Extracellular Adenosine 5'-Triphosphate Modulates Interleukin-6 Production by Human Thyrocytes through Functional Purinergic P2 Receptors
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内分泌学杂志 2005年第7期
Department of Internal Medicine (N.C., F.M., E.S., S.C., E.F., A.S.), University of Pisa, I-56100 Pisa; and Department of Experimental and Diagnostic Medicine (D.F., M.G.C., S.G., C.P., F.D.V.), Section of General Pathology, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, I-44100 Ferrara, Italy
Address all correspondence and requests for reprints to: Anna Solini, M.D. Ph.D., Department of Internal Medicine University of Pisa, Section of Internal Medicine III, Via Roma 67, I-56100 Pisa. Italy. E-mail: a.solini@med.unipi.it.
Abstract
We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X3, P2X5, P2X6, P2X7, and P2Y1, P2Y2, P2Y4, and P2Y11. Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca2+ signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca2+ ([Ca2+]i) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca2+]i) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.
Introduction
EXTRACELLULAR NUCLEOTIDES are emerging as ubiquitous mediators of cell-to-cell communication (1) and potent stimuli for release of bioactive factors from many different cell types (2, 3). Furthermore, for their unusual ability to drive chemotaxis and differentiation of dendritic cells, nucleotides are attracting increasing attention as danger signals released during the initial phases of inflammation (4, 5). Nucleotides are not only exteriorized as a consequence of cell injury or cell death, but also via nonlytic mechanisms involving secretory exocytosis or plasma membrane transporters (6). In their function of extracellular messengers, nucleotides are ligated by selective plasma membrane receptors, P2 receptors (P2Rs), belonging to the purinergic receptor family, together with receptors for extracellular adenosine (P1Rs). The P2Rs are further classified into ionotropic P2XRs and metabotropic P2YRs. P2XRs, of which seven subtypes have been so far cloned (P2X1–7), are cation-selective channels directly gated by extracellular ATP, the only known physiological agonist. They were originally identified and characterized in neurons, and later found in nonexcitable cells (7). The P2YRs are typical seven membrane-spanning, G protein-coupled receptors, with a broader agonist selectivity than the P2XRs. So far, eight P2Y subtypes have been cloned, but their number will likely increase as more orphan receptors are screened for ability to respond to extracellular nucleotides (8). Among P2YRs, P2Y11 is the only ATP-selective, whereas P2Y1, P2Y12, and P2Y13 prefer ADP, at P2Y2 ATP and uridine 5'-triphosphate (UTP) are equipotent, P2Y4 prefers UTP and P2Y6 is uridine 5'-diphosphate selective. At P2Y14, the preferred ligand is uridine 5'-diphosphate-glucose. As true danger signals, extracellular nucleotides are potent stimuli for release of key proinflammatory cytokines such as IL-1?, IL-6, and TNF (4). Although it is generally thought that the main receptor involved in these responses is P2X7, clear evidence also supports a role for P2Y6.
The role of extracellular nucleotides and their receptors has been so far investigated in a few endocrine glands. P2Rs activation either stimulates or inhibits, depending on nucleotide concentration, insulin release from INS1 cells and rat pancreatic islets (9), and triggers estradiol secretion from rat Sertoli cells (10). In endocrine tissues, a nucleotide-based autocrine/paracrine loop seems to be operating because endocrine cells not only express P2Rs but also secrete ATP via nonlytic mechanisms (11). Little is known of the expression and function of P2Rs in human thyroid, despite the potential role that nucleotides might play in this endocrine gland thanks to their immunomodulatory role. The thyroid gland is in fact both a target for immune-mediated responses and a source of cytokines, among which IL-6 is of paramount relevance.
Over the last years, our understanding of the role and mechanism of action of cytokines has greatly expanded, and it is now generally accepted that, besides their functions in the immune and hematopoietic system, cytokines participate in an extended neuro-immune-endocrine network. IL-6, in particular, is a pleiotropic factor with strong differentiating and growth-promoting effects in endocrine tissues: its immunoreactivity has been detected in adrenal cells, in Leydig and Sertoli cells in the testis, and in 12% of pituitary adenomas, whereas pancreatic islets are variably positive (12, 13). To further underscore the tight links with the endocrine system, IL-6 synthesis and release are suppressed by steroids and estrogens (14), and stimulated by catecholamines (15).
In the thyroid, IL-6 seems to play a pivotal role in the pathogenesis of the euthyroid sick syndrome, most likely by inhibiting 5'-deiodinase (16), and an increased IL-6 production has been claimed as one of the mechanisms by which amiodarone exerts its toxic effect on the thyroid gland (17). A significant role of IL-6 in the pathogenesis of Graves’ disease has also been suggested, whereas antithyroid drugs have been shown to block IL-6 response to sublethal complement attack in thyrocytes from Graves’ glands (18). Furthermore, IL-6 secretion was found strongly increased in anaplastic thyroid carcinoma cells (19). There is also evidence, albeit controversial, that TSH supports IL-6 secretion (14, 17, 20).
Overall, despite ample evidence that human thyrocytes synthesize and secrete IL-6 (20, 21), the mechanisms regulating its synthesis and release are poorly understood. The aim of the present study was to investigate the possible role in IL-6 secretion from thyroid tissue of an autocrine/paracrine loop based on extracellular ATP and P2Rs.
Materials and Methods
Cell cultures
Human thyrocytes were obtained from freshly isolated thyroid tissue samples from six euthyroid patients subjected to total thyroidectomy for cold nodular goiter. Tissue samples were carefully dissected and minced with scissors into small pieces and incubated with 200 U/ml type II collagenase (Invitrogen, Grand Island, NY) in a shaking water bath at 37 C for 4–6 h. Thyroid cells were resuspended in DMEM containing 10% fetal calf serum, 100 IU/ml penicillin, and 50 μg/ml streptomycin (all from Sigma-Aldrich, Milano, Italy). Cells were counted and plated in 25-cm2 Falcon primary tissue culture flasks (Becton Dickinson, Franklin Lanes, NJ), and incubated at 37 C in a humidified incubator, in the presence of 5% CO2. Cells were used for experiments at 70–80% confluence.
