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-Inducible 
-Chemokine CXCL10 Serum Levels in Patients with Active Graves’ Disease or Toxic Nodular GoiterMetabolism Unit (A.A., P.F., S.M.F., E.F.), Department of Medicine, and Regional Center of Nuclear Medicine (M.G., G.M., G.B.), University of Pisa Medical School, I-56100 Pisa, Italy; Department of Clinical Pathophysiology (M.R., P.R., M.S.), Endocrinology Unit, University of Florence, 50139 Florence, Italy; and Consiglio Nazionale delle Ricerche Institute of Clinical Physiology (E.F.), 56126 Pisa, Italy
Address all correspondence and requests for reprints to: Alessandro Antonelli, M.D., Department of Internal Medicine, University of Pisa Medical School, Via Roma, 67, I-56100 Pisa, Italy. E-mail: a.antonelli{at}med.unipi.it.
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Abstract |
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Objective, Design, and Patients: The aim of the present study was to measure the serum CXCL10 levels in 20 patients with GD and 10 patients with toxic nodular goiter (TNG) before and 6 months after 131I treatment, when patients had achieved euthyroidism. Forty healthy subjects and 40 patients with autoimmune thyroiditis served as control groups.
Results: Before 131I, mean CXCL10 was significantly higher in patients with GD and thyroiditis than controls or those with TNG. Serum CXCL10 levels significantly decreased in GD patients 6 months after 131I treatment, whereas they remained within normal limits in TNG patients after restoration of euthyroidism by 131I.
Conclusions: In conclusion, our results demonstrate that high serum CXCL10 levels are associated with the hyperthyroid phase in GD but not TNG, providing further evidence for a minimal role of hyperthyroidism per se in determining high CXCL10 levels and showing a strong association with the autoimmune process. The reduction of CXCL10 levels after 131I treatment in GD only shows that the thyroid gland itself is the main source of circulating CXCL10.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Chemokines are a group of low-molecular-weight peptides that are able to recruit specific leukocyte subtypes to inflammation sites (4), but they also play a role in tumoral growth, angiogenesis, and organ sclerosis (5, 6). At present, more than 50 chemokines have been described, and classified into four main families (4). The CXC chemokines (CXCL9, CXCL10, CXCL11) inducible by interferon-
(IFN-g) are associated with Th1-mediated immune responses. CXCL10 is a prototype of this class, its serum levels being increased in several endocrine autoimmune conditions (7, 8, 9, 10, 11, 12). It has recently been demonstrated that CXC chemokines, and in particular CXCL10, play an important pathophysiological role in the initial phases of autoimmune thyroid disorders (7, 9, 13). Furthermore, increased serum levels of CXCL10 in patients with autoimmune thyroiditis (AT) are associated with hypothyroidism; thus, this chemokine can be considered a marker of a more aggressive autoimmune process leading to thyroid destruction (10, 11).
The IFN-g inducible chemokine status in autoimmune and nonautoimmune hyperthyroidism has not yet been evaluated systematically in relation to radioiodine treatment. This issue also has some clinical interest in view of the recent demonstration that serum CXCL10 levels are increased in GD patients with active Graves’ ophthalmopathy (GO), thus supporting the concept that CXCL10 is involved in the initial phase of GO when the inflammatory process is mainly sustained by the T-helper lymphocyte-1 immune phenotype (14). In fact, GO activity must be carefully evaluated before performing 131I therapy because several studies have demonstrated GO progression after radioiodine treatment (1, 15, 16).
In the present study, we assessed the variations of serum CXCL10 levels in patients with GD and with TNG before and after 131I therapy, to correlate them with thyroid function and radioiodine treatment and evaluate the associated changes of the T-helper lymphocyte-1-mediated immune response.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We prospectively studied 20 consecutive Caucasian GD patients without ophthalmopathy (Table 1
). The diagnosis of GD (1) was established on the basis of clinical presentation (presence of a diffuse goiter, varying in size from normal to very large), thyroid hormones, and thyroid autoantibodies serum levels [especially antithyrotropin-receptor autoantibodies (TRAbs)], thyroid ultrasonography (decreased or dyshomogeneous echogenicity, and diffuse goiter), and/or thyroid scan (diffusely increased uptake). TRAbs were present in 17 of 20 patients. All these patients had goiter, and three of 20 patients underwent fine-needle aspiration of thyroid nodules that were cold on the thyroid scan to exclude the presence of thyroid cancer or lymphoma; in these cases, cytology excluded the presence of a malignancy.
