Terry F. Davies
Graves' disease is a uniquely human autoimmune disease with an interesting immunopathogenesis, because unique stimulating autoantibodies to the thyrotropin (TSH) receptor are the major pathogenic feature of the disease. Unlike many autoimmune diseases, these antibodies are not just markers of the disease but are responsible for the hyperthyroidism that is the predominant feature of the disease. In most patients with Graves' disease, both B- and T-lymphocytes are directed at all three well-characterized thyroid autoantigens—thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSH receptor (TSH-R)—as well as a variety of minor antigens such as the sodium iodide transporter (NIS). However, much evidence suggests that only the TSH-R is the primary autoantigen of Graves' disease, and that the other thyroid antigens are only secondarily involved (1).
Definition of an Autoantigen
There are a number of simple rules concerning the self-molecules (autoantigens) with which T cells and autoantibodies interact. Autoantigens are present from birth and do not just appear during later development. In fact, autoantigens are highly conserved structural proteins coded for by genes with low mutation rates. Hence, autoantigens are not abnormal molecules, but they may be coded for by polymorphic genes within the population. Such polymorphisms may cause structural and functional variations, which may be important such as reported for Tg (2). However, autoimmune diseases are thought primarily to involve defects in immune surveillance pathways. The molecular recognition sites (epitopes) for autoantibodies versus T cells usually differ, and immunization of animals with the appropriate purified antigen always induces antigen-specific T- and B-cell responses against both T- and B- cell-directed epitopes.
The Nature of T Cells
The T cells that survive both intrathymic deletion and peripheral deletion are a complex mixture of cells of different phenotypes. Both CD4+ and CD8+ cells consist of many subsets; therefore, a full discussion of them is not possible here. Furthermore, many such T cells are in transition between immature and mature forms, and this may occur within an autoimmune infiltrate. However, knowledge of biologic function is probably more important than phenotype. On the whole, CD4+ cells tend to be regulatory cells (especially the CD4+/CD25+ subset) (3), and CD8+ cells tend to be cytotoxic cells capable of lysing target cells. Many T cells exert their function by the secretion of cytokines, and studies of cytokine secretion in mice have provided useful criteria to help understand the way T cells initiate and control the immune response. The CD4+ T cells have been shown to be of two principal types, T helper 1 and T helper 2 (Th1 and Th2), which differ functionally in their pattern of cytokine secretion and differ phenotypically in their pattern of chemokine receptor expression (4,5). In fact, Th1 and Th2 cells represent two polarized forms of the T-cell response, while other T cells fall outside this pattern. Th1 cells secrete principally interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-β and induce target cell cytotoxicity, while Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are particularly effective at inducing antibody secretion. In humans, this dichotomy is not as strict as in mice, and much cellular interchange has been shown. However, there is general agreement that human Th2 cells may be best defined as those that always secrete IL-4, while Th1 cells usually, but not always, secrete IFN-γ. This Th1/Th2 concept has been most helpful in understanding immunopathology.
Antibody and T-Cell Interactions with Antigen
Antibodies are immunoglobulins that may be present in the serum or expressed on the surface of B cells and are able to bind to their specific antigenic molecules directly. The strength of this binding is measured as the affinity of the antibody and is directly dependent, to a large degree, on the number of antigen-binding sites (Fig. 23A.1). Hence, the binding energy is usually greater for conformational antigens with multiple binding sites than for the binding to a small linear antigen (6). Pathogenic antibodies of high affinity are therefore most likely to interact with conformational antigens (7), and this is an important concept in understanding Graves' disease where stimulating TSH-R antibodies (TSHR-Ab) only interact appropriately with the extracellular domain of TSH-Rs. In contrast to immunoglobulins, T cells recognize the complexity of antigen and major histocompatibility antigens (HLA) (Fig. 23A.2). The T-cell antigen receptor is also a member of the immunoglobulin family, but it has a transmembrane domain that anchors it within the cell surface. The CD8+ T cells recognize antigens complexed with HLA class I molecules (A, C, and B), and CD4+ T cells recognize antigens with HLA class II molecules (DR, DP, and DQ). T cells, through their T-cell antigen receptor, recognize small linear peptides complexed with an appropriate HLA molecule and therefore are termed HLA restricted. In thyroid autoimmunity, thyroid antigens are first engulfed by antigen-presenting cells (APCs) such as macrophages and dendritic cells, then digested by proteases within these cells. Antigen breakdown products (peptides) are then bound to intracellular HLA molecules, and the complexes are transported onto the cell surface by transporters associated with antigen processing (TAPs). Thyroid cells may act as APCs (see later in the chapter).
