N-Acetylcysteine and 15 Deoxy-Δ12,14-Prostaglandin J2 Exert a Protective Effect Against Autoimmune Thyroid Destruction in Vivo but Not Against Interleukin-1α/Interferon γ-Induced Inhibitory Effects in Thyrocytes in Vitro
2010; Elsevier BV; Volume: 177; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.091253
ISSN1525-2191
AutoresSylvie Poncin, Ides M. Colin, Brigitte Decallonne, Isabelle Clinckspooor, Marie‐Christine Many, Jean‐François Denef, A Gérard,
Tópico(s)NF-κB Signaling Pathways
ResumoReactive oxygen species (ROS) are crucial for thyroid hormonogenesis, and their production is kept under tight control. Oxidative stress (OS) is toxic for thyrocytes in an inflammatory context. In vitro, Th1 pro-inflammatory cytokines have already been shown to decrease thyroid-specific protein expression. In the present study, OS level and its impact on thyroid function were analyzed in vitro in Th1 cytokine (interleukin [IL]-1α/interferon [IFN] γ)-incubated thyrocytes (rat and human), as well as in vivo in thyroids from nonobese diabetic mice, a model of spontaneous autoimmune thyroiditis. N-acetylcysteine (NAC) and prostaglandin, 15 deoxy-Δ12,14-prostaglandinJ2 (15dPGJ2), were used for their antioxidant and anti-inflammatory properties, respectively. ROS production and OS were increased in IL-1α/IFNγ-incubated thyrocytes and in destructive thyroiditis. In vitro, NAC not only reduced ROS production below control levels, but further decreased the expression of thyroid-specific proteins in addition to IL-1α/IFNγ-inhibitory effects. Thus, besides ROS, other intracellular intermediaries likely mediate Th1 cytokine effects. In vivo, NAC and 15dPGJ2 reduced OS and the immune infiltration, thereby leading to a restoration of thyroid morphology. It is therefore likely that NAC and 15dPGJ2 mainly exert their protective effects by acting on infiltrating inflammatory cells rather than directly on thyrocytes. Reactive oxygen species (ROS) are crucial for thyroid hormonogenesis, and their production is kept under tight control. Oxidative stress (OS) is toxic for thyrocytes in an inflammatory context. In vitro, Th1 pro-inflammatory cytokines have already been shown to decrease thyroid-specific protein expression. In the present study, OS level and its impact on thyroid function were analyzed in vitro in Th1 cytokine (interleukin [IL]-1α/interferon [IFN] γ)-incubated thyrocytes (rat and human), as well as in vivo in thyroids from nonobese diabetic mice, a model of spontaneous autoimmune thyroiditis. N-acetylcysteine (NAC) and prostaglandin, 15 deoxy-Δ12,14-prostaglandinJ2 (15dPGJ2), were used for their antioxidant and anti-inflammatory properties, respectively. ROS production and OS were increased in IL-1α/IFNγ-incubated thyrocytes and in destructive thyroiditis. In vitro, NAC not only reduced ROS production below control levels, but further decreased the expression of thyroid-specific proteins in addition to IL-1α/IFNγ-inhibitory effects. Thus, besides ROS, other intracellular intermediaries likely mediate Th1 cytokine effects. In vivo, NAC and 15dPGJ2 reduced OS and the immune infiltration, thereby leading to a restoration of thyroid morphology. It is therefore likely that NAC and 15dPGJ2 mainly exert their protective effects by acting on infiltrating inflammatory cells rather than directly on thyrocytes. Thyrocytes continuously produce H2O2 and various reactive oxygen species (ROS) that are physiologically required for normal thyroid hormone synthesis. To control the toxicity resulting from ROS, thyrocytes possess several protective mechanisms. During thyroid hormone synthesis, H2O2 is produced in a limited area at the apical membrane and is immediately consumed in the peroxidation reaction catalyzed by thyroperoxidase (TPO). When ROS are produced in higher amounts, they are systematically eliminated by potent antioxidant systems such as peroxiredoxins, catalase, and glutathione peroxidases.1Gerard AC Many MC Daumerie C Knoops B Colin IM Peroxiredoxin 5 expression in the human thyroid gland.Thyroid. 