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Effect of Radioactive Iodine Therapy on Cytokine Production in Graves’ Disease: Transient Increases in Interleukin-4 (IL-4), IL-6, IL-10, and Tumor Necrosis Factor-, with Longer Term Increases in Interferon- Production¹

Departments of Pathology, Clinical Oncology (C.C.H.K.) and Medicine (A.W.C.K.), University of Hong Kong, Queen Mary Hospital, Hong Kong

Address all correspondence and requests for reprints to: Dr. B. M. Jones, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail: bmjones{at}ha.org.hk

	Abstract

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Spontaneous and mitogen-stimulated production of interleukin-4 (IL-4), IL-6, IL-10, IL-12, interferon- (IFN), and tumor necrosis factor- were evaluated by enzyme-linked immunospot assay of peripheral blood mononuclear cells from patients with Graves’ disease immediately before and at 4, 17, and 59 days after treatment with radioactive iodine. Patients had significantly reduced IL-4 and IFN production before treatment compared with healthy controls. Both cytokines were increased to normal levels by day 17 after treatment, and IFN remained at normal levels on day 59, whereas IL-4 returned to subnormal levels at this time. IL-12 production was initially normal and was not significantly altered by therapy. IL-6, IL-10, and tumor necrosis factor- were also normal before radiotherapy, but increased significantly on day 17, returning to pretreatment levels by day 59. Thus, radioiodine treatment induced a transient increase in both proinflammatory and antiinflammatory cytokines and a more prolonged increase in IFN production, the latter representing a definite shift toward a type 1 cytokine profile.

	Introduction

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HYPERTHYROIDISM affects approximately 2% of women and 0.2% of men (1), the commonest cause being Graves’ disease (GD), an autoimmune disorder associated with antibodies that bind to and stimulate the TSH receptor (2, 3). Radioiodine (¹³¹I; half-life, 8 days) is increasingly being used as first-line therapy for hyperthyroidism; it is readily and selectively taken up by the thyroid gland, reduces thyroid volume by as much as 80%, and inhibits thyroid hormone production, initially normalizing these hormones but eventually inducing hypothyroidism in a high proportion of patients (4).

Cytokines produced by intrathyroidal lymphocytes are likely to be involved in the induction and maintenance of the autoimmune process in GD. Type 1 cytokines, including interleukin-2 (IL- 2) and interferon- (IFN), promote cell-mediated immune responses, whereas type 2 cytokines, including IL-4 and IL-10, promote antibody production and allergy (5, 6). Paschke et al. (7) found a variable pattern of increased accumulation of transcripts for IL-2, IL-4, IL-10, and IFN in surgical thyroid specimens from GD patients, indicative of neither type 1 nor type 2 cytokine polarization, whereas Roura-Mir et al. (8) found that thyroid-infiltrating lymphocytes produced both type 1 and type 2 cytokines, although large activated T cells, presumably responsible for autoimmune damage, produced predominantly IL-4. Heuer et al. (9) demonstrated elevated levels of messenger ribonucleic acid for type 2 cytokines IL-4 and IL-10 in thyroid biopsies of patients with GD who had high levels of antithyroid antibodies, while Guo et al. (10) found IL-4 and IL-10, but not IFN, complementary DNA in intact thyroid tissue from GD patients. In contrast, Watson et al. (11) found IL-2, IFN, tumor necrosis factor- (TNF), and IL-10, but not IL-4, messenger ribonucleic acid in intrathyroidal lymphocytes from GD patients.

When peripheral blood lymphocytes were examined, untreated GD patients produced more IL-4 than normal controls after phytohemagglutinin (PHA) stimulation (12), whereas another study found less TNF and IL-4 in GD patients than controls, although the ratio between IFN or TNF and IL-4 or IL-10 indicated type 2 cytokine dominance (13). An informative study showed that exogenous type 1 cytokines IFN, IL-2, and TNF all suppressed the production of antithyroid autoantibodies by thyroid B cells in vitro (14). Although the evidence is somewhat conflicting, the majority opinion is that GD is promoted by type 2 cytokines and regulated by type 1 cytokines.