FIGURE 23A.1. Antigen-antibody interactions. Left: An immunoglobulin (Ig)G antibody interacts with a conformational (nonlinear) determinant involving noncontiguous parts of the antigen. Right: An IgG antibody reacts with a linear (accessible) determinant; an inaccessible determinant is buried within the antigen. (From Abbas AK, Lichtman AH, Pober JS. Anonymous cellular and molecular immunology. Philadelphia: WB Saunders, 1991, adapted with permission.)
FIGURE 23A.2. Diagram of the mechanism of antigen presentation by an antigen-presenting cell to the T-cell antigen receptor of a T cell. APC, antigen-presenting cell.
Both B cells and T cells rely on secondary signals once antigen has been identified in order to enter a proliferative and secretory state (8). A variety of cytokines secreted by T cells and APCs serve as second B-cell signals. In contrast, T-cell second signals are supplied by signal transduction from cell-surface molecules (such as the B7 family and CD40) (Table 23A.1), which are activated by ligands on the surface of the APCs (CD28 and CD40L). B cells and T cells that interact with specific antigen in the absence of a second signal become desensitized, a state referred to as anergy. Hence, anergy is one of the mechanisms that may be used for suppressing an immune response (9).
TABLE 23A.1. SECOND SIGNALS FOR T-CELL ACTIVATION
B7–1 > B7–2 (CD80) (e.g., dendritic cells)
B7h (GL50, ICOS-L) (e.g., fibroblasts)
PD-1/PD-1L (e.g., hematopoietic cells)
CD40/CD40L (e.g., B cells, fibroblasts, and thyroid cells)
ICAM/LFA-1 (e.g., thyroid cells)
Criteria for an Autoimmune Disease
Three types of evidence can be marshaled to establish that a human disease is autoimmune in origin:
1. Direct evidence is provided if the disease, or manifestations of the disease, arise from transfer of pathogenic antibody or pathogenic T cells to humans or animals.
2. Indirect evidence may be based on studies in experimental animals by immunization with candidate antigens, and a disorder similar to the human disease ensues.
3. Circumstantial evidence may be obtained from clinical studies demonstrating, for example, an immune infiltrate at the site of disease or associated autoimmune diseases.
In Graves' disease, the antibody and cell-mediated thyroid antigen-specific immune responses are well defined, and there are now induced animal models of this uniquely human disease. Historically, the induction of thyroid hyperfunction by TSHR-Ab in normal subjects by the transfer of serum from patients with Graves' disease (10) was the first direct proof for an autoimmune origin of a human disease.
Restriction vs. Polyclonality
It is now possible to learn whether autoimmune reactions are multireactive and more likely to be representative of a secondary polyclonal immune response, or whether the immune response is much more focused, involving restricted sets of B cells and T cells (11). In an autoimmune disease, the immune system is where the abnormality is to be found and, therefore, the primary autoimmune response at the onset of the disease should be oligoclonal. This has, indeed, proven to be the case for most of the human autoimmune diseases, including Graves' disease, as discussed later in the chapter.
Intrathyroidal B cells
The B cells that accumulate within the thyroid gland of patients with Graves' disease have reduced proliferative responses to B-cell mitogens and greater basal immunoglobulin secretion than peripheral blood B cells, indicative of their activated state. B cells from Graves' thyroid tissue may also secrete thyroid autoantibodies spontaneously in vitro, again implying preactivation (12). Hence, the thyroid gland is a primary, but not exclusive, site of thyroid autoantibody secretion in autoimmune thyroid disease (AITD), perhaps best described by studies in mice with severe combined immunodeficiency (SCID) (Fig. 23A.3). Transplantation of Graves' thyroid tissue into T cell– and B cell–deficient mice resulted in the detection of human thyroid autoantibodies in the serum of the recipient mice (13). Additional direct evidence came from studies with animal models of thyroiditis (see later in the chapter) and, indirectly, from the decline in thyroid autoantibodies after antithyroid drug treatment, thyroidectomy, and, in the long term, radioiodine therapy (14,15,16).
FIGURE 23A.3. Production of antibodies in mice with subacute combined immunodeficiency disease after thyroid tissue engraftment or intraperitoneal injection of peripheral blood mononuclear cells (PBMC) from a patient undergoing surgery for the treatment of Graves' disease. Thyroid antibodies were measured in mouse serum 4 to 6 weeks after transplantation. In the absence of thyroid tissue and PBMC, no thyroid antibodies were detectable (upper right). In contrast, transplantation of thyroid tissue resulted in the production of thyroglobulin (Tg), thyroid peroxidase (TPo), and thyrotropin-receptor (TSH-R) antibodies (Ab) by the mice.