2005; 15: 205-209Crossref PubMed Scopus (41) Google Scholar, 2Poncin S Gerard AC Boucquey M Senou M Calderon PB Knoops B Lengele B Many MC Colin IM Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.Endocrinology. 2008; 149: 424-433Crossref PubMed Scopus (94) Google Scholar, 3Mano T Shinohara R Iwase K Kotake M Hamada M Uchimuro K Hayakawa N Hayashi R Nakai A Ishizuki Y Nagasaka A Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders.Horm Metab Res. 1997; 29: 351-354Crossref PubMed Scopus (51) Google Scholar, 4Mutaku JF Many MC Colin I Denef JF van den Hove MF Antigoitrogenic effect of combined supplementation with dl-alpha-tocopherol, ascorbic acid, and beta-carotene and of dl-alpha-tocopherol alone in the rat.J Endocrinol. 1998; 156: 551-561Crossref PubMed Scopus (18) Google Scholar, 5Nadolnik LI Valentyukevich OI Peculiarities of the antioxidant status of the thyroid gland.Bull Exp Biol Med. 2007; 144: 529-531Crossref PubMed Scopus (8) Google Scholar Thus, a basal ROS production, which we define as oxidative load, is required to safeguard thyroid hormone synthesis, as recently demonstrated.6Poncin S Colin IM Gerard AC Minimal oxidative load: a prerequisite for thyroid cell function.J Endocrinol. 2009; 201: 161-167Crossref PubMed Scopus (26) Google Scholar Likewise, an harmless oxidative stress (OS) may also be important for cell division during goiter formation when thyrocytes are facing iodine deprivation.7Poncin S Van ES Humblet K Colin IM Gerard AC Oxidative stress: a required condition for thyroid cell proliferation.Am J Pathol. 2010; 176: 1355-1363Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar The context is quite different in the case of thyroid inflammation. Thus, in models of thyroiditis (transient or permanent), high amounts of ROS are produced and may become toxic.8Many MC Mestdagh C van den Hove MF Denef JF In vitro study of acute toxic effects of high iodide doses in human thyroid follicles.Endocrinology. 1992; 131: 621-630PubMed Google Scholar, 9Belshaw BE Becker DV Necrosis of follicular cells and discharge of thyroidal iodine induced by administering iodide to iodine-deficient dogs.J Clin Endocrinol Metab. 1973; 36: 466-474Crossref PubMed Scopus (52) Google Scholar, 10Wollman SH Breitman TR Changes in DNA and weight of thyroid glands during hyperplasia and involution.Endocrinology. 1970; 86: 322-327Crossref PubMed Scopus (73) Google Scholar, 11Mahmoud I Colin I Many MC Denef JF Direct toxic effect of iodide in excess on iodine-deficient thyroid glands: epithelial necrosis and inflammation associated with lipofuscin accumulation.Exp Mol Pathol. 1986; 44: 259-271Crossref PubMed Scopus (100) Google Scholar, 12Many MC Maniratunga S Varis I Dardenne M Drexhage HA Denef JF Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone non-obese diabetic mice: effects of iodine-induced cell necrosis.J Endocrinol. 1995; 147: 311-320Crossref PubMed Scopus (85) Google Scholar, 13Mutaku JF Poma JF Many MC Denef JF van den Hove MF Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution.J Endocrinol. 