibroblasts (A). IFNγ (IFNg, 1000 IU/ml) significantly induced the CCL2 release (ANOVA, P<0.001), while TNFα (TNFa, 10 ng/ml) alone had no effect. The combination of IFNγ (1000 IU/ml) and TNFα (10 ng/ml) had a significant synergistic effect on CCL2 secretion (ANOVA, P<0.001). Similar results were observed in preadipocytes (B; ANOVA, P<0.001). Bars are mean±s.e.m. *P<0.05 or less vs control (CT) by Bonferroni–Dunn test; °P<0.05 or less vs IFNg or TNFa by Bonferroni–Dunn test.

Citation: Journal of Endocrinology 213, 2; 10.1530/JOE-11-0488

Treating orbital fibroblasts with fenofibrate (Fig. 4A), gemfibrozil (Fig. 4B), or ciprofibrate (Fig. 4C), together with IFNγ and TNFα stimulation, CCL2 release was dose dependently inhibited. Similar results were observed in preadipocytes, in which fenofibrate, gemfibrozil, or ciprofibrate dose dependently inhibited the IFNγ- and TNFα-stimulated CXCL10 release (ANOVA, P<0.0001; data not shown).

Figure 4
Figure 4

Increasing doses of fenofibrate (Feno; 5, 10, 50, and 100 μmol/l) (A), gemfibrozil (Gem; 50, 100, 200, and 400 μmol/l; B), or ciprofibrate (Cipro; 50, 100, 200, and 400 μmol/l; C) inhibit CCL2 release from orbital fibroblasts stimulated with IFNγ (IFNg, 1000 IU/ml) and TNFα (TNFa, 10 ng/ml), (IFNg+TNFa; P<0.0001 by ANOVA). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test.

Citation: Journal of Endocrinology 213, 2; 10.1530/JOE-11-0488

The inhibition of the CCL2 chemokine secretion induced by PPARα agonists at the highest dosage in GO fibroblasts averaged 65% (Fig. 5A). Treating orbital fibroblasts (Fig. 5A) or preadipocytes (Fig. 5B) with rosiglitazone or pioglitazone, together with IFNγ and TNFα stimulation, had no significant effect on the CCL2 release.

Figure 5
Figure 5

The inhibition of the CCL2 chemokine secretion induced by PPARα agonists at the maximum dosage in GO fibroblasts (A; fenofibrate (Feno; 100 μmol/l), gemfibrozil (Gem; 400 μmol/l), ciprofibrate (Cipro; 400 μmol/l)) ranged from about 60 to 70%. Treating GO fibroblasts with pioglitazone (Pio 20; 20 μmol/l) or rosiglitazone (Rosi 20; 20 μmol/l), together with IFNγ (IFNg; 1000 IU/ml) and TNFα (TNFa; 10 ng/ml) stimulation, had no significant effect on the CCL2 release. Similar results were observed in preadipocytes (B; ANOVA, P<0.001). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test. °P<0.05 or less vs IFNg+TNFa+Rosi 20 or IFNg+TNFa+Pio 20 by Bonferroni–Dunn test.

Citation: Journal of Endocrinology 213, 2; 10.1530/JOE-11-0488

Rosiglitazone or pioglitazone, fenofibrate, ciprofibrate, or gemfibrozil alone had no effect and did not affect cell viability or total protein content (data not shown). The data obtained with orbital fibroblasts or preadipocytes were not significantly different from those obtained with control fibroblasts or preadipocytes (data not shown).

Discussion

This study shows that IFNγ alone, or in combination with TNFα, stimulated the secretion of the CC chemokine CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO. IFNγ and TNFα stimulated the CXC chemokine secretion as expected. The presence of PPARα in primary fibroblasts or preadipocytes from patients with GO has been confirmed (Antonelli et al. 2012). PPARα activators inhibited the secretion of both CXC (CXCL10) and CC chemokines (CCL2), while PPARγ activators were confirmed to inhibit CXC chemokines but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists.

Recently, some studies have suggested a possible role for CCL2 in GO. Hwang et al. (2009) demonstrated that orbital fibroblasts from patients with GO overexpress CD40 and that CD154 (the ligand for CD40) upregulated the expression of CCL2. Furthermore, Chen et al. (2008) found that the expression of CCL2 was higher in GO orbital fat than in the orbital fat of controls and suggested that macrophage infiltration may play an important role in the pathogenesis of GO via overexpression of CCL2.