Autoantibodies to the Human Thyrotropin Receptor
Long-acting thyroid stimulator (LATS) was discovered by Adams and Purves almost 50 years ago during a search for thyroid-stimulating activity in the serum of patients with Graves' disease using a bioassay for pituitary TSH (17). The patients' serum stimulated radioiodine release from the prelabeled thyroid glands of guinea pigs for a much longer time period than did a pituitary TSH preparation (hence the term “long-acting”). This prolonged stimulating activity was then found to reside in the immunoglobulin (Ig)G fraction of serum. With the advent of biologically active radiolabeled TSH, it became possible to detect TSH-Rs on thyroid membranes, and subsequently this IgG activity in Graves' disease patients was found to compete with TSH for receptor occupancy, proving that it contained TSHR-Ab acting as TSH agonists (18). In patients with Graves' disease, the thyroid gland is no longer under the control of pituitary TSH but is continuously stimulated by circulating antibodies with TSH-like activity. Hence, it is not possible to put the thyroid to “sleep” using exogenous thyroxine suppression of any remaining endogenous TSH because of the presence of thyroid-stimulating antibodies.
Bioactivity of Thyrotropin-Receptor Antibodies
We now know that antibodies that bind to the TSH-R may or may not initiate an intracellular signal. Those that induce signal transduction are referred to as TSHR-stimulating, and those that do not are TSHR-blocking or TSHR-neutral antibodies (19). Further complicating this issue has been the observation of the simultaneous presence of TSHR-stimulating and TSHR-blocking antibodies in the same serum samples from patients with Graves' disease. The effective degree of thyroid stimulation under such circumstances is dependent on the relative concentration and bioactivities of the different antibodies (20). The original self-infusion of serum from patients with Graves' disease by Adams and colleagues and the resulting thyroid stimulation was the first direct evidence for the role of TSHR-Ab in the induction of hyperthyroidism in humans (10). Another early demonstration of the in vivo effects of TSHR-Ab came from studies demonstrating stimulation of the thyroid in neonates by transplacental passage of TSHR-Ab from their mothers (21).
Prevalence of Thyrotropin-Receptor Antibodies in Graves' Disease
TSHR-Ab are detectable only in patients with AITD. Such antibodies are therefore disease specific in great contrast to the high prevalence of Tg and TPO-antibodies in people with apparently normal thyroid function. Eighty to 100% of untreated patients with thyrotoxicosis caused by Graves' disease have detectable TSHR-Ab in their serum (22,23,24).The titers of TSHR-Ab may be reduced by treatment of thyrotoxicosis, and when they persist in higher concentrations they often predict a recurrence after withdrawal of antithyroid drug treatment (25,26,27,28) (Table 23A.2). TSHR-blocking antibodies may, in time, become the more prevalent antibody after treatment of thyrotoxic patients with Graves' disease, contributing to the development of later thyroid failure (29).
TABLE 23A.2. FACTORS INFLUENCING RECURRENCE OF GRAVES' DISEASE
High thyrotropin-receptor antibody concentration
Large iodine intake
Marked residual goiter
Short duration of antithyroid drug treatment
Failure to normalize low serum thyrotropin concentrations
Immunologic Characteristics of Thyrotropin-Receptor Antibodies
TSHR-Ab are oligoclonal. Patients with Graves' disease have TSHR-Ab with light chain restriction, and the TSH agonist bioactivity is found mostly in the IgG1 subclass (30). However, this evidence of a pauciclonal B-cell response may not be typical of all patients with Graves' disease. For example, it is in contrast to the variable biologic nature of the antibodies when examined in vitro, and as discussed earlier in the chapter, with many patients having stimulating and blocking antibodies at the same time.
Thyrotropin Antibody Epitopes
The cloning of the TSH-R has permitted the initiation of detailed studies of its epitopes and structure-function relationships (31,32,33). The large extracellular domain of the TSH-R is the major immunogenic region, and TSHR-Ab bind to this domain of the receptor (Fig. 23A.4). The difference in functional activity of different TSHR-Ab may relate to molecular binding characteristics dependent on conformational changes and affinity. Many studies have now indicated that high affinity–stimulating antibodies to the TSH-R are dependent on the receptor being in its normal conformation (34,35) and interact with highest affinity to the extracellular domain of the receptor alone (36). However, some TSHR-Ab recognize linear epitopes, but with low affinity, and many of these may be blocking or neutral in their activity. To date, there is suggestive evidence that stimulating antibodies bind to a restricted conformational epitope in the extracellular domain, but that blocking antibodies bind to more than one epitope. This conclusion is based on three primary conformational epitopes on the TSH-R identified using mouse monoclonal antibodies to the TSHR (37) (Fig. 23A.5). However, the location of such epitopes in the normally conformed TSH-R is uncertain.
FIGURE 23A.4. A model of the thyrotropin receptor (TSH-R). Note the α- and β-subunits of the receptor, which are disulfide linked and formed by protease cleavage of the intact molecule. The α-subunit is subsequently thought to be shed into the circulation, where it most likely contributes to the maintenance of tolerance to the TSH-R.