2002; 172: 375-386Crossref PubMed Scopus (35) Google Scholar Using one of these models, we recently showed that increased OS associated with a strong inflammatory reaction can be controlled by 15 deoxy-Δ12,14-prostaglandin J2 (15dPGJ2),2Poncin S Gerard AC Boucquey M Senou M Calderon PB Knoops B Lengele B Many MC Colin IM Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.Endocrinology. 2008; 149: 424-433Crossref PubMed Scopus (94) Google Scholar an anti-inflammatory prostaglandin14Straus DS Glass CK Cyclopentenone prostaglandins: new insights on biological activities and cellular targets.Med Res Rev. 2001; 21: 185-210Crossref PubMed Scopus (550) Google Scholar that prevents OS-induced cytotoxicity.15Garg TK Chang JY 15-deoxy-delta 12, 14-Prostaglandin J2 prevents reactive oxygen species generation and mitochondrial membrane depolarization induced by oxidative stress.BMC Pharmacol. 2004; 4: 6Crossref PubMed Scopus (45) Google Scholar Iodine administration to goitrous thyrocytes produces an inflammatory reaction that is transient in most cases. However, in individuals genetically prone to develop autoimmune thyroiditis, this transient inflammation may become permanent, thereby evolving toward destructive thyroiditis. A model of destructive thyroiditis can be obtained in nonobese diabetic (NOD) mice. In this model of Hashimoto’s-like thyroiditis, the ongoing inflammatory reaction relies on pro-inflammatory Th1 cytokines16Rapoport B McLachlan SM Thyroid autoimmunity.J Clin Invest. 2001; 108: 1253-1259Crossref PubMed Scopus (155) Google Scholar, 17Stassi G De MR Autoimmune thyroid disease: new models of cell death in autoimmunity.Nat Rev Immunol. 2002; 2: 195-204Crossref PubMed Scopus (224) Google Scholar, 18Colin IM Isaac J Dupret P Ledant T D'Hautcourt JL Functional lymphocyte subset assessment of the Th1/Th2 profile in patients with autoimmune thyroiditis by flowcytometric analysis of peripheral lymphocytes.J Biol Regul Homeost Agents. 2004; 18: 72-76PubMed Google Scholar that inhibit the expression of thyroid-specific proteins such as thyroglobulin, TPO, Na+/I− symporter (NIS), and dual oxidases (Duoxs).19Ajjan RA Watson PF Findlay C Metcalfe RA Crisp M Ludgate M Weetman AP The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity.J Endocrinol. 1998; 158: 351-358Crossref PubMed Scopus (100) Google Scholar, 20Rasmussen AK Bendtzen K Feldt-Rasmussen U Thyrocyte-interleukin-1 interactions.Exp Clin Endocrinol Diabetes. 2000; 108: 67-71Crossref PubMed Scopus (22) Google Scholar, 21Caraccio N Giannini R Cuccato S Faviana P Berti P Galleri D Dardano A Basolo F Ferrannini E Monzani F Type I interferons modulate the expression of thyroid peroxidase, sodium/iodide symporter, and thyroglobulin genes in primary human thyrocyte cultures.J Clin Endocrinol Metab. 2005; 90: 1156-1162Crossref PubMed Scopus (49) Google Scholar, 22Gerard AC Boucquey M van den Hove MF Colin IM Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.Am J Physiol Endocrinol Metab. 2006; 291: E242-E253Crossref PubMed Scopus (28) Google Scholar, 23Sato K Satoh T Shizume K Ozawa M Han DC Imamura H Tsushima T Demura H Kanaji Y Ito Y Inhibition of 125I organification and thyroid hormone release by interleukin-1, tumor necrosis factor-alpha, and interferon-gamma in human thyrocytes in suspension culture.J Clin Endocrinol Metab. 1990; 70: 1735-1743Crossref PubMed Scopus (157) Google Scholar, 24Poncin S Lengele B Colin IM Gerard AC Differential interactions between Th1/Th2, Th1/Th3, and Th2/Th3 cytokines in the regulation of TPO and DUOX expression, and of thyroglobulin secretion in thyrocytes in vitro.Endocrinology. 