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Endocrinology, doi:10.1210/en.2008-0078
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Endocrinology Vol. 149, No. 7 3626-3634
Copyright © 2008 by The Endocrine Society

Elocalcitol Inhibits Inflammatory Responses in Human Thyroid Cells and T Cells

E. Borgogni1, E. Sarchielli1, M. Sottili, V. Santarlasci, L. Cosmi, S. Gelmini, A. Lombardi, G. Cantini, G. Perigli, M. Luconi, G. B. Vannelli, F. Annunziato, L. Adorini, M. Serio and C. Crescioli

Departments of Clinical Pathophysiology (E.B., M.So., S.G., A.L., G.C., M.L., M.Se., C.C.) and Internal Medicine (V.S., L.C., F.A.), Center for Research Transfer and High Education "DENOthe" (De Novo Therapies), Department of Anatomy, Histology and Forensic Medicine (E.S., G.B.V.), General Surgery Medical School (G.P.), University of Florence, 6-50139 Florence, Italy; and BioXell (L.A.), 20132 Milan, Italy

Address all correspondence and requests for reprints to: Dr. Clara Crescioli, Department of Clinical Pathophysiology, Unit of Endocrinology, University of Florence, Viale Pieraccini, 6-50139 Florence, Italy. E-mail: c.crescioli{at}dfc.unifi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves’ disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN){gamma} levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFN{gamma} and TNF{alpha}-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFN{gamma} pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH GRAVES’ disease (GD) has long been considered a T-helper 2 (Th2)-mediated autoimmune disease, a large body of evidence suggests Th1 rather than Th2 dominance in its initial phase. The predominant cell pattern involved in GD might, indeed, change throughout the disease course, with Th1 dominance at disease onset, and Th2 later on (1). CXCR3-expressing Th1 lymphocytes and the corresponding ligand, interferon (IFN){gamma}-inducible protein (CXCL10), were detected in the thyroid gland of patients with recent disease onset (2). In addition, CXCL10 serum levels were significantly increased in GD patients at disease initiation (2) and with active Graves’ ophthalmopathy (3). CXCL10 is a CXC chemokine inducible by IFN{gamma}, as CXCL9 and CXCL11, which controls leukocyte recruitment from blood (4) and is associated with Th1-mediated immune responses (5). In particular, CXCL10 has been identified as a prototypic chemokine involved in the pathogenesis of glandular autoimmunity (2, 6, 7, 8, 9), and the thyroid itself seems the main site of its secretion, perpetuating the Th1-mediated autoimmune cascade in autoimmune thyroid diseases (AITDs) (2, 8, 10, 11, 12, 13). In isolated thyrocytes CXCL10 secretion is synergistically sustained by IFN{gamma} and TNF{alpha} (3, 13, 14), and the mechanism underlying cytokine synergy is linked to a significant up-regulation of IFN{gamma} receptor (IFN{gamma}R) driven by TNF{alpha} (14).

We have previously shown that methimazole (MMI) can revert Th1 cytokine-mediated CXCL10 secretion in thyroid cells by dampening the mechanism underlying cytokine synergy (14). Antithyroid drugs have interfered with immunological signals associated with GD hyperthyroidism (15, 16). In particular, decreased CXCL10 serum levels (17) and a transition from Th1 to Th2 dominance have been reported in hyperthyroid patients with GD in the active phase (18) treated with MMI. However, its mechanism of action still remains to be elucidated fully.

Recently, vitamin D receptor (VDR) agonists have exerted pleiotropic activities in immune regulation (19, 20). VDR agonists have been effective in several models of autoimmune disease (21). They are currently clinically exploited for the topical treatment of psoriasis, a Th1 cell-mediated autoimmune disease of the skin (22), but recent advances in understanding their immunomodulatory mechanisms suggest a wider applicability in the treatment of autoimmune diseases. In particular, the antiinflammatory properties of elocalcitol (BXL-628), a nonhypercalcemic vitamin D analog, presently in phase II trial for benign prostate hyperplasia treatment (23), have been recently demonstrated in an experimental model of autoimmune prostatitis (24).

