The Prevalence and Characteristics of Allergy in Autoimmune Thyroid Diseases

Objective: The relationship between autoimmunity and allergy is well known. The immunoregulation can be modified towards T helper 2 dominance by cytokines and regulatory T cells, which participate in both diseases. Autoimmune thyroid diseases possess different immunoregulatory and sympathoadrenal activity, whose features can be reflected by their association with allergic attacks and the degree of allergen-specific IgE levels. Methods: Two-hundred-fifty-nine patients, 149 with Graves’ disease (57 had ophthalmopathy), 110 with Hashimoto’s thyroiditis and 65 controls with small euthyroid goitre were investigated. Allergen-specific IgE levels to 20 respiratory and 20 food allergens were detected by AllergySreen immunoblot method and given in IU/ml. Thyroid hormones and autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Htg) were measured in a fully automated way, but TSH receptor antibodies with radioimmunoassay. Results: The prevalences of respiratory and food allergen-specific IgE levels were higher in Graves’ disease in comparison with Hashimoto’s thyroiditis and controls. The seasonal allergen-specific IgE levels increased more in Graves’ patients without ophthalmopathy, than in patients who had eye symptoms and Hashimoto’s thyroiditis. Mugwortand soybean-specific IgE levels elevated significantly in the absence of anti-Htg and the presence of TSH receptor antibodies, respectively. In turn, the presence of allergen-specific IgE levels was associated with alterations in thyroid hormone and antithyroid antibody levels too. Conclusion: The elevated prevalences of respiratory and food allergies reflected Th2-derived cytokine productions in autoimmune thyroid diseases. The lower respiratory and food allergen-specific IgE levels highlighted a role of prominent proinflammatory cytokine productions in Graves’ ophthalmopathy and Hashimoto’s thyroiditis. Allergy due to increased Th2-derived and proinflammatory cytokine productions may aggravate or induce AITDs, as well as via modified thyroid hormone and antithyroid antibody levels may contribute to a slow remission rate of Graves’ hyperthyroidism.


Introduction
Graves' disease and Hashimoto's thyroiditis are member of autoimmune thyroid diseases (AITDs). Graves' disease is characterized by diffuse goitre, hyperthyroidism and ophthalmopathy in 25-50% of patients [1]. Stimulating TSH receptor antibodies are regarded as factors for hyperthyroidism, and autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Htg) can produce too. The exact causes, which lead to alterations in both thyroid glands and orbital tissues, have not been cleared yet. In Graves' disease, the immune regulation is characterized by the schift to T helper 2 (Th2) dominance with the productions of cytokines and autoantibodies directing to thyroidal and non-thyroidal antigens [2]. Hashimoto's thyroiditis is characterized by the presence of anti-TPO and anti-Htg autoantibodies, as well as by the rare occurrence of TSH receptor antibodies, in 3-5% only. The immune processes lead to thyroid tissue destruction due to the productions of proinflammatory IL-1, IL-6, IL-17 and TNFα cytokines and the increased apoptosis demonstrating Th1 dominance [3]. Elevated IgE levels in Graves' disease, can be regarded as inducing factors for hyperthyroidism and connect to higher TSH receptor antibody levels [4]. IgE deposits were demonstrated in eye muscle tissue derived from patients with ophthalmopathy [5]. Seasonal allergic attacks showed a strong association with higher prevalence of Graves' hyperthyroidism [6]. Th2-derived IL-5 and IL-13 cytokines were elevated in Graves' disease and also that of IL-5 in Hashimoto's thyroiditis [7][8]. The main regulatory factors of immune responses are mast cells, T cell dysregulation and cytokines, which are involved in both allergy and autoimmunity. Our previous reports highlighted the pathognomic role of serum total IgE, IL-5 and IL-12 cytokine levels, as well as nerve growth factor (NGF) in Graves' ophthalmopathy [9][10][11].
The objective of the study was • To investigate the prevalence of respiratory and food allergic events and the presence of allergen-specific IgE levels in autoimmune and nonimmune thyroid diseases. • To demonstrate any difference in Graves' disease between the presence and absence of ophthalmopathy. • To reveal any linkage between allergic events and thyroid status.
The difference in allergen-specific IgE levels may reflect a distinct immune regulation in AITDs. The results may contribute to a better understanding of the association of thyroid diseases with ophthalmopathy. In turn, the investigations give an answer, whether the presence of allergen-specific IgE levels via modifying effect on thyroid hormone and autoantibody levels can contribute to a slow remission rate of Graves' hyperthyroidism.

