Werner & Ingbar's The Thyroid: A Fundamental & Clinical Text, 9th Edition

15.Thyroid-Directed Antibodies

Claudio Marcocci

Michele Marino

Humoral and cellular immune responses are involved in the two main types of autoimmune thyroid disease (AITD), Graves' disease and chronic autoimmune thyroiditis. The humoral immune response is dominant in Graves' disease, as indicated by the fact that thyrotoxicosis in patients with the disease is caused by the action of antibodies that activate the thyrotropin receptor (TSHR). In contrast, the cellular immune response is dominant in chronic autoimmune thyroiditis. Although nearly all patients with chronic autoimmune thyroiditis have high serum concentrations of antibodies against several thyroid antigens, for the most part the disorder appears to be the consequence of tissue damage initiated by T cells (1). Whatever the pathophysiologic role of antibodies, in clinical practice measurements of serum antibodies against thyroid antigens are useful for the evaluation of patients suspected of having either Graves' thyrotoxicosis or chronic autoimmune thyroiditis (see section on Pathogenesis of Graves' Disease in Chapter 23 and Chapter 47).

The major thyroid antigens are thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSHR (Table 15.1). Other thyroid antigens include the thyroid hormones thyroxine (T4) and triiodothyronine (T3), as components of Tg; the sodium iodide symporter (NIS); megalin (gp330); uncharacterized membrane antigens other than TPO and TSHR; and antigens cloned from the complementary DNA (cDNA) of human thyroid libraries.

TABLE 15.1. THYROID ANTIGEN ANTIBODY SYSTEMS AND INDICATIONS FOR MEASUREMENTS OF SERUM ANTITHYROID ANTIBODIES


Antigen

Antibody

indication for Measurement


Tg

Anti-Tg antibodies

Diagnosis of autoimmune thyroid disease in anti-TPO-negative cases when serum Tg is measured

TPO

Anti-TPO antiboides

Diagnosis of chronic autoimmune thyroiditis

 TSHR antibodies

TBII

Diagnois of Graves' thyrotoxicosis and euthyroid ophthalmopathy

TSHR

Third trimestre of pregnancy in women with Graves' disease or chronic autoimmune thyroiditis

 TSHR-SAb

TSHR-BAb

 Tg+TPO

Tg-TPOAb

CA2

CA2-Ab

Megalin

Megalin-Ab

 Thyroid hormones

Anti-T3 and anti-T4antibodies

 NIS

NIS-Ab

 


Ab, antibody; CA2, second colloid antigen; NIS, sodium iodide symporter; Tg, thyroglobulin; TPO, thyroid peroxidase; TSH, thyrotropin; TSHR, TSH receptor; TSHR-BAb, TSH-blocking antibodies; TSHR-SAb, thyroid-stimulating antibodies.

ANTITHYROGLOBULIN ANTIBODIES

Structure and Forms of Thyroglobulin

Thyroid follicles, the functional units of the thyroid, are composed of a single layer of epithelial cells (thyrocytes) surrounding a lumen that contains a viscous substance known as colloid. Thyroglobulin, both the precursor and storage form of thyroid hormone, is the major protein component of colloid (see Chapter 5) (2). In its major, mature form, Tg is a large 660 kDa dimeric glycoprotein composed of two identical 330 kDa monomers (2).

Iodination of tyrosyl residues of Tg and coupling of iodotyrosyl residues within Tg at the cell-colloid interface result in the formation of T4 and T3 within the Tg molecule, a process catalyzed by TPO (2). The hormonal content of Tg depends on iodide availability, and, on average, a molecule of human Tg contains 2.3 molecules of T4 and 0.3 molecule of T3 (2). Thyroglobulin molecules within the colloid are heterogeneous in terms of hormone content, extent of glycosylation, and size (some are present as monomers, and others as polymers or proteolytic fragments) (2). Newly secreted Tg molecules adjacent to the apical surface of thyrocytes are readily available for reabsorption into thyrocytes and proteolysis, with release of T4 and T3 (3). Those Tg molecules that are not reabsorbed are stored in the colloid (4).

Thyroglobulin Recognition by Antibodies

The immune properties of Tg depend, at least in part, on its hormone content (5). Thus, formation of T4 and T3 within Tg changes its conformation so that certain epitopes are masked and others are unmasked (5,6). As a consequence, the binding ability of antibodies is variably affected by the hormonal content of Tg (5). Anti-Tg antibodies found in the serum of normal subjects usually recognize only highly conserved epitopes located in the hormone-containing regions of Tg, whereas among patients with AITD and mice with experimental thyroiditis the anti-Tg antibodies are less restricted (5,6,7,8). This implies some sort of epitopes spreading. In addition, the epitopes recognized by anti-Tg antibodies from patients with chronic autoimmune thyroiditis, Graves' disease, and differentiated thyroid carcinoma differ in some respects (5). In general, anti-Tg antibodies recognize conformational epitopes preferentially, and linear epitopes are less important (5), indicating that the immunogenic potential of intact Tg is greater than that of fragments of Tg that have lost the ability to form conformational epitopes.