Cytoplasmic free Ca2+ measurement
Changes in [Ca2+]i were measured with the fluorescent indicator Fura-2/AM (Molecular Probes, Leiden, The Netherlands), using an LS50 PerkinElmer fluorometer (PerkinElmer Ltd., Beaconsfield, UK). Cells were detached from flasks, seeded onto glass coverslips, and incubated with 2 μM fura-2/AM and 250 μM sulfinpyrazone (Sigma-Aldrich) in a saline solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 5 mM NaHCO3, 1 mM CaCl2, and 20 mM HEPES (pH 7.4 with NaOH), hereafter also referred to as standard saline solution. In some experiments, CaCl2 was omitted and 0.5 mM EGTA added (Ca2+ free saline solution). After 20 min of incubation at 37 C, coverslips were rinsed twice with the same solution. [Ca2+]i changes were determined in a thermostated cuvette equipped with a magnetic stirrer. Excitation ratio was 340/380 at an emission wavelength of 505 nm. Experiments were performed either in the presence or absence of extracellular calcium, as indicated.
RT-PCR
Total RNA was extracted using a RNeasy Mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. High-purity RNA was eluted in 30 μl of water and treated with ribonuclease-free deoxyribonuclease to guarantee elimination of DNA contamination from RNA samples. RNA was quantified spectrophotometrically, and a constant amount of total RNA (5 μg) was reverse transcribed at 42 C for 60 min in a total reaction volume of 20 μl, using first-strand cDNA Synthesis Kit (Roche Diagnostics Corp., Indianapolis, IN). cDNA was incubated at 95 C for 5 min to inactivate the reverse transcriptase and served as a template DNA for 30 amplification rounds in a Cycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA).
RT-PCR was performed in a standard 25-μl reaction mixture, consisting of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM deoxynucleotide triphosphates, 20 pmol of each sense and antisense primer, and 2.5 U of AmpliTaq DNA polymerase (Laboratoires Eurobio, Les Ulis Cedex, France). Table 1 shows primer sequences and experimental conditions for RT-PCR, as well as size of the cDNA fragments. As negative control, the DNA template was omitted in the reaction. The presence of a single band amplified with specific primers for ?-actin with the same cDNA was used as internal control under identical conditions. Each experiment was replicated at least three times. RT-PCR products were run on a 2% agarose gel containing 0.5 μg/ml ethidium bromide and photographed under UV light. For semiquantitative analysis, the film was subjected to densitometry, using Kodak Digital Science 1D system (Eastman-Kodak Co., Fullerton, CA).
TABLE 1. P2X and P2Y receptors found to be expressed in human thyrocytes: primer sequences, size of fragments, and PCR conditions
IL-6 determinations
For Western blot analysis, cells were resuspended in a buffer containing 300 mM sucrose, 1 mM MgSO4, 1 mM K2HPO4, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, 0.2 μM deoxyribonuclease, and 0.2 μg of ribonuclease and lysed by repeated homogenizations. Proteins were separated on 14% sodium dodecyl sulfate-polyacrylamide gel, and blotted overnight onto nitrocellulose paper. The biotinilated specific rat monoclonal antibody (eBioscience, San Diego, CA) was diluted 1:250 in Tween-Tris-buffered saline. Neutravidin diluted 1:6000 in Tween-Tris-buffered saline was used as internal control. IL-6 release was measured by enzyme-linked immunosorbent assay using a biotin-conjugated monoclonal anti-IL-6 antibody (Bioscience, San Diego, CA). The assay sensitivity was less than 2 pg/ml, and the interassay coefficient of variation was 5.2%. Apyrase from potato (Sigma-Aldrich) to inhibit cell stimulation by spontaneously released extracellular ATP was used at a concentration of 5 U/ml. Inactivation was performed by boiling for 15 min.
ATP determinations
ATP levels were measured by luminometric assay, using the ATP-Lite Luminescence ATP Detection Assay System (PerkinElmer Ltd., Beaconsfield, UK), a high-sensitivity detection system (down to 5 cells in 100 μl medium) with high reproducibility and long half-life of the light emission (>5 h). Moreover, this system ensures an efficient inactivation of ecto-ATPases, thus avoiding underestimation of extracellular ATP concentration. Thyrocytes were seeded at a concentration of 5 x 103 cells/well in microtiter plastic dishes in a total volume of 100 μl of culture medium. Before ATP measurements, cells were rinsed and supplemented with 100 μl of ATP-Lite (buffer solution + substrate solution with luciferase-luciferin assay; PerkinElmer). Cells were directly placed into the test chamber of the luminometer, light emission was recorded, and converted into ATP concentration (extracellular ATP). Cells were then shaken for 5 min in the presence of lysis solution, and measurements repeated to determine total ATP levels; intracellular ATP was calculated by subtraction.
Results
Human thyrocytes express the mRNA for P2X and P2Y receptor subtypes
Beside a few reports in rat thyroid cells (22), no information on P2Rs expression and function is available for human primary thyrocytes so far. RT-PCR shows that human thyrocytes express mRNA for the following P2XR and P2YR subtypes: P2X3, P2X4, P2X5, P2X6, and P2X7, and P2Y1, P2Y2, P2Y4, P2Y11 (Fig. 1). Some of these receptors, however, were weakly expressed, and their expression varied among subjects.
FIG. 1. mRNA for P2X and P2Y receptor subtypes expressed by thyrocytes. RT-PCR was performed as described in Materials and Methods. Lanes 1–6 are thyrocyte samples from six different donors; in lane 7 FB2 thyroid carcinoma cells were used as positive control. In lane 8, DNA template was omitted as negative control.
Stimulation with ATP or UTP induces [Ca2+]i transients
As anticipated by the expression of both P2YRs andP2XRs, and as previously reported in rat thyrocytes and FRTL cells (22, 23), stimulation with ATP caused a [Ca2+]i increase (Fig. 2, A and B) characterized by an early rise, presumably due to [Ca2+]i release from intracellular stores, and a delayed plateau, likely due to influx across the plasma membrane. Incubation in a Ca2+-free, 0.5 mM EGTA-containing saline solution (dotted lines), showed little effect on the early [Ca2+]i increase, whereas it fully abrogated the delayed plateau. Obliteration of the late [Ca2+]i increase was particularly striking upon stimulation with 1 mM ATP. This is not surprising because the contribution of Ca2+ influx to the [Ca2+]i rise is much more relevant at high (millimolar) rather than low (nanomolar) nucleotide concentrations.