Patients
Two-hundred-fifty-nine cases formed the patient groups: 149 patients had Graves' disease (mean age of 49 ± 13 years, 57 cases had ophthalmopathy), 110 Hashimoto's thyroiditis (mean age of 50 ± 14 years) and 65 cases with euthyroid goitre were the controls (mean age of 48 ± 13 years) ( Table 1). Thirty-eight patients with Graves' disease were hyperthyroid, 87 euthyroid and 24 hypothyroid at the time of the study. Hyperthyroid Graves' patients were treated with thyrostatic therapy (methimazole or propylthiouracil) in 26 cases, radioiodine in 8 cases and surgical removal in 4 cases. In hypothyroid Graves' patients, 3 patients did not receive any therapy, 4 patients were treated with thyrostatic drugs, 12 patients with radioiodine and 5 with surgery treatments. Eleven hypothyroid patients were insufficiently substituted with levothyroxine therapy at the time of the study. Only 7 out of 24 hypothyroid patients had no autoantibodies to thyroid peroxidase and/or thyroglobulin. In Graves' ophthalmopathy, the exact diagnosis based on the investigation of ophthalmologist. All patients with Hashimoto's thyroiditis were treated with levothyroxine supplementation and showed euthyroid state demonstrating autoantibodies to thyroid peroxidase and/or thyroglobulin. Four patients with Hashimoto's thyroiditis had surgical removal in history. The control group consisted of healthy patients without immune, infection, cancer and endocrine diseases in the history, and they had normal biochemical data, but the thyroid ultrasound detected a small (<10 mm) nodule in thyroid glands. None of patients were treated with steroid and antihistamine medications at the time of the study. The diagnosis of seasonal allergies or food sensitivities was based on the physical examination and the typical allergic symptoms in the history with the presence of allergen-specific IgE levels.

Detection of allergen-specific IgE levels
Allergen-specific IgE levels were detected with AllergyScreen immunoblot method (MEDIWISS Analytic GmbH, Germany) against the following respiratory and food allergens: I. Crab, F10: Sesame seed, F20: Almond. The strips were handled with 250 µl nondiluted sera at room temperature for 45 minutes and shaked. After washing buffer (TRIS/NaCl, pH=7.5), 250 µl of biotinylated detector antibodies were added for 45 minutes. The strips were washed and 250 µl of streptavidin conjugates were measured into the membranes for 20 minutes. After washing, 250 µl of substrate solutions were added for 20 minutes in dark and terminated by briefly rising under flowing water. The membranes were dried and evaluated with Kodak camera. The results were given in IU/ml. The sensitivity and specificity of AllergenScreen were the following according to the instruction manual in comparison with Prick and ELISA tests: 95.1% and 81.2% for skin-Prick test; 84.3% and 95% for ELISA system, respectively.

Detection of thyroid hormone and anti-thyroid autoantibody levels
Thyroid hormone serum levels (TSH, FT4 and FT3), anti-thyroid peroxidase (TPO) and anti-thyroglobulin (Htg) antibodies were measured with chemiluminescence method (Immulite, USA) in a fully automated way, in Kenézy Hospital laboratory. The outpatient sera were sampled, stored at 4°C and measured within 24 hours. The normal ranges were the following: 0.35-4.94 µU/ml for TSH, 0.7-1.48 ng/dl for FT4, 1.45-3.48 pg/ml for FT3, 0-115 IU/ml for anti-TPO antibodies and 0-65 IU/ml for anti-Htg antibodies. TSH receptor antibody detection was carried out with radioimmunoassay (Brahms, Germany), the normal range was below 1.5 U/l.

Statistical analysis
The data were exhibited as mean ± standard error (SE). The distribution of data was non-normally, therefore Chi-squared with Yates' correction and nonparametric Mann-Whitney tests were applied for comparison of two groups.

Modifying effects of allergen-specific IgE levels on thyroid hormone and antithyroid antibody levels in Graves' disease
The relationship between the presence of allergen-specific IgE levels and thyroid hormone or anti-thyroid antibody levels highlighted that respiratory and food allergens can influence the thyroid immunoregulation in Graves' disease. Three food and 4 seasonal allergen-specific IgE levels were associated with increased TSH [ (Figure 4).