2008; 149: 1534-1542Crossref PubMed Scopus (18) Google Scholar Mechanisms responsible for this inhibition are not yet known. In autoimmune processes targeting other cell types, such as pancreatic β cells, Th1 cytokine effects are mediated by nitric oxide (NO).25Corbett JA Wang JL Hughes JH Wolf BA Sweetland MA Lancaster Jr, JR McDaniel ML Nitric oxide and cyclic GMP formation induced by interleukin 1 beta in islets of Langerhans: evidence for an effector role of nitric oxide in islet dysfunction.Biochem J. 1992; 287: 229-235PubMed Google Scholar, 26Suarez-Pinzon WL Strynadka K Schulz R Rabinovitch A Mechanisms of cytokine-induced destruction of rat insulinoma cells: the role of nitric oxide.Endocrinology. 1994; 134: 1006-1010Crossref PubMed Scopus (72) Google Scholar In human, but not in rat thyrocytes, NO has also been identified as mediating the inhibitory actions of Th1 cytokines, but only partially.22Gerard AC Boucquey M van den Hove MF Colin IM Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.Am J Physiol Endocrinol Metab. 2006; 291: E242-E253Crossref PubMed Scopus (28) Google Scholar, 27Kasai K Hattori Y Nakanishi N Manaka K Banba N Motohashi S Shimoda S Regulation of inducible nitric oxide production by cytokines in human thyrocytes in culture.Endocrinology. 1995; 136: 4261-4270Crossref PubMed Google Scholar, 28Motohashi S Kasai K Banba N Hattori Y Shimoda S Nitric oxide inhibits cell growth in cultured human thyrocytes.Life Sci. 1996; 59: L227-L234Crossref PubMed Scopus (16) Google Scholar, 29Reimers JI Rasmussen AK Karlsen AE Bjerre U Liang H Morin O Andersen HU Mandrup-Poulsen T Burger AG Feldt-Rasmussen U Nerup J Interleukin-1 beta inhibits rat thyroid cell function in vivo and in vitro by an NO-independent mechanism and induces hypothyroidism and accelerated thyroiditis in diabetes-prone BB rats.J Endocrinol. 1996; 151: 147-157Crossref PubMed Scopus (15) Google Scholar, 30van den Hove MF Stoenoiu MS Croizet K Couvreur M Courtoy PJ Devuyst O Colin IM Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes.J Endocrinol. 2002; 173: 177-185Crossref PubMed Scopus (21) Google Scholar Intracellular factors other than NO should therefore mediate Th1 cytokine-induced inhibitory effects. ROS may represent, among others, alternative candidates, as suggested by previous studies. For instance, Th1 cytokines are known to increase ROS generation in the respiratory tract,31Harper RW Xu C Eiserich JP Chen Y Kao CY Thai P Setiadi H Wu R Differential regulation of dual NADPH oxidases/peroxidases: Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium.FEBS Lett. 2005; 579: 4911-4917Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar in osteoarthritis,32Darlington LG Stone TW Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders.Br J Nutr. 2001; 85: 251-269Crossref PubMed Google Scholar, 33Davies CM Guilak F Weinberg JB Fermor B Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis.Osteoarthritis Cartilage. 2008; 16: 624-630Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and in pancreatic islets.34Suarez-Pinzon WL Strynadka K Rabinovitch A Destruction of rat pancreatic islet beta-cells by cytokines involves the production of cytotoxic aldehydes.Endocrinology. 1996; 137: 5290-5296Crossref PubMed Scopus (55) Google Scholar, 35Rabinovitch A Suarez-Pinzon WL Strynadka K Lakey JR Rajotte RV Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production.J Clin Endocrinol Metab. 