FIG. 2. ATP induces [Ca2+]i increases in the presence as well as in the absence of extracellular Ca2+. Cells were loaded with the Ca2+ indicator fura-2/AM as reported in Materials and Methods. Thyrocytes were stimulated with ATP (A and B) in the presence (continuous line) or absence (dotted line) of extracellular Ca2+. For Ca2+-free conditions, cells were incubated in Ca2+-free, 0.5 mM EGTA-supplemented medium. For nucleotide dose-dependency curves, cells were incubated in the presence of increasing ATP (C and D) or UTP (E) concentrations. Experiments were performed in Ca2+ containing standard saline (C and E), or Ca2+-free, 0.5 mM EGTA-supplemented solution (D).
Dose-dependency curve performed in a standard saline solution (i.e. in the presence of 1 mM Ca2+) (Fig. 2C) shows that half-maximal effect is obtained with an ATP concentration of 3 μM, whereas plateau is reached at 300 μM. Half-maximal ATP stimulatory concentration in the absence of extracellular Ca2+ is about 2 μM, whereas maximal stimulatory concentration is about 100 μM (Fig. 2D). Thyrocytes were also sensitive to stimulation with UTP. Half-maximal and maximal Ca2+ responses were obtained with 2 and 100 μM UTP, respectively (Fig. 2E).
TSH is known to modulate many thyroid functions; thus, we investigated the effect of TSH treatment on the nucleotide-triggered [Ca2+]i increase (Fig. 3). At low nucleotide concentrations (Fig. 3, A and C) incubation in the presence of TSH 10 mU/ml had little effect on the Ca2+ transient (Fig. 3A, [Ca2+]i increase of 70 ± 12 and 62 ± 10 nM, P = not significant in the presence or absence of TSH, respectively; Fig. 3C, [Ca2+]i increase of 150 ± 13 and 145 ± 7 nM, P = not significant in the presence or absence of TSH, respectively). However, at higher concentrations both the early and delayed Ca2+ increments were substantially reduced (Fig. 3, B and D). With ATP as a stimulus, the [Ca2+]i increase was 280 ± 32 and 122 ± 24 nM, P < 0.001 in the presence or absence of TSH, respectively. With UTP as a stimulus, the [Ca2+]i increase was 170 ± 26 and 70 ± 14 nM, P < 0.001 in the absence or presence of TSH, respectively.
FIG. 3. Nucleotide-induced [Ca2+]i increases are modulated by TSH. Cells were treated as in Fig. 2. Thyrocytes were stimulated in a standard saline solution, with ATP (A and B) or UTP (C and D). Continuous line, Control cells; dotted line, TSH-stimulated cells. Each trace is representative of 12 similar performed in each experimental condition.
Autocrine-secreted ATP induces IL-6 release from human thyrocytes
As already pointed out, IL-6 appears to be an important inflammatory mediator in the thyroid. Given recent evidence supporting a role for nucleotides in IL-6 release (3), we first explored the role of ATP in modulating spontaneous IL-6 release. IL-6 is measurable in the surnatants of resting cells, showing that nonstimulated thyrocytes spontaneously release this cytokine. As previously reported (14), IL-6 release is substantially increased by treating cells with phorbol 12-myristate 13-acetate 100 nM. Incubation in the presence of 5 U/ml of apyrase, an ecto-ATP/ADPase that hydrolyzes extracellular ATP/ADP to AMP and inorganic phosphate, decreases spontaneous IL-6 release by about 20–30% (Fig. 4).
FIG. 4. Spontaneous IL-6 release in human thyrocytes. IL-6 release in the supernatants was measured by ELISA as described in Materials and Methods; black bars, untreated cells; gray bars, apyrase; white bars, heat-inactivated apyrase; hatched bars, phorbol 12-myristate 13-acetate-stimulated cells. Data are averages ± SD of 24 determinations from the six different donors. *, P < 0.05 vs. untreated cells.
To confirm that this effect was specifically due to enzymatic activity, we repeated the experiment with the heat-inactivated enzyme: this treatment prevented the inhibitory effect of apyrase on IL-6 release. Accordingly, extracellular ATP was 71 ± 10 nM in the supernatants from unstimulated cells and decreased to undosable levels in cells incubated with apyrase, whereas it was 86 ± 15 nM in cells treated with the heat-inactivated enzyme.
Stimulation with exogenous nucleotides increases IL-6 synthesis and release from thyrocytes
We next investigated whether exogenous ATP could stimulate IL-6 release. As shown by Western blot in Fig. 5, ATP (B) or UTP (C) treatment induced synthesis of the IL-6 protein. Accordingly, cytokine externalization was also stimulated by ATP in a dose-dependent fashion (Fig. 6A). Dose-dependency curve was bell shaped, with an optimal ATP dose between 0.25 and 0.5 mM. At ATP concentrations higher than 0.5 mM, IL-6 secretion was inhibited. UTP, a nucleotide preferentially active at the P2Y2 and P2Y4 receptors, also caused IL-6 release (Fig. 6B, left); conversely BzATP, a potent P2X7 agonist, was fully inactive (Fig. 6B, right).
FIG. 5. Effect of ATP and UTP on IL-6 protein expression. Western blot was performed as described in Materials and Methods and is representative of six different experiments. A, Control cells; B, 0.25 mM ATP; C, 0.25 mM UTP. Lane 1, Neutravidin; lane 2, neutravidin + specific IL-6 antibody.
FIG. 6. Effect of ATP, UTP, or BzATP on IL-6 secretion. Cells were stimulated with increasing nucleotide concentrations for 6 h. IL-6 in the supernatants was measured by ELISA as described in Materials and Methods. Data are averages ± SD of 30 determinations from the six different donors. *, P < 0.05; °, P < 0.001 vs. basal.