Our results show that CCL2 chemokine was secreted basally by normal fibroblasts or preadipocytes such as those from GO, responded to IFNγ alone, and a synergistic action of IFNγ in combination with TNFα was observed inducing the secretion of the chemokine in primary fibroblasts or preadipocytes from patients with GO. This last result is unexpected because Th2 chemokines are poor responders to IFNγ, but it is in agreement with the results obtained by other studies on murine (Yamana et al. 2009) and human fibroblasts (Struyf et al. 1998).

The fact that CCL2 can be produced by GO fibroblasts or preadipocytes, under the influence of cytokines such as IFNγ or TNFα (released by activated Th1 lymphocytes), suggests a possible role of these cells on recruitment of monocytes and T lymphocytes to the tissue from the blood in GO.

IFNγ and TNFα also stimulated CXC chemokine secretion as expected (Antonelli et al. 2006c, 2009a). These results, on the whole, are in agreement with the view that autoimmune disorders evolve from an initial Th1 phase to a later Th2 prevalent immune response with progression toward fibrosis (Romagnani 1997, Antonelli et al. 2008). For example, the early expression of CXCL10 and CCL2 suggests their involvement in the initiation and perpetuation of disease in severe granulomatous experimental autoimmune thyroiditis, which progresses to fibrosis (Chen et al. 2005).

PPARα has been confirmed to be expressed more in GO fibroblasts or preadipocytes than in controls. These results are in agreement with our data (Antonelli et al. 2012) and the ones obtained by Pasquali et al. (2004). In our studies, no significant adipocyte change was observed in fibroblasts after a 24 h incubation period with PPARα, according to the results obtained by Pasquali et al. (2004) that showed that few subpopulations (1–2%) of fibroblasts from Graves' ophthalmopathy (FGOs) exposed to fenofibrate changed their typical fibroblastic appearance after 24 h, such as in controls. Our results suggest that PPARα agonists are able to modulate both the Th1 and Th2 immune responses as suggested by their powerful action on the inhibition of chemokines. In fact, PPARα can modulate both CXC (CXCL10) and CC (CCL2) chemokines.

The anti-inflammatory action of PPARα has been widely investigated by many studies, and some studies advocate PPARα agonists as therapy for autoimmune disease (Cuzzocrea et al. 2004, Gervois et al. 2004, Lovett-Racke et al. 2004, Lo Verme et al. 2005, Okamoto et al. 2005, Lee et al. 2007, Oliveira et al. 2007). The effect of PPARα on CXC chemokines has been less deeply investigated. In the study by Lee et al. (2007), fenofibrate repressed the expression of the gene encoding CXCL10 and repressed CXCL10 gene promoter activity in TNFα-treated HT-29 cells. Our results are in agreement with a previous study that has demonstrated that CXCL9 and CXCL11 chemokines are modulated by PPARα agonists in Graves' and normal thyrocytes (Antonelli et al. 2010).

The results of this study shed new light on the effect of PPARα on CXC chemokines, demonstrating a powerful effect on the secretion of CXCL10 both in normal fibroblasts and in preadipocytes, such as in cells obtained by GO, inhibiting the stimulatory effect of IFNγ and TNFα on the secretion of these chemokines. Interestingly, the effect of PPARα has been exerted at near-therapeutic doses as also suggested by the study of Lovett-Racke et al. (2004). The potency of the PPARα agonist used is maximum on the secretion of CXCL10 reaching about a 60% of inhibition by fenofibrate, while about a 40–50% of inhibition was observed with the others. This may suggest that the different PPARα agonist compounds may act through different pathways that remain to be explored.

In the study by Lee et al. (2007), fenofibrate repressed the expression of a CXCL10 promoter–luciferase construct, indicating that the repression of CXCL10 mRNA expression occurs at least partly by a transcriptional mechanism. TNFα-stimulated transcription of the CXCL10 gene is largely mediated by nuclear factor κB (NF-κB) binding to 2 κB binding sites located in the CXCL10 proximal promoter (Majumder et al. 1998).

PPAR ligands repress transcriptional activation by NF-κB via a mechanism known as ligand-dependent transrepression (Delerive et al. 1999, Pascual et al. 2005); thus, NF-κB is a likely target for repression of CXCL10 transcription by fenofibrate.