Here, we investigate the modulation by elocalcitol, compared with MMI, of Th1-type cytokine-induced CXCL10 secretion in thyroid cells. Furthermore, we assess the effect of both elocalcitol and MMI on purified CD4+ T cells in terms of Th1- and Th2-type cytokine production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
DMEM/Ham’s F-12 medium (1:1) with and without phenol red, RPMI 1640, PBS Ca2+/Mg2+-free, BSA fraction V, glutamine, antibiotics, collagenase type IV, NaOH, Bradford reagent, Ficoll-Hypaque, Phorbol 12-myristate 13-acetate (P), Ionomycin (I), and MMI were from Sigma-Aldrich (St. Louis, MO). The protein measurement kit was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Fetal bovine serum was purchased from Unipath (Bedford, UK). Fetal calf serum was from HyClone (Logan, UT). L-Glutamine, nonessential amino acids, pyruvate, and 2-mercaptoethanol were from Life Technologies, Inc. Laboratories (Grand Island, NY). IFN{gamma}, TNF{alpha}, and ELISA kits for human CXCL10, IFN{gamma}, TNF{alpha}, and IL-17 measurement were from R&D Systems, Inc. (Minneapolis, MN). The ELISA kit for human IL-5 measurement was from Arcus (Modena, Italy). For the quantitation of IL-4, a homemade ELISA using commercial monoclonal antibody (mAb) (BD Biosciences PharMingen, San Diego, CA) was used (25). Elocalcitol was from BioXell (Milan, Italy). Mouse anti-thyroglobulin (Tg) mAb was from Cell Marque Corp. (Hot Spring, AR). Goat anti-Pax8 polyclonal Ab was from Abcam plc (Cambridge, UK). For flow cytometry analysis, phycoerythrin-conjugated anti-CD119 (GIR-208, mouse IgG1) mAb was from BD Biosciences (Mountain View, CA); conjugated isotype-matched control Abs were from Southern Biotechnology Associated Inc. (Birmingham, AL), (mouse IgG1:clone 15H6). For RNA extraction, the RNeasy Mini reagent kit was from QIAGEN Italy (Milan, Italy). The TaqMan Reverse Transcription Reagents kit, all primer/probe mixes (TaqMan Gene Expression Assays), CXCL10 [identification (ID) no. Hs00171042-m1], IFN{gamma} (ID no. Hs00989290-m1), TNF{alpha} (ID no. Hs00219742-m1), IL-17 (ID no. Hs00174383-m1), IL-4 (ID no. Hs00929862-m1), IL-5 (ID no. Hs00174200-m1), IFN{gamma}R (ID no. Hs00166223-m1), and 1x Universal Master Mix were from Applied Biosystems (Foster City, CA). Quantitative PCR human reference total RNA was purchased from Stratagene (La Jolla, CA). For Western blot and/or immunocytochemistry analysis, primary Abs rabbit anti-phospho Tyr701 signal transducer and activator of transcription 1 (pStat1), mouse anti-phospho Ser536 nuclear factor-{kappa}B (pNF-{kappa}B), mouse antiphospho-inhibitor {kappa}B, and anti-inhibitor {kappa}B (I{kappa}B) were from Cell Signaling (Danvers, MA); anti-signal transducer and activator of transcription (Stat) 1 and anti-β-actin, rabbit polyclonal antihuman primary Ab against nuclear factor (NF)-{kappa}B p65 (C-20) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Alexa Fluor 488 goat antirabbit conjugate Ab was from Molecular Probes (Eugene, OR); peroxidase-secondary Abs were from Sigma-Aldrich. All reagents for SDS-PAGE were from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). Plasticware for cell cultures and disposable filtration units for growth media preparation were purchased from Corning (Milan, Italy).

Cell cultures of thyrocytes
Primary cultures of thyrocytes were obtained from the internodular parenchyma of thyroid tissues derived from 15 patients who underwent surgery for multinodular goiter as previously described (14). Certificates of consent were obtained. Patients did not receive any specific treatment for thyroid disease; thyroid hormones and thyroid autoantibody measurements were in the normal range.

Preparation and isolation of CD4+ T lymphocytes
Buffy coats from healthy adult anonymous donors were obtained in accordance with local ethical committee approval. Peripheral blood mononuclear cells were isolated as previously described (26). CD4+ T cells were selected from peripheral blood mononuclear cells by immunomagnetic cell sorting, using the CD4 isolation kit (Miltenyi Biotec, Bisley, Germany) as described elsewhere (27) and maintained in RPMI 1640 supplemented with 2 mmol/liter L-glutamine, 1% nonessential amino acids, 1% pyruvate, 2 x 10–5 M 2-mercaptoethanol, and 10% fetal calf serum. The purity of the sorted population was constantly more than 95%.

CXCL10 secretion assay in thyrocytes
For CXCL10 secretion assays, 4000 cells per well were seeded and maintained as previously described (14). Cells in phenol red- and serum-free medium containing 0.1% BSA and vehicle (absolute ethanol, 0.47%, vol/vol) were used as a control. For dose-response assays, cells were incubated for 24 h with IFN{gamma} (1000 U/ml) plus TNF{alpha} (10 ng/ml) in the absence or in presence of elocalcitol (10–13–10–6 M) or MMI (8.76 x 10–9, 8.76 x 10–8, 4.38 x 10–7, 8.76 x 10–7, 1.75 x 10–6, 2.63 x 10–6, and 4.38 x 10–6 M). The supernatant was harvested and kept frozen at –20 C until performing the CXCL10 ELISA. Experiments were performed in esaplicate with four different cell preparations.

Cytokine secretion assay in CD4+ T lymphocytes
For cytokine secretion assays, 200,000 cells per well were seeded onto 96-well round bottom plates in their growth medium (200 µl) and stimulated with P/I (1 µM/10 ng/ml) with or without elocalcitol (10–8 M) or MMI (300 ng/ml = 2.63 x 10–6 M). After 48 h the supernatant was harvested, centrifuged to remove cells, and stored at –20 C until performing ELISAs. Experiments were performed in esaplicate with six different cell preparations.