The effect of local irradiation of the thyroid on peripheral cytokine secretion profiles has not been thoroughly examined. Initial suppression of cytokines could be expected due to destruction of infiltrating activated lymphocytes and macrophages, whereas the inflammatory reaction to damaged thyroid tissue could initiate a new wave of thyroid colonization by leukocytes and renewed production of cytokines by these and other cell types. As we were not able to study thyroid-infiltrating lymphocytes per se, we evaluated cytokine production by peripheral blood mononuclear cells (PBM). The circulating lymphocyte population changes phenotypically after radioiodine treatment, with increases in numbers of T cells and T helper (15), activated T cell, memory T cell, and contrasuppressor T cell (16) subsets. It would therefore be expected that cytokine profiles might also be affected and that such changes might influence the autoimmune process.

The present study used sensitive enzyme-linked immunospot (ELISPOT) assays to measure numbers of cytokine-secreting cells (17, 18), and it was found that GD patients initially had low capacity for IL-4 and IFN production. Radioiodine therapy induced short term increases in IL-4, IL-10, and proinflammatory cytokines IL-6 and TNF, but longer term normalization of IFN.

	Experimental Subjects

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Diagnosis of GD was based on the presence of soft diffuse goiter, raised serum free T₄ (fT₄) and suppressed TSH. Cytokine profiles were evaluated in 13 unselected patients (11 women and 2 men), aged 32–71 yr (mean, 49.5 yr). Three were newly diagnosed and untreated, and 10 had previously received carbimazole (30–60 mg/day) but had relapsed. They were treated with a single oral dose of 200–400 megabecquerels ¹³¹I. Ten patients were available for retesting 4 and 17 days after treatment, and 8 were tested at 59 days. A single patient with nodular goiter (NG) who received 350 MBq ¹³¹I was also tested before and 4, 17, and 59 days after therapy. The control group comprised 15 female and 3 male subjects, aged 27–59 yr (mean, 38.9 yr) who were all in good health. All subjects were of Chinese ethnic origin and gave informed consent. The study was approved by ethical committee review.

	Materials and Methods

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Blood samples

Blood was obtained from patients shortly before radioiodine therapy and at 4, 17, and 59 days afterward. Healthy controls matched approximately for age and sex were tested at intervals throughout the study period. PBM were separated within 30 min by centrifugation over Lymphoprep (Nycomed, Oslo, Norway).

Thyroid function tests

Free T₄ was measured by competitive immunoassay on the ACS180 (Ciba Corning Diagnostics Corp., Medfield, MA). The interassay coefficient of variation (CV) was 10% at 14 pmol/L and 7% at 58 pmol/L. TSH was measured by a two-site chemiluminometric immunoassay on the ACS180. The interassay CV was 8% at 0.70 mIU/L and 5% at 18 mIU/L.

Anti-TSH receptor (TSHR) antibody

Anti-TSHR was determined as TSH binding inhibitory Ig (TBII) by a radioreceptor assay (RSR, Cardiff, UK). Intra- and interassay CVs were 5.0% and 8.6%, respectively.