Interestingly, however, in the study by Lee et al. (2007), all five chemokine genes tested are known to be activated by NF-κB, but two of these genes, CXCL8 (IL8) and CXCL1 (GRO1), were not repressed significantly by fenofibrate. This suggests that the promoter context and perhaps the presence or absence of corepressor complexes associated with promoters in the inactive state may determine which NF-κB-activated promoters are repressed by fenofibrate (Pascual et al. 2005).

In addition to repressing TNFα-mediated activation of the CXCL10 promoter, fenofibrate also repressed activation of the promoter by IFNγ. This repression very likely acts via IFN response factor-containing complexes that bind to the IFN-stimulated response element of the CXCL10 promoter after stimulation with IFNγ (Majumder et al. 1998).

PPARγ ligands show an inhibitory activity on CXC chemokines, confirming the results of our previous studies (Antonelli et al. 2006c, 2009a). However, the relative potency of PPARα agonists on the inhibition of the secretion of CXC chemokine is higher than that of PPARγ ligands. This could suggest that PPARα agonists may act, at least in part, through different pathways than PPARγ agonists (Marx et al. 2000, Schaefer et al. 2005, Lombardi et al. 2008, Antonelli et al. 2009b).

The effect of PPARα agonists on CCL2 has been investigated but not in GO cells. In fact, PPARα activators suppress lipopolysaccharide-stimulated Ccl2 transcription and release in activated rat glia (Lee et al. 2005). Furthermore, PPARα activators inhibit CCL2 expression induced by high glucose concentration in human endothelial cells (Dragomir et al. 2006).

The results of this study show a potent inhibition (more than 60%) of the cytokine-stimulated CCL2 secretion by PPARα agonists both in normal and in GO cells. Interestingly, PPARγ agonists show no effect on the secretion of CCL2 in GO cells. This discrepancy is in agreement with the results of other studies on different cells (Nie et al. 2005). The different effects of PPARα and PPARγ agonists on the secretion of chemokines in GO cells may be, at least in part, explained by the different pathways involved.

Recently, it has been demonstrated that rosiglitazone was associated with an increased risk of stroke, heart failure, and all-cause mortality in elderly patients (Graham et al. 2010), and the European Medicines Agency (EMA) recommended on September 2010 that rosiglitazone be suspended from the European market. More recently, the EMA extended the review of safety to pioglitazone (Moynihan 2011). Even if these arguments cannot be automatically translated in GO field, they do not actually advise PPARγ agonists for the therapy of GO.

The presence of PPARα in GO fibroblasts or preadipocytes and the immunomodulatory effect of PPARα agonists on the production of Th1 and Th2 chemokines suggest that PPARα may be involved in the modulation of the immune response both in normal and GO cells, probably by interaction with endogenous ligands. A therapeutic use of PPARα ligands in GO remains to be explored, but it is suggested by the fact that their effect on chemokines has been exerted at near-therapeutic doses. Despite anti-inflammatory actions of PPARα, its proadipogenic functions in the orbit might worsen the disease, contraindicating the use of these agents in GO (Pasquali et al. 2004). Thus, further studies are necessary to establish whether in active GO the anti-inflammatory effects of PPARα activation can be exploited without the risk of expanding retrobulbar fat mass, for example, combining PPARα agonists and non-steroidal anti-inflammatory drugs or glucocorticoids that may synergize to exert their anti-inflammatory action.

In conclusion, this study shows that CCL2 is modulated by IFNγ and TNFα in GO. PPARα activators are able to inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

References

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  • Antonelli A, Rotondi M, Ferrari SM, Fallahi P, Romagnani P, Franceschini SS, Serio M & Ferrannini E 2006c Interferon-gamma-inducible alpha-chemokine CXCL10 involvement in Graves' ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. Journal of Clinical Endocrinology and Metabolism 91 614620. doi:10.1210/jc.2005-1689.

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  • Antonelli A, Rotondi M, Fallahi P, Grosso M, Boni G, Ferrari SM, Romagnani P, Serio M, Mariani G & Ferrannini E 2007 Iodine-131 given for therapeutic purposes modulates differently interferon-gamma-inducible alpha-chemokine CXCL10 serum levels in patients with active Graves' disease or toxic nodular goiter. Journal of Clinical Endocrinology and Metabolism 92 14851490. doi:10.1210/jc.2006-1571.

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  • Antonelli A, Ferri C, Fallahi P, Ferrari SM, Giuggioli D, Colaci M, Manfredi A, Frascerra S, Franzoni F & Galetta F 2008 CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosis – a longitudinal study. Rheumatology 47 4549. doi:10.1093/rheumatology/kem313.