ELISAs
CXCL10, IFN{gamma}, TNF{alpha}, IL-17, and IL-5 levels were measured in cell culture supernatants using commercially available kits, according to manufacturer’s recommendations, whereas IL-4 was measured using a homemade ELISA (25). The sensitivity was from 0.41–4.46 pg/ml for CXCL10, less than 8.0 for IFN{gamma}, from 0.5–5.5 pg/ml for TNF{alpha}, less than 15 pg/ml for IL-17 and IL-4, and less than 4 pg/ml for IL-5. The intraassay and interassay coefficients of variation were 3.1 and 6.7% for CXCL10, 2.6 and 6.4% for IFN{gamma}, 5.3 and 6.8% for TNF{alpha}, 4.1 and 8.6% for IL-17, and 3.6 and 6.8%, respectively, for IL-5. Samples were assayed in quadruplicate for thyrocytes and in duplicate for CD4+ T lymphocytes. Quality control pools of low, normal, or high concentrations for all parameters were included in each assay. The obtained results were expressed as pg/ml for CD4+ T lymphocytes and pg/µg total protein amount for thyrocytes. Protein extraction and measurement were performed as reported elsewhere (14).

Flow cytometry analysis
For cytometry analysis, thyrocytes were seeded and maintained in the same conditions detailed elsewhere (14). Cells were stimulated for 24 h with TNF{alpha} (10 ng/ml) in phenol red- and serum-free medium with 0.1% BSA. Cells in phenol red- and serum-free medium with 0.1% BSA and vehicle (absolute ethanol, 0.47%, vol/vol) were used as a control. Flow cytometry analysis was performed as detailed elsewhere (28). Cells were analyzed on a BDLSRII cytofluorimeter using the Diva software (BD Biosciences). A total of 10,000 events for each sample were acquired. The area of positivity was determined using an isotype-matched mAb. Experiments were performed four times with different cell preparations.

RNA extraction
For mRNA analysis, thyrocytes, plated and maintained as previously described (14), were incubated with TNF{alpha} (10 ng/ml), alone or combined with IFN{gamma} (1000 U/ml), with or without elocalcitol (10–8 M) or MMI (2.63 x 10–6 M). Cells in phenol red- and serum-free medium 0.1% BSA and vehicle (absolute ethanol, 0.47%, vol/vol) were used as a control. After trypsinization, cells were processed as reported elsewhere (14). Experiments were performed three times with different cell preparations. For mRNA analysis of lymphocytes, cells, incubated in the same conditions as reported previously, were collected by centrifugation and pelletted before lysis, then processed as reported elsewhere (14). Experiments were performed four times with different cell preparations.

Real-time PCR
Total RNA (400 ng) was reverse transcribed using the TaqMan Reverse Transcription Reagents kit, the measurement of gene expression was performed by quantitative real-time PCR (TaqMan), and samples were processed as previously reported (14). The amount of target, normalized to an endogenous reference (18s, predeveloped TaqMan Assay Reagents) and relative to a calibrator (quantitaive PCR human reference total RNA), was given by 2–{Delta}{Delta}Ct calculation (29).

Western blot analysis
For protein analysis, thyrocytes (800,000 in 100-mm dishes) were maintained in phenol red- and serum-free medium for 24 h and incubated for 15 min in phenol red- and serum-free medium containing 0.1% BSA with IFN{gamma} (1,000 U/ml), or TNF{alpha} (10 ng/ml), with or without elocalcitol (10–8 M) or MMI (2.63 x 10–6 M). Cells were then processed as previously described (30). Briefly, after protein concentration measurement with the Coomassie protein assay kit (Bio-Rad Laboratories), protein aliquots (20 µg), processed and loaded onto 10% SDS-PAGE, were transferred on nitrocellulose membranes. Thereafter, membranes were incubated with primary Abs appropriately diluted in Tween Tris-buffered saline (TTBS) (for pStat1, pNF-{kappa}B, Stat1 1:1,000; for phospho-I{kappa}B 1:500; and for anti-β actin 1:10,000), followed by peroxidase-conjugated secondary IgG (1:3,000). Proteins were revealed by the enhanced chemiluminescence system (ECL Plus; Amersham Biosciences, Buckinghamshire, UK). Image acquisition and densitometric analysis were performed with Quantity One software on a ChemiDoc XRS instrument (Bio-Rad Laboratories). Western blot analysis was performed for at least three independent experiments. To confirm equal protein loading, membranes were stripped (Pierce Biotechnology, Rockford, IL) and reprobed with the appropriate primary Ab.

Immunofluorescence microscopy
A total of 10,000 cells was seeded onto glass coverslips in growth medium. After 24 h, cells were processed for Tg and Pax8 detection. To evaluate Stat1 or NF-{kappa}B activation, cells were incubated with serum-free medium overnight, before treatment with TNF{alpha} (10 ng/ml), or IFN{gamma} (1000 U/ml), in the presence or absence of elocalcitol (10–8 M) or MMI (2.63 x 10–6 M) for 30 min. Cells in phenol red- and serum-free medium containing 0.1% BSA and vehicle (absolute ethanol, 0.47%, vol/vol) were used as a control. For method specificity, slides lacking the primary Abs or stained with the corresponding nonimmune serum were processed. Slides were processed as reported elsewhere (31) using primary Abs against Tg (1:200), Pax8 (1:200), NF-{kappa}B p65 (1:100), and p-Stat1 (1:100), followed by Alexa Fluor 488 conjugate secondary Ab (1:200). The percentage of positive cells was calculated by counting the number of stained cells over the total cells in at least 15 separate fields per slide. The slides were examined with a phase contrast microscope (Nikon Microphot-FX microscope; Nikon, Tokyo, Japan). Experiments were performed three times with different cell preparations.