FIGURE 23A.5. A scheme for thyrotropin (TSH)-receptor epitopes based on monoclonal antibody binding to the wild type receptor. Three major sites are illustrated. Monoclonal antibodies to one site do not bind to any of the other sites. (From Ando T, Latif R, Davies T. First identification of three binding sites for monoclonal antibodies to the native TSH receptor. Thyroid 2003; 13:734(abst), with permission.)
Thyrotropin Receptor Regulation in Graves' Disease
Similar to TSH, TSHR-stimulating antibodies activate both cyclic adenosine monophosphate (cyclic AMP)-mediated signal transduction and the phosphoinositol cascade (38). The result is release of thyroid hormone and Tg, and stimulation of iodine uptake, protein synthesis, and thyroid cell growth. Although desensitization of the thyroidal cyclic AMP response by prolonged exposure to TSHR-Ab occurs in vitro and in vivo, this effect is highly concentration dependent. The low levels of antibody in patients therefore ensure that this does not occur to a great extent in vivo or they would not remain thyrotoxic (39,40). Resistance to desensitization by lower concentrations of TSH and of TSHR-Ab allows the hyperthyroid state to persist. In addition, although the TSH-R is not transcriptionally regulated, at lower levels of stimulation there is evidence for positive regulation of the TSH-R by TSH, perhaps on the basis of messenger RNA stabilization (41,42).
Lessons from Monoclonal Thyrotropin-Receptor Antibodies with Stimulating Activity
For many years, attempts to induce Graves' disease in animals resulted in TSHR-Ab that blocked the TSH-R. Recently, several groups have isolated high-affinity TSHR-stimulating antibodies (43,44) (Fig. 23A.6), allowing new probes to be developed for the TSH-R. Such stimulating antibodies only interact with the natural, fully conformed receptor and appear to share the same conformational epitopes. Chronic treatment of mice with stimulating TSHR-Ab, however, did not produce severe thyrotoxicosis as expected, but a relatively mild degree of thyroid overactivity secondary to desensitization of the host thyroid (45). The low levels of TSHR-Ab in Graves' disease patients (46,47) may explain why the thyroid is able to continue to oversecrete thyroid hormone for many years.
FIGURE 23A.6. In vivo thyroid stimulation by monoclonal thyroid-stimulating antibody. 20 µg of antibody (MS-1) was administered intraperitoneally, and the serum thyroid hormone response measured at the illustrated time intervals. The insets show the histologic response of the thyroid gland with thyroid cell hypertrophy and colloid droplet formation.
Other Thyroid Antibodies in Graves' Disease
The majority of patients with Graves' disease have circulating antibodies to additional thyroid autoantigens, not just the TSH-R. In particular, Tg and TPO antibodies, sometimes in high titers, are found in the majority of such patients. Since these antibodies are polyclonal in nature, and the role of TSHR-Ab is so important in the disease, the Tg and TPO antibodies appear to reflect a coincidental but controlled autoimmune thyroiditis (48). Autoantibodies to the iodide transporter have also been demonstrated in patients with Graves' disease, but are likely to be similar signs of coincidental thyroiditis (49). Hence, Graves' disease appears to develop on a background of or is coincidental with autoimmune thyroiditis. This explains why many patients not treated with destructive therapy become hypothyroid in time (29).
The Intrathyroidal Lymphocytic Infiltrate
Much of the early evidence that Graves' disease was an autoimmune disease was based on the discovery of TSHR-Ab by Adams and Kennedy (50). However, supporting evidence was in fact found much earlier within the thyroid gland itself. The thyroid in Graves' disease is characterized by a lymphocytic infiltration, that is nonhomogeneous, suggestive of differing degrees of antigenicity between follicles. Also suggestive of this explanation is thyroid follicular hyperplasia, which tends to be more extensive in areas of infiltration (51,52) (Fig. 23A.7). Antithyroid drug treatment may markedly reduce the degree of lymphocytic infiltration, which should be kept in mind when examining individual patient samples. Although the intrathyroidal lymphocyte population is mixed, immunohistologic staining and functional studies have shown that the majority of cells are T cells and that B-cell germinal centers are much less common than in chronic autoimmune thyroiditis (Hashimoto's disease) (53,54). Intra-epithelial T cells and plasma cells can be seen both adjacent to and within the thyroid follicles. Staining for cytokines has suggested the presence of both Th1 and Th2 T cells, sometimes with a predominance of Th2 (55). There is none of the follicular destruction seen in Hashimoto's disease, despite the lymphocytic infiltration (see Chapter 21). In fact, as mentioned earlier, thyroid follicular epithelial cell size has been correlated with the intensity of the local infiltrate, which suggests local thyroid cell stimulation by TSHR-Ab (52).
FIGURE 23A.7. Histologic section from a patient with Graves' disease showing mild thyroid hyperplasia and a patchy lymphocytic infiltrate, which is characteristic of the disease.