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  • Antonelli A, Ferrari SM, Fallahi P, Frascerra S, Santini E, Franceschini SS & Ferrannini E 2009a Monokine induced by interferon gamma (IFNgamma) (CXCL9) and IFNgamma inducible T-cell alpha-chemoattractant (CXCL11) involvement in Graves' disease and ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. Journal of Clinical Endocrinology and Metabolism 94 18031809. doi:10.1210/jc.2008-2450.

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  • Antonelli A, Ferrari SM, Fallahi P, Frascerra S, Piaggi S, Gelmini S, Lupi C, Minuto M, Berti P & Benvenga S 2009b Dysregulation of secretion of CXC alpha-chemokine CXCL10 in papillary thyroid cancer: modulation by peroxisome proliferator-activated receptor-gamma agonists. Endocrine-Related Cancer 16 12991311. doi:10.1677/ERC-08-0337.

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  • Antonelli A, Ferrari SM, Frascerra S, Pupilli C, Mancusi C, Metelli MR, Orlando C, Ferrannini E & Fallahi P 2010 CXCL9 and CXCL11 chemokines modulation by peroxisome proliferator-activated receptor-alpha agonists secretion in Graves' and normal thyrocytes. Journal of Clinical Endocrinology and Metabolism 95 E413E420. doi:10.1210/jc.2010-0923.

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  • Antonelli A, Ferrari SM, Frascerra S, Ruffilli I, Gelmini S, Minuto M, Pupilli C, Miccoli P & Sellari-Franceschini S 2012 Peroxisome proliferator-activated receptor-α agonists modulate CXCL9 and CXCL11 chemokines in Graves' ophthalmopathy fibroblasts and preadipocytes. Molecular and Cellular Endocrinology 349 255261. doi:10.1016/j.mce.2011.11.001.

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  • Bahn RS, Gorman CA, Johnson CM & Smith TJ 1989 Presence of antibodies in the sera of patients with Graves' disease recognizing a 23 kilodalton fibroblast protein. Journal of Clinical Endocrinology and Metabolism 69 622662. doi:10.1210/jcem-69-3-622.

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  • Chen K, Wei Y, Alter A, Sharp GC & Braley-Mullen H 2005 Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis. Journal of Leukocyte Biology 78 716724. doi:10.1189/jlb.0205102.

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  • Chen MH, Liao SL, Chang TC & Chuang LM 2008 Role of macrophage infiltration in the orbital fat of patients with Graves' ophthalmopathy. Clinical Endocrinology 69 332337. doi:10.1111/j.1365-2265.2008.03219.x.

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  • Cuzzocrea S, Di Paola R, Mazzon E, Genovese T, Muià C, Centorrino T & Caputi AP 2004 Role of endogenous and exogenous ligands for the peroxisome proliferators activated receptors alpha (PPAR-alpha) in the development of inflammatory bowel disease in mice. Laboratory Investigation 84 16431654. doi:10.1038/labinvest.3700185.

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  • Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G & Staels B 1999 Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. Journal of Biological Chemistry 274 3204832054. doi:10.1074/jbc.274.45.32048.

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  • Delerive P, Gervois P, Fruchart JC & Staels B 2000 Induction of IkappaBalpha expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators. Journal of Biological Chemistry 275 3670336707. doi:10.1074/jbc.M004045200.

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  • Dragomir E, Tircol M, Manduteanu I, Voinea M & Simionescu M 2006 Aspirin and PPAR-alpha activators inhibit monocyte chemoattractant protein-1 expression induced by high glucose concentration in human endothelial cells. Vascular Pharmacology 44 440449. doi:10.1016/j.vph.2006.02.006.

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  • Garcià-Lòpez MA, Sancho D, Sànchez-Madrid F & Marazuela M 2001 Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3+ lymphocytes. Journal of Clinical Endocrinology and Metabolism 86 50085016. doi:10.1210/jc.86.10.5008.

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    • Export Citation
  • Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B & Kooistra T 2004 Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate. Journal of Biological Chemistry 279 1615416160. doi:10.1074/jbc.M400346200.

    • Search Google Scholar
    • Export Citation
  • Graham DJ, Ouellet-Hellstrom R, MaCurdy TE, Ali F, Sholley C, Worrall C & Kelman JA 2010 Risk of acute myocardial infarction, stroke, heart failure, and death in elderly Medicare patients treated with rosiglitazone or pioglitazone. Journal of the American Medical Association 304 411418. doi:10.1001/jama.2010.920.