Finally, we explored whether TSH, besides decreasing the [Ca2+]i rise, also down-regulated IL-6 release. Figure 7 shows that acute challenge with TSH had no significant stimulatory effect on basal IL-6 secretion; chronic incubation with TSH had little effect on IL-6 release induced by low nucleotide concentrations (1100 ± 120 vs. 1130 ± 160 pg/ml/3 x 103 cells, n = 30, in samples stimulated with 0.1 mM ATP in the absence or presence of TSH, respectively), whereas it significantly decreased IL-6 secretion triggered by higher ATP concentrations (P < 0.001 for TSH-less vs. TSH-treated samples at all ATP concentrations above 0.1 mM).
FIG. 7. Effect of TSH on ATP-induced IL-6 secretion. Cells were maintained in the presence of 10 mU/ml TSH alone or together with increasing ATP concentrations for 6 h. IL-6 in the supernatants was measured by ELISA as described in Materials and Methods. Data are averages ± SD of 30 determinations from the six different donors.
Discussion
P2Rs modulate ion conductance and activate different signaling cascades in several cell types. However, little is known on their presence and function in primary human thyroid cells, besides a few reports showing that extracellular ATP activates phospholipase C and regulates DNA synthesis in the rat FRTL-5 thyroid cell line (23, 24), or can induce generation of inositols in human thyrocytes (25).
In the present study, we show that human primary thyrocytes, despite an individual variability among different subjects, express mRNA for the P2X3–7 and P2Y1, P2Y2, P2Y4, and P2Y11 receptor subtypes. Among P2XRs, the P2X4 subtype was robustly expressed in samples from all donors, whereas the P2X7R was weakly expressed in four of six subjects, and basically absent in the remaining two. Among P2YRs, P2Y1, P2Y2 and P2Y4 were present in all samples, whereas P2Y11 was detected in four of six. Stimulation with either ATP or UTP triggered a rise in [Ca2+]i and release of IL-6 in cells from all donor subjects, whereas the selective P2X ligand BzATP was ineffective, thus indicating a main role of P2YRs in cytokine release. The P2X7R was previously shown to stimulate IL-6 and IL-1? secretion in vitro and in vivo (1, 26, 27); the minor, if any, role of P2X7 in thyrocytes might be due to its low expression. Although the ATP dose dependency for IL-6 release was slightly shifted to the right compared with that for the [Ca2+]i rise, there was a fairly good correlation between the two events, suggesting a role of [Ca2+]i changes in ATP-stimulated IL-6 secretion.
Thyroid cells constitutively release IL-6 and other cytokines (28) by a complex process involving several mutually interacting stimulatory and inhibitory pathways as yet incompletely understood. In the presence of the nucleotide-hydrolyzing enzyme apyrase, basal IL-6 release was substantially, although not entirely, curtailed. This suggests that also in the thyroid, as previously shown in other tissues, cytokine secretion can be modulated by an autocrine/paracrine purinergic loop. In support of this hypothesis, we show that cultured thyrocytes spontaneously secrete ATP. The ATP concentration in the culture supernatants is in the 50–80 nM range, well below the threshold for IL-6 release. However, it is necessary to remind that the ATP concentration in the bulk solution does not accurately reflect ATP levels near the release site, i.e. at the plasma membrane surface, especially if secretion occurs at restricted, specialized sites. In the few instances where probes capable of measuring the ATP concentration close to the plasma membrane were used, ATP levels 10- to 20-fold higher than those in the supernatants were found (29), a concentration that is sufficient to support tonic stimulation of P2YRs.
The thyroid is a paradigmatic example of the close relationship between the endocrine and the immune system because this gland is a preferential target of immune-mediated diseases. A host of lymphocyte-derived factors are known to deeply affect thyroid function (28). The ability of ATP to support IL-6 release points to this nucleotide as an additional participant in the cross-talk between the endocrine and immune systems. In the thyroid, ATP can be released directly by the thyrocytes themselves, as shown in the present work, by vascular endothelium, or by infiltrating lymphocytes or monocytes (30, 31). Increasing evidence suggests that extracellular nucleotides can act as danger signals to alert the immune system of cell and tissue damage, and as such nucleotides have the ability to drive secretion of proinflammatory cytokines, and trigger activation and differentiation of dendritic cells (4, 32). In the light of their purported role as a proinflammatory mediators, or as danger signals (4), it is not surprising that extracellular nucleotides are also potent inducers of IL-6 release.
IL-6, together with IL-1? (which is also constitutively produced by thyrocytes), might be an amplification signal to spread the inflammatory response initiated by drugs and/or autoimmune aggression.
IL-6 is also known to directly damage thyrocytes; however, under our experimental conditions we did not observe any short-term direct cell damage, probably because thyrocytes themselves seldom express receptors for IL-6 (33).
A further interesting finding is that signaling by ATP is modulated by TSH. This hormone decreases the ATP-induced Ca2+ increase as well as IL-6 release. Both the early and delayed phase of the [Ca2+]i rise are reduced by TSH, suggesting that both release from stores and influx across the plasma membrane are affected. The effect of TSH on IL-6 secretion is controversial: previous studies showed a stimulatory action (20, 34, 35), whereas a later article reported that TSH does not stimulate IL-6 release from thyrocytes obtained from either Graves’ patients or from multinodular goiters (14). Our data suggest a novel pathway for modulation of IL-6 release in the thyroid based on the inhibitory activity of TSH on ATP-stimulated responses. The observation that TSH is unable to down-regulate IL-6 release under basal conditions, i.e. in the presence of low extracellular ATP levels, although it is effective at high ATP concentrations, suggests that this modulatory pathway is inactive in the healthy thyroid, becoming operational only under conditions like stress or inflammation, when the local ATP concentration increases dramatically due to enhanced release from the damaged thyrocytes or infiltrating immune cells. This antiinflammatory role of TSH could also explain the already described negative correlation between IL-6 and T3 or T4 (36).
The interaction between TSH and IL-6 may have several clinical implications, for example in the regulation of sodium/iodide symporter (NIS) expression. The gene encoding NIS, a transporter that plays a critical role in iodide transport in the thyroid and in the production of the iodine-containing thyroid hormones, is up-regulated by TSH and down-regulated by IL-6 (37). Our results suggest an additional pathway, via a reduction of ATP-induced IL-6 release, by which TSH could promote NIS expression.