Functional Analysis of Intrathyroidal T Cells
As would be expected, activated B cells and T cells are more frequent in intrathyroidal lymphocyte cultures than peripheral blood cultures. T cells in patients with AITD are reactive to processed thyroid antigens (as peptides) (56). Such activated T cells enhance antibody (anti-Tg, anti-TPO, and TSHR-Ab) production and have helper and regulatory functions. About 10% of activated T cells infiltrating the thyroid gland in patients with AITD proliferate in response to thyroid cell antigens (57). In Graves' disease, intrathyroidal T-cell clones, when grown under appropriate conditions, are primarily Th2 with considerable T-cell helper activity (58,59) (Table 23A.3). This supports the concept that the functional role of T cells in Graves' disease is primarily a helper role rather than a suppressor or cytotoxic role.
TABLE 23A.3. CHARACTERISTICS OF INTRATHYROIDAL T-CELL CLONES
Data were expressed as % cells exhibiting autologous mixed lymphocyte reactions.
aProliferation in response to crude thyroid antigen (thyroid), or lysis of autologous thyroid cells (cytotoxic).
MLR, mixed lymphocyte reaction; ND, not done; PBMC, peripheral blood mononuclear cells.
From Mackenzie WA, Schwartz AE, Friedman EW, et al. Intrathyroidal T-cell clones from patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1987;64:818, with permission.
Apoptosis in Graves' Disease
It is now clear that deletion of T cells is achieved via apoptosis, and there are a number of pathways that signal this mechanism via T-cell surface molecules such as Fas antigen. Such signaling is initiated by binding of Fas ligand expressed, for example, by thymic epithelium; regulatory T cells, and even thyroid cells, may be involved in this pathway (see later in the chapter) (60). Autoimmune disease is a reflection of the failure to delete or anergize antigen-specific T cells, in other words, a failure to achieve tolerance of thyroid self. However, controversy has arisen over the importance of the role of thyroid cell apoptosis in autoimmune thyroiditis, and this will be dealt with elsewhere in this volume (see Chapter 47). There is, however, evidence that thyroid cells may become apoptosed even in hypertrophied Graves' thyroid glands (61), but this is a minor population of cells and may be the normal response to coincidental thyroiditis and dividing thyroid cells.
The Intrathyroidal T-Cell Receptor V Gene Repertoire
Most antigen receptors on the surface of T cells consist of two noncovalently linked chains (α and β), each with variable (V), diversity (D) (mainly β), and junctional (J) regions with common constant (Cα and Cβ) regions (62). Other T cells of less-certain function have γ/δ receptors. The V, D, and J genes code for recognition of the antigen-HLA complex by the T-cell receptor, affording antigen specificity. In addition to the many V (> 100) and J (> 50) genes present in the genome, random nucleotide (N) additions and deletions to the D region add immense complexity to the T-cell antigen receptor repertoire, causing this region, referred to as the third complementarity determining region (CDR3), to be of prime importance in antigen recognition (63). Most studies of intrathyroidal T cells have demonstrated bias in V gene utilization by T cells from within the thyroid as compared with peripheral blood from the same patient (11,64) (Fig. 23A.8). Evidence has also been sought for clonal expansion of T-cell populations within the thyroid gland of patients by direct sequencing of the CDR3 regions of the T-cell antigen receptors of intrathyroidal T cells generated by reverse transcription–polymerase chain reaction (RT-PCR). The most prominent V-gene families were indeed representative of clonally expanded T cells, based on the evidence of multiple identical sequences within the generated fragments (65). Such information once again supports the concept of pauciclonal intrathyroidal T-cells in Graves' disease and points to the importance of T-cells in disease etiology. Identical T-cell antigen receptors have also been shown to occur within the thyroid and retroorbital tissues of patients with Graves' thyroid and eye disease (64). A similar T-cell receptor bias has been observed in many other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis (Fig. 23A.8). We, and others, have suggested that highly restricted T-cell responses occur early in autoimmune disease, but that, as the pathologic process progresses, the response is less restricted secondary to “determinant spreading” and bystander activation (see later in the chapter) (66,67,68).
FIGURE 23A.8. Results of Southern blot analyses of thyroid tissue from a normal fetus, two patients with Graves' disease, and two patients with autoimmune thyroiditis. Results are shown as densitometric measurements of Vα gene fragments. Note the reduced V gene use in the tissue from the four patients as compared with the widespread use in the fetal thyroid. n, number of V-gene families detected.