    • Search Google Scholar
    • Export Citation
  • Hwang CJ, Afifiyan N, Sand D, Naik V, Said J, Pollock SJ, Chen B, Phipps RP, Goldberg RA & Smith TJ 2009 Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1. Investigative Ophthalmology and Visual Science 50 22622268. doi:10.1167/iovs.08-2328.

    • Search Google Scholar
    • Export Citation
  • Lee SM, Yung WH, Chen L & Chow BK 2005 Expression and spatial distribution of secretin and secretin receptor in human cerebellum. Neuroreport 16 219222. doi:10.1097/00001756-200502280-00003.

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    • Export Citation
  • Lee JW, Bajwa PJ, Carson MJ, Jeske DR, Cong Y, Elson CO, Lytle C & Straus DS 2007 Fenofibrate represses interleukin-17 and interferon-gamma expression and improves colitis in interleukin-10 deficient mice. Gastroenterology 133 108123. doi:10.1053/j.gastro.2007.03.113.

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  • Lombardi A, Cantini G, Piscitelli E, Gelmini S, Francalanci M, Mello T, Ceni E, Varano G, Forti G & Rotondi M 2008 A new mechanism involving ERK contributes to rosiglitazone inhibition of tumor necrosis factor-alpha and interferon-gamma inflammatory effects in human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology 28 718724. doi:10.1161/ATVBAHA.107.160713.

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  • Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A & Piomelli D 2005 The nuclear receptor peroxisome proliferator activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Molecular Pharmacology 67 1519. doi:10.1124/mol.104.006353.

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    • Export Citation
  • Lovett-Racke AE, Hussain RZ, Northrop S, Choy J, Rocchini A, Matthes L, Chavis JA, Diab A, Drew PD & Racke MK 2004 Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease. Journal of Immunology 172 57905798.

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  • Majumder S, Zhou LZ, Chaturvedi P, Babcock G, Aras S & Ransohoff RM 1998 p48/STAT-1 alpha-containing complexes play a predominant role in induction of IFN-gamma-inducible protein, 10 kDa (IP-10) by IFN-gamma alone or in synergy with TNF-alpha. Journal of Immunology 161 47364744.

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  • Marx N, Mach F, Sauty A, Leung JH, Sarafi MN, Ransohoff RM, Libby P, Plutzky J & Luster AD 2000 Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. Journal of Immunology 164 65036508.

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    • Export Citation
  • Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA & O'Rahilly S 2006 International union of pharmacology LXI peroxisome proliferator-activator receptors. Pharmacological Reviews 58 726741. doi:10.1124/pr.58.4.5.

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  • Moynihan R 2011 European drug agency extends review of safety of pioglitazone. BMJ 342 d4105 doi:10.1136/bmj.d4105.

  • Nie M, Corbett L, Knox AJ & Pang L 2005 Differential regulation of chemokine expression by peroxisome proliferator-activated receptor gamma agonists: interactions with glucocorticoids and beta2-agonists. Journal of Biological Chemistry 280 25502561. doi:10.1074/jbc.M410616200.

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  • Okamoto H, Iwamoto T, Kotake S, Momohara S, Yamanaka H & Kamatani N 2005 Inhibition of NF-kappaB signaling by fenofibrate, a peroxisome proliferator-activated receptor-alpha ligand, presents a therapeutic strategy for rheumatoid arthritis. Clinical and Experimental Rheumatology 23 323330.

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    Increasing doses of fenofibrate (Feno; 5, 10, 50, and 100 μmol/l; A), gemfibrozil (Gem; 50, 100, 200, and 400 μmol/l; B), or ciprofibrate (Cipro; 50, 100, 200, and 400 μmol/l; C) inhibit CXCL10 release from orbital fibroblasts stimulated with interferon (IFNγ) (IFNg, 1000 IU/ml) and tumor necrosis factor (TNFα) (TNFa, 10 ng/ml), (IFNg+TNFa; P<0.0001 by ANOVA). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test.

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    The inhibition of the CXCL10 chemokine secretion induced by PPARα agonists at the maximum dosage in GO fibroblasts (A) or preadipocytes (B; fenofibrate (Feno; 100 μmol/l), gemfibrozil (Gem; 400 μmol/l), and ciprofibrate (Cipro; 400 μmol/l)) was stronger than that induced by PPARγ activators (pioglitazone (Pio 20; 20 μmol/l) and rosiglitazone (Rosi 20; 20 μmol/l)). Bars are mean±s.e.m. *P<0.05 or less vs IFNγ (IFNg)+TNFα (TNFa) by Bonferroni–Dunn test. °P<0.05 or less vs IFNg+TNFa+Rosi 20 or IFNg+TNFa+Pio 20 by Bonferroni–Dunn test.