Statistical analysis
The statistical analysis was performed using the SPSS 12.0 software package (SPSS for Windows 12.0; SPSS, Inc., Chicago, IL). The Kolmogorov-Smirnov test was used to test for normal distribution of the data. One-way ANOVA was applied. A P value less than 0.05 was considered significant and was corrected for comparisons using the Dunnett or Bonferroni’s post hoc test where appropriate. Data are expressed as mean ± SE. The ALLFIT program (National Institutes of Health, Bethesda, MD) (32) was used for the analysis of sigmoid dose-response curves to calculate elocalcitol and MMI IC50. Data were expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To investigate the possible role of elocalcitol on CXCL10 secretion induced by proinflammatory cytokines in human thyrocytes, cells were simultaneously incubated with a combination of IFN{gamma} (1000 U/ml) and TNF{alpha} (10 ng/ml), and increasing concentrations of elocalcitol (10–13–10–6 M) or MMI (8.76 x 10–9, 8.76 x 10–8, 4.38 x 10–7, 8.76 x 10–7, 1.75 x 10–6, 2.63 x 10–6, 4.38 x 10–6, and 8.76 x 10–6 M) used for comparison. As shown in Fig. 1AGo, elocalcitol dose dependently decreased cytokine-induced CXCL10 secretion significantly from 10–11 M (P < 0.01 vs. IFN{gamma} plus TNF{alpha}-treated cells). MMI exerted a similar inhibitory effect on CXCL10 secretion [maximum inhibition: elocalcitol 51.51 ± 5.40% at 10–8 M; MMI 46.28 ± 3.04% at 2.63 x 10–6 M (14)], however, elocalcitol was several times more potent, as shown by the simultaneous fitting of their inhibitory curves (elocalcitol: IC50 = 5.69 x 10–13 ± 3.92 x 10–13 M; MMI: IC50 = 1.3 x 10–7 ± 0.6 x 10–7 M; P < 0.01) using the ALLFIT program (32). The inset of Fig. 1AGo depicts cell characterization shown by positive staining for Tg (89.76 ± 1.86%) and Pax8 (88.34 ± 1.86%).


Figure 1
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  		FIG. 1. Elocalcitol inhibits CXCL10 protein secretion by human thyrocytes more potently than MMI and down-regulates CXCL10 mRNA expression. A, Human thyrocytes were stimulated with IFN{gamma} (1000 U/ml) plus TNF{alpha} (10 ng/ml) and increasing doses of elocalcitol (10–13–10–6 M). Elocalcitol inhibited dose-dependently cytokine-induced CXCL10 secretion (**, P < 0.01 vs. IFN{gamma} plus TNF{alpha}-treated cells). The maximum inhibitory concentration was 10–8 M (maximum inhibition 51.51 ± 5.4%). The inhibitory effect of MMI (8.76 x 10–9, 8.76 x 10–8, 4.38 x 10–7, 8.76 x 10–7, 1.75 x 10–6, 2.63 x 10–6, 4.38 x 10–6, and 8.76 x 10–6 M) is depicted for comparison (*, P < 0.05; **, P < 0.01 vs. IFN{gamma} plus TNF{alpha}-treated cells). ALLFIT interpolation indicated that elocalcitol (open squares) exerted its effect with a greater potency in respect to MMI (closed squares) (elocalcitol: IC50 5.69 x 10–13 ± 3.92 x 10–13 M; MMI: IC50 = 1.3 x 10–7 ± 0.6 x 10–7 M; P < 0.01). The drug concentrations were selected on the basis of their near therapy dose, according to pharmacokinetics (maximum concentration and area under the time-concentration curve). Inset, Staining for Tg (left picture) and Pax8 (right picture) in human thyrocytes, evaluated by immunocytochemistry. Magnification, x20. B, Elocalcitol (10–8 M), but not MMI (2.63 x 10–6 M), significantly reduced IFN{gamma} plus TNF{alpha}-induced mRNA expression of CXCL10 (P < 0.05 vs. cytokine-induced expression; **, P < 0.01 vs. control). A ordinate, CXCL10 secretion expressed as percentage of IFN{gamma} plus TNF{alpha}-treated cells; columns are mean ± SE. Data are derived from four separate experiments using distinct cell preparations (n = 4). B ordinate, CXCL10 mRNA fold increase vs. expression in control cells, taken as one; columns are mean ± SE. Data are derived from three separate experiments using distinct cell preparations (n = 3).

 
In addition, elocalcitol (10–8 M) was able to significantly reduce CXCL10 mRNA expression induced by IFN{gamma} (1000 U/ml) plus TNF{alpha} (10 ng/ml) (P < 0.05 vs. IFN{gamma} plus TNF{alpha}-induced expression, Fig. 1BGo); at variance, MMI (2.63 x 10–6 M) did not exert any significant effect.