1996; 81: 3197-3202PubMed Google Scholar Up to now, nothing is known about the eventual involvement of ROS as intracellular mediators of Th1 cytokine-induced inhibitory actions in thyrocytes. In the present study we aimed to evaluate the impact of Th1 cytokines (interleukin [IL]-1α/interferon [IFN] γ) on ROS production and how they may influence thyroid cell function in vitro. Likewise, the role played by OS in thyroiditis was analyzed in vivo in the aforementioned NOD mouse model of spontaneous autoimmune thyoiditis. In both in vitro and in vivo models, the roles of ROS were evaluated by using N-acetylcysteine (NAC), a potent antioxidant, and 15dPGJ2 for its anti-inflammatory properties. We also investigated how antioxidant systems behave in these conditions. PCCL3 cells, a continuous line of nontransformed rat thyroid follicular cells,36Fusco A Berlingieri MT Di Fiore PP Portella G Grieco M Vecchio G One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.Mol Cell Biol. 1987; 7: 3365-3370Crossref PubMed Scopus (246) Google Scholar were a gift of Dr. F. Miot (Université Libre de Bruxelles, Institut de recherche interdisciplin aire en biologie humaine et moléculaire, Brussels, Belgium). They were grown to 80% to 90% confluence in Coon’s modified Ham’s F12 medium (BRL-Gibco, Paisley, Strathclyde, UK) supplemented with 5% newborn calf serum, penicillin (50 U/ml), streptomycin (50 μg/ml), fungizone (2.5 μg/ml; BRL-Gibco), 1 mU/ml thyroid stimulating hormone, 10 μg/ml insulin, and 5 μg/ml transferin (Sigma, Bornem, Belgium), in a humidified atmosphere (5% CO2). Recombinant rat IL-1α (2 ng/ml, Chemicon International, Temecula, CA) and recombinant rat IFNγ (100 U/ml, Chemicon International) were added for three additional days, in combination or not with NAC (1 mmol/L, Sigma) or 15dPGJ2 (2.5 μmol/L, Sigma) in the same medium containing 0.5% newborn calf serum and 1 mU/ml thyroid stimulating hormone. NAC, 15dPGJ2, or vehicle was added 2 hours before the cytokine cocktail. As a control, NAC or 15dPGJ2 were added on thyroid cells in the absence of cytokines. Human thyroid tissues from patients who underwent thyroid surgery for benign multinodular goiter were obtained from the anatomopathology department after patients gave their informed consent. Thyrocytes were isolated according to Nilsson et al37Nilsson M Husmark J Nilsson B Tisell LE Ericson LE Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function.Eur J Endocrinol. 1996; 135: 469-480Crossref PubMed Scopus (31) Google Scholar and suspended in modified Earle’s medium without phenol red containing 5% newborn calf serum, penicillin (50 U/ml), streptomycin (50 μg/ml), and fungizone (2.5 μg/ml; BRL-Gibco). They were plated in 6-well plates (50 μg DNA/well) or in multichamber glass slide (Nunc International, Naperville, IL; 7 μg DNA/chamber) and cultured in a humidified atmosphere (5% CO2) with 1 mU/ml thyroid stimulating hormone. After 1 week, cells were incubated for three additional days with cytokines in combination or not with NAC (1 mmol/L) or 15dPGJ2 (2.5 μmol/L), as described for PCCL3 cells. All experiments were repeated at least twice. Thyrocytes were incubated in multichamber glass slides in appropriate medium. ROS production was measured by using a fluorescent dye, 2′, 7′ dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Paisley, UK). PBS-washed (pH 7.4) thyroid cells were incubated in Krebs-Ringer HEPES medium, pH 7.4, containing DCFH-DA (25 μmol/L) at 37°C for 1 hour. The excess of dye was removed by two washes with PBS. Cells were stained with Hoechst for 20 minutes and rinsed in PBS. Cover slides were mounted in fluorescent mounting medium (DakoCytomation, Carpinteria, CA) for microscopic observation. ROS production was visualized on a fluorescent microscope equipped with a digital camera. Cell viability was assessed by using the Alamar blue assay (Biosource International, Camarillo, CA), as previously described.22Gerard AC Boucquey M van den Hove MF Colin IM Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.Am J Physiol Endocrinol Metab. 