Evaluation of cytokine production by ELISPOT assay

The ELISPOT assay for determining numbers of PBM secreting IFN, IL-4, IL-6, IL-10, IL-12, or TNF was based on the method described by Hagiwara et al. (19) with modifications. Optimal, standardized conditions for ELISPOT development were used throughout the study. Multiscreen 96-well filtration plates (Millipore Corp., Bedford, MA) were coated overnight at 4 C with capture anticytokine antibodies at 2 µg/mL in 0.1 mol/L carbonate-bicarbonate buffer, pH 9.6. Antibodies to IFN, IL-4, IL-6, IL-10, and TNF were purchased from PharMingen (San Diego, CA), and anti-IL-12 was obtained from Endogen, Inc. (Woburn, MA). Wells were washed with phosphate-buffered saline (PBS) and blocked with tissue culture medium RPMI 1640 containing 5% FCS (Sigma Chemical Co., St. Louis, MO). Duplicate cultures of PBM at 10⁵ (for IFN, IL-4, IL-10, and IL-12) or 10⁴ (for IL-6 and TNF) cells/well were left unstimulated or were stimulated with the T cell activators PHA (Bacto, Detroit, MI; 10 µg/mL), concanavalin A (Sigma Chemical Co.; 20 µg/mL), or anti-CD3 monoclonal antibody (OKT3, Orthoclone, Raritan, NJ) coated onto M280 sheep antimouse IgG-coated Dynabeads (Dynal A.S., Oslo, Norway), 2 x 10⁵ OKT3 beads/mL, or the monocyte activator Staphylococcus aureus Cowan I (SAC; Calbiochem, La Jolla, CA) at 1:100,000 (w/v) for 18–22 h at 37 C in 5% CO₂. Cells were washed out with PBS plus 0.05% Tween-20 and biotinylated detection antibodies at 2 µg/mL (anti-IFN, -IL-4, -IL-6, -IL-10, and -TNF; all from PharMingen) or 0.25 µg/mL (anti-IL-12; Endogen, Inc.) in PBS plus 0.05% Tween-20, added for 3 h at room temperature. After further washings, streptavidin-alkaline phosphatase (Sigma Chemical Co.; 1 µg/mL) was added for 2 h, plates were again washed extensively, and then substrate 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (Calbiochem) was added for 20 min. Plates were thoroughly washed with running tap water and allowed to dry overnight, and spots of insoluble blue formazan, each corresponding to a single cytokine-secreting cell, were counted by microscopy. Up to 2000 spots/well could be counted accurately with the aid of eyepiece graticules corresponding to 1/8th and 1/72th of the well area. Spots were of characteristic smooth, round appearance, with stronger coloration at the center than at the edges, and could be readily distinguished from pseudospots, which were smaller and more intense and did not show lighter edges. Pseudospots occasionally appeared in control wells lacking capture or detection antibodies, whereas true spots never developed in these wells. Results are expressed as ELISPOTS per10⁶ PBM.

The intraassay CV of the ELISPOT assay was determined using PBM from four normal control subjects, each of whom was tested in quadruplicate cultures. Combined data for IFN, IL-4, IL-6, IL-10, IL-12, and TNF and all stimuli gave CVs of 8.8 ± 5.8%. Interassay variation (four subjects tested on two occasions, 3 weeks apart) was 13.2 ± 4.9% when each subject was considered individually, but only 5.3 ± 3.8% when comparing mean values for the two time points.

Statistical analysis

Differences between patients and controls, between patients at different time points before and after treatment, and between hypothyroid and euthyroid or mildly hyperthyroid patients were analyzed by two-tailed unpaired t test. Correlations between cytokine production and dose of radioiodine, thyroid function, and autoantibody levels were examined by Pearson’s test. Prism version 2.0 software (GraphPad Software, Inc., San Diego, CA) was used.

	Results

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fT₄ levels immediately before therapy were elevated in all the newly diagnosed patients (mean, 84.3 ± 50.2 pmol/L; normal, 12–23) and fell successively on days 4, 19, and 59 after therapy to 51.1 ± 27.7, 29.2 ± 7.0, and 13.8 ± 7.5, respectively. TSH was very low or undetectable in 11 patients before treatment and became normal (0.35–5.5 mIU/L) in one and elevated in 2 patients by day 59. TBII was positive in 11 of 13 subjects before treatment (142 ± 104 U/L) and increased significantly in 4 of 8 patients by day 59 (301 ± 363 U/L).

Therapy-induced changes in numbers of unstimulated and stimulated cytokine-producing cells were measured by ELISPOT assay. IFN- and IL-4-secreting cells were not seen in patient or control unstimulated PBM. The numbers of PBM producing IFN (Fig. 1A) or IL-4 (Fig. 1B) in response to PHA, concanavalin A, or OKT3 were significantly lower in newly diagnosed GD patients shortly before radioiodine therapy than in normal controls. Production of both cytokines remained lower than control values on day 4 after treatment, but increased to levels similar to those in controls by day 17. Of major interest, IFN production in response to all stimuli remained at normal control levels on day 59, whereas numbers of IL-4-producing cells had returned to the subnormal pretreatment value at this time.