Western blot analysis (Fig. 2Go) revealed that, in human thyrocytes, elocalcitol (10–8 M) blocked both Stat1 phosphorylation induced by IFN{gamma} (1000 U/ml, upper panel), and NF-{kappa}B phosphorylation induced by TNF{alpha} (10 ng/ml, middle panel). NF-{kappa}B activation is preceded by phosphorylation, ubiquitination, and proteolytic degradation of its inhibitor I{kappa}B. Cell extract analysis with I{kappa}B-specific Ab revealed that TNF{alpha} treatment caused a rapid degradation of I{kappa}B that was blocked by elocalcitol (lower panel). Conversely, MMI (2.63 x 10–6 M) acted only on Stat1 (upper panel) without affecting the NF-{kappa}B/I{kappa}B pathway (middle and lower panels).


Figure 2
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  		FIG. 2. Elocalcitol impairs Stat1 and NF-{kappa}B phosphorylation, whereas MMI is effective only on Stat1. Sodium dodecyl sulfate extracts of thyrocytes stimulated with IFN{gamma} (1000 U/ml, upper panel) or TNF{alpha} (10 ng/ml, middle and lower panels) with or without elocalcitol (10–8 M) and MMI (2.63 x 10–6 M) were immunoblotted with specific antibodies to assess Stat1 (upper panel), NF-{kappa}B (middle panel), or I{kappa}B (lower panel) activation. Stat1 or NF-{kappa}B/I{kappa}B phosphorylation were induced by IFN{gamma} or TNF{alpha} treatment, respectively. A simultaneous degradation of I{kappa}B was observed in the presence of TNF{alpha}. Elocalcitol inhibited both cytokine pathways blocking Stat1 and NF-{kappa}B/I{kappa}B phosphorylation and I{kappa}B degradation (upper, middle, and lower panels). MMI, instead, acted only on Stat1 (upper panel) without affecting NF-{kappa}B/I{kappa}B pathway (middle and lower panels). Stat1 (upper and lower panels) or β-actin (middle panel) was used as a loading control. Results are derived from four to six separate experiments, using distinct cell preparations (n = 4/6). pI{kappa}B, Phospho-inhibitor {kappa}B.

 
The capacity of elocalcitol to block Stat1 and NF-{kappa}B activation was also confirmed with a nuclear translocation assay (Fig. 3Go). Stimulation with IFN{gamma} (1000 U/ml, Fig. 3AGo, second picture from the top) or TNF{alpha} (10 ng/ml, Fig. 3BGo, second picture from the top) resulted in a compact increase of pStat1 or NF-{kappa}B translocation from the cytoplasmic to the nuclear compartment (pStat1 nuclear staining: 60.45 ± 7.07% vs. 0.58 ± 0.4% in control cells; NF-{kappa}B nuclear staining: 92.90 ± 3.43% vs. 1.07 ± 0.58% in control cells, Fig. 3CGo). Coincubation with elocalcitol (10–8 M) significantly reduced cytokine-stimulated pStat1 (Fig. 3AGo, third picture from the top) or NF-{kappa}B (Fig. 3BGo, third picture from the top) translocation (pStat1 nuclear staining: 0.90 ± 0.23%, P < 0.01 vs. IFN{gamma}-treated cells; NF-{kappa}B nuclear staining: 35.28 ± 5.06%, P < 0.01 vs. TNF{alpha} -treated cells, Fig. 3CGo). Similar results were obtained after incubation with MMI (2.63 x 10–6 M) onto pStat1 translocation (Fig. 3AGo, fourth picture from the top), whereas no significant effect was observed on NF-{kappa}B activation (Fig. 3BGo, fourth picture from the top), which is consistent with our previous results (14), (pStat1 nuclear staining: 1.02 ± 0.55% P < 0.01 vs. IFN{gamma}-treated cells; NF-{kappa}B nuclear staining: 81.8 ± 4.41%, Fig. 3CGo).