Importance of the Presence of Thyroglobulin in the Circulation

Thyroglobulin is uniquely produced by thyroid cells, and most of it is stored within thyroid follicles, but small amounts are present in the circulation (9). In this regard, exposure of the immune system to Tg may be responsible for T-cell reactivity or antibody production. However, this does not seem to be crucial for the production of anti-Tg antibodies, because many patients with high serum Tg concentrations have no circulating anti-Tg antibodies (10,11). On the other hand, massive release of Tg after destruction of the thyroid can be followed by the appearance of anti-Tg antibodies in the serum (11). Indeed, exposure of the immune system to thyroid antigens is probably necessary for maintaining an autoimmune response. Thus, in patients with thyroid carcinoma, both anti-Tg antibodies and anti-TPO antibodies disappear from the circulation after the complete elimination of thyroid tissue by thyroidectomy and radioiodine treatment (12).

Thyroglobulin can reach the circulation by several mechanisms (9). One is transepithelial transport (transcytosis), by which Tg is internalized, in conjunction with the endocytic receptor megalin, at the apical surface of thyrocytes and transported through the thyrocyte to be released into the extracellular space (13). This is the main mechanism of Tg secretion under conditions in which thyroid follicular structure is intact (9,13). Another mechanism is leakage of Tg because of disruption of the thyroid follicles, as it occurs in destructive processes (14). Finally, Tg may be directly secreted by thyrocytes immediately after synthesis; this seems to be the cause of high serum Tg concentrations in thyroid carcinoma (13). Whether the mechanism of Tg release affects anti-Tg antibody production is not known.

Importance of Antithyroglobulin Antibodies in the Pathogenesis of Autoimmune Thyroid Disease

Most anti-Tg antibodies are IgG antibodies. They may be of any subclass, but in patients with AITD they are predominantly IgG 4 and IgG 2 (15). IgA and IgM anti-Tg antibodies can also be found (15). Anti-Tg antibodies do not fix complement, probably because the immunodominant epitopes of Tg are widely spaced in the Tg molecule, thereby preventing IgG cross-linking (16). The lack of defined biological actions of anti-Tg antibodies implies that they have no pathogenic role. This is supported by the presence of high serum anti-Tg antibody concentrations in some otherwise healthy subjects (Fig. 15.1) (10) and in patients with monoclonal gammopathy (17), as well as the lack of correlation between serum anti-Tg antibody concentrations and disease activity in patients with chronic autoimmune thyroiditis and mice with experimental autoimmune thyroiditis (10,11,18).

FIGURE 15.1. Frequency of high serum anti-Tg and anti-TPO antibody concentrations in patients with autoimmune thyroidi tis (AT), Graves~ disease (GD), differentiated thyroid cancer (DTC), and nodular goiter (NG), and in normal subjects (N). (From Mariotti S, Pisani S, Russova A, et al. A new solid-phase immuno radiometric assay for anti-thyroglobulin autoantibody. J Endo crinol Invest 1982;5:227; and Mariotti S, Caturegli P, Piccolo P, et al. Antithyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab 1990;71:661, with modifications.)

There is, however, some evidence for a role of anti-Tg antibodies in chronic autoimmune thyroiditis. Serum anti-Tg antibodies are oligoclonal in patients with chronic autoimmune thyroiditis, whereas they are polyclonal in normal subjects (15,19). There is deposition of Tg–anti-Tg immune complexes in kidney basal membranes in some patients with glomerulonephritis (20). Pregnant women who have high serum anti-Tg antibody concentrations are more likely to have postpartum thyroiditis than pregnant women who have normal serum anti-Tg antibody concentrations (21). Finally, passive transfer of anti-Tg antibodies can result in autoimmune thyroiditis in animals (22).

Serum Antithyroglobulin Antibody Concentrations in Various Conditions

Anti-Tg antibodies have been measured in serum using several techniques (11,23). Initially, they were measured by passive agglutination, and the results were reported simply as positive or negative or as the dilution of serum giving a positive response (titer). Subsequently, much more sensitive methods, including radioimmunoassays (RIAs), immunoradiometric assays (IRMAs), enzyme-linked immu nosorbent assays (ELISAs), and chemiluminescence assays were developed. The latter methods are now used in clinical practice, and the results are usually reported quantitatively, in units per milliliter of serum.

Anti-Tg antibodies are rarely present in children without thyroid disease. Among normal adults, about 10% have high serum anti-Tg antibody concentrations, and among women age 60 years and older 15% may have high concentrations (Fig. 15.1) (10,11,24). Among patients with chronic autoimmune thyroiditis, 80% to 90% have high concentrations. The concentrations are high in 50% to 60% of patients with thyrotoxicosis caused by Graves' disease (20,21), and in an even higher percentage of patients with Graves' thyrotoxicosis who become hypothyroid after radioiodine treatment (25). They also are high in about 20% to 25% of patients with thyroid carcinoma (26,27), and in 10% to 20% of patients with nodular goiter (10,11), but are undetectable or present in low concentrations in patients with subacute thyroiditis (11). The presence of high serum anti-Tg antibody concentrations in patients with thyroid carcinoma limits the value of measurements of serum Tg as a tumor marker in these patients (see Chapter 14).