View larger version (32K): [in this window] [in a new window]   Figure 1. A, Increase in IFN secretion by mitogen-activated PBM from patients with GD up to 59 days after 131I therapy; B, increase in IL-4 secretion up to 17 days after therapy followed by return to subnormal levels at day 59; C, lack of alteration in IL-12 secretion after therapy. Cytokine-secreting cells per 106 PBM are shown in control subjects (C) and patients with Graves’ disease prior to (P) and on days 4, 17, and 59 after treatment with 131I. Error bars represent SEMs. 1) P < 0.001 vs. C; 2) P < 0.05 vs. P; 3) P < 0.01 vs. C; 4) P < 0.05 vs. C; 5) P < 0.01 vs. P; 6) P < 0.05 vs. 0.17 (by unpaired t test).

IL-10 production by GD patients was initially similar to that of controls, but there were slight increases on day 4 and major increases by day 17 after treatment, the latter being significantly above pretreatment levels in unstimulated and PHA- or SAC-stimulated cultures. However, IL-10 production then fell to values that on day 59 were somewhat lower than control or pretreatment values and significantly lower than day 17 values (Fig. 2A). Numbers of IL-6 (Fig. 2B)- and TNF (Fig. 2C)-producing cells in unstimulated and PHA- or SAC-stimulated cultures of GD PBM were slightly and not significantly higher than control values before treatment and on day 4 after treatment. Major increases in IL-6 and TNF occurred by day 17, when values were significantly higher than pretreatment levels under all culture conditions. Both cytokines returned to pretreatment levels by day 59 and were then significantly lower than on day 17 and similar to control and pretreatment values.

View larger version (35K): [in this window] [in a new window]   Figure 2. Increases in IL-10 (A), IL-6 (B), and TNF (C) secretion by unstimulated or mitogen-activated PBM from patients with GD, most evident 17 days after 131I therapy, with return to near-normal levels on day 59. For further explanation, see Fig. 1. 1) P < 0.05 vs. P; 2) P < 0.01 vs. P; 3) P < 0.01 vs. 17; 4) P < 0.0001 vs. 17; 5) P < 0.0001 vs. C, P <0.01 vs. P; 6) P < 0.05 vs. 17; 7) P < 0.0001 vs. C and P (by unpaired t test).

Three of the patients studied at 59 days after radioiodine became hypothyroid (fT₄, <10 pmol/L), whereas the other five were euthyroid or mildly hyperthyroid (fT₄, 15–25 pmol/L) at this time. The production of IL-12 was higher in the first group than in the second before treatment [unstimulated, 299 ± 34 vs. 90 ± 60 ELISPOTs/10⁶ PBM; OKT3-stimulated, 2687 ± 1214 vs. 252 ± 103 (P < 0.05); SAC-stimulated, 425 ± 25 vs. 116 ± 31 (P < 0.001)], on day 4 after ¹³¹I (OKT3-stimulated, 2440 ± 1061 vs. 384 ± 206; P < 0.05) and on day 17 (SAC-stimulated, 267 ± 24 vs. 106 ± 31; P < 0.05).

	Discussion

Top Abstract Introduction Experimental Subjects Materials and Methods Results Discussion References

 
There is currently great interest in the possibility of moderating autoimmune diseases via immunological deviation, i.e. by altering cytokine production profiles in such a way that pathogenic influences are lessened or neutralized (20, 21). Treatment with radioactive iodine alters the production of thyroid hormones and autoantibodies in GD, and we wondered whether altered cytokine production might contribute to these benefits.

To evaluate changes in cytokine production that are related to disease events or therapeutic interventions, reproducible assays are required that remain stable over time in the absence of such events. The intraassay variation in the cytokine ELISPOT assay employed in the present study was acceptably low (8.8 ± 5.8%), and although interassay variation for individual control subjects was higher (13.2 ± 4.9%), day to day variation was low when data for each time point were pooled (5.3 ± 3.8%). Thus, although cytokine production per se is affected by daily variability, the ELISPOT assay should be capable of identifying any significant changes in cytokine production in a group of GD patients after radioiodine treatment.