Collectively, our data are in keeping with the hypothesis that the local production of IL-6 in the microenvironment may be part of an homeostatic mechanism and/or cellular alarm system regulating thyrocyte function. Extracellular ATP participates in the modulation of IL-6 synthesis and release by thyrocytes: therefore, the purinergic system may have a role in the regulation of thyroid function and may be involved in the propagation and progression of autoimmune thyroid diseases.
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Address all correspondence and requests for reprints to: Anna Solini, M.D. Ph.D., Department of Internal Medicine University of Pisa, Section of Internal Medicine III, Via Roma 67, I-56100 Pisa. Italy. E-mail: a.solini@med.unipi.it.
Abstract
We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X3, P2X5, P2X6, P2X7, and P2Y1, P2Y2, P2Y4, and P2Y11. Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca2+ signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca2+ ([Ca2+]i) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca2+]i) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.
Introduction
EXTRACELLULAR NUCLEOTIDES are emerging as ubiquitous mediators of cell-to-cell communication (1) and potent stimuli for release of bioactive factors from many different cell types (2, 3). Furthermore, for their unusual ability to drive chemotaxis and differentiation of dendritic cells, nucleotides are attracting increasing attention as danger signals released during the initial phases of inflammation (4, 5). Nucleotides are not only exteriorized as a consequence of cell injury or cell death, but also via nonlytic mechanisms involving secretory exocytosis or plasma membrane transporters (6). In their function of extracellular messengers, nucleotides are ligated by selective plasma membrane receptors, P2 receptors (P2Rs), belonging to the purinergic receptor family, together with receptors for extracellular adenosine (P1Rs). The P2Rs are further classified into ionotropic P2XRs and metabotropic P2YRs. P2XRs, of which seven subtypes have been so far cloned (P2X1–7), are cation-selective channels directly gated by extracellular ATP, the only known physiological agonist. They were originally identified and characterized in neurons, and later found in nonexcitable cells (7). The P2YRs are typical seven membrane-spanning, G protein-coupled receptors, with a broader agonist selectivity than the P2XRs. So far, eight P2Y subtypes have been cloned, but their number will likely increase as more orphan receptors are screened for ability to respond to extracellular nucleotides (8). Among P2YRs, P2Y11 is the only ATP-selective, whereas P2Y1, P2Y12, and P2Y13 prefer ADP, at P2Y2 ATP and uridine 5'-triphosphate (UTP) are equipotent, P2Y4 prefers UTP and P2Y6 is uridine 5'-diphosphate selective. At P2Y14, the preferred ligand is uridine 5'-diphosphate-glucose. As true danger signals, extracellular nucleotides are potent stimuli for release of key proinflammatory cytokines such as IL-1?, IL-6, and TNF (4). Although it is generally thought that the main receptor involved in these responses is P2X7, clear evidence also supports a role for P2Y6.
The role of extracellular nucleotides and their receptors has been so far investigated in a few endocrine glands. P2Rs activation either stimulates or inhibits, depending on nucleotide concentration, insulin release from INS1 cells and rat pancreatic islets (9), and triggers estradiol secretion from rat Sertoli cells (10). In endocrine tissues, a nucleotide-based autocrine/paracrine loop seems to be operating because endocrine cells not only express P2Rs but also secrete ATP via nonlytic mechanisms (11). Little is known of the expression and function of P2Rs in human thyroid, despite the potential role that nucleotides might play in this endocrine gland thanks to their immunomodulatory role. The thyroid gland is in fact both a target for immune-mediated responses and a source of cytokines, among which IL-6 is of paramount relevance.
Over the last years, our understanding of the role and mechanism of action of cytokines has greatly expanded, and it is now generally accepted that, besides their functions in the immune and hematopoietic system, cytokines participate in an extended neuro-immune-endocrine network. IL-6, in particular, is a pleiotropic factor with strong differentiating and growth-promoting effects in endocrine tissues: its immunoreactivity has been detected in adrenal cells, in Leydig and Sertoli cells in the testis, and in 12% of pituitary adenomas, whereas pancreatic islets are variably positive (12, 13). To further underscore the tight links with the endocrine system, IL-6 synthesis and release are suppressed by steroids and estrogens (14), and stimulated by catecholamines (15).
In the thyroid, IL-6 seems to play a pivotal role in the pathogenesis of the euthyroid sick syndrome, most likely by inhibiting 5'-deiodinase (16), and an increased IL-6 production has been claimed as one of the mechanisms by which amiodarone exerts its toxic effect on the thyroid gland (17). A significant role of IL-6 in the pathogenesis of Graves’ disease has also been suggested, whereas antithyroid drugs have been shown to block IL-6 response to sublethal complement attack in thyrocytes from Graves’ glands (18). Furthermore, IL-6 secretion was found strongly increased in anaplastic thyroid carcinoma cells (19). There is also evidence, albeit controversial, that TSH supports IL-6 secretion (14, 17, 20).
Overall, despite ample evidence that human thyrocytes synthesize and secrete IL-6 (20, 21), the mechanisms regulating its synthesis and release are poorly understood. The aim of the present study was to investigate the possible role in IL-6 secretion from thyroid tissue of an autocrine/paracrine loop based on extracellular ATP and P2Rs.
Materials and Methods
Cell cultures
Human thyrocytes were obtained from freshly isolated thyroid tissue samples from six euthyroid patients subjected to total thyroidectomy for cold nodular goiter. Tissue samples were carefully dissected and minced with scissors into small pieces and incubated with 200 U/ml type II collagenase (Invitrogen, Grand Island, NY) in a shaking water bath at 37 C for 4–6 h. Thyroid cells were resuspended in DMEM containing 10% fetal calf serum, 100 IU/ml penicillin, and 50 μg/ml streptomycin (all from Sigma-Aldrich, Milano, Italy). Cells were counted and plated in 25-cm2 Falcon primary tissue culture flasks (Becton Dickinson, Franklin Lanes, NJ), and incubated at 37 C in a humidified incubator, in the presence of 5% CO2. Cells were used for experiments at 70–80% confluence.
Cytoplasmic free Ca2+ measurement
Changes in [Ca2+]i were measured with the fluorescent indicator Fura-2/AM (Molecular Probes, Leiden, The Netherlands), using an LS50 PerkinElmer fluorometer (PerkinElmer Ltd., Beaconsfield, UK). Cells were detached from flasks, seeded onto glass coverslips, and incubated with 2 μM fura-2/AM and 250 μM sulfinpyrazone (Sigma-Aldrich) in a saline solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 5 mM NaHCO3, 1 mM CaCl2, and 20 mM HEPES (pH 7.4 with NaOH), hereafter also referred to as standard saline solution. In some experiments, CaCl2 was omitted and 0.5 mM EGTA added (Ca2+ free saline solution). After 20 min of incubation at 37 C, coverslips were rinsed twice with the same solution. [Ca2+]i changes were determined in a thermostated cuvette equipped with a magnetic stirrer. Excitation ratio was 340/380 at an emission wavelength of 505 nm. Experiments were performed either in the presence or absence of extracellular calcium, as indicated.
RT-PCR
Total RNA was extracted using a RNeasy Mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. High-purity RNA was eluted in 30 μl of water and treated with ribonuclease-free deoxyribonuclease to guarantee elimination of DNA contamination from RNA samples. RNA was quantified spectrophotometrically, and a constant amount of total RNA (5 μg) was reverse transcribed at 42 C for 60 min in a total reaction volume of 20 μl, using first-strand cDNA Synthesis Kit (Roche Diagnostics Corp., Indianapolis, IN). cDNA was incubated at 95 C for 5 min to inactivate the reverse transcriptase and served as a template DNA for 30 amplification rounds in a Cycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA).
RT-PCR was performed in a standard 25-μl reaction mixture, consisting of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM deoxynucleotide triphosphates, 20 pmol of each sense and antisense primer, and 2.5 U of AmpliTaq DNA polymerase (Laboratoires Eurobio, Les Ulis Cedex, France). Table 1 shows primer sequences and experimental conditions for RT-PCR, as well as size of the cDNA fragments. As negative control, the DNA template was omitted in the reaction. The presence of a single band amplified with specific primers for ?-actin with the same cDNA was used as internal control under identical conditions. Each experiment was replicated at least three times. RT-PCR products were run on a 2% agarose gel containing 0.5 μg/ml ethidium bromide and photographed under UV light. For semiquantitative analysis, the film was subjected to densitometry, using Kodak Digital Science 1D system (Eastman-Kodak Co., Fullerton, CA).
TABLE 1. P2X and P2Y receptors found to be expressed in human thyrocytes: primer sequences, size of fragments, and PCR conditions
IL-6 determinations
For Western blot analysis, cells were resuspended in a buffer containing 300 mM sucrose, 1 mM MgSO4, 1 mM K2HPO4, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, 0.2 μM deoxyribonuclease, and 0.2 μg of ribonuclease and lysed by repeated homogenizations. Proteins were separated on 14% sodium dodecyl sulfate-polyacrylamide gel, and blotted overnight onto nitrocellulose paper. The biotinilated specific rat monoclonal antibody (eBioscience, San Diego, CA) was diluted 1:250 in Tween-Tris-buffered saline. Neutravidin diluted 1:6000 in Tween-Tris-buffered saline was used as internal control. IL-6 release was measured by enzyme-linked immunosorbent assay using a biotin-conjugated monoclonal anti-IL-6 antibody (Bioscience, San Diego, CA). The assay sensitivity was less than 2 pg/ml, and the interassay coefficient of variation was 5.2%. Apyrase from potato (Sigma-Aldrich) to inhibit cell stimulation by spontaneously released extracellular ATP was used at a concentration of 5 U/ml. Inactivation was performed by boiling for 15 min.
ATP determinations
ATP levels were measured by luminometric assay, using the ATP-Lite Luminescence ATP Detection Assay System (PerkinElmer Ltd., Beaconsfield, UK), a high-sensitivity detection system (down to 5 cells in 100 μl medium) with high reproducibility and long half-life of the light emission (>5 h). Moreover, this system ensures an efficient inactivation of ecto-ATPases, thus avoiding underestimation of extracellular ATP concentration. Thyrocytes were seeded at a concentration of 5 x 103 cells/well in microtiter plastic dishes in a total volume of 100 μl of culture medium. Before ATP measurements, cells were rinsed and supplemented with 100 μl of ATP-Lite (buffer solution + substrate solution with luciferase-luciferin assay; PerkinElmer). Cells were directly placed into the test chamber of the luminometer, light emission was recorded, and converted into ATP concentration (extracellular ATP). Cells were then shaken for 5 min in the presence of lysis solution, and measurements repeated to determine total ATP levels; intracellular ATP was calculated by subtraction.
Results
Human thyrocytes express the mRNA for P2X and P2Y receptor subtypes
Beside a few reports in rat thyroid cells (22), no information on P2Rs expression and function is available for human primary thyrocytes so far. RT-PCR shows that human thyrocytes express mRNA for the following P2XR and P2YR subtypes: P2X3, P2X4, P2X5, P2X6, and P2X7, and P2Y1, P2Y2, P2Y4, P2Y11 (Fig. 1). Some of these receptors, however, were weakly expressed, and their expression varied among subjects.
FIG. 1. mRNA for P2X and P2Y receptor subtypes expressed by thyrocytes. RT-PCR was performed as described in Materials and Methods. Lanes 1–6 are thyrocyte samples from six different donors; in lane 7 FB2 thyroid carcinoma cells were used as positive control. In lane 8, DNA template was omitted as negative control.
Stimulation with ATP or UTP induces [Ca2+]i transients
As anticipated by the expression of both P2YRs andP2XRs, and as previously reported in rat thyrocytes and FRTL cells (22, 23), stimulation with ATP caused a [Ca2+]i increase (Fig. 2, A and B) characterized by an early rise, presumably due to [Ca2+]i release from intracellular stores, and a delayed plateau, likely due to influx across the plasma membrane. Incubation in a Ca2+-free, 0.5 mM EGTA-containing saline solution (dotted lines), showed little effect on the early [Ca2+]i increase, whereas it fully abrogated the delayed plateau. Obliteration of the late [Ca2+]i increase was particularly striking upon stimulation with 1 mM ATP. This is not surprising because the contribution of Ca2+ influx to the [Ca2+]i rise is much more relevant at high (millimolar) rather than low (nanomolar) nucleotide concentrations.
FIG. 2. ATP induces [Ca2+]i increases in the presence as well as in the absence of extracellular Ca2+. Cells were loaded with the Ca2+ indicator fura-2/AM as reported in Materials and Methods. Thyrocytes were stimulated with ATP (A and B) in the presence (continuous line) or absence (dotted line) of extracellular Ca2+. For Ca2+-free conditions, cells were incubated in Ca2+-free, 0.5 mM EGTA-supplemented medium. For nucleotide dose-dependency curves, cells were incubated in the presence of increasing ATP (C and D) or UTP (E) concentrations. Experiments were performed in Ca2+ containing standard saline (C and E), or Ca2+-free, 0.5 mM EGTA-supplemented solution (D).
Dose-dependency curve performed in a standard saline solution (i.e. in the presence of 1 mM Ca2+) (Fig. 2C) shows that half-maximal effect is obtained with an ATP concentration of 3 μM, whereas plateau is reached at 300 μM. Half-maximal ATP stimulatory concentration in the absence of extracellular Ca2+ is about 2 μM, whereas maximal stimulatory concentration is about 100 μM (Fig. 2D). Thyrocytes were also sensitive to stimulation with UTP. Half-maximal and maximal Ca2+ responses were obtained with 2 and 100 μM UTP, respectively (Fig. 2E).
TSH is known to modulate many thyroid functions; thus, we investigated the effect of TSH treatment on the nucleotide-triggered [Ca2+]i increase (Fig. 3). At low nucleotide concentrations (Fig. 3, A and C) incubation in the presence of TSH 10 mU/ml had little effect on the Ca2+ transient (Fig. 3A, [Ca2+]i increase of 70 ± 12 and 62 ± 10 nM, P = not significant in the presence or absence of TSH, respectively; Fig. 3C, [Ca2+]i increase of 150 ± 13 and 145 ± 7 nM, P = not significant in the presence or absence of TSH, respectively). However, at higher concentrations both the early and delayed Ca2+ increments were substantially reduced (Fig. 3, B and D). With ATP as a stimulus, the [Ca2+]i increase was 280 ± 32 and 122 ± 24 nM, P < 0.001 in the presence or absence of TSH, respectively. With UTP as a stimulus, the [Ca2+]i increase was 170 ± 26 and 70 ± 14 nM, P < 0.001 in the absence or presence of TSH, respectively.
FIG. 3. Nucleotide-induced [Ca2+]i increases are modulated by TSH. Cells were treated as in Fig. 2. Thyrocytes were stimulated in a standard saline solution, with ATP (A and B) or UTP (C and D). Continuous line, Control cells; dotted line, TSH-stimulated cells. Each trace is representative of 12 similar performed in each experimental condition.
Autocrine-secreted ATP induces IL-6 release from human thyrocytes
As already pointed out, IL-6 appears to be an important inflammatory mediator in the thyroid. Given recent evidence supporting a role for nucleotides in IL-6 release (3), we first explored the role of ATP in modulating spontaneous IL-6 release. IL-6 is measurable in the surnatants of resting cells, showing that nonstimulated thyrocytes spontaneously release this cytokine. As previously reported (14), IL-6 release is substantially increased by treating cells with phorbol 12-myristate 13-acetate 100 nM. Incubation in the presence of 5 U/ml of apyrase, an ecto-ATP/ADPase that hydrolyzes extracellular ATP/ADP to AMP and inorganic phosphate, decreases spontaneous IL-6 release by about 20–30% (Fig. 4).
FIG. 4. Spontaneous IL-6 release in human thyrocytes. IL-6 release in the supernatants was measured by ELISA as described in Materials and Methods; black bars, untreated cells; gray bars, apyrase; white bars, heat-inactivated apyrase; hatched bars, phorbol 12-myristate 13-acetate-stimulated cells. Data are averages ± SD of 24 determinations from the six different donors. *, P < 0.05 vs. untreated cells.
To confirm that this effect was specifically due to enzymatic activity, we repeated the experiment with the heat-inactivated enzyme: this treatment prevented the inhibitory effect of apyrase on IL-6 release. Accordingly, extracellular ATP was 71 ± 10 nM in the supernatants from unstimulated cells and decreased to undosable levels in cells incubated with apyrase, whereas it was 86 ± 15 nM in cells treated with the heat-inactivated enzyme.
Stimulation with exogenous nucleotides increases IL-6 synthesis and release from thyrocytes
We next investigated whether exogenous ATP could stimulate IL-6 release. As shown by Western blot in Fig. 5, ATP (B) or UTP (C) treatment induced synthesis of the IL-6 protein. Accordingly, cytokine externalization was also stimulated by ATP in a dose-dependent fashion (Fig. 6A). Dose-dependency curve was bell shaped, with an optimal ATP dose between 0.25 and 0.5 mM. At ATP concentrations higher than 0.5 mM, IL-6 secretion was inhibited. UTP, a nucleotide preferentially active at the P2Y2 and P2Y4 receptors, also caused IL-6 release (Fig. 6B, left); conversely BzATP, a potent P2X7 agonist, was fully inactive (Fig. 6B, right).
FIG. 5. Effect of ATP and UTP on IL-6 protein expression. Western blot was performed as described in Materials and Methods and is representative of six different experiments. A, Control cells; B, 0.25 mM ATP; C, 0.25 mM UTP. Lane 1, Neutravidin; lane 2, neutravidin + specific IL-6 antibody.
FIG. 6. Effect of ATP, UTP, or BzATP on IL-6 secretion. Cells were stimulated with increasing nucleotide concentrations for 6 h. IL-6 in the supernatants was measured by ELISA as described in Materials and Methods. Data are averages ± SD of 30 determinations from the six different donors. *, P < 0.05; °, P < 0.001 vs. basal.
Finally, we explored whether TSH, besides decreasing the [Ca2+]i rise, also down-regulated IL-6 release. Figure 7 shows that acute challenge with TSH had no significant stimulatory effect on basal IL-6 secretion; chronic incubation with TSH had little effect on IL-6 release induced by low nucleotide concentrations (1100 ± 120 vs. 1130 ± 160 pg/ml/3 x 103 cells, n = 30, in samples stimulated with 0.1 mM ATP in the absence or presence of TSH, respectively), whereas it significantly decreased IL-6 secretion triggered by higher ATP concentrations (P < 0.001 for TSH-less vs. TSH-treated samples at all ATP concentrations above 0.1 mM).
FIG. 7. Effect of TSH on ATP-induced IL-6 secretion. Cells were maintained in the presence of 10 mU/ml TSH alone or together with increasing ATP concentrations for 6 h. IL-6 in the supernatants was measured by ELISA as described in Materials and Methods. Data are averages ± SD of 30 determinations from the six different donors.
Discussion
P2Rs modulate ion conductance and activate different signaling cascades in several cell types. However, little is known on their presence and function in primary human thyroid cells, besides a few reports showing that extracellular ATP activates phospholipase C and regulates DNA synthesis in the rat FRTL-5 thyroid cell line (23, 24), or can induce generation of inositols in human thyrocytes (25).
In the present study, we show that human primary thyrocytes, despite an individual variability among different subjects, express mRNA for the P2X3–7 and P2Y1, P2Y2, P2Y4, and P2Y11 receptor subtypes. Among P2XRs, the P2X4 subtype was robustly expressed in samples from all donors, whereas the P2X7R was weakly expressed in four of six subjects, and basically absent in the remaining two. Among P2YRs, P2Y1, P2Y2 and P2Y4 were present in all samples, whereas P2Y11 was detected in four of six. Stimulation with either ATP or UTP triggered a rise in [Ca2+]i and release of IL-6 in cells from all donor subjects, whereas the selective P2X ligand BzATP was ineffective, thus indicating a main role of P2YRs in cytokine release. The P2X7R was previously shown to stimulate IL-6 and IL-1? secretion in vitro and in vivo (1, 26, 27); the minor, if any, role of P2X7 in thyrocytes might be due to its low expression. Although the ATP dose dependency for IL-6 release was slightly shifted to the right compared with that for the [Ca2+]i rise, there was a fairly good correlation between the two events, suggesting a role of [Ca2+]i changes in ATP-stimulated IL-6 secretion.
Thyroid cells constitutively release IL-6 and other cytokines (28) by a complex process involving several mutually interacting stimulatory and inhibitory pathways as yet incompletely understood. In the presence of the nucleotide-hydrolyzing enzyme apyrase, basal IL-6 release was substantially, although not entirely, curtailed. This suggests that also in the thyroid, as previously shown in other tissues, cytokine secretion can be modulated by an autocrine/paracrine purinergic loop. In support of this hypothesis, we show that cultured thyrocytes spontaneously secrete ATP. The ATP concentration in the culture supernatants is in the 50–80 nM range, well below the threshold for IL-6 release. However, it is necessary to remind that the ATP concentration in the bulk solution does not accurately reflect ATP levels near the release site, i.e. at the plasma membrane surface, especially if secretion occurs at restricted, specialized sites. In the few instances where probes capable of measuring the ATP concentration close to the plasma membrane were used, ATP levels 10- to 20-fold higher than those in the supernatants were found (29), a concentration that is sufficient to support tonic stimulation of P2YRs.
The thyroid is a paradigmatic example of the close relationship between the endocrine and the immune system because this gland is a preferential target of immune-mediated diseases. A host of lymphocyte-derived factors are known to deeply affect thyroid function (28). The ability of ATP to support IL-6 release points to this nucleotide as an additional participant in the cross-talk between the endocrine and immune systems. In the thyroid, ATP can be released directly by the thyrocytes themselves, as shown in the present work, by vascular endothelium, or by infiltrating lymphocytes or monocytes (30, 31). Increasing evidence suggests that extracellular nucleotides can act as danger signals to alert the immune system of cell and tissue damage, and as such nucleotides have the ability to drive secretion of proinflammatory cytokines, and trigger activation and differentiation of dendritic cells (4, 32). In the light of their purported role as a proinflammatory mediators, or as danger signals (4), it is not surprising that extracellular nucleotides are also potent inducers of IL-6 release.
IL-6, together with IL-1? (which is also constitutively produced by thyrocytes), might be an amplification signal to spread the inflammatory response initiated by drugs and/or autoimmune aggression.
IL-6 is also known to directly damage thyrocytes; however, under our experimental conditions we did not observe any short-term direct cell damage, probably because thyrocytes themselves seldom express receptors for IL-6 (33).
A further interesting finding is that signaling by ATP is modulated by TSH. This hormone decreases the ATP-induced Ca2+ increase as well as IL-6 release. Both the early and delayed phase of the [Ca2+]i rise are reduced by TSH, suggesting that both release from stores and influx across the plasma membrane are affected. The effect of TSH on IL-6 secretion is controversial: previous studies showed a stimulatory action (20, 34, 35), whereas a later article reported that TSH does not stimulate IL-6 release from thyrocytes obtained from either Graves’ patients or from multinodular goiters (14). Our data suggest a novel pathway for modulation of IL-6 release in the thyroid based on the inhibitory activity of TSH on ATP-stimulated responses. The observation that TSH is unable to down-regulate IL-6 release under basal conditions, i.e. in the presence of low extracellular ATP levels, although it is effective at high ATP concentrations, suggests that this modulatory pathway is inactive in the healthy thyroid, becoming operational only under conditions like stress or inflammation, when the local ATP concentration increases dramatically due to enhanced release from the damaged thyrocytes or infiltrating immune cells. This antiinflammatory role of TSH could also explain the already described negative correlation between IL-6 and T3 or T4 (36).
The interaction between TSH and IL-6 may have several clinical implications, for example in the regulation of sodium/iodide symporter (NIS) expression. The gene encoding NIS, a transporter that plays a critical role in iodide transport in the thyroid and in the production of the iodine-containing thyroid hormones, is up-regulated by TSH and down-regulated by IL-6 (37). Our results suggest an additional pathway, via a reduction of ATP-induced IL-6 release, by which TSH could promote NIS expression.
Collectively, our data are in keeping with the hypothesis that the local production of IL-6 in the microenvironment may be part of an homeostatic mechanism and/or cellular alarm system regulating thyrocyte function. Extracellular ATP participates in the modulation of IL-6 synthesis and release by thyrocytes: therefore, the purinergic system may have a role in the regulation of thyroid function and may be involved in the propagation and progression of autoimmune thyroid diseases.
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