Among patients with chronic autoimmune thyroiditis, serum anti-Tg antibody concentrations are high less often than are serum anti-TPO antibody concentrations (Fig. 15.1) (10,11), and therefore serum anti-Tg antibodies should not be measured routinely in these patients. A few patients with this disorder, however, have normal serum anti-TPO antibody concentrations but high serum anti-Tg antibody concentrations (27,28).

ANTITHYROID PEROXIDASE ANTIBODIES

Structure and Functions of Thyroid Peroxidase

Thyroid peroxidase (TPO) is a poorly glycosylated membrane-bound enzyme that contains a heme group (29). The human TPO gene encodes an approximately 100 kDa protein composed of 933 amino acids (29). TPO is expressed on the apical membrane of thyrocytes, directly facing the colloid, where it catalyzes iodine oxidation and iodination of tyrosyl residues of Tg (see section on thyroid hormone synthesis in Chapter 4 (2,29).

Antibodies reacting with an antigen located in the cytoplasm and at the apical surface of thyrocytes were originally identified by immunofluorescence (29). The antigen was originally called the “microsomal antigen,” based on the apparent subcellular localization of most of the staining. Subsequent studies revealed the antigen to be TPO, and the antibodies are measured now as TPO antibodies.

Immunodominant Epitopes of Thyroid Peroxidase

Anti-TPO antibodies have an IgG subclass distribution similar to that of anti-Tg antibodies (29,30). The major immunogenic region of TPO is located near the carboxyl-terminal end of the molecule. Most human anti-TPO antibodies bind to conformational epitopes in this region of TPO, although some react with linear epitopes (30,31,32,33). There are some differences in epitope recognition between the anti-TPO antibodies found in patients with AITD and those found in occasional normal subjects, suggesting that recognition of specific epitopes may have pathogenic importance (34). Unlike anti-Tg antibodies, anti-TPO antibodies do not display a high degree of epitope spreading (30).

Importance of Antithyroid Peroxidase Antibodies in the Pathogenesis of Autoimmune Thyroid Disease

There is little evidence that anti-TPO antibodies play a role in the pathogenesis of AITD, but they are more likely to be of pathogenic importance than anti-Tg antibodies, for several reasons. Serum anti-TPO antibody concentrations are correlated with the active phase of the disease in patients with chronic autoimmune thyroiditis (29,30). More patients with chronic autoimmune thyroiditis have high serum anti-TPO antibody concentrations than high anti-Tg antibody concentrations (Fig. 15.1) (10,11,27). Anti-TPO antibodies can fix complement, and they can bind to thyrocytes and kill them in vitro; if similarly active in vivo, they could contribute to the development of hypothyroidism (35,36,37). However, in one study there was no correlation between the level of cytotoxic activity and anti-TPO antibody concentration in serum (36).

Formation of immune complexes containing thyroid-directed antibodies may be involved in cell damage (38,39,40). Deposits of immune complexes have been found along the basal membrane of thyrocytes, both in Graves' disease and chronic autoimmune thyroiditis, and the complexes contained terminal components of complement, suggesting the formation of membrane attack complexes (38). Circulating soluble immune complexes containing small amounts of Tg and anti-Tg antibodies, but not TPO and anti-TPO antibodies, have been found in patients with AITD (38). These immune complexes could activate the killer T cells that mediate antibody-dependent cell-mediated cytotoxicity (38). Passive infusion of serum from patients with chronic autoimmune thyroiditis that contained high concentrations of complement-fixing anti-TPO antibodies into monkeys did not cause thyroid injury (41).

With respect to possible anti-TPO antibody-mediated inhibition of TPO activity, infants of mothers with chronic autoimmune thyroiditis and high serum anti-TPO antibody concentrations have normal thyroid function, despite transplacental transfer of the antibodies to the infants (42). Serum containing anti-TPO antibodies inhibited the enzymatic activity of TPO in vitro in some but not other studies (43,44,45,46). However, when present, the inhibition was not dose dependent and was unrelated to the thyroid function of the donors.

Serum Antithyroid Peroxidase Antibody Assay in Various Conditions

Several methods are available to measure anti-TPO antibodies in serum (47). Most early studies were done using immunofluorescence or passive agglutination, and, as for anti-Tg antibodies, the results were reported as positive or negative or as antibody titers. These methods have been replaced by RIA, IRMA, ELISA and chemiluminescence methods, with the results being reported quantitatively (usually as units per milliliter of serum) (47).