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    CCL2 was detectable in the supernatants collected from orbital fibroblasts (A). IFNγ (IFNg, 1000 IU/ml) significantly induced the CCL2 release (ANOVA, P<0.001), while TNFα (TNFa, 10 ng/ml) alone had no effect. The combination of IFNγ (1000 IU/ml) and TNFα (10 ng/ml) had a significant synergistic effect on CCL2 secretion (ANOVA, P<0.001). Similar results were observed in preadipocytes (B; ANOVA, P<0.001). Bars are mean±s.e.m. *P<0.05 or less vs control (CT) by Bonferroni–Dunn test; °P<0.05 or less vs IFNg or TNFa by Bonferroni–Dunn test.

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    Increasing doses of fenofibrate (Feno; 5, 10, 50, and 100 μmol/l) (A), gemfibrozil (Gem; 50, 100, 200, and 400 μmol/l; B), or ciprofibrate (Cipro; 50, 100, 200, and 400 μmol/l; C) inhibit CCL2 release from orbital fibroblasts stimulated with IFNγ (IFNg, 1000 IU/ml) and TNFα (TNFa, 10 ng/ml), (IFNg+TNFa; P<0.0001 by ANOVA). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test.

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    The inhibition of the CCL2 chemokine secretion induced by PPARα agonists at the maximum dosage in GO fibroblasts (A; fenofibrate (Feno; 100 μmol/l), gemfibrozil (Gem; 400 μmol/l), ciprofibrate (Cipro; 400 μmol/l)) ranged from about 60 to 70%. Treating GO fibroblasts with pioglitazone (Pio 20; 20 μmol/l) or rosiglitazone (Rosi 20; 20 μmol/l), together with IFNγ (IFNg; 1000 IU/ml) and TNFα (TNFa; 10 ng/ml) stimulation, had no significant effect on the CCL2 release. Similar results were observed in preadipocytes (B; ANOVA, P<0.001). Bars are mean±s.e.m. *P<0.05 or less vs IFNg+TNFa by Bonferroni–Dunn test. °P<0.05 or less vs IFNg+TNFa+Rosi 20 or IFNg+TNFa+Pio 20 by Bonferroni–Dunn test.

  • Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Buonamano A, Ferrannini E & Serio M 2004 High levels of circulating CXCL10 are associated with chronic autoimmune thyroiditis and hypothyroidism. Journal of Clinical Endocrinology and Metabolism 89 54965499. doi:10.1210/jc.2004-0977.

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  • Antonelli A, Fallahi P, Rotondi M, Ferrari SM, Romagnani P, Grosso M, Ferrannini E & Serio M 2006a Increased serum CXCL10 in Graves' disease or autoimmune thyroiditis is not associated with hyper- or hypothyroidism per se, but is specifically sustained by the autoimmune, inflammatory process. European Journal of Endocrinology 154 651658. doi:10.1530/eje.1.02137.

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  • Antonelli A, Fallahi P, Rotondi M, Ferrari SM, Serio M & Miccoli P 2006b Serum levels of the interferon-gamma-inducible alpha chemokine CXCL10 in patients with active Graves' disease, and modulation by methimazole therapy and thyroidectomy. British Journal of Surgery 93 12261231. doi:10.1002/bjs.5401.

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  • Antonelli A, Rotondi M, Ferrari SM, Fallahi P, Romagnani P, Franceschini SS, Serio M & Ferrannini E 2006c Interferon-gamma-inducible alpha-chemokine CXCL10 involvement in Graves' ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. Journal of Clinical Endocrinology and Metabolism 91 614620. doi:10.1210/jc.2005-1689.

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  • Antonelli A, Rotondi M, Fallahi P, Grosso M, Boni G, Ferrari SM, Romagnani P, Serio M, Mariani G & Ferrannini E 2007 Iodine-131 given for therapeutic purposes modulates differently interferon-gamma-inducible alpha-chemokine CXCL10 serum levels in patients with active Graves' disease or toxic nodular goiter. Journal of Clinical Endocrinology and Metabolism 92 14851490. doi:10.1210/jc.2006-1571.