2006; 291: E242-E253Crossref PubMed Scopus (28) Google Scholar Caspase activity was measured by using a CaspACE fluorescein isothiocyanate-VAD-fmk in situ marker (Promega, Madison, WI), which binds activated caspases, according to the manufacturer’s instructions. Briefly, cells were incubated with 20 μmol/L fluorescein isothiocyanate-VAD-fmk at 37°C for 20 minutes. Cells were then washed twice with PBS, fixed in 10% buffered formalin for 30 minutes, and rinsed with PBS. Coverslides were mounted in fluorescent mounting medium for microscopic observation. Cells treated with staurosporine (5 μmol/L; Sigma) were used as positive control. Nitrite accumulation in the medium of human thyrocytes was measured by the Griess reaction by using a commercially available kit (Promega). Cells were suspended in TriPure isolation reagent (Roche Diagnostics GmbH, Mannheim, Germany), and total RNA was purified according to the manufacturer’s protocol. Reverse transcription was performed by incubating 2 μg RNA with 200 U Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen, Merelbeke, Belgium) in the recommended buffer containing 1 μl RNasin (Promega), 0.5 mmol/L dNTP (Promega), 2 μmol/L oligodT (Sigma), and 10 mmol/L dithiothreitol (20 μl final volume) overnight at 42°C. CDNA was diluted 1:5 in water for use in real time PCRs. CDNAs (2 μl) were mixed with 500 nmol/L of each selected primer (Table 1) and SYBR Green reaction mix (BioRad, Herts, UK) in a final volume of 25 μl. Reactions were performed by using a iCycler apparatus (BioRad) as follows: 95°C for 1 minute, followed by 40 cycles of 95°C for 15 seconds, annealing temperature for 45 seconds (Table 1), and 81°C for 15 seconds. Amplification levels were normalized to that of β-actin.Table 1Forward and Reverse Primers and Annealing Temperatures UsedTargetPrimer forwardPrimer reverseAnnealing temperature, °CActin5′-CATCCTGCGTCTGGACCT-3′5′-AGGAGGAGCAATGATCTTGAT-3′62rDuox5′-GTGGCTGGAGGGAGCCAT-3′5′-CCGTGAACAGACTCCTGT-3′60rDuox15′-CCTGCAAGCCAAAAGAAGAC-3′5′-CCACTGAAGTTTTCCCGTACA-3′60rDuox25′-AGAGGGAGCCATTACCCTGT-3′5′-CGCATAGCTGAGATGGATGA-3′60rTPO5′-CAGGTTTTGGTGGGAGAA-3′5′-CTGCACACTCATTAACATCTT-3′58rNIS5′-GCGCTGCGACTCTCCCACTGAC-3′5′-GGCGGTAGAAGATCGGCAAGAAGA-3′60hDuox5′-GTGGCTGGCTGACATCAT-3′5′-TGCAGGGAGTTGAAGAA-3′58h Duox15′-GGACCCCCAGGACCAGGAT-3′5′-CTTACACTCACCGCCCCAACAC-3′60hDuox25′-AACCCAAACGTCCATCAACA-3′5′-CCTTGTACCCCCTTCCACTT-3′58hTPO5′-CACGATGCAGAGAAACCTCAA-3′5′-ATAGACTGGAGGGAGCCAT-3′60hNIS5′-ACCGCGCCCCACCTCTTTCTTATT-3′5′-CCCCCTCCTGATTCTGGTTGTTG-3′62 Open table in a new tab Thyrocytes were suspended in Laemmli buffer (50 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol), containing a protease inhibitor cocktail (Sigma), and were sonicated during 30 seconds. Protein concentration was determined by using a bicinchoninic acid protein assay kit (Pierce, Rockfort, IL). Duox (antibody provided by F. Miot, IRIBHN, Brussels), TPO (antibody provided by J. Ruf, Université de la Méditerranée, Marseille, France), catalase (Sigma), peroxiredoxin 3 and 5 (PRDX3, PRDX5; antibodies provided by B. Knoops, Université catholique de Louvain, Louvain La Neuve), and β-actin (Sigma) Western blottings were performed as previously described.22Gerard AC Boucquey M van den Hove MF Colin IM Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.Am J Physiol Endocrinol Metab. 