About 10% of normal adults have high serum anti-TPO antibody concentrations, and the prevalence increases to up to 30% in the elderly (Fig. 15.1) (see Chapter 19) (24,47,48). The concentrations are high in 10% to 20% of patients with nodular goiter or thyroid carcinoma, about 75% of patients with Graves' thyrotoxicosis, and nearly all (> 90%) patients with chronic autoimmune thyroiditis (47). Serum anti-TPO antibody concentrations are not correlated with thyroid function or type of thyroid disease; the concentrations can be similarly high in patients with chronic autoimmune thyroiditis and Graves' disease (47). The concentrations decrease during antithyroid drug therapy in patients with Graves' thyrotoxicosis, but measurements are not helpful in predicting the outcome of thyrotoxicosis after withdrawal of antithyroid drug therapy (49). Serum anti-TPO antibody concentrations levels may decrease during T4 therapy in patients with chronic autoimmune thyroiditis, especially in those with atrophic thyroiditis (see Chapter 47) (50). In contrast, the concentrations rise transiently in patients with thyrotoxicosis after radioiodine therapy, probably due to massive release of thyroid antigens (51). The presence of a high serum anti-TPO antibody concentration during the first trimester of pregnancy may predict the occurrence of postpartum thyroiditis, and the concentrations are high in women with postpartum thyroiditis (see Chapter 27).

Regardless of the assay used, high serum anti-TPO antibody concentrations are more commonly found than high serum anti-Tg antibody concentrations in patients with AITD (Fig. 15.1) (10,11,27). In addition, as mentioned earlier, some patients have high serum anti-TPO antibody concentrations but normal serum anti-Tg antibody concentrations, although the opposite is rare (27,28). Therefore, measurement of serum anti-TPO antibodies is a more sensitive test of thyroid autoimmunity, and it should be the main diagnostic tool for the identification of the autoimmune origin of thyroid disease (Table 15.1).

ANTIBODIES THAT REACT WITH BOTH THYROGLOBULIN AND THYROID PEROXIDASE

Some antibodies react with both Tg and TPO. In mice, these Tg-TPO antibodies can be induced by immunization with Tg, but not with TPO (52). In a large multicenter study, high serum Tg-TPO antibody concentrations were found in approximately 40% of patients with chronic autoimmune thyroiditis, 35% of patients with Graves' disease, 15% of those with postpartum thyroiditis, and 20% of patients with thyroid carcinoma (53). The proportions were slightly higher in another, smaller study (54). These antibodies may react with other unrelated antigens besides Tg and TPO, e.g., a Tg-TPO antibody cloned from an IgG gene combinatory library constructed from B cells infiltrating the thyroid of a patient with a high serum Tg-TPO antibody concentration bound Tg, TPO, and other unrelated thyroid antigens (55).

ANTIBODIES TO THE SECOND COLLOID ANTIGEN

Antibodies that bind to a colloid antigen other than Tg, known as the second colloid antigen (CA2), have been found in the serum of patients with AITD and occasionally other thyroid disorders (56). These antibodies were detected by immunofluorescence on sections of fixed thyroid tissue. The biochemical properties and function of this antigen have not been characterized.

ANTIBODIES TO CELL SURFACE ANTIGENS OTHER THAN THYROID PEROXIDASE AND THYROTROPIN RECEPTORS

Antibodies capable of binding to antigens on the surface of thyrocytes have been detected by immunofluorescence and by mixed hemadsorption using cultured thyrocytes (57). TPO antibodies and TSHR antibodies would be expected to be detected by these techniques, as would megalin, a recently identified thyroid protein (58).

Antibodies to Megalin

Megalin, also known as gp330, is a multiligand receptor expressed in several organs (9); in the thyroid it can be found on the apical surface of thyrocytes, where it functions as a receptor for Tg and is then responsible for Tg transcytosis across thyrocytes (59). Thyroglobulin that is carried across thyrocytes by megalin enters the circulation complexed with components of megalin (13). Thus, when transcytosis is increased, the immune system may be exposed to large amounts of megalin, which may induce antibody production. Indeed, the ability of megalin to induce antibodies in rats with Heymann nephritis prompted the search for megalin antibodies in the serum of patients with AITD (58). Using flow cytometry to measure binding of serum IgG to megalin expressed on cultured cells, antibodies to megalin were detected in 50% of patients with chronic autoimmune thyroiditis and in some patients with Graves' disease and thyroid carcinoma, but not in normal subjects. These IgGs also precipitated megalin extracted from cells. The pathogenic role or clinical importance of megalin antibodies is not known.

ANTIBODIES TO THYROID HORMONES

Antibodies reacting with T4 and T3 are present in the serum of some patients with chronic autoimmune thyroiditis and Graves' disease, and rarely in patients with other thyroid diseases (60,61). The appearance of these antibodies in the serum seems to be related to massive exposure of the immune system to the hormonogenic epitopes of Tg (62,63). Anti-T4 and anti-T3 antibodies can also be found in the serum of patients with non-organ-specific autoimmune diseases, including Sjögren's syndrome and rheu matoid arthritis, suggesting that cross-reacting epitopes are present in connective tissue (61).

Anti-T4 and anti-T3 antibodies are not commonly measured in the clinical practice, and their importance is related mainly to the fact that they can interfere with measurement of T4 and T3, thereby causing either falsely high or low values, depending on the method used for measurements of serum T4 and T3 (Table 15.1) (see Chapter 13).