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  • Antonelli A, Ferri C, Fallahi P, Ferrari SM, Giuggioli D, Colaci M, Manfredi A, Frascerra S, Franzoni F & Galetta F 2008 CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosis – a longitudinal study. Rheumatology 47 4549. doi:10.1093/rheumatology/kem313.

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  • Antonelli A, Ferrari SM, Fallahi P, Frascerra S, Santini E, Franceschini SS & Ferrannini E 2009a Monokine induced by interferon gamma (IFNgamma) (CXCL9) and IFNgamma inducible T-cell alpha-chemoattractant (CXCL11) involvement in Graves' disease and ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. Journal of Clinical Endocrinology and Metabolism 94 18031809. doi:10.1210/jc.2008-2450.

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  • Antonelli A, Ferrari SM, Fallahi P, Frascerra S, Piaggi S, Gelmini S, Lupi C, Minuto M, Berti P & Benvenga S 2009b Dysregulation of secretion of CXC alpha-chemokine CXCL10 in papillary thyroid cancer: modulation by peroxisome proliferator-activated receptor-gamma agonists. Endocrine-Related Cancer 16 12991311. doi:10.1677/ERC-08-0337.

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  • Antonelli A, Ferrari SM, Frascerra S, Pupilli C, Mancusi C, Metelli MR, Orlando C, Ferrannini E & Fallahi P 2010 CXCL9 and CXCL11 chemokines modulation by peroxisome proliferator-activated receptor-alpha agonists secretion in Graves' and normal thyrocytes. Journal of Clinical Endocrinology and Metabolism 95 E413E420. doi:10.1210/jc.2010-0923.

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  • Antonelli A, Ferrari SM, Frascerra S, Ruffilli I, Gelmini S, Minuto M, Pupilli C, Miccoli P & Sellari-Franceschini S 2012 Peroxisome proliferator-activated receptor-α agonists modulate CXCL9 and CXCL11 chemokines in Graves' ophthalmopathy fibroblasts and preadipocytes. Molecular and Cellular Endocrinology 349 255261. doi:10.1016/j.mce.2011.11.001.

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  • Bahn RS, Gorman CA, Johnson CM & Smith TJ 1989 Presence of antibodies in the sera of patients with Graves' disease recognizing a 23 kilodalton fibroblast protein. Journal of Clinical Endocrinology and Metabolism 69 622662. doi:10.1210/jcem-69-3-622.

    • Search Google Scholar
    • Export Citation
  • Chen K, Wei Y, Alter A, Sharp GC & Braley-Mullen H 2005 Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis. Journal of Leukocyte Biology 78 716724. doi:10.1189/jlb.0205102.

    • Search Google Scholar
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  • Chen MH, Liao SL, Chang TC & Chuang LM 2008 Role of macrophage infiltration in the orbital fat of patients with Graves' ophthalmopathy. Clinical Endocrinology 69 332337. doi:10.1111/j.1365-2265.2008.03219.x.

    • Search Google Scholar
    • Export Citation
  • Cuzzocrea S, Di Paola R, Mazzon E, Genovese T, Muià C, Centorrino T & Caputi AP 2004 Role of endogenous and exogenous ligands for the peroxisome proliferators activated receptors alpha (PPAR-alpha) in the development of inflammatory bowel disease in mice. Laboratory Investigation 84 16431654. doi:10.1038/labinvest.3700185.

    • Search Google Scholar
    • Export Citation
  • Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G & Staels B 1999 Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. Journal of Biological Chemistry 274 3204832054. doi:10.1074/jbc.274.45.32048.

    • Search Google Scholar
    • Export Citation
  • Delerive P, Gervois P, Fruchart JC & Staels B 2000 Induction of IkappaBalpha expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators. Journal of Biological Chemistry 275 3670336707. doi:10.1074/jbc.M004045200.

    • Search Google Scholar
    • Export Citation
  • Dragomir E, Tircol M, Manduteanu I, Voinea M & Simionescu M 2006 Aspirin and PPAR-alpha activators inhibit monocyte chemoattractant protein-1 expression induced by high glucose concentration in human endothelial cells. Vascular Pharmacology 44 440449. doi:10.1016/j.vph.2006.02.006.

    • Search Google Scholar
    • Export Citation
  • Garcià-Lòpez MA, Sancho D, Sànchez-Madrid F & Marazuela M 2001 Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3+ lymphocytes. Journal of Clinical Endocrinology and Metabolism 86 50085016. doi:10.1210/jc.86.10.5008.

    • Search Google Scholar
    • Export Citation
  • Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B & Kooistra T 2004 Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate. Journal of Biological Chemistry 279 1615416160. doi:10.1074/jbc.M400346200.

    • Search Google Scholar
    • Export Citation
  • Graham DJ, Ouellet-Hellstrom R, MaCurdy TE, Ali F, Sholley C, Worrall C & Kelman JA 2010 Risk of acute myocardial infarction, stroke, heart failure, and death in elderly Medicare patients treated with rosiglitazone or pioglitazone. Journal of the American Medical Association 304 411418. doi:10.1001/jama.2010.920.

    • Search Google Scholar
    • Export Citation
  • Hwang CJ, Afifiyan N, Sand D, Naik V, Said J, Pollock SJ, Chen B, Phipps RP, Goldberg RA & Smith TJ 2009 Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1. Investigative Ophthalmology and Visual Science 50 22622268. doi:10.1167/iovs.08-2328.

    • Search Google Scholar
    • Export Citation
  • Lee SM, Yung WH, Chen L & Chow BK 2005 Expression and spatial distribution of secretin and secretin receptor in human cerebellum. Neuroreport 16 219222. doi:10.1097/00001756-200502280-00003.

    • Search Google Scholar
    • Export Citation
  • Lee JW, Bajwa PJ, Carson MJ, Jeske DR, Cong Y, Elson CO, Lytle C & Straus DS 2007 Fenofibrate represses interleukin-17 and interferon-gamma expression and improves colitis in interleukin-10 deficient mice. Gastroenterology 133 108123. doi:10.1053/j.gastro.2007.03.113.

    • Search Google Scholar
    • Export Citation
  • Lombardi A, Cantini G, Piscitelli E, Gelmini S, Francalanci M, Mello T, Ceni E, Varano G, Forti G & Rotondi M 2008 A new mechanism involving ERK contributes to rosiglitazone inhibition of tumor necrosis factor-alpha and interferon-gamma inflammatory effects in human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology 28 718724. doi:10.1161/ATVBAHA.107.160713.

    • Search Google Scholar
    • Export Citation
  • Lo Verme J, Fu J, Astarita G, La Rana G, Russo R, Calignano A & Piomelli D 2005 The nuclear receptor peroxisome proliferator activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Molecular Pharmacology 67 1519. doi:10.1124/mol.104.006353.

    • Search Google Scholar
    • Export Citation
  • Lovett-Racke AE, Hussain RZ, Northrop S, Choy J, Rocchini A, Matthes L, Chavis JA, Diab A, Drew PD & Racke MK 2004 Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease. Journal of Immunology 172 57905798.

    • Search Google Scholar
    • Export Citation
  • Majumder S, Zhou LZ, Chaturvedi P, Babcock G, Aras S & Ransohoff RM 1998 p48/STAT-1 alpha-containing complexes play a predominant role in induction of IFN-gamma-inducible protein, 10 kDa (IP-10) by IFN-gamma alone or in synergy with TNF-alpha. Journal of Immunology 161 47364744.

    • Search Google Scholar
    • Export Citation
  • Marx N, Mach F, Sauty A, Leung JH, Sarafi MN, Ransohoff RM, Libby P, Plutzky J & Luster AD 2000 Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. Journal of Immunology 164 65036508.

    • Search Google Scholar
    • Export Citation
  • Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA & O'Rahilly S 2006 International union of pharmacology LXI peroxisome proliferator-activator receptors. Pharmacological Reviews 58 726741. doi:10.1124/pr.58.4.5.

    • Search Google Scholar
    • Export Citation
  • Moynihan R 2011 European drug agency extends review of safety of pioglitazone. BMJ 342 d4105 doi:10.1136/bmj.d4105.

  • Nie M, Corbett L, Knox AJ & Pang L 2005 Differential regulation of chemokine expression by peroxisome proliferator-activated receptor gamma agonists: interactions with glucocorticoids and beta2-agonists. Journal of Biological Chemistry 280 25502561. doi:10.1074/jbc.M410616200.

    • Search Google Scholar
    • Export Citation
  • Okamoto H, Iwamoto T, Kotake S, Momohara S, Yamanaka H & Kamatani N 2005 Inhibition of NF-kappaB signaling by fenofibrate, a peroxisome proliferator-activated receptor-alpha ligand, presents a therapeutic strategy for rheumatoid arthritis. Clinical and Experimental Rheumatology 23 323330.

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