Regulatory Effects of T Cells in Autoimmune Thyroid Disease
With the identification of the CD4+/CD25+ T-cell subset as a major regulatory cell population (3,69), these cells have been reported to be abnormal in some immune diseases, including experimental autoimmune thyroiditis (69). Their importance in human AITD is uncertain because other mechanisms are in place to maintain thyroid-specific tolerance. These include:
1. The secretion of inhibitory cytokines by immune cells.
2. The induction of anergized T cells because of the absence of second signals.
3. The induction of apoptosis leading to immune-cell deletion.
Positive and negative selection of T cells and B cells occurs in the thymus and in the peripheral immune system (70,71). TSH-R and Tg have been shown to be expressed by thymic epithelial cells (72). Thyroid-reactive T cells should, in principal, therefore, be removed or anergized within the thymus, and if they escape deletion at that site, they should normally be deleted by peripheral mechanisms. Anergy occurs when T cells and B cells bind antigen in the absence of second signals, as discussed earlier. The resulting anergized T cells have potent suppressive actions and may act as regulatory cells (73), but are also subject to apoptosis. Early data demonstrated that certain HLA-DR haplotypes conferred a reduced nonspecific regulatory T-cell function. For example, normal subjects with HLA-DR3 had reduced suppressor T-cell activity as compared with non-DR3 subjects (74,75), associated with reduced IL-2 secretion and impaired lymphocyte apoptosis (76,77).
IMMUNE MECHANISMS IN THE PATHOGENESIS OF GRAVES' DISEASE
Several possible explanations for the onset of all autoimmune diseases, including Graves' disease, have been hypothesized on the basis of how the immune system works (Table 23A.3). However, only recently have clear experimental data been generated in the exploration of these hypotheses. Here we review just a few of these hypotheses and the evidence for, or against, their involvement in Graves' disease.
Viral infection may lead to the virus becoming a persistent endogenous antigen, or in theory may lead to overexposure of previously sequestered or unexposed antigens. For example, transgenic mice expressing lymphocytic chorismeningitis (LCM) virus antigens in pancreatic β cells, when challenged with LCM virus, developed lymphocytic infiltrates in their β cells and then diabetes mellitus (78). Expression of retroviral protein has been reported in thyroid tissue from patients with AITD, but confirmation has not been forthcoming (see later in the chapter).
Failure of Deletion
TSH-R and Tg are expressed in the thymus and lead to central deletion of autoreactive T cells. In autoimmune diabetes (type 1), variations in the thymic expression of insulin have been related to a failure to delete insulin-reactive T cells (79). However, this was not related to the insulin itself, but rather to transcriptional regulation of its expression. Similar phenomena may play a role in other autoimmune diseases. The autoimmune regulator gene (AIRE) has been shown to be a major regulator of intrathymic gene expression (80). However, this gene has not been linked to or associated with AITD.
Specificity Crossover (Molecular Mimicry)
Structural similarity between antigens encoded by different genes can lead to crossover of specificity (81). Antigenic similarity between infectious agents and host-cell proteins is common, and in one analysis of 600 monoclonal antibodies raised against a large variety of viruses, 4% crossreacted with host determinants expressed in uninfected tissues (82). Mice infected with reovirus type 1 develop an autoimmune polyendocrinopathy and generate antibodies directed against normal pancreas, pituitary, thyroid, and gastric mucosa, which suggests antigenic similarity between a retroviral antigen and a tissue antigen expressed in multiple endocrine tissues (83). Molecular mimicry has also been reported between Yersinia enterocolitica and the TSH-R, based on the crossreaction between Y. enterocolitica and serum from patients with Graves' disease. Similar observations have been made between retroviral sequences and the TSH-R (84). However, there is no evidence that either Y. enterocolitica or retroviral infection leads to Graves' disease.
A potential mechanism for inducing thyroid autoimmunity in vivo would be a local viral infection that would induce an inflammatory reaction and stimulate production of γ-interferon and/or other cytokines by nonthyroid-specific (bystander) immune cells infiltrating the thyroid gland. This activity would have an effect on any resident thyroid-specific T cells within the gland. Such activity within the gland would also induce expression of HLA class II molecules on thyroid epithelial cells. HLA class II expression would endow the thyroid cell with the potential ability to self-present its own autoantigens in a unique way (see later in the chapter) and would induce further activation of resident thyroid-specific autoreactive T cells and development of disease. This bystander hypothesis has experimental support in NOD mice, in which pancreatic inflammation induced with Coxsackie B4 virus (85) was followed by autoimmune diabetes. Of course, this explanation assumes there are thyroid-specific T cells standing by in the gland of susceptible individuals. There is evidence of the failure to lose thyroid-specific T cells from the Graves' thyroid gland (86), but there are no data analyzing the glands of susceptible individuals before the onset of disease. We have used a murine model of experimental autoimmune thyroiditis to examine bystander effects. When we transferred Tg-specific T cells, labeled with a green fluorescent tag into naive hosts, they accumulated in the host thyroids. However, the infiltrate was not just of green cells but also of host lymphocytes, indicating they had been attracted into the gland as a result of bystander activation (87).