Figure 3
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  		FIG. 3. Elocalcitol inhibits NF-{kappa}B and Stat1 nuclear accumulation in human thyrocytes. A, pStat1 nuclear accumulation in thyrocytes treated for 30 min with medium or with IFN{gamma} (1000 U/ml) in the absence or presence of elocalcitol (10–8 M) and MMI (2.63 x 10–6 M). In control cells (first picture from the top), pStat1 was virtually absent. IFN{gamma} treatment induced pStat1 to accumulate in the nucleus (second picture from the top); in cells stimulated with IFN{gamma} pStat1 nuclear accumulation was inhibited by elocalcitol (third picture from the top) and by MMI (fourth picture from the top). Magnification, x20. B, NF-{kappa}B p65 subunit nuclear accumulation in thyrocytes treated for 30 min with medium or TNF{alpha} (10 ng/ml) in the absence or presence of elocalcitol (10–8 M) and MMI (2.63 x 10–6 M). In control cells, p65 was distributed throughout the cytosol (first picture from the top). After stimulation with TNF{alpha}, p65 consistently disappeared from the cytosol and accumulated in the nucleus, in most of the cells (second picture from the top); the simultaneous presence of elocalcitol (third picture from the top) inhibited p65 nuclear translocation induced by TNF{alpha}, whereas MMI (fourth picture from the top) did not exert any effect. Magnification, x20. C, Cells were scored as either positive or negative for pStat1 and NF-{kappa}B p65 subunit, and results were expressed as the percentage of positive cells ± SE (calculated by counting the number of stained cells over total in 15 separate fields in each stained slide). Nuclear stained cells were virtually absent in control cells (pStat1: 0.58 ± 0.4%; NF-{kappa}B: 1.07 ± 0.58%). The percentage of pStat1 or NF-{kappa}B positive cells after stimulation with IFN{gamma} or TNF{alpha} was 60.45 ± 7.07% and 92.90 ± 3.43%, respectively (**, P < 0.01 vs. control cells). After elocalcitol (10–8 M) treatment, pStat1 nuclear staining was 0.90 ± 0.23% (°°, P < 0.01 vs. IFN{gamma}-treated cells), and NF-{kappa}B nuclear staining was 35.28 ± 5.06% (°°, P < 0.01 vs. TNF{alpha}-treated cells). After MMI (2.63 x 10–6 M) treatment, pStat1 nuclear staining was 1.02 ± 0.55% (°°, P < 0.01 vs. IFN{gamma}-treated cells), and NF-{kappa}B nuclear staining was 81.8 ± 4.41%. C ordinate, pStat1 or NF-{kappa}B nuclear positive cells (percentage over total cells); columns are mean ± SE. Results are derived from three separate experiments using distinct cell preparations (n = 3).

 
To investigate whether elocalcitol was able to affect the mechanism underlying cytokine synergy, we analyzed IFN{gamma}R expression induced by TNF{alpha} (10 ng/ml) in the presence of elocalcitol (10–8 M). Flow cytometric analysis showed that the treatment of thyrocytes with elocalcitol significantly reduced TNF{alpha}-induced IFN{gamma}R membrane protein expression (13.9 ± 3.2% vs. 30.0 ± 1.5% induced by TNF{alpha} treatment; P < 0.01; Fig. 4AGo). Real-time PCR analysis confirmed that elocalcitol significantly reduced IFN{gamma}R transcripts induced by TNF{alpha} (P < 0.05 vs. TNF{alpha}-induced expression, Fig. 4BGo).


Figure 4
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  		FIG. 4. Elocalcitol decreases IFN{gamma}R protein and gene expression in human thyrocytes. A, As revealed by flow cytometry, 24-h treatment with elocalcitol (10–8) significantly reduced IFN{gamma}R membrane protein expression up-regulated by TNF{alpha} (10 ng/ml) (13.9 ± 3.2% vs. 30.0 ± 1.5%; °°, P < 0.01 vs. TNF{alpha}-induced expression; **, P < 0.01 vs. control). B, Quantitative analysis using RT-PCR revealed that elocalcitol (10–8 M) inhibited IFN{gamma}R mRNA up-regulation induced by TNF{alpha} (10 ng/ml) (°, P < 0.05 vs. TNF{alpha}-induced expression; **, P < 0.01 vs. control). A ordinate, IFN{gamma}R membrane protein expression (percentage of positive cells over total); columns are mean ± SE. Results are derived from four separate experiments using distinct cell preparations (n = 4). B ordinate, IFN{gamma}R mRNA expression (fold increase vs. expression in control cells, taken as one); columns are mean ± SE. Results are derived from three separate experiments using distinct cell preparations (n = 3).

 
The treatment for 48 h with P/I (1 µM/10 ng/ml) induced purified human CD4+ T lymphocytes to secrete large amounts of CXCL10 (244.54 ± 46.67 pg/ml), IFN{gamma} (103,558.11 ± 14,294.40 pg/ml), TNF{alpha} (50,920.56 ± 6,203.17 pg/ml), IL-17 (5,418.58 ± 151.40 pg/ml), IL-4 (859.83 ± 22.34 pg/ml), and IL-5 (568.78 ± 66.32 pg/ml) over an almost undetectable level in control cells (for each secreted protein P < 0.01 vs. control cells).

Coincubation with elocalcitol (10–8 M) or MMI (2.63 x 10–6 M) significantly decreased P/I-induced CXCL10 (elocalcitol: 62.33 ± 18.80 pg/ml; MMI: 92.06 ± 17.96 pg/ml), IFN{gamma} (elocalcitol: 50,326.39 ± 6,918 pg/ml; MMI: 48,140.49 ± 9,490 pg/ml), and TNF{alpha} (elocalcitol: 24,400.14 ± 6,087.76 pg/ml; MMI: 24,973.81 ± 7,256.25 pg/ml) secretion by CD4+ T cells (P < 0.01 or P < 0.05 vs. P/I-treated cells, Fig. 5Go, A–C). As shown in Fig. 5DGo, elocalcitol significantly reduced P/I-induced IL-17 secretion (3704.13 ± 88.5 pg/ml; P < 0.05), whereas MMI did not. Elocalcitol significantly increased P/I-induced IL-4 (1421.20 ± 69.77 pg/ml; P < 0.01) and IL-5 (1028.11 ± 101.00 pg/ml; P < 0.01) secretion by CD4+ T cells, whereas MMI did not significantly modify the Th2 cytokine secretion pattern in activated CD4+ T cells (Fig. 5Go, E and F). Real-time PCR mRNA analysis confirmed the cytokine secretion pattern profile obtained by ELISA (Fig. 5GGo).


Figure 5
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  		FIG. 5. Effect of elocalcitol and MMI on Th1/Th17 and Th2-type protein and gene expression in CD4+ T cells. A–F, CD4+ T lymphocytes were stimulated with P/I (1 µM/10 ng/ml) for 48 h with or without elocalcitol (10–8 M) or MMI (2.63 x 10–6 M). Elocalcitol and MMI significantly decreased CXCL10, IFN{gamma}, and TNF{alpha} secretion induced by P/I in CD4+ T lymphocytes (**, P < 0.01 vs. control; °, P < 0.05; °°, P < 0.01 vs. P/I-induced secretion, A–C). Differently from MMI, elocalcitol was able to decrease significantly IL-17, and significantly induce IL-4 and IL-5 secretion by P/I-activated CD4+ T lymphocytes (**, P < 0.01 vs. control; °, P < 0.05; °°, P < 0.01 vs. P/I-induced secretion, D–F). G, Specific mRNA expression in CD4+ T cells under different treatments is depicted. A–F ordinate, Protein secretion is expressed as pg/ml. Columns are mean ± SE. Data derived from six separate experiments using distinct cell preparations (n = 6). G ordinate, Specific mRNA expression (fold increase vs. P/I-induced expression, taken as one); columns are mean ± SE. Results are derived from four separate experiments using distinct cell preparations (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows, for the first time, that elocalcitol impairs Th1-mediated inflammatory responses in thyrocytes, where it targets different intracellular pathways. In addition, elocalcitol inhibits in CD4+ T cells the production of Th1-type cytokines and of IL-17, while shifting the T-cell response toward a Th2 phenotype, whereas MMI inhibits only Th1-type cytokine production.

In thyrocytes elocalcitol decreased IFN{gamma} plus TNF{alpha}-induced CXCL10 protein secretion more potently than MMI, and it significantly reduced CXCL10 transcripts. Elocalcitol impaired the mechanism underlying cytokine synergy, and inhibited both IFN{gamma} and TNF{alpha} pathways, by targeting Stat1 and NF-{kappa}B, at variance with MMI, which affected only Stat1. Finally, elocalcitol targeted isolated CD4+ T cells as well, decreasing Th1- and increasing Th2-related cytokine secretion, whereas MMI specifically suppressed Th1-type responses.

The inflammatory response is a multistep process, characterized by different cytokines and chemokines, whose balance determines Th1 or Th2 dominance. The first predominates in inflammatory response (33), whereas the latter is credited with tolerance (i.e. of xenograft or of the fetus during pregnancy). Overactivation of either pattern can lead to disease. In particular, an overactive Th1 response is associated with autoimmunity, characterized by the presence of CXCR3-bearing T cells and the expression of IFN{gamma}-inducible CXC chemokines (CXCL9, CXCL10, CXCL11) (34). The released chemokines determine, in sequence, a massive recruitment of leukocytes at the site of inflammation.

CXCL10, in particular, enhances Th1 immunity promoting inflammatory loop, driven by the recruited CD4+ T lymphocytes at inflammation sites, supporting T-cell proliferation and IFN{gamma} secretion (35, 36, 37). In addition to endothelial cells and T cells (38, 39, 40), thyroid resident cells have been shown as the main site of CXCL10 secretion for the recruitment and activation of inflammatory cells (2, 8, 12, 13, 14). Therefore, interfering with CXCR3 expression or CXCL10 production might result in a significant inhibition of the inflammatory process (41). Neutralizing the in vivo activity of CXCL10 can alter the Th1/Th2 balance due, at least in part, to a direct effect of CXCL10 on T-cell polarization toward Th1 cell subset (42). Targeted CXCL10 vaccines could be used to avoid pro-Th1 polarization of T cells (42), as in the case of a phase I, multicenter, dose-escalation trial using an anti-CXCL10 human mAb in patients with ulcerative colitis (see http://www.clinicaltrials.gov/ct/show/NCT00295282).

Interestingly, elocalcitol was able to interrupt this self-promoting inflammatory loop by impairing CXCL10 production by thyrocytes and by CD4+ T cells, targeting both IFN{gamma} and TNF{alpha}-induced pathways. A cross talk between IFN{gamma} and VDR has been described in immune cells, albeit with opposite effects (43, 44). At variance with MMI (14), elocalcitol inhibited in thyrocytes NF-{kappa}B nuclear translocation, in line with previous data obtained with VDR agonists in the pancreatic islet cells of nonobese diabetic mice, spontaneously developing type 1 diabetes (45), and in myeloid dendritic cells (46). The TNF{alpha} pathway has been implicated in the cytotoxic mechanisms that characterize the thyroid destruction in AITDs (47, 48). We have also recently highlighted the relevance of TNF{alpha} in GD pathogenesis because in thyrocytes this cytokine alone elicited a dose-dependent CXCL10 protein secretion and synergized with IFN{gamma}, up-regulating IFN{gamma}R membrane expression (14). The latter effect seems particularly relevant because it affects the magnitude of the cell response to IFN{gamma}, at least in terms of CXCL10 secretion, in line with our recent data reported in human cardiomyocytes (49). Like MMI (14), elocalcitol is able to revert CXCL10 secretion by inhibiting cytokine synergy, decreasing TNF{alpha}-induced IFN{gamma}R membrane protein and mRNA expression. However, elocalcitol is several times more potent than MMI. This different potency might be explained, at least in part, by the intracellular pathways targeted by the two drugs.

