SZKOLENIE PODYPLOMOWE/POSTGRADUATE EDUCATION

The role of the immune system and cytokines involved in the pathogenesis of autoimmune thyroid disease (AITD)

Rola układu immunologicznego oraz udział cytokin w patomechanizmie autoimmunologicznej choroby tarczycy (AITD)

Hanna Mikoś, Marcin Mikoś, Monika Obara-Moszyńska, Marek Niedziela

II Katedra Pediatrii, Klinika Endokrynologii i Reumatologii Dziecięcej, Uniwersytet Medyczny im. Karola Marcinkowskiego w Poznaniu, Poland

Prof. Marek Niedziela M.D., Ph.D., II Katedra Pediatrii, Klinika Endokrynologii i Reumatologii Dziecięcej, Uniwersytet Medyczny im. Karola Marcinkowskiego w Poznaniu, Poland, Szpitalna St. 27/33, 60–572 Poznań, tel. +48 61 849 14 24, tel./fax: +48 61 848 02 91, tel. kom.: 606 640 392, e-mail: mniedzie@ump.edu.pl

The immune system and autoimmune thyroid disease

Autoimmune thyroid dysfunction forms a clinical picture in which Graves’ disease (GD), characterised by hyperthyroidism, is at one pole, and chronic autoimmune thyroiditis (cAIT), manifested by hypothyroidism, is at the opposite one.

Antibodies against thyroid antigens are present in both these diseases, but their specific epitopes are different, resulting in different functional antibodies. AITD development occurs due to loss of immune tolerance to autoantigens and reactivity of the thyroid, which leads to infiltration of the gland by T cells and B cells, which in turn produce antibodies specific for clinical manifestations of hyperthyroidism and hypothyroidism, GD and cAIT, respectively. Moreover, T cells in cAIT induce apoptosis in thyroid follicular cells, leading to the destruction of the gland [1, 2].

Antigen-presenting cells (APCs) or accessory cells, belonging to major histocompatibility complex (MHC) class II, especially dendritic cells, accumulate within the thyroid gland. APCs present specific thyroid antigens to lymphocytes within the lymph nodes, which leads to activation and proliferation of autoreactive lymphocytes T and B. In this way, activated antigen-specific T-helper lymphocytes CD4+ induce the formation of cytotoxic CD8+ T cells, and activate B cells, that produce autoantibodies, creating ectopic proliferation centers. Gland infiltration by cytotoxic T cells is primarily responsible for the destruction of thyroid parenchyma [3, 4].

The presence of an immunologically competent cell subset with suppressive activity, preventing a potentially harmful autoimmune response, is due to regulatory T cells CD4+ CD25+ (Treg). Lack of T regulatory cells in humans and mice results in the development of a number of systemic autoimmune disorders such as thyroiditis, arthritis, gastritis, multiple sclerosis, inflammation of the ovaries, and others [5, 6].

Currently, there are documented three different populations of CD4+ T lymphocytes: Th1, Th2, and Th17. The development and differentiation of native CD4+ Th cells in populations diagnosing antigen is preceded, in cooperation with MHC class II signalling, by dendritic cells DC, with toll receptors similar (TLRs) and C-type lectin receptors (CLR). The development of Th1 cells is dependent on IL-12, Th2 cells from IL-4, Th17 cells by TGF-β and IL-6 [7].

Th1 cells produce pro-inflammatory cytokines IL-2, IFN-γ, TNF, IL-1β, which leads to the activation of macrophages and cytotoxic effects. Th2 cells produce IL-4, IL-5, IL-6, IL-10, IL-13, which can inhibit the production of Th1 cytokines, but primarily stimulate B cells to produce antibodies and activation of anti-apoptotic molecules [8].

Hashimoto’s thyroiditis (HT), a hyperplastic subtype of cAIT, is predominantly Th1 immune response, favouring the development of cell-mediated immunity, and thyroid follicular cell death by apoptosis [9, 10]. Proapoptotic death ligands and receptors, such as TNF, FasL and TRAIL present on thyroid cells, under physiological conditions are inactive [9]. However, expression of the Fas/FasL-induced in response to pro-inflammatory Th1 cytokines by infiltrating IFN-γ TNF and IL-1β activates thyroid cell apoptosis [11–13].

The GD promotes Th2 predominantly humoral response, increased production of antibodies by B cells increases concentration of immunoglobulin G (IgG) and the cytokines produced by Th2 cells, which promote humoral response, thus inhibiting the expression of Fas/FasL and results in activation of the anti-apoptotic molecule Bcl-2, that protects thyrocytes from apoptosis, but increases apoptosis of cytotoxic lymphocytes, infiltrating thyroid tissue [10, 12]. Schematic pathogenesis of AITD illustrates the diagram in Figure 1.

Figure 1. Pathogenesis of chronic autoimmune thyroiditis (cAIT) and Graves’ disease (GD)

Rycina 1. Patogeneza przewlekłego autoimmunologicznego zapalenia tarczycy (cAIT) i choroby Gravesa-Basedowa (GD)

A new subtype of Th17 response may also be involved in the pathogenesis of GD [14]. Th17 cells, which have been described recently, secrete proinflammatory cytokines (IL-17, IL-17F, IL-21, IL-22) and play an important role in chronic inflammatory diseases such as asthma and systemic lupus erythematosus [15]. The percentage of Th17 cells in patients with GD was first described by Nanba et al., showing a higher rate of Th17 lymphocytes in patients not treated with anti-thyroid drugs, compared to remission in patients with GD [16].

Study profile of Th17 cells in HT revealed increased expression of the gene RORC2 responsible for the differentiation subpopulation Th17 phenotype, and increased number of Th17 cells in peripheral blood and thyroid tissue in patients with HT, without proof of a similar situation in patients with GD [17]. Similar observations have been made by Idzkowska and Bossowski among children with newly diagnosed HT, to yield a statistically significant elevated levels of IL-17 and IL-23, which supports the participation of the Th17 in the pathogenesis of HT [18].

Extracellular mechanism of peripheral tolerance is mediated by regulatory T cells CD4+ CD25+, mainly type 1 (Tr1). T regulatory cells were first described in the 1970s and 80s, when they were called suppressor cells. Recent studies have shown the importance of the key T lymphocytes CD4+ CD25+ cells in the immune response, in which the suppressor mechanism of action is based on direct effect on thyroid follicular cells. It is assumed now that all T regulatory cells are characterised by expression of the transcription factor Foxp3, which is an essential element in the development and functioning. Regulatory T cells play an important role in disorders of immune tolerance to self antigens. Deficiency and impaired function of these cells may be the cause of autoimmune thyroid disease. Nakano et al. found that the percentage of regulatory T cells within the thyroid lymphocyte cells was lower in patients with autoimmune thyroid disease [19, 20].

In human and mouse T lymphocytes, CD4+ CD25+ represent about 5–10% of the population of CD4+ T lymphocytes. Regulatory T cells mediate suppression mechanisms, by inhibiting IL-2 secretion and the secretion of anti-inflammatory cytokines, such as IL-4, IL-10, and TGF-β. Thus the action of T regulatory lymphocytes inhibits and modulates immune response Th1, Th2 and Th17 [21, 22].

Therefore, the clinical manifestation of autoimmune thyroid phenotype in the direction to GD and cAIT largely depends on the balance of the immune response induced Th1 or Th2 by the APCs, and the cytokine profile, which dominates at this point in thyroid parenchyma. The fact that these two functionally opposite states can develop in the same person at different periods suggests that the Th1/Th2 balance and the related cytokine profile is a dynamic process, evolving under the influence of external factors operating in the local environment of the thyroid gland [23, 24].

The participation of antibodies in the pathogenesis of AITD

Autoimmune thyroid disease is an autoimmune disease that affects approximately 5% of the population and is the most common organ-specific autoimmune disease. Activated B cells secrete antibodies directed against several major thyroid antigens. The occurrence of autoantibodies is characteristic of the disease, although these antibodies are also found in healthy individuals [25].

Anti-thyroid antibodies (ATG, ATPO)

Thyroid peroxidase antibodies (ATPO) are a marker of autoimmune thyroid disease. ATPO are present in nearly all patients with Hashimoto’s thyroiditis, in two thirds of patients with postpartum thyroiditis, and in 75% of patients with hyperthyroidism in the course of Graves’ disease. Thyroid peroxidase is a key enzyme involved in thyroid hormonogenesis. This antigen was identified in 1959 as the thyroid microsomal antigen. ATPO levels are associated with the expression of MHC on thyrocytes and with degree of infiltration by lymphocytes, which may ‘sensitise’ and trigger the synthesis of autoantibodies [26, 27].

Antibodies are produced mainly by lymphocytes infiltrating the thyroid gland, and only to a small extent by the local lymph nodes or bone marrow. It turns out that the presence of gammaglobulin antibodies is ascertained in 12–26% of asymptomatic patients with thyroid disease and is a risk factor for the future onset of hypothyroidism. Anti-TPO antibodies, in contrast to anti-thyroglobulin antibodies (ATG), are able to induce the complement system and cellular cytotoxicity. There is a high correlation between the concentration of anti-thyroid peroxidase antibodies and the presence of lymphocytic infiltration of the thyroid gland [28, 29].

ATPO are polyclonal, heterogeneous and directed towards different parts of TPO molecules, most of which recognise epitopes located on the surface of the enzyme TPO. Two major conformational immunomodulatory A and B regions (IDR-A and IDR-B) on the surface of the TPO are of particular importance, since they are in close proximity or even overlap each other [30]. Studies have shown that antibodies to the IDR-B domain are confirmed in most patients with HT and GD [31].

ATPO are also present in 12–16% of euthyroid patients, in 20% of type 1 diabetics and also in other autoimmune diseases: Addison’s disease — 50%, rheumatoid arthritis — 30%, Sjogren’s syndrome — 17–40%, SLE — 23%, scleroderma — 19%, and fibromyalgia — 24% [27].

In the NHANES III study population, the incidence of ATPO in the United States in healthy Caucasians was 12.3%. It was similar in the population of Mexican origin — 10.1%, but significantly lower among African-Americans — 4.5%. Female gender is associated with a 2–4-fold higher incidence of ATPO. The percentage of antibodies increases with age for both sexes to almost 30% in chronically ill elderly patients. Antibodies against thyroglobulin are present in 97% of patients with Hashimoto’s thyroiditis, and about 50% of cases of Graves’ disease and in 10–20% of patients with subacute thyroiditis (de Quervaine’s disease)[32].

In a multicentre study performed in 2007–2010 in Poland (PolSenior), in age groups ranging from 65 to 90 years of age, increased ATPO was revealed in 19% of the cohort. Prevalence of ATPO positivity was higher in females than in males (26.6% v.15.5%). Among 1,542 subjects with known concentrations of both ATPO and thyroid function, 1,110 were euthyroid (72%), 317 hypothyroid (20.6%), and 115 hyperthyroid (7.4%) [33].

Thyrotropin receptor antibodies (TRAb)

TSH receptor antibodies (TRAb) mimic the function of TSH and cause disease by binding to the TSH receptor and stimulate or inhibit thyroid cells in terms of T3 and T4 production. Patients with AIT may present both stimulating and blocking antibodies. The clinical picture of the disease is a result of the relative strength of each antibody type. While Graves’ disease is characterised by the production of stimulating autoantibodies TSHRTSAb, another group of patients with autoimmune thyroid disease may have autoantibodies that block the activation of TSHR-TBAb [34, 35].

The determination of concentrations of TRAb is helpful in the differential diagnosis of hyperthyroidism, Graves’ disease, autoimmune disease aetiology and in predicting relapse after anti-thyroid drug treatment. Patients with high levels of TRAb (> 10 IU/L) have a 8.7 to 31.1 times higher risk of severe course of GO, and are likely to relapse after initial treatment with anti-thyroid drugs [35].

Currently, the detection of TRAb antibodies is widely used in Europe and Japan, the second and third generation of competitive tests TBII were developed for the first time by Smith et al [36]. However, this test does not differentiate the biological activity of the antibodies, TSAb and TBAb, which is possible using a bioassay based on cell culture and recombinant human TSHR, but is limited due to much efforts and costs. Second generation TBII tests have 100% specificity and 98% diagnostic sensitivity [37–39].

Participation of cytokines in the pathogenesis of autoimmune thyroid disease

Cytokines are glycoproteins released by activated cells from different tissues. They provide a network control system of many processes such as proliferation, differentiation, secretion of biologically important substances. They play a key role in modulating the immune response and influence the balance between maintaining tolerance and initiating autoimmunity. In addition to hormone-like properties, they are involved in the process of haematopoesis, affect the functions of other cells and are mediators of inflammation and immune response, repair processes and wound healing.

Cytokines are involved in the pathogenesis of thyroid diseases, working in both the immune system and directly targeting the thyroid follicular cells. They participate in the induction and effector phase of the immune response and inflammation and play a key role in the pathogenesis of autoimmune thyroid disease, including cAIT and Graves’ disease. Within the inflammatory cells and thyroid follicular cells, the presence of multiple cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, TNF-α and IFN-γ has been demonstrated [40, 41].

Cytokines in cytokine network play different functions: some effects are promoted by cytokines and inhibited by others. Cytokine secretion profile can be considered as a pro-inflammatory or anti-inflammatory, pro-apoptotic or anti-apoptotic. Under the influence of chronic antigen challenge, macrophages and CD4+ T cells can be divided into cells Th1 producing cytokines involved in the cellular response (IL-2, IFN-γ, TNF-α, IL-1β), or subpopulations of Th2 lymphocytes, which secrete cytokines associated with humoral response (IL-4, IL-5, IL-6, IL-10, IL-13), and Th17 producing IL-17, IL-21, IL-22. Th3 immune cells produce mainly TGF and play an important role in protecting against the occurrence of autoimmune diseases [42–44]. Despite attempts to classify autoimmune thyroid disease as a classical Th1or Th2-dependent, there is mixed Th1/Th2 immune response in both HT and in GD [16].

Cytokines are produced by both lymphocytes and thyroid follicular cells. Experiments conducted in vitro have confirmed the production of cytokines by the stimulation of thyroid follicular cells by IL-1, IFN-γ and TNF-α, suggesting an increase of activity and an increased infiltration of pro-inflammatory cells in vivo. These cytokines increase the expression of adhesion molecules on the surface of thyroid follicular cells, and may also stimulate production of nitric oxide (NO) and prostaglandin (PG), increasing the inflammatory response in AITD. Thyroid tissue cytokines also play a role in antigen presentation to T cells by increasing the expression of MHC class I and II on the surface of the thyroid follicular cells, leading to destruction of the thyroid gland by T cell cytotoxicity. In thyroid ophthalmopathy, pathogenic action of cytokines: IL-β, TNF-α and IFN-γ, exacerbate inflammation and proliferation of fibroblasts, resulting in the accumulation of glycosaminoglycans within the orbit. In Graves’ disease, IL-1β stimulates the production of hyaluronic acid in thyroid epithelial cells and fibroblasts, thus contributing to the development of the goitre [45–48].

On the basis of thyroid tissue immunohistochemistry, the presence of IFN-γ in cells infiltrating the thyroid gland, IL-1 in endothelial thyroid cells, and increased the amount of IL-1, IL-6 and TNF-α in thyroid follicular cells was revealed. In cell cultures, IL-1 and IL-6 increase the proliferation of the thyroid follicular cells, but also have inhibitory effects on thyrocytes during stimulation of these cells by TSH [49].

Cytokines in many cases exhibit a pleiotropic action and often may function as both an immunostimulatory and an immunosuppressive agent, depending on the type and location of the target. For example, the cytokine IL-2, IFN-γ and TNF-α activity may exhibit both inhibitory and excitatory function in autoimmune diseases [50].

IFN-γ and TNF-α inhibit the growth and proliferation of the thyroid follicular cells, but do not affect the viability of cells [49]. Pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α are generally associated with the stimulation of inflammation and autoimmunity, and may also stimulate production of T regulatory lymphocytes, inhibiting development of autoimmune diseases [51].

Cytokines enhance the inflammatory response by stimulation of both T and B lymphocytes, resulting in the production of antibodies and thyroid tissue damage by apoptosis, in particular in HT [52]. Cytokines can modulate the growth and function of thyroid follicular cells themselves in AITD, playing an important role in the extra-thyroid AITD complications, particularly ophthalmopathy. Exogenous administration of cytokines such as IFN-α is associated with the appearance of autoimmune thyroid disorders and the emergence of antibodies ATPO and ATG [40]. Krupinska et al. reported an elevated concentration of antithyroid antibodies in 17.14% of patients, with no signs of AIT at baseline, detected during therapy with interferon, after six and 12 months of treatment. After six months of treatment in this group of patients, 11 had hypothyroidsm and six had hyperthyroidism [53].

Conclusions

The role of cytokines in the development of autoimmune thyroid disease and their participation in the processes leading to the development of hypothyroidism in chronic autoimmune thyroiditis and hyperthyroidism in Graves’ disease is still not clear. The demonstration of immune cells and anti-thyroid antibodies within the thyroid gland, and the determination of the levels of cytokines in peripheral blood, provide information about their involvement in the pathogenesis of AITD. These observations serve to confirm the effect of cytokines and their important role in maintaining the balance between health and disease.