2006; 291: E242-E253Crossref PubMed Scopus (28) Google Scholar Proteins (30 μg/lane) were heated at 95°C for 5 minutes in the loading buffer (Laemmli buffer containing 100 mmol/L dithiothreitol and 0.1% bromophenol blue), separated by 8% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Hybond ECL, Amersham Biosciences, Rosenthaal, The Netherlands). Membranes were blocked for 1 hour at room temperature in PBS (pH 7.4), 5% nonfat dry milk, 0.1% Tween, and incubated overnight at 4°C with the primary antibody at a dilution of 1:4000 (Duox, TPO), 1:10,000 (PRDX3, PRDX5), or 1:2000 (catalase, β-actin). Membranes were incubated for 1 hour at room temperature with EnVision (1:200, DakoCytomation) peroxidase-labeled secondary antibody and visualized with enhanced chemiluminescence (SuperSignal West Pico, Pierce) on CLXposure TM films (Pierce). Western blots were scanned and quantified by densitometry using the NIH Scion Image Analysis Software (NIH, Bethesda, MD). Values were normalized by reporting the signal intensity to β-actin expression. Thyrocytes were cultured in multichamber glass slides in appropriate medium. Thyrocytes were fixed for 30 minutes in 4% paraformaldehyde, rinsed once with PBS, permeabilized for 15 minutes in a PBS-Triton 1% solution at room temperature, and washed with PBS supplemented with 1% bovine serum albumin. Cells were then incubated overnight with PRDX5 primary antibody (1:75) at room temperature. After being washed in PBS, fluorescein isothiocyanate-conjugated secondary antibody was added for 1 hour at room temperature at a dilution of 1:30 (anti-rabbit; DakoCytomation). Coverslides were mounted in fluorescent mounting medium for microscopic observation. Three-month-old female NOD mice, under a standard diet and kept under semibarrier conditions, were originally obtained from Professor Wu (Beijing, China) and inbreeded since 1989 (Proefdierencentrum, Leuven, Belgium). Animals were injected intraperitoneally with a saline solution of NAC (100 mg/kg/day) or with a saline solution of 15dPGJ2 (40 μg/kg/day) for 4 days. NMRI mice were used as control. Mice were housed and handled according to Belgian Regulation of Laboratory Animal Welfare. Five animals of each group were anesthetized with pentothal, and thyroid lobes were dissected. One thyroid lobe was fixed in paraformaldehyde (4% in PBS) for 24 hours and embedded in paraffin. The second lobe was frozen for cryostat sections. Thick sections (5 μm) were used for morphology analysis and for immunohistochemistry. Morphometric measurements were performed by using the point-counting method described by Weibel et al.38Weibel ER Kistler GS Scherle WF Practical stereological methods for morphometric cytology.J Cell Biol. 1966; 30: 23-38Crossref PubMed Scopus (1460) Google Scholar For each thyroid, 1000 points were counted, and the relative volumes of epithelium, colloid, vessels, and interstitium were measured. 4-hydroxynonenal (4-HNE), PRDX5, and catalase immunostainings were performed on paraffin sections. Sections were dewaxed and rehydrated. Except for PRDX5 detection, paraffin sections were pretreated in a microwave oven in citrate buffer (pH 6.6) for one cycle of 3 minutes at 750 W, followed by four cycles of 3.5 minutes, each at 350 W. Endogenous peroxidases were quenched with 1% H2O2 for 30 minutes. Then, paraffin sections were washed with PBS supplemented with 1% or 5% bovine serum albumin and thereafter incubated in PBS supplemented with 1% or 5% bovine serum albumin containing 2% or 5% normal goat serum at room temperature. Sections were incubated with the first antibody (4-HNE, PRDX5, and catalase) at room temperature (Table 2). The binding of antibodies was detected by using a second antibody conjugated to a peroxidase-labeled polymer (EnVision detection, DakoCytomation, Carpinteria, CA) or a biotinylated second antibody for 30 minutes followed by an avidin-biotin peroxidase complex for 30 minutes (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The peroxidase activity was measured with 3-amino-9-ethylcarbazole substrate (DakoCytomation). Sections were counterstained with Mayer’s hematoxylin, rinsed, and mounted in Faramount Aqueous mounting medium (DakoCytomation). To verify the binding specificity, some sections were incubated with the second antibody alone.Table 2Experimental Conditions for ImmunohistochemistryAntibodyFirst antibodySecond antibodyRevelation substrate4-HNE (Calbiochem, Darmstadt, Germany)Rabbit polyclonalEnVision rabbit (DakoCytomation)AEC (DakoCytomation)Dilution: 1:800Incubation time: overnightPRDX5 (Polyclonal rabbit, B. Knoops, Université catholique de Louvain)Rabbit polyclonalEnVision rabbit (DakoCytomation)AEC (DakoCytomation)Dilution: 1:200Incubation time: 1 hourPRDX3 (Polyclonal rabbit, B. Knoops, Université catholique de Louvain)Rabbit polyclonalEnVision rabbit (DakoCytomation)AEC (DakoCytomation)Dilution: 1:500Incubation time: 1 hourCatalase (Sigma)Monoclonal mouseEnVision mouse (DakoCytomation)AEC (DakoCytomation)Dilution: 1:200Incubation time: 3 hours Open table in a new tab Data were expressed as mean ± SEM, n = 6 for all experiments. Each experiment was repeated at least twice. Statistical analyses were performed by using analysis of variance followed by Tukey-Kramer Multiple Comparison Test (GraphPad InStat, San Diego, CA), or by unpaired t-test. P < 0.05 was considered as statistically significant. Although Th1 cytokines are known to induce ROS production in various cell types,31Harper RW Xu C Eiserich JP Chen Y Kao CY Thai P Setiadi H Wu R Differential regulation of dual NADPH oxidases/peroxidases: Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium.FEBS Lett. 2005; 579: 4911-4917Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 32Darlington LG Stone TW Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders.Br J Nutr. 2001; 85: 251-269Crossref PubMed Google Scholar, 34Suarez-Pinzon WL Strynadka K Rabinovitch A Destruction of rat pancreatic islet beta-cells by cytokines involves the production of cytotoxic aldehydes.Endocrinology. 1996; 137: 5290-5296Crossref PubMed Scopus (55) Google Scholar, 35Rabinovitch A Suarez-Pinzon WL Strynadka K Lakey JR Rajotte RV Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production.J Clin Endocrinol Metab. 1996; 81: 3197-3202PubMed Google Scholar this has not been yet described in thyrocytes. IL-1α/IFNγ-induced ROS, and nitrite production was therefore analyzed both in rat and human thyroid cells. In rat PCCL3 control cells, ROS detected by DCFH-DA fluorescence were observed as granules within the cytoplasm (Figure 1A). The staining was greatly enhanced in Th1 cytokine-treated cells (Figure 1B), whereas ROS were detected both in the cytoplasm and in nuclei. In cells treated with NAC alone, and in accordance with NAC anti-oxidant properties, ROS fluorescence was strongly reduced compared with control cells (Figure 1C). In cells co-incubated with IL-1α/IFNγ together with NAC, ROS fluorescence was below control levels (Figure 1D). By contrast, 15dPGJ2 influenced ROS production, neither in control cells (data not shown) nor in Th1 cytokine-treated cells (Figure 1E). Similar results were obtained in human primary cells (data not shown). According to previous results, nitrite levels, the stable end-product of NO generation, were low in media from control human thyroid cells, but greatly enhanced in Th1 cytokine-treated cells (Figure 1F).22Gerard AC Boucquey M van den Hove MF Colin IM Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.Am J Physiol Endocrinol Metab. 20
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