VDR agonists appear primarily to inhibit proinflammatory, pathogenic T cells like Th1 and Th17 T cells, and under appropriate conditions, may favor a deviation to the Th2 pathway (19). These effects could be, in part, a consequence of direct T-cell targeting, but modulation of dendritic cells (DC) function by VDR agonists certainly plays an important role in shaping the development of T-cell responses. Thus, VDR agonists can target T cells both directly and indirectly, selectively inhibiting T-cell subsets able to mediate inflammation and tissue damage (19). Notably, elocalcitol administration has decreased IL-17, in addition to IFN{gamma}, production in prostate-draining lymph node T cells from nonobese diabetic mice affected by experimental autoimmune prostatitis (50). IL-17 is a proinflammatory cytokine with a major pathogenic role in many autoimmune diseases (51). It is produced by CD4+ Th cell effectors Th17, distinct from the classic Th1 and Th2, albeit very recently a new subset of IFN{gamma}-producing Th17, named Th17/Th1 cells, sharing features with both Th1 and Th17, has been described in humans (52). We found that, in addition to its effects on thyrocytes, elocalcitol significantly decreased IL-17; it reduced, to a higher extent, CXCL10, the prototypic chemokine involved in the pathogenesis of glandular autoimmunity (2, 6, 7, 8, 9), and decreased IFN{gamma} and TNF{alpha}, the proinflammatory cytokines that synergistically induce its secretion. Thus, our data support the concept that elocalcitol inhibits both Th1 and Th17 cells, even if a role for those latter in the pathogenesis of GD has been suggested but not fully understood (53). Furthermore, elocalcitol increased IL-4 and IL-5 secretion by isolated CD4+ T cells. In conclusion, elocalcitol is a potent inhibitor of inflammatory responses both in thyrocytes and T cells, where it inhibits Th1- and Th17-type cytokine production while shifting the response toward a Th2 phenotype. The latter effect would seem to be particularly intriguing because the immune shift toward Th2 is accompanied by a progressive decrease of serum TSH receptor antibodies that belong to Th1 type (18, 54). The capacity of VDR ligands to skew T cells toward a Th2 pathway has been previously suggested, however, their ability to regulate Th2 cell differentiation is still under debate (19).

Both Th1 and Th2 cells can be targets of VDR agonists, likely depending on the cell activation and differentiation status (19). In addition, vitamin D may play an important role also in the maintenance of B-cell homeostasis by regulating autoantibody production, and the correction of vitamin D deficiency may be even useful in the treatment of B-cell-mediated autoimmune disorders (55).

MMI, instead, significantly decreased only CXCL10, IFN{gamma}, and TNF{alpha} production, without any effect on IL-17 and Th2-type cytokine secretion. Therefore, MMI appears to be a selective inhibitor of Th1-mediated responses both in thyroid cells, by targeting the IFN{gamma} pathway, and in CD4+ T cells. Thus, we can hypothesize that the Th1 to Th2 dominance shift observed in hyperthyroid GD patients (18) treated with MMI is due to a specific suppression of the Th1 response.

These observations encourage further exploration of the immunomodulatory properties of elocalcitol in the development of therapies for AITDs, and suggest a possible combination of drugs with different intracellular targets to decrease therapeutic doses and minimize side effects.


   Acknowledgments
 
We thank Dr. Fabiana Rosati, Department of Clinical Pathophysiology, Unit of Endocrinology, University of Florence, Florence, Italy, for her help with sigmoid curves analysis.


   Footnotes
 
This research was supported by Tuscany Regional Study On Rosiglitazone and Ministero dell’Università e della Ricerca.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 27, 2008

1 E.B. and E.S. contributed equally to this work. Back

Abbreviations: Ab, Antibody; AITD, autoimmune thyroid disease; GD, Graves’ disease; I, Ionomycin; ID, identification; IFN, interferon; IFN{gamma}R, IFN{gamma} receptor; I{kappa}B, inhibitor {kappa}B; mAb, monoclonal antibody; MMI, methimazole; NF, nuclear factor; P, Phorbol 12-myristate 13-acetate; pNF-{kappa}B, phospho Ser536 nuclear factor-{kappa}B; pStat1, phospho Tyr701 signal transducer and activator of transcription 1; Stat, signal transducer and activator of transcription; Tg, thyroglobulin; Th1, Th2, or Th17, T-helper 1, 2, or 17; VDR, vitamin D receptor.

Received January 16, 2008.

Accepted for publication March 17, 2008.


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