ANTIBODIES TO THE SODIUM IODIDE SYMPORTER

The sodium iodide symporter (NIS) is a membrane protein belonging to the superfamily of ion transporters. It is expressed on the basolateral surface of thyrocytes, where it mediates entry of iodide ions into the cells (see section of thyroid iodine transport in Chapter 4) (64). Investigations on NIS as a thyroid autoantigen stemmed from findings suggesting that serum immunoglobulins inhibited iodide uptake by thyrocytes (64). However, subsequent studies indicated that the inhibition of iodide transport was not mediated by immunoglobulins (65,66).

Antibodies against the NIS protein have been detected in serum from patients with AITD using several techniques (64). The frequency of detection of these antibodies in the serum of patients with thyroid disease has varied, from approximately 5% to 25%, depending on the criteria used to define a positive test (67); when present, their epitope specificity is limited (68). Thus, anti-NIS antibodies seem to be rather rare and of little pathophysiologic importance.

OTHER ANTIBODIES

Antibodies to Yersinia Enterocolitica

Antibodies against constituents of Yersinia enterocolitica have been implicated in the pathogenesis of Graves' disease, based on similarities between the envelope of this organism and the TSHR (69). However, anti-Yersinia antibodies can be found not only in patients with Graves' disease, but also in patients with chronic autoimmune thyroiditis (69), and therefore they are not clearly related to TSHR antibodies. In addition, there is no evidence that Graves' disease or any other AITD is more common in patients with Yersinia infection, as compared with those with no evidence of Yersinia infection (69).

Antibodies to Antigens Shared by Thyroid and Orbital Tissue

Based on the hypothesis that the pathogenesis of Graves' ophthalmopathy is related to immunologic cross-reactivity against antigenic determinants shared by thyroid and orbital tissue (70), several investigators have attempted to identify and characterize the responsible antigens and the antibodies directed against these antigens. The search has led to the identification of several possible autoimmune targets in extraocular muscle and thyroid tissue, among which at least three have been characterized: a 67 kDa protein, the flavoprotein subunit of mitochondrial succinate dehydrogenase (71); a 55 kDa protein, G2s (72); and a 63 kDa protein, calsequestrin (73). Serum antibodies against these proteins have been detected in some patients with Graves' ophthalmopathy, patients with Graves' thyrotoxicosis but no ophthalmopathy, patients with chronic autoimmune thyroiditis, and normal subjects (71,72,73). These antibodies could be markers of muscle damage in patients with ophthalmopathy, but their prevalence in patients with ophthalmopathy is relatively low (30% to 70% in various studies), it is almost as high in patients with the other disorders listed in the preceding sentence, and it is as high as 15% of normal subjects and patients with unrelated diseases.

Antibodies to Thyrotropin

Serum antibodies against thyrotropin (TSH) have been detected in a few patients (74), usually in patients who had some type of AITD and in whom the measured serum TSH concentration was not the expected value.

Thyrotropin Receptor Antibodies

TSHR antibodies have a direct pathogenic role in AITD. In patients with Graves' thyrotoxicosis, TSHR antibodies act as a TSH agonist and activate TSHRs in the same way TSH does. In a proportion of patients with chronic autoimmune thyroiditis, TSHR antibodies have a TSH antagonist action. As a result of these different properties, and variations in methods of detection, the nomenclature of TSHR antibodies is complex (Table 15.1). TSHR antibodies that are measured by their ability to inhibit the binding of TSH to TSH receptors in vitro are called TSH-binding inhibitory immunoglobulins (TBII). TSHR antibodies that are measured by their ability to stimulate cyclic adenosine monophosphate production (cyclic AMP) in cultured cells expressing TSHRs (either thyrocytes or other cell types transfected with TSHR cDNA) are called thyroid-stimulating antibodies (TSHR-SAb). TSHR antibodies that are measured by their ability to inhibit the action of TSH in cultured cells expressing TSH receptors are called thryoid-stimulating-blocking antibodies (TSHR-BAb).

THYROTROPIN RECEPTOR STRUCTURE AND FUNCTIONS

The TSH receptor is a member of the G-protein-coupled family of receptors, and, like other members of this fam ily, its structure is characterized by seven transmembrane-spanning domains linked by three sets of alternating in tra cellular and extracellular loops, a relatively large amino-terminal extracellular tail, and a carboxyl-terminal cytoplasmic tail (75,76). The human TSHR gene is located on chromosome 14, and it encodes a 744-amino-acid molecule, with a molecular weight of 84 kDa, approximately 35 kDa of which is carbohydrate. The receptor is highly homologous among different species, which allows the use of TSH receptors from species other than humans for in vitro assays of TSHR antibodies (see section on the thyrotropin recaptor in Chapter 10 and the section on pathogenesis of Graves' disease in Chapter 23) (75,76).

High-affinity TSH binding sites are located in the extracellular domain of the TSHR, whereas its transmembrane and intracellular regions are involved in signal transduction (75,76). Binding of TSH to the extracellular domain of the receptor at the basolateral surface of thyrocytes results in conformational modifications that lead to high-affinity interactions of the cytoplasmic tail with guanine nucleotide-binding (G) proteins, which ultimately triggers intracellular signaling (see section on biological actions of thyrotropin in Chapter 10) (75,76).