Thyroid Cell Expression of Histocompatibility Antigen Molecules
Major histocompatibility complex (MHC) class II molecules (HLA-DR, DP, DQ in humans) are expressed on thyroid cells from patients with AITD but not on the thyroid cells from normal subjects (Fig. 23A.9) (88). Because T-cell derived cytokines, such as γ-interferon, are well known to be able to overexpress HLA class I molecules and to induce the expression of class II molecules on thyroid epithelial cells, this thyroid cell phenomenon has been considered to be a secondary response. For example, in vivo induction of MHC class II molecules on thyroid follicular cells by γ-interferon can induce autoimmune thyroiditis in susceptible mice (89). However, viruses may also be able to directly induce the expression of class II MHC molecules independently of cytokine secretion (90). Cultured rat thyroid cells infected with reovirus types 1 and 3 expressed MHC class II antigens in a dose-dependent manner in the absence of T cells (91). Cytomegalovirus infection of primary cultures of human thyroid cells also resulted in induction of HLA-DR expression on the cells (92). This approach would fit the original hypothesis of aberrant HLA expression of Bottazzo et al. (93). In support of this hypothesis is one of the mouse models of Graves' disease (94,95). The Shimojo model is dependent on the constitutive expression of MHC class II antigens on TSH-expressing L-fibroblast cells used as immunizing antigen. Such a model provides additional evidence for this potential mechanism of thyroid antigen presentation. Nevertheless, there are currently no data to support a direct induction of thyroid cell HLA expression as the initiator of human thyroid disease.
FIGURE 23A.9. HLA-DR expression (dark immuno-peroxidase-stained areas) in thyroid tissue from a patient with Graves' disease (200× magnification).
Evidence for Thyroid Cells as Antigen-Presenting Cells
In order for many of these hypotheses to be attractive, the thyroid antigen presentation to the immune system must occur within the gland itself. All cells that express HLA molecules have the potential to present antigen directly to T cells. Efficient APCs such as dendritic cells and B cells are plentiful in the Graves' thyroid gland (96,97). However, it is also possible that the thyroid epithelial cells themselves present thyroid antigen directly (see Fig. 23A.2; Table 23A.4). Thyroid follicular cells bearing HLA class II determinants can present preprocessed viral peptide antigens to cloned human T cells (98). In addition, thyroid antigen-specific T-cell clones react specifically with cloned autologous thyroid cells in the absence of conventional antigen-presenting cells, suggesting the presence of secondary signals as well as presentation (99). The observation of CD40 cofactor expression on thyroid cells also supports this potential property of human thyroid cells (100). However, as discussed earlier, HLA antigen expression in the absence of second signal would have the opposite effect, exerting a suppressive influence on the local immune response, and there is evidence of such protection in certain circumstances (101,102).
TABLE 23A.4. SUMMARY OF EVIDENCE THAT THYROID CELLS MAY ACT AS ANTIGEN-PRESENTING CELLS
HLA-DR positive thyroid cells will stimulate an autologous mixed lymphocyte reaction with proliferation of helper T cells.
Coculture of thyroid cells and peripheral blood mononuclear cells from patients with Graves' disease leads to γ production and thyroid cell HLA-DR expression.
Human thyroid epithelial cells were able to present an influenza-specific peptide to a peptide-specific human T-cell clone a reaction that was blocked by HLA class II antibody. However, the thyroid cells were unable to process complex antigen (intact influenza virus) for presentation.
Thyroid epithelial cells were capable of phagocytosis but at a slower rate than macrophages. This function was inhibited by interleukin-1, methimazole, and dexamethasone, but enhanced by interleukin-2 and interferon-γ.
A cloned line of thyroid cells from Wistar rats was able to interact directly with cloned antigen-specific T cells in the absence of other antigen presenting cells.
Expression of CD40 antigen on thyroid epithelial cells.
HLA, histocompatibility antigens.
From Davies TF. Co-culture of human thyroid monolayer cells and autologous T cells: impact of HLA class II antigen expression. J Clin Endocrinol Metab 1985;61:418 (152); Eguchi K, Otsubo T, Kawabe K, et al. The remarkable proliferation of helper T cell subset in response to autologous thyrocytes and intrathyroidal T cells from patients with Graves' disease. Isr J Med Sci 1987;70:403 (153); Iwatani Y, Gerstein HC, Iitaka M, et al. Thyrocyte HLA-DR expression and gamma interferon production in autoimmune thyroid disease. J Clin Endocrinol Metab 1986;63:695 (154); Londei M, Lamb JR, Bottazzo GF, et al. Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells. Nature 1984;312:639; Matsunaga M. The effects of cytokines, antithyroidal drugs, and glucocorticoids on phagocytosis by thyroid cells. Acta Endocrinol (Copenh) 1988;119: 413 (155); Kimura H, Davies TF. Thyroid-specific T cells in the normal Wistar rat. II. T cell clones interact with cloned Wistar rat thyroid cells and provide direct evidence for autoantigen presentation by thyroid epithelial cells. Clin Immunol Immunopathol1991;58:195; and Metcalfe RA, McIntosh RS, Marelli-Berg F, et al. Detection of CD40 on human thyroid follicular cells: analysis of expression and function. J Clin Endocrinol Metab 1998;83:1268, with permission.