Discussion
Our results highlighted that the increased prevalences of respiratory and food allergen-specific IgE levels were connected rather to Graves' disease. In this study, the manifestation of allergic conjunctivitis (and not rhinitis) was more frequent in Graves' disease suggesting that the seasonal allergic attacks can play as aggravating or inducing role in hyperthyroidism. The relationship between Graves' disease and allergy is not surprising, because thyroid autoimmunity and hyperthyroidism is characterized by Th2 dominance. The prevalence of asthma increased in women with elevated anti-TPO antibodies [12]. Th2 dominance associating with elevated nerve growth factor (NGF) levels represents an increased sympathoadrenal activity and connects to hyper-and hypothyroidism [10]. The orbital tissues are special sources for NGF release and these regions are densely innervated by sympathetic nerve fibers [13]. Ocular tissues are targets for systemic anaphylaxis or allergy due to the increased number of resident mast and eosinophil cells. The concomitant release of vasoactive amines leads to the cosecretion of NGF [14,15]. AITDs can be regarded as two endpoints of thyroid autoimmunity [16]. The difference in allergenspecific IgE levels between Graves' patients without and with ophthalmopathy highligthed that the absence of eye symptoms was associated with higher increased IgE serum levels, particularly in seasonal allergy. IL-10 elevation via influencing immunoregulation can be responsible for the stronger immunosuppressive effect in patients with ophthalmopathy [17]. In another way, the elevated levels of IL-17 and IL-6 proinflammatory cytokines can contribute to lower allergen-specific IgE levels in Graves' ophthalmopathy [18,19]. Increased IL-17 levels are also present in allergic events, Hashimoto's thyroiditis and smoking induced inflammatory processes [20][21][22]. IL-33 is another cytokine, which is involved in both allergic and autoimmune diseases [23]. Activation of IL-33 receptor -expressed on dendritic cells, T and B lymphocytes, natural killer (NK), mast, basophil, eosinophil and macrophage cells -leads to Th2-and NGFmediated cytokine productions [24]. IL-33 serum levels increase significantly and correlate positively with thyroid hormone levels in Graves' disease in comparison with Hashimoto's thyroiditis and controls [25]. In house dust mite allergy, the airway inflammation was induced by Treg cells and IgA production,which immune responses could be attenuated by probiotics [26]. The prevalences of food allergic sensitizations and the degrees of allergen-specific IgE levels were similarly high in Graves' disease, particularly in patients without eye symptoms, but lower in Hashimoto's thyroiditis and Graves' ophthalmopathy. Carrot-and potato-specific IgE levels showed significant differences between Graves' patients without and with ophthalmopathy. The presence of TSH receptor antibodies was associated with elevated soybean-specific IgE levels in Graves' ophthalmopathy. Food allergy represents mucosal immunity against food allergens [27]. The role of IL-17 cytokine was demonstrated in food allergy too [28]. Gastrointestinal autoimmunity, such as coeliac disease and autoimmune gastritis were associated with Graves' ophthalmopathy rather than Graves' patients without eye symptoms [29]. Estrogenic isoflavones in soyabeans possess goitrogenic activity inhibiting iodine uptake via decreasing its organification [30]. Genistein inhibits thyroxine hormone synthesis and acts as a potent stimulator for T and B cell-mediated immune processes [31]. In our study, the immune effects of genistein may be explained by the increased anti-TPO and anti-Htg serum levels in patients with soybean-specific IgE levels. The clinical importance of modifying effect of respiratory or food allergen-specific IgE levels on thyroid hormone or antibody levels needs futher investigations. This fact highlights, that allergy similarly to smoking can contribute to a slow remission rate of Graves' hyperthyroidism.
Summarizing, the prevalences and the degrees of food and respiratory allergen-specific IgE levels increased significantly in Graves' disease highlighting the dominance of Th2-derived cytokines. In turn, the elevation of proinflammatory cytokines can be responsible for the lower degree of allergen-specific IgE levels in Graves'ophthalmopathy and Hashimoto's thyroiditis. Surprisingly, Hashimoto's thyroiditis has also Th2-derived feature with respect to respiratory allergy but failed for food allergy. Some allergen-specific IgE level had modifying effect on thyroid hormone and antithyroid antibody levels contributing to a slow remission rate in Graves' disease.