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), seen in our unit in the same period, were also studied. All these patients were hyperthyroid, and the majority of them had goiter (70%). The diagnosis of TNG (1) was established on the basis of clinical presentation (presence of nodular goiter); thyroid hormone serum levels; and absence of circulating thyroid autoantibodies (especially TRAb), thyroid ultrasonography (presence of nodules), and/or thyroid scan (nodular uptake). All these patients underwent fine-needle aspiration of cold nodules on the thyroid scan to exclude the presence of thyroid cancer; in all these cases, cytology excluded the presence of a malignancy. The prevalence of patients with increased TRAb was 85% [antithyroglobulin antibodies (AbTg), 55%, antithyroperoxidase antibodies (AbTPO), 70%)], and thyroid volume greater than 20 ml was 75%. The diagnosis of GD in the three patients with negative TRAb was made on the base of positivity for AbTg and/or AbTPO, in presence of diffuse iodine uptake at the thyroid scan and a hypoechoic pattern at ultrasonography. All GD and TNG patients were hyperthyroid at presentation [low TSH associated with high levels of free T3 (FT3) and/or free T4 (FT4)].
131I therapy was advised for the following reasons: 1) because of relapse of hyperthyroidism after a prior methimazole (MMI) course in GD patients without ophthalmopathy and with a thyroid volume less than 70 ml; 2) in all TNG patients with a thyroid volume less than 70 ml.
All patients were treated with MMI for hyperthyroidism until reaching normal TSH, FT3, and FT4 values (euthyroidism) (after 1–3 months of treatment), and subsequently (1–2 months after reaching euthyroidism) submitted to 131I treatment.
Controls
Two control groups (matched by age and gender with the GD patients) were considered (Table 1).
The first control group consisted of 40 subjects extracted from a random sample of the general population from the same geographic area (17, 18) in whom a complete thyroid work-up (history, physical examination, TSH, FT3, FT4, AbTg, and AbTPO measurements and ultrasonography) was available and excluded the presence of thyroid disorders.
A second control group included 40 patients with euthyroid chronic AT (Table 1). The diagnosis of AT was established on the basis of the clinical presentation (presence of a firm goiter, varying in size from small to very large, with a lobulated surface), thyroid hormones and thyroid autoantibody measurements, and thyroid ultrasonography (decreased, dyshomogeneous echogenicity).
In all patients and controls, a blood sample was collected in the morning after overnight fasting, and serum was kept frozen until thyroid hormones, TSH, thyroid autoantibodies, and CXCL10 measurements. Blood samples for CXCL10 measurement were collected at baseline (before any treatment) in all subjects and in the AT patients; in the GD and TNG patients, serum CXCL10 was again evaluated at 2, 4, and 6 months after 131I treatment. After 2 months from 131I therapy, 13 of 20 were under MMI treatment, whereas after 4 months, seven of 20 were still on MMI. At the last control (6 months), 16 of 20 GD patients were euthyroid on levothyroxine substitutive therapy, whereas the other four were euthyroid but still treated with MMI; among the 10 TNG patients, eight were euthyroid without any treatment, one had subclinical hyperthyroidism (TSH < 0.3 µU/ml, with normal FT4 and FT3) off therapies, and one was euthyroid but treated with MMI.
To avoid previously reported biases due to changes of thyroid function (from hyper- to hypo- or to euthyroidism) or change of treatments (MMI) as well as the inflammatory effect induced by the radioiodine itself (19), serum CXCL10 was assayed in patients in euthyroidism after 131I and not before the sixth month after therapy.
All study subjects gave their informed consent to the study, which was approved by the local ethical committee.
Laboratory evaluation
Thyroid function and thyroid autoantibodies were measured as previously described (17). Circulating FT3 [reference range: 2.3–5.6 pg/ml (3.5–8.6 pmol/l)] and FT4 [5.6–13.0 pg/ml (7.2–16.7 pmol/l)] were measured by commercial RIA kits (AMERLEX-MAB FT3/FT4 kit; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Serum TSH (0.3–3.6 µU/ml) (DiaSorin, Stillwater, MN), AbTPO, and AbTg (ICN Pharmaceuticals, Costa Mesa, CA) were evaluated by immunoradiometric assay methods. TRAb autoantibodies were measured with a radioreceptor assay (Radim, Pomezia, Italy) (normal range 0–1 UI/ml). For AbTg and AbTPO, positivity was set at greater than 50 and greater than 10 UI/ml, respectively.
CXCL10 ELISA
Serum CXCL10 levels were assayed by a quantitative sandwich immunoassay using a commercially available kit (R&D Systems, Minneapolis, MN), with an analytical sensitivity ranging from 0.41 to 4.46 pg/ml and a mean minimum detectable level of 1.67 pg/ml. The intra- and interassay coefficients of variation were 3.0 and 6.9%, respectively.