Lessons from Animal Models
Induced animal models of autoimmune disease rely on mechanisms that differ from the apparently spontaneous disease in patients, but have provided much of our basic understanding of disease mechanisms. Immunization of mice with recombinant TSH-R extracellular domain resulted in thyroid failure rather than thyroid overactivity (103). However, both Shimojo et al (94,95) and Costagliola et al (104) reported new approaches to the induction of Graves' disease in mice, the former using fibroblasts expressing the human TSH-R, as discussed earlier in the chapter, and the latter using human TSH-R complementary DNA immunizations. More recently, a highly effective immunizing agent has been shown to be an adenovirus construct incorporating the full length TSH-R (46). The immunized mice became hyperthyroid and had many, but not all, of the features of Graves' disease, including thyroid-stimulating antibodies and markedly increased serum triiodothyronine and thyroxine levels, and weight loss and hyperactivity. Their thyroids had the histological features of thyroid hyperactivity, including thyroid cell hypertrophy and colloid droplet formation—all consistent with Graves' disease (Fig. 23A.10)—and some investigators have observed retroorbital tissue involvement. However, the only model to have an intrathyroidal lymphocytic infiltrate has been a hamster model—perhaps because of their outbred nature (105). Monoclonal antibodies to the TSH-R obtained in these models were discussed earlier. These results have demonstrated that immunization with human TSH-R expressed on mammalian cells, in the presence of MHC class II antigen, induced functional TSH-R autoantibodies that stimulated the host thyroid gland. Interestingly, some animals developed gross thyroid failure rather than hyperthyroidism, and both TSH-R-stimulating and -blocking antibodies were formed, again reminiscent of human Graves' disease.
FIGURE 23A.10. A model of Graves' disease. A. Thyroid histology of a normal mouse. B. Thyroid histology of a mouse immunized with thyrotropin (TSH) receptors. (From Kita M, Ahmad L, Marians RC, et al. Regulation and transfer of a murine model of thyrotropin receptor mediated Graves' disease. Endocrinology 1999;140:1392, with permission.)
KNOWN RISK FACTORS FOR GRAVES' DISEASE
Susceptibility Genes and the Precipitation of Graves' Disease
The genetics of AITD are discussed at length in Chapter 20. Graves' disease is a complex genetic disease with several gene loci involved (Table 23A.5). However, it is important to note here that for Graves' disease to occur, the individual must be susceptible and that a large part of this susceptibility is inherited, as shown by a sibling recurrence risk (λs) of 11.6 for Graves' disease (106,107). Presumably, this inherited susceptibility is expressed via the presence of T cells and B cells, which induce AITD under appropriately abnormal conditions secondary to molecular mimicry or bystander stimulation. Only some of the factors likely to precipitate Graves' disease are known; these include infection, thyroid autoantibodies, trauma and injury, stress, sex steroids, pregnancy, iodine, and irradiation (Table 23A.5).
TABLE 23A.5. POTENTIAL PATHOGENIC MECHANISMS IN GRAVES' DISEASE
Self-antigen expression of a viral antigen or a previously hidden antigen
Specificity crossover between self-antigens or an infectious agent
Indirect or direct induction of thyroid cell HLA class II antigens
Pregnancy (microchimerism, the postpartum period)
The X chromosome
HLA, histocompatibility antigens.
For infection to be defined as the cause of Graves' disease, an identifiable agent should, in theory, be present in the majority of patients and transfer of the agent should transfer the disease. As discussed earlier, some data have directly and indirectly implicated infectious agents in the possible immune mechanisms involved in the pathogenesis of Graves' disease (108). The disease has been associated with a variety of infectious agents (e.g., Y. enterocolitica; 109,110,111), but there is no evidence that such infections lead directly to it. Infection of the thyroid gland itself (e.g., subacute thyroiditis, congenital rubella) is associated with thyroid autoimmune phenomena, including thyroid autoantibody secretion and the development of AITD (for review, see reference 108), and, therefore, AITD may be the common endpoint of many different infections working via their bystander effect (see earlier in the chapter). However, the causative role of infectious agents in Graves' disease is unproven. Autoimmune thyroiditis can be induced in experimental animals by certain viral infections, but Graves' disease does not ensue. Reports of retroviral sequences in the thyroid glands of patients with Graves' disease have not been substantiated (112,113,114,115,116).
The Whickham survey in the northeast of England followed a community for 20 years and confirