Thyroid Autoantibodies: Thyroid Peroxidase and Thyroglobulin Antibodies
Barbara Czarnocka,1 Deirdre Cocks Eschler,2 Marlena Godlewska,1 and Yaron Tomer2,3 1Medical
Center of Postgraduate Education, Department of Biochemistry and Molecular Biology, Warsaw, Poland, 2Division of Endocrinology, Mount Sinai Medical Center, New York, NY, 3James J. Peters VA Medical Center, New York, NY
Historical note The thyroid gland is the target for two autoimmune diseases – Graves disease (GD) and Hashimoto thyroiditis (HT). GD was first described in 1835 by Dr. Robert Graves when he reported three patients with hyperthyroidism, one of whom had exophthalmos. Nearly 80 years later, in 1912, Hakaru Hashimoto, while still a medical student in Japan, reported for the first time four patients with goiter showing lymphocytic infiltration, which he called “struma lympomatosa” (reviewed in ). Today, these two disorders are collectively referred to as autoimmune thyroid diseases (AITD). Autoantibodies reactive to a thyroid gland-specific antigen, distinct from thyroglobulin, were first described in the sera of patients with HT by Belyavin and Trotter in 1959; this so-called “microsomal antigen” was identified almost 30 years later as thyroid peroxidase (TPO), the key enzyme in the biosynthesis of the thyroid hormones thyroxin (T4) and tri-iodothyronine (T3) (reviewed in ).
Thyroid peroxidase autoantibodies Structural and functional characteristics of thyroid peroxidase Human thyroid peroxidase (hTPO) is a membrane-bound type I glycosylated protein composed of 933 amino acids (105 kDa in size) that is expressed on thyrocytes as a homodimer at the apical pole facing the colloidal lumen, where the main steps of hormonogenesis take place. Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones. It catalyzes both the iodination of tyrosine residues to form monoiodotyrosine (MIT) and diiodotyrosine (DIT) and the coupling of hormonogenic iodotyrosine residues in the thyroglobulin (Tg) molecule to form tri-iodothyronine (T3) and thyroxin (T4). Thus, TPO is essential for normal thyroid function [3,4]. TPO has a large extracellular ectodomain containing the catalytic site with a heme prosthetic group that projects into the follicular lumen, a short trans-membrane domain, and a 61-amino acid cytoplasmic tail (Figure 44.1). Newly synthesized TPO polypeptide undergoes extensive post-translational modifications, including glycosylation, heme incorporation, dimer formation, and proteolytic trimming Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00044-7 Copyright © 2014 Elsevier B.V. All rights reserved.
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FIGURE 44.1 Three-dimensional ribbon diagram showing the predicted structure of thyroid peroxidase (TPO). The diagram shows the location of contact amino acid residues within immunodominant region (IDR)-A (green) and IDR-B (red). The myeloperoxidase (MPO)-like (amino acids 142–738), Complement control protein (CCP)-like (739–795), epidermal growth factor (EGF)-like (796–841), transmembrane, and intracellular domains (845–933) are marked. The flexibility of the hinge regions is indicated by an arrow. Reproduced with permission from Dr. JP Banga and Dr. BJ Sutton on 15 January 2013; the model adjusted using Swiss-PDB Viewer 4.0.2 freeware.
Thyroid peroxidase autoantibodies
of the N-terminal region. These steps are followed by intracellular trafficking of the native form to its final location on the apical membrane of the thyrocyte. The majority of TPO molecules are degraded intracellularly, mostly due to improper maturation, and only about 2% of TPO molecules synthesized become the enzymatically active form found at the apical membrane–colloid interface, where the prosthetic group is exposed to the colloidal lumen [3,4]. The three-dimensional structure of TPO has yet to be solved. Therefore, the current threedimensional model of hTPO was created on the basis of the known structure of myeloperoxidase (MPO), a granulocyte enzyme that shares 42% sequence homology with TPO. According to this putative model, the ectodomain of TPO is composed of three distinct modules: an MPO-like domain at the N terminus, a Complement control protein (CCP)-like domain towards the C terminus, and an epidermal growth factor (EGF)-like domain at the boundary with the transmembrane domain (Figure 44.1). A full understanding of the precise arrangement of these domains on the membrane surface, and the organization of TPO dimers, awaits determination of the three-dimensional TPO structure. Therefore, it is currently not possible to map the exact locations of autoepitopes interacting with TPO antibodies (Ab) and the immunodominant regions (IDR) of hTPO [3,4].
Characteristics of thyroid peroxidase autoantibodies TPO Abs are one of the hallmarks of autoimmune thyroid diseases (AITD), and they are found in both Graves disease (GD) and Hashimoto thyroiditis (HT). TPO Abs are detected in the sera of the majority of patients with GD (∼85%), HT (> 90%), postpartum thyroiditis (PPT) (∼67%), and 10–20% of nonAITDs, as well as in up to 26% of euthyroid healthy subjects (Table 44.1) [5,6]. The prevalence of TPO Abs in the general population increases with age. These antibodies are mainly produced by B lymphocytes infiltrating the thyroid gland, and their titers reflect the severity of lymphocytic infiltration. Circulating TPO Abs are often present at high concentrations, and levels quantified in milligrams per milliliters have been reported in some patients. TPO Abs are not restricted to a single immunoglobulin (Ig)G subclass, although circulating antibodies are predominantly of the IgG1 and IgG4 subclasses, and the kappa light chain is usually dominant. However, IgG2 and IgG3 subclasses and lambda chain-containing TPO Abs have also been detected in some patients [5,7]. In recent years, a large number of TPO Abs have been isolated from combinatorial Ig gene libraries derived mainly from B cells infiltrating the thyroid gland or lymph nodes of patients with AITD. Analysis of human monoclonal TPO Abs produced in the form of Fab fragments has provided important information about the TPO Ab repertoire: (i) there is restriction in the IgV gene usage, (ii) the VDJ recombination process preferentially uses the inverted D gene, (iii) there is greater preference for J gene Table 44.1 Prevalence of Thyroid Peroxidase (TOP) and Thyroglobulin (Tg) Autoantibodies in Healthy Adults Versus in Patients with Autoimmune Thyroid Disease
Healthy adults Hashimoto thyroiditis Graves disease
Up to 26% females Up to 9% males > 90% ∼85%
10–27% 20–90% 50–60%
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usage and limited somatic mutation of J proximal light chain genes, suggesting a defect in receptor editing in AITD, and (iv) certain somatic mutations are observed in most TPO Abs irrespective of the library [5,6].
Epitope mapping Polyclonal heterogeneous TPO Abs present in the sera of patients with AITD react with several B-cell epitopes located on the surface of hTPO. The first epitopic map of human TPO was defined using competition assays between a panel of murine anti-TPO monoclonal antibodies and autoantibodies from sera of patients with AITD. These studies demonstrated that TPO Abs interact with epitopes located on the overlapping A and B domains. These findings were subsequently confirmed in studies using human TPO monoclonal antibodies expressed in the form of Fab fragments that are similar to Abs present in patients’ sera. Therefore, it is now widely accepted that TPO Abs in the sera of patients with AITD react with the overlapping domains A (IDR-A) and B (IDR-B) that form the IDR on the TPO surface. Epitopic recognition profiles seem to be unrelated to thyroid status; they are conserved over time and appear to be genetically determined [3–5]. Moreover, there is no statistically significant difference between the sera of HT and GD patients in the IDR-A and IDR-B autoantibody level. However, it is still not clear whether the TPO Ab repertoire of AITD patients is different from that of healthy individuals or patients with non thyroid autoimmune diseases. TPO Abs mostly recognize conformational epitopes that are dependent on the three-dimensional structure and folding of TPO. In addition, a small minority of TPO Abs recognize linear epitopes that are formed by continuous amino acid sequences in the TPO polypeptide and are most commonly found outside the IDR [3–5]. Many attempts have been made to locate the IDR-A and IDR-B on the TPO molecule. However, the exact location and structure of the discontinuous IDR of TPO is yet to be determined. Several approaches have been used to identify TPO epitopes involved in TPO Abs binding. These included competition studies with monoclonal mouse, human Fab, and polyclonal TPO Abs raised against peptides predicted to be exposed on the surface of hTPO. Using these methods, several TPO amino acids and peptides have been identified to participate in TPO Ab binding – residues 646, 707, 620, 624, 627, 630, 210–225, 353–363, 377–386, 549–563, 599–617, 713–720, and 766–775. These amino acids are located within the IDR-A and IDR-B, mainly in the MPO-like domain, with some located in the CCP-like domain (Figure 44.1) [3–5]. The location of the reactive amino acids and peptides on the predicted TPO antigenic surface supports the discontinuous nature of the IDR. This is not unexpected given that native TPO has a densely folded structure, with the MPO- and CCP-like domains lying in close proximity to form the conformational surface recognized by TPO Abs found in the majority of patients with AITD. Following a recent analysis of the TPO IDR using the available human, mouse, and rabbit antibodies, it was proposed that the IDR (A and B) forms a single complex on TPO, centered around residues 599–617 within the MPOlike domain, whereas the EGF-like domain, transmembrane fragment, and homodimer contact regions may not be involved in Ab binding. However, the CCP and EGF domains and the hinge region help maintain the three-dimensional structure of TPO required for Ab binding [4,5,8].
Thyroid peroxidase antibodies and thyroid dysfunction TPO Abs may be involved in autoimmune thyroid cell death via two mechanisms: antibody-dependent cytotoxicity (ADCC) involving natural killer (NK) cells and C3 complement-mediated cytotoxicity (CDC). They also influence the diversity of the pathogenic T-cell epitope repertoire. Some TPO Abs
have been reported to bind to TPO and inhibit its enzymatic activity. This effect was observed in vitro and most likely does not occur in vivo due to the inability of Abs to penetrate the follicles and reach TPO on the apical pole. Furthermore, this finding was not reproduced using human monoclonal antibodies. It has recently been suggested that the effects of TPO Abs may require the involvement of FcRn, an Ig receptor expressed on thyrocytes, which is implicated in transcytosis of IgG across epithelia [3–5].
Detection of thyroid peroxidase antibodies TPO Abs were initially described as antimicrosomal antibodies after they were found to react with crude homogenized thyroid membrane preparations. The older microsomal Ab assays used passive tanned red cell agglutination tests or immunofluorescence assays. Once TPO was identified as the microsomal antigen, purified or recombinant TPO was used to improve the sensitivity and specificity of the assays. However, the prevalence and normal cut-off values depend on the assay method. The data from current TPO Ab assays are given in international units and all tests are standardized using the Medical Research Council (MRC) 66/387 reference preparation .
Thyroglobulin autoantibodies The thyroglobulin gene Tg is encoded by a gene on chromosome 8q24. It is the major thyroidal protein, accounting for approximately 80% of total thyroidal proteins. Tg is the precursor of the thyroid hormones, T3 and T4. The Tg molecule undergoes important post-translational modifications, the most important one being iodination of tyrosines, which is critical for the formation of thyroid hormones. We and other researchers have shown that Tg is a major gene for AITD. Whole genome linkage studies identified a locus on chromosome 8q24 that was linked with AITD, and fine mapping of this locus identified Tg as the susceptibility gene at this region. Sequencing of the Tg gene identified three nonsynonymous single nucleotide polymorphisms (SNPs) (causing amino acid changes) and one promoter SNP that showed strong association with AITD . Moreover, further analysis showed strong interaction between one Tg variant and HLA-DR3 in predisposing to AITD. This interaction can be explained by formation of pathogenic Tg peptide repertoire that can bind to the disease-associated HLA-DR3. Indeed, we recently identified four Tg peptides that bind with high affinity to the disease-associated DR3 pocket. One of these peptides, Tg.2098, was shown to be a major T-cell epitope (reviewed in ).
Measurement of thyroglobulin antibodies Due to the heterogeneity of the Tg molecule even within the same individual and to differences in the autoantibodies to Tg in AITD patients compared to normal subjects, there are inherent difficulties in standardizing the measurement of anti-Tg Abs. New immunoassays have improved the sensitivity and specify of anti-Tg Ab testing; however, there is still no standardization in laboratory technique for identifying anti-Tg Abs. Finally, high levels of Tg in the serum can interfere with assay measurements (reviewed in [1,11]).
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Epidemiology of thyroglobulin antibodies While Tg Abs are one of the hallmarks of AITD, they are also quite frequent in healthy individuals. According to different reports, 10–27% of the normal adult population has measurable Tg Abs, depending on the sensitivity of the assay used (reviewed in [11–13]). Small amounts of Tg leak to the systemic circulation from the thyroid follicles by release from thyrocytes after its synthesis or by transepithelial export from the thyrocyte after thyroid hormone is released from Tg (reviewed in ). This systemic exposure of the immune system to Tg is likely necessary to maintain an immune response in patients who develop AITD. Indeed in mouse models of AITD induced by immunization with Tg+adjuvant, the disease is transient due to lack of continued immune stimulation (reviewed in ). While Tg Abs are not specific to AITD, their levels are often higher than those in the general population and may be used to diagnose AITD in the setting of abnormal thyroid function tests. Tg Abs are reported to be present in 20–50% of patients with HT by some (reviewed in ), while others report that 80–90% patients with HT have elevated Tg Abs (reviewed in ). In GD, 50–60% of patients have positive Tg Abs (reviewed in ) (Table 44.1). Tg Abs are less frequent in AITD compared with anti-TPO Abs, which are positive in over 90–95% of patients with HT and approximately 85% patients with GD (reviewed in ). Thus, Tg Abs are less predictive of overt thyroid dysfunction than TPO Abs, and the latter have a better predictive value for the development of hypothyroidism (reviewed in ). In fact, anti-TPO Abs are the most sensitive test in predicting AITD . Therefore, currently, it is not recommended to test for Tg Abs as a diagnostic test for AITD in iodine sufficient areas (reviewed in ). In contrast, in iodine deficient areas, Tg Abs may be helpful in identifying patients with AITD in the presence of a goiter (reviewed in ). In thyroid cancer follow-up, Tg levels are measured to assess disease recurrence (as Tg is produced only by thyroid cells). In addition, Tg Abs must be measured in this patient population since 20% of thyroid cancer patients have Tg Abs and the presence of Tg Abs will falsely lower measured Tg levels . Furthermore, in those thyroid cancer patients with positive Tg Abs, an increase in serum Tg Ab titers can be the first indication of recurrent cancer .
Do thyroglobulin autoantibodies play a role in the pathogenesis of autoimmune thyroid disease? Most Tg Abs are of the IgG subclass and do not fix complement (reviewed in ). Analysis of Tg revealed 40 putative antigenic epitopes, but of them only four to six are thought to be recognized by B cells and, therefore, involved in the autoimmune process in AITD (reviewed in ). It remains unclear as to whether Tg Abs have a role in the disease process or whether they are merely surrogate markers that are formed due to thyroid cell apoptosis . As mentioned, up to 25% of the adult population has been found to have Tg Abs without thyroid disease using sensitive assays. Tg Abs are also found in patients with monoclonal gammopathies (reviewed in ). Moreover, in patients with AITD, Tg Abs levels do not correlate with disease activity; this has also been shown in animals with experimental autoimmune thyroiditis (EAT) (reviewed in ). While these all may suggest that Tg Abs are not pathologic, there are data suggesting a difference in the epitope specificity of Tg Abs found in healthy adults compared to those in patients with AITD. Furthermore, pregnant women with positive Tg Abs are more likely to experience first trimester fetal loss and to suffer from PPT (reviewed in ). Tg undergoes several key post-translational modifications (reviewed in [1,12]). These modifications in Tg are different in patients with AITD compared with healthy subjects and likely contribute to its
antigenicity, creating different epitopes for Tg Ab binding . Disease-specific Tg Abs also differ from those found naturally in normal individuals: Tg Abs in AITD are oligoclonal and bind to specific, more recently evolved, conformational epitopes on the Tg molecule. In contrast, Tg Abs in normal subjects are polyclonal and bind to more evolutionarily conserved epitopes (reviewed in ). Thus, autoantibodies resulting in thyroid disease differ in both idiotype and epitope specificity . The data on EAT in animals argues both in favor of and against Tg Abs being causative and not just secondary to the autoimmune damage in the thyroid. The ability of fragments of the Tg molecule to induce EAT were tested by multiple authors. Some were able to induce thyroiditis in such a manner, though anti-Tg Abs were not produced in the animal’s sera, perhaps because these epitopes are naturally not accessible in a folded Tg molecule (reviewed in ). Additionally, some studies were able to induce EAT in animals by passive transfer of anti-Tg Abs, while others were unable to reproduce this. Finally, with the knowledge of idiotype specificity of the anti-Tg Abs in disease states, transfer of anti-idiotypes of the anti-Tg Abs was shown to induce EAT in some experimental models (reviewed in ). Interestingly, it has recently been noted that epitope spreading from Tg to TPO may occur in AITD, with Tg being the primary antigen responsible for breaking B-cell tolerance and TPO being responsible for the maintenance of autoimmunity in AITD [15,16]. In experimental models, most mice developed anti-Tg Abs but not anti-TPO Abs; however, those mice that developed anti-TPO Abs all also had antiTg Abs. In other studies, age correlated with Ab development, with anti-Tg Abs developing first, then 100% subsequently developing anti-TPO Abs. In humans, specifically in families with juvenile HT, similar observations were made; anti-Tg Abs developed first, but once anti-TPO Abs were present, they became the dominant antibodies . Moreover, the interaction of iodide with TPO and hydrogen peroxidase has been postulated to generate pathogenic thyrgolgobulin peptides that could trigger thyroid autoimmunity . TPO Abs can then develop through epitope spreading.