Thyrotropin Receptor Antibody Interactions with Thyrotropin Receptors

Proposed models of the TSHR suggest the existence of a TSH-binding pocket in the extracellular domain, which contains major conformational binding sites for both TSH and TSHR antibodies (75,76). Although the binding sites for TSHR antibodies and TSH overlap and encompass much of the extracellular domain of the receptor, they are not identical. Thus, the major functional epitopes for TSHR-SAb binding are located in the extreme amino-terminal portion of the extracellular domain of the receptor, whereas TSHR-BAb bind more to the carboxyl-terminal portion of the extracellular domain closer to the cell membrane (75,76,77,78,79,80,81). However, TSHR antibodies are heterogeneous in terms of epitope recognition, possibly due to epitope spreading during the immune response (75,76,77,78,79,80,81,82). Whether and to what extent glycosylation of the extracellular domain of the TSHR affects its recognition by antibodies is uncertain. Although glycosylation is certainly fundamental for correct folding of the receptor during biosynthesis, variations in oligosaccharide content of the receptor do not much alter the binding of TSHR-SAb from patients with Graves' thyrotoxicosis (83), but some glycosylation of the extracellular domain of the receptor is necessary for antibody recognition (84).

Shedding of the amino-terminal 310 amino-acid residues of the extracellular domain of the TSHR (the A subunit) may either initiate or amplify the immune response against the receptor, and epitopes recognized by TSHR-SAb are partially sterically hindered on the holoreceptor on the plasma membrane (85,86). Thus, TSHR-SAb from patients with Graves' thyrotoxicosis react to a greater extent with a chimeric A subunit-fusion protein, as compared with the wild type holoreceptor (both transfected in cultured cells), suggesting better recognition of the A subunit by TSHR-SAb when this portion of the receptor is not in its native conformation, as should occur after shedding (85). In addition, immunization of mice with the A subunit of the receptor results in a greater proportion of TSHR antibodies with stimulating activity, as compared with immunization with the wild type holoreceptor (86).

Biological Actions of Thyrotropin Receptor Antibodies

Thyrotropin Receptor–Stimulating Antibodies

TSHR-SAb are TSH agonists, and they are the cause of thyrotoxicosis and goiter in patients with Graves' disease. TSHR-SAb are oligoclonal or pauciclonal, based on isoelectric focusing studies, IgG1-subclass restriction, and light-chain restriction (75,76). Indeed, monoclonal TSHR-SAb have been reported to be produced by lymphocytes from patients with Graves' thyrotoxicosis (87), a human monoclonal TSHR-SAb has been purified and characterized (88), and several monoclonal TSHR-SAb have been produced in mice immunized with the TSHR or TSHR cDNA (89,90,91). TSHR-SAb that are IgG4 subclass also exist (92).

TSHR-SAb stimulate adenylyl cyclase activity, iodide uptake, TPO and Tg synthesis, and T4 and T3 release by thyrocytes, as well as growth, DNA accumulation, and mitotic activity in thyrocytes (93,94,95,96). Like TSH, TSHR-SAb exert most of their biological effects via the cyclic AMP pathway, and to a lesser extent via the phosphoinositol pathway (75,76).

Thyrotropin Receptor–Blocking Antibodies

TSHR-BAb bind to TSHRs and block the action of TSH. They contribute to the development of hypothyroidism in some patients with atrophic chronic autoimmune thyroiditis and occasional patients with goitrous chronic autoimmune thyroiditis (97). Unlike TSHR-SAb, TSHR-BAb are not subclass restricted and are therefore likely to have a polyclonal origin (98). TSHR-BAb act by blocking binding of TSH to TSHRs, thereby reducing the stimulating actions of TSH on thyroid cells, such as cyclic AMP production, iodine uptake and organification, and cell growth (75,76,99).

Serum Assays for Thyrotropin Receptor Antibodies in Various Conditions

As mentioned above, serum TSHR antibodies can be measured by two major types of assays, radioreceptor assays, by which it is not possible to distinguish whether the antibodies are stimulating or blocking antibodies, and in vitro bioassays, by which it is possible to determine the biologic activity (stimulating or blocking) of the antibodies.

Radioreceptor Assays

Radioreceptor assays measure the inhibition of binding of labeled TSH to TSHRs; as mentioned above, TSHR antibodies measured in this way are called TSHR-binding inhibitory antibodies (TBII). The assays for TBII are based on the ability or serum or the IgG fraction of serum to inhibit the binding of labeled TSH to porcine TSHRs, TSHRs purified from Chinese hamster ovary (CHO) cells transfected with recombinant human TSHR cDNA (100), or TSHRs expressed in leukemic cells transfected with human TSHR cDNA (101). With the latter assay, TBII can be detected in up to 99% of patients with untreated Graves' thyrotoxicosis, approximately 90% of patients with Graves' thyrotoxicosis treated with methimazole, 50% of patients with Graves' thyrotoxicosis who are in remission after cessation of methimazole therapy, 15% of patients with chronic autoimmune thyroiditis, and 0.5% of normal subjects (Fig. 15.2) (101). The prevalence of high values in patients with Graves' disease using the latter assay is clearly higher than that reported in studies in which older methods were used (99,102).

FIGURE 15.2. Frequency of high serum TSH-binding inhibitory immunoglobulin (TBII) activity in patients with Graves' thyrotoxicosis, autoimmune thyroiditis (AT), non-autoimmune thyroid disease (N-AITD), and in normal subjects (N). On MMI, patients studied during methimazole therapy; Eu after MMI, patients remaining euthyroid after cessation of methimazole therapy. (From Costagliola S, Morgenthaler NG, Hoer mann R, et al. Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease. J Clin Endocrinol Metab 1999;84:90, with modifications.)

In Vitro Bioassays for Thyrotropin Receptor Antibodies

In vitro bioassays for TSHR antibodies are mainly based on measurements of cyclic AMP production in rat thyrocytes (FRTL5 cells) or in CHO cells transfected with human TSHR cDNA (103,104,105). Unlike assays for TBII, these assays are not widely available.

TSHR-SAb assays measure the ability of serum or serum IgG to increase cyclic AMP production by cultured cells (103,104,105). Using these assays TSHR-SAb can be detected in the serum of about 90% of patients with Graves' thyrotoxicosis at the time of diagnosis (103,104,105). Assays using CHO cells transfected with human TSHR cDNA are more sensitive than those using FRTL5 cells, probably because of the use of human rather than rat TSHRs (104). Using transfected CHO cells, TSHR-SAb can be detected in up to 95% of patients with Graves' thyrotoxicosis at the time of diagnosis; approximately 90% of patients with Graves' thyrotoxicosis treated with methimazole, but still thyrotoxic; 60% of patients with Graves' thyrotoxicosis who are taking methimazole and are euthyroid; and 35% of patients with Graves' thyrotoxicosis who remain euthyroid after cessation of methimazole (Fig. 15.2) (104).

In general, there is a good correlation between serum TBII and TSHR-SAb values in patients with Graves' thyrotoxicosis (106). The presence of TBII activity but not TSHR-SAb activity is indicative of atrophic chronic autoimmune thyroiditis, and many of these patients have high serum TSHR-BAb activity (107).

TSHR-BAb assays measure the ability of the whole serum or of serum IgG to inhibit the TSH-stimulated increase of cyclic AMP production in cultured cells expressing

TSHRs (99,105). For TSHR-SAb assays, the cell types used are FRTL5 cells and CHO cells transfected with TSHR cDNA (99,105). In addition, primary cultures of human or porcine thyrocytes can be used. As for TSHR-SAb, the CHO cell-based system provides greater sensitivity (99). With this type of assay, TSHR-BAb can be detected in the serum of approximately 65% of patients with atrophic chronic autoimmune thyroiditis, 25% of patients with goitrous chronic autoimmune thyroiditis who have overt hypothyroidism, and 10% of patients with goitrous chronic autoimmune thyroiditis who have subclinical hypothyroidism, whereas they are undetectable in normal subjects (Fig. 15.2) (97).

Coexistence of Thyrotropin Receptor–Stimulating and–Blocking Antibodies

TSHR antibodies with stimulating and blocking activity have been found at the same or different times in some patients with AITD (107,108). These patients may have hypothyroidism, and high serum TSHR-BAb activity, at one time, and thyrotoxicosis, and high serum TSHR-SAb activity, at another time (107,108). In rare patients, most of whom have thyrotoxicosis, both activities can be detected at the same time. The coexistence of TSHR-SAb and TSHR- BAb is suggested by a biphasic dose-response curve in the TSHR-SAb assay.

Clinical Value of Measurements of Thyrotropin Receptor Antibodies

Most patients with Graves' thyrotoxicosis have positive serum tests for TBII and TSHR-SAb (Fig. 15.2 and Fig. 15.3)(99,100,101,102,103,104,105,106). Nevertheless, the diagnostic value of measurements of serum TSHR antibodies in serum is rather limited (Table 15.1). Thus, the presence of a diffuse goiter, and especially signs of Graves' ophthalmopathy, and measurements of serum TSH and free T4 are sufficient to confirm a diagnosis of Graves' thyrotoxicosis in the majority of patients.

FIGURE 15.3. Frequency of high serum TSH receptor antibody activity in patients with Graves' thyrotoxicosis (top) and chronic autoimmune thyroiditis (bottom). In the top panel, the anti bodies were measured as TSH receptor stimulating antibodies (TSHR-SAb). In the bottom panel, the antibodies were measured as TSH receptor blocking antibodies (TSHR-BAb). AT, autoimmune thyroiditis; Eu, euthyroid; Hypo, hypothyroidism; MMI, methimazole; Sub Hypo, subclinical hypothyroidism. (From Chiovato L, Vitti P, Bendinelli G, et al. Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor. J Endocrinol Invest 1994;17:809, with modifications; and Costagliola S, Morgenthaler NG, Hoermann R, et al. Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease. J Clin Endocrinol Metab 1999;84:90, with modifications.)

Serum assays for TBII may, however, be useful in certain patients, for example euthyroid patients with ophthalmopathy, patients with the nodular variant of Graves' disease, and pregnant women with either Graves' thyrotoxicosis of chronic autoimmune thyroiditis. In euthyroid patients with ophthalmopathy and in those with nodular goiter and thyrotoxicosis, the presence of TBII in the serum helps to demonstrate that the patient's ophthalmopathy or thyrotoxicosis is caused by Graves' disease (70). In pregnant women, transplacental passage of TSHR-SAb or TSHR-BAb can result in transient fetal and neonatal thyrotoxicosis or hypothyroidism (109,110,111,112); measurements of maternal serum TBII are helpful in assessing the likelihood of fetal or neonatal thyrotoxicosis in pregnant women with Graves' thyrotoxicosis (even if treated with thyroidectomy or radioiodine and taking T4) and the likelihood of neonatal hypothyroidism in pregnant women with hypothyroidism and chronic autoimmune thyroiditis (see section on congenital hypothyroidism in Chapter 75 and Chapter 76). The tests should be done in the third trimester. If the maternal serum TBII value is high, serum TSHR-SAb or TSHR-BAb can be measured. The risk of neonatal thyrotoxicosis is very low unless maternal serum TBII and TSHR-SAb activity is very high (112).

Among postpartum women with thyrotoxicosis, measurements of serum TSHR antibodies may help to distinguish between Graves' thyrotoxicosis and postpartum thyroiditis as the cause of the thyrotoxicosis (see Chapter 27) (113). Women with postpartum thyroiditis may have high serum TSHR-BAb activity during the hypothyroid phase of the illness (113), but measurements of TBII or TSHR-BAb are rarely indicated.

The occasional presence of serum TSHR-SAb in euthyroid patients with diffuse goiter or a family history of AITD can forecast the development of Graves' thyrotoxicosis (114). Similarly, occasional patients with subclinical thyrotoxicosis have high levels of serum TBII or TSHR-SAb activity, indicating that the cause of the subclinical thyrotoxicosis is Graves' disease (115). Among patients with Graves' thyrotoxicosis, serum TBII and TSHR-SAb activity decline during antithyroid drug therapy (116), and the decline is greater in patients who remain euthyroid after cessation of therapy. The risk of later recurrence of thyrotoxicosis may be lower in those patients in whom serum TBII or TSHR-SAb activity disappears, but the issue is debated (102,117,118,119,120). Overall, the presence of high levels of serum TSHR antibodies at the end of antithyroid drug therapy has a high positive predictive value (98%) and specificity (73% to 99%) for relapse of thyrotoxicosis, but a low negative predictive value (47%) and sensitivity (28% to 49%) (117,119). Whether measurements of serum TBII or TSHR-SAb activity at the start antithyroid drug therapy have predictive value is a matter of debate (102,117,120,121).

Thyroid Growth–Promoting and–Blocking Antibodies

The extent of thyroid enlargement varies substantially among patients with Graves' disease and those with chronic autoimmune thyroiditis, and years ago some investigators obtained evidence for the presence of serum antibodies that stimulated thyroid growth or inhibited the growth-stimulating action of TSH, without affecting cyclic AMP production (122). However, subsequent studies have not confirmed the existence of thyroid growth-promoting or growth-blocking antibodies (96,122).

MEASUREMENT OF THYROID-DIRECTED ANTIBODIES IN CLINICAL PRACTICE

Measurements of thyroid-directed antibodies in serum can be clinically useful in the evaluation of some patients with chronic autoimmune thyroiditis or Graves' disease (Table 15.1). Measurements of serum anti-TPO antibodies are more useful in evaluating patients suspected to have chronic autoimmune thyroiditis than are measurements of serum anti-Tg antibodies, and the latter measurement is indicated only if suspicion of the disorder is high and the patient's serum anti-TPO antibody concentration is normal. Measurements of serum anti-TPO antibodies in pregnant women are useful in estimating the risk of postpartum thyroiditis, but it is debated whether it should be performed routinely. Serum anti-Tg antibodies should be measured whenever serum Tg is measured, especially in patients with thyroid carcinoma, because serum Tg cannot be measured reliably when anti-Tg antibodies are present (see Chapter 14).

Measurements of serum TBII can help to identify, or exclude, Graves' disease as a cause of thyrotoxicosis, but other clinical parameters serve the same purpose in most instances. However, these measurements may be helpful in confirming, or excluding, the presence of Graves' disease in patients with a nodular goiter and euthyroid patients with ophthalmopathy, and for assessing the risk of fetal or neonatal thyrotoxicosis or hypothyroidism in pregnant women with Graves' disease or chronic autoimmune thyroiditis, respectively.

ACKNOWLEDGMENTS

We are grateful to Dr. Francesco Latrofa for his critical reading of the manuscript and helpful suggestions.

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