Polarization of intrathyroidal cytokines toward a type 2 profile probably contributes to the production of pathogenic autoantibodies in GD (9), and type 1 cytokines appear to suppress their production (14). The present studies using ELISPOT assays to measure the secretion of protein by PBM demonstrated significant deficiencies in both IFN and IL-4 in preirradiation GD patients and did not, therefore, indicate a systemic imbalance of the major type 1 and type 2 cytokines. Both cytokines increased to normal levels by 17 days posttreatment, so that at this time also no polarization of cytokine production by PBM toward type 1 or type 2 could be identified. However, on day 59 IFN production clearly predominated over IL-4, as the former remained at normal levels whereas the latter decreased to levels similar to those obtained before treatment.

IL-12 is a powerful inducer of IFN and type 1 responses (22, 23), but we found no evidence for this cytokine being affected by either the disease process or therapy; spontaneous and stimulated IL-12 production remained normal throughout the study period. The proinflammatory cytokines IL-6 and TNF were also normal initially, but increased significantly by day 17 after treatment. This confirms that therapy provided a strong inflammatory stimulus in the thyroid and that evaluations performed on peripheral blood cells were sufficiently sensitive to register this event. It also agrees with a previous study showing increased serum IL-6 after radioiodine or other thyroid-destructive treatments (24). Recent evidence supports the chronic effect of thyroid hormone excess rather than autoimmune inflammation as the cause of elevated serum IL-6 in GD (25). IL-6 and TNF returned to normal pretreatment levels by day 59, which could be because of resolution of ¹²⁵I-induced inflammatory changes or reduction of thyroid hormone levels by this time.

IL-10 is a potent stimulator of B cells (26) and an important negative regulator of macrophages and T cells (27) and is likely to have a major influence on autoantibody production in GD. Before treatment there were normal numbers of unstimulated, PHA-stimulated, and SAC-stimulated IL-10-secreting PBM, indicating dominance of this type 2 cytokine over IFN, which was reduced at this time. IL-10 increased markedly in parallel with IL-6 and TNF, in response, presumably, to thyroidal inflammation. However, by day 59, IL-10 production, like that of IL-4, fell back to pretreatment levels, leaving normalized IFN and still normal IL-12 as the dominant cytokines.

Changes in cytokine profiles induced by radioiodine could have been due to the inflammatory process itself, with IFN perhaps being of central importance in suppressing IL-4 and IL-10 at times later than day 17 posttherapy and in stabilizing its own normal production. Alterations in levels of thyroid hormones might also influence cytokine production profiles, but we found no correlation of numbers of cytokine-producing cells to fT₄ or TSH at any time point. The single ¹³¹I-treated euthyroidal NG patient available for study showed only small fluctuations in numbers of cytokine-secreting cells over the study period, and values remained essentially within normal limits (results not shown). Additional NG patients should be studied to confirm that radioiodine fails to induce cytokine fluctuations in the absence of thyroid hormone abnormalities.

Cytokine production characteristics of individual GD patients could also have influenced autoantibody levels, thyroid function, and disease manifestations such as opthalmopathy. None of the patients developed the latter complication, so the possible role of cytokines could not be evaluated. Four subjects had significantly increased TBII at 59 days after radioiodine compared with pretreatment levels, and four had stable or decreased TBII, but cytokine levels were not noticeably different in the two groups.

Despite only eight patients being available for study on day 59, plasma fT₄ at this time appeared to be negatively associated with IL-12 production. High levels of IL-12 might support a more powerful thyroid-damaging cell-mediated response after radioiodine-induced release of immune stimulatory sequestered antigens. It will be important to examine additional patients to determine whether IL-12 is truly involved in the development of ¹³¹I-induced hypothyroidism.

	Acknowledgments

 
We are grateful to the Clinical Biochemistry Laboratory, Queen Mary Hospital, for performing thyroid hormone assays.

	Footnotes

 
¹ This work was supported by the Committee for Research and Conference Grants, University of Hong Kong.

Received February 22, 1999.

Revised May 19, 1999.

Revised July 13, 1999.

Accepted July 29, 1999.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jones, B. M. Articles by Kung, A. W. C. Search for Related Content PubMed PubMed Citation Articles by Jones, B. M. Articles by Kung, A. W. C.