Clinical utilities TPO Abs are a hallmark of AITD and are present in 90–95% of patients with HT and in about 85% of patients with GD (Table 44.1). TPO Abs are also detected in up to 26% of healthy women and up to 9% of healthy men, but the clinical significance of low levels of TPO Abs in euthyroid healthy subjects is unknown. AITD are associated with a spectrum of other autoimmune diseases such as type 1 diabetes and autoimmune polyglandular syndromes, and circulating TPO Abs are present in the sera of these patients and are used to diagnose concurrent AITD. The detection of TPO Abs is important not only for the diagnosis of AITD predicting thyroid autoimmunity since it also represents a risk factor for the development of autoimmune thyroiditis in several conditions (Table 44.2) [6,9]. In contrast, Tg Abs are present in 20–90% of patients with HT (reviewed in [13,14]), in 50–60% of patients with GD (reviewed in ), and in 10–26% of healthy adults (Table 44.1) (reviewed in [11–13]). Thus, Tg Abs are less predictive of overt thyroid dysfunction than TPO Abs and have less predictive value for the development of hypothyroidism. Therefore, measuring Tg Abs levels may not be necessary for the diagnosis of AITD in iodine-sufficient areas. However, in patients with high clinical suspicion of AITD and negative TPO Abs, measuring Tg Abs is useful in establishing the presence of thyroid autoimmunity. Tg Abs are also useful in the follow-up of thyroid cancer patients since 20% of thyroid cancer patients have Tg Abs and the presence of Tg Abs in the serum interferes with the measurement of Tg levels, a marker of disease recurrence (Table 44.2) (reviewed in ).
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Table 44.2 Indications for Thyroid Autoantibody Measurement Indications for measurement of TPO antibodies • D iagnosis of autoimmune thyroid diseases (Graves disease, Hashimoto thyroiditis, subclinical hypothyroidism) • Diagnosis of PPT • As a risk factor for AITD in patients with nonthyroid tissue-specific and systemic autoimmune diseases • As a risk factor for thyroid dysfunction or hypothyroidism before and during amiodarone, lithium, interferon-alfa, or aldesleukin therapy • As a risk factor for hypothyroidism in Down syndrome patients • As a risk factor for thyroid dysfunction during pregnancy and for PPT • As a risk factor for spontaneous miscarriage Indications for measurement of Tg antibodies • D iagnosis of Hashimoto thyroiditis and PPT in patients who are negative for TPO antibodies • May be helpful in identifying patients with AITD in the presence of a goiter in iodine deficient areas • In thyroid cancer follow-up to assess disease recurrence AITD: autoimmune thyroid disease; PPT: postpartum thyroiditis; Tg: thyroglobulin; TPO: thyroid peroxidase.
Take-home messages • T PO and Tg are major antigens in AITDs, GD, and HT. • The measurement of TPO and Tg autoantibodies has clinical value, including the prediction of thyroid dysfunction in several risk groups. • TPO Ab epitopes are limited to a conformational IDR located predominantly on an MPO-like fragment comprising two closely related domains A and B. • The Tg gene is a major gene for thyroid autoimmunity, suggesting that it is the primary antigen triggering thyroid autoimmunity. • Further investigation would enhance our understanding not only of the diagnostic role of TPO and Tg antibodies in patients with AITD but also of their role in the pathogenesis of AITD.
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