Ultrasonography of the neck
Neck ultrasonography was performed by the same operator, who was unaware of the results of thyroid hormones, autoantibodies, and CXCL10 measurements, using a high-resolution probe with 7.5-MHz transducer (AU5; Esaote, Florence, Italy). Thyroid volume (20, 21), thyroid or nodular mass, the presence of hypoechoic and dyshomogeneous echogenicity (10, 11, 22), and thyroid blood flow assessed by color-flow Doppler (19, 10, 11, 23) were evaluated as previously shown (19, 10, 11, 23).
Nuclear medicine procedures
All patients were advised to stay on a generic low-iodine diet for at least 2 wk before measurements and were taken off antithyroid drug therapy before radioiodine administration (average 5–7 d for GD and 15–20 d for TNG). Antithyroid drug treatment was restarted at least 7 d after 131I therapy. Besides possible refusal of radioiodine therapy by the patient, exclusion criteria included an age younger than 18 yr, suspicion of pregnancy, and the presence of any suspicious thyroid nodule on ultrasound examination.
Radioiodine uptake (RAIU) and determination of 131I kinetics
The RAIU and 131I kinetics in the patients were evaluated as follows: 1) the radioactivity count rate from a diagnostic dose of 131I (1.85 MBq) placed in a thyroid phantom was measured using a properly collimated and calibrated NaI(Tl) probe (reference measurement); 2) thyroid uptake at 4 and 24 h after administration of the diagnostic 131I dose was measured in each patient in the upright position with the anterior surface of the neck at 25 cm from the same NaI(Tl) probe (120 sec counting time); RAIU values were calculated after correcting for background activity, as follows: RAIU = (neck – background)/(reference – background); and 3) the patterns of 131I kinetics in the thyroid were identified after calculating the 4- and 24-h fractional thyroidal uptake by deriving from such uptake values estimates of the half-time of 131I in the thyroid. The median effective half-life was 5.04 d in controls.
Thyroid scintigraphy
Twenty-four hours after receiving the 131I tracer dose (1.85 Mbq), all patients underwent 131I scintigraphy immediately after performing the 24-h RAIU measurement and before receiving the therapeutic dose. A single-head circular large field-of-view -camera equipped with a medium-energy collimator (3000 CAMSTAR; GE Medical System, Chicago, IL) was used for imaging, acquiring 100,000 counts with a 128 x 128 matrix, zoom 1.00 (peak energy setting: 364 keV with a ± 10% window) to confirm the GD or the TNG diagnosis and evaluate the extranodular vs. nodular activity.
Radioiodine therapy
The required amount of radioiodine to achieve the target dose (150 Gy) for GD patients (24) was calculated as previously described (25, 26), whereas the Marinelli’s formula was used in the TNG patients to achieve a target dose of 300 Gy (27). The average radioiodine activity administered to all patients was 449 ± 215 MBq, with a range of 111–740 Mbq (12.1 ± 5.8 mCi; range 3–20 mCi). The corresponding average thyroid absorbed dose, as calculated by the medical internal radiation dose equation was 148 ± 26 Gy for GD and 295 ± 52 Gy for TNG.
Data analysis
Values are given as mean ± SD for normally distributed variables, otherwise as median and interquartile range. Mean group values were compared by using one-way ANOVA for normally distributed variables. Proportions were compared by the Pearson’s 
2 test. Post hoc comparisons on normally distributed variables were carried out using the Bonferroni-Dunn test.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. The mean baseline CXCL10 levels were significantly higher in patients with GD and euthyroid AT than the controls or TNG patients (167 ± 121, 142 ± 107, 78 ± 46, 100 ± 24, respectively), without statistical difference between the GD and the AT groups.
When stratifying the GD patients according to various clinical and laboratory/imaging parameters (Table 2), the pretreatment serum CXCL10 levels were significantly higher in patients older than 50 yr or with a hypoechoic pattern (79%) and in those with hypervascularity (72%), whereas no significant difference was observed in relation to the presence of goiter, AbTPO, AbTg, or TRAb positivity. Finally, no relation was observed with duration of the disease.
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). Two months after radioiodine therapy, 13 of 20 were under MMI treatment and CXCL10 serum levels were 138 ± 96 pg/ml, whereas after 4 months seven of 20 were still on MMI and CXCL10 serum levels were 112 ± 79 pg/ml. In the four patients with GD on MMI treatment when reviewed at 6 months, serum CXCL10 levels were not significantly different from that of the other 16 patients (92 ± 37 vs. 78 ± 36 pg/ml; P = not significant). None of the GD patients developed ophthalmopathy after radioiodine. On the other hand, only a slight but not significant decrease in serum CXCL10 levels vs. baseline was observed in the patients with TNG 6 months after treatment with radioiodine (Fig. 1B).
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, such as hypoechoic pattern, hypervascularity, or thyroid autoantibodies (data not shown). |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). Similarly high levels of CXCL10 were observed in the patients with autoimmune thyroid disease not associated with hyperthyroidism. After 131I therapy, the high CXCL10 levels of GD patients almost completely normalized, thus suggesting that the destruction of thyroid follicular cells in these patients is associated with a reduction of CXCL10 production and/or an immunomodulation of the autoimmune inflammatory process.
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The possibility of some immunological flare up after 131I treatment has been demonstrated by the observation of a surge in the TSH receptor antibodies (peaking approximately 3 months after treatment) and by appearance and/or worsening of thyroid-associated ophthalmopathy (1, 29). Although the pathogenesis of this immunological activation is still not fully elucidated, several evidences suggest that this occurrence is secondary to the release of thyroid antigens induced by destruction of thyroid cells by 131I (1). Furthermore, circulating lymphocytes change phenotypically after radioiodine treatment, with increased numbers of the T cells and the T helper (2), activated T cell, memory T cell, and contrasuppressor T cell (3) subsets. It is therefore reasonable to expect that cytokine profiles might also be affected and that such changes might influence the autoimmune process. Few previous studies have evaluated the effect of radioactive iodine therapy on cytokine production in GD patients, with contrasting results (28, 30).
In a previous study evaluating the IL-1ß, IL-6, and intercellular adhesion molecule-1 serum levels in GD patients basally and 4, 7, and 21 d and 3 months after 131I treatment, the circulating levels of none of the above cytokines changed at any time (30). On the other hand, another study assessed the production of IL-4, IL-6, IL-10, TNF-
, and IFN-g by peripheral blood mononuclear cells before and after radioiodine treatment in GD patients. A transient increase in both proinflammatory and antiinflammatory cytokines was observed by d 17 after therapy, returning to pretreatment levels by d 59. Overall, cytokines profiles expressed by peripheral blood mononuclear cells showed significant differences when assessed on d 4, 17, and 59 after 131I vs. baseline (19).
The present study was designed to minimize any effect of possible confounding factors in determining modifications of circulating CXCL10 levels. To this purpose, 131I-induced CXCL10 modifications were evaluated in GD and TNG patients after reaching euthyroidism and only after a prolonged washout period (6 months) to avoid possible biases due to recent change of thyroid function (from hyper- to hypo- or euthyroidism) or treatments (MMI) as well as the inflammatory effect of radioiodine itself.
Although suggesting that the thyroid is the main source of abnormal CXCL10 levels in patients with autoimmune thyroid disorders with (GD) or without (AT) hyperthyroidism, normalization of circulating CXCL10 levels after destruction of thyroid tissue by 131I therapy can be explained by removal of the large part of either intrathyroidal lymphocytes and/or thyrocytes.
In conclusion, the results of this study demonstrate that abnormally increased production of IFN-g inducible chemokine CXCL10 is associated with the hyperthyroid phase of GD, but not TNG, providing further evidence for a minimal role of hyperthyroidism per se in determining high CXCL10 levels and showing a strong association with the autoimmune process. The reduction of the CXCL10 circulating levels in GD patients treated with 131I may be related to intrathyroidal lymphocytes and/or thyrocytes destruction induced by 131I, suggesting that the gland itself is the main source of CXCL10.
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Footnotes |
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First Published Online January 23, 2007
Abbreviations: AbTg, Antithyroglobulin antibody; AbTPO, antithyroperoxidase antibody; AT, autoimmune thyroiditis; FT3, free T3; FT4, free T4; GD, Graves’ disease; GO, Graves’ ophthalmopathy; 131I, iodine-131; IFN-g, interferon-; MMI, methimazole; RAIU, radioiodine uptake; TNG, toxic nodular goiter; TRAb, thyrotropin-receptor autoantibody.
Received July 19, 2006.
Accepted January 17, 2007.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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inducile chemokines in endocrine autoimmunity: an expanding field. J Endocrinol Invest 26:177–180[Medline] inducible a chemokine CXCL10 but not ß chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol 152:171–177-inducible chemokine-10/CXC chemokine ligand-10 in autoimmune primary adrenal insufficiency and in vitro expression in human adrenal cells primary cultures after stimulation with proinflammatory cytokines. J Clin Endocrinol Metab 90:2357–2363-inducible -chemokine CXCL10 involvement in Graves’ ophthalmopathy: modulation by peroxisome proliferator-activated receptor- agonists. J Clin Endocrinol Metab 9:614–620, with longer term increases in interferon- production. J Clin Endocrinol Metab 84:4106–4110This article has been cited by other articles:
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 A. Antonelli, C. Ferri, P. Fallahi, S. M. Ferrari, D. Giuggioli, M. Colaci, A. Manfredi, S. Frascerra, F. Franzoni, F. Galetta, et al. CXCL10 ({alpha}) and CCL2 ( ) chemokines in systemic sclerosis a longitudinal study Rheumatology, January 1, 2008; 47(1): 45 - 49. [Abstract] [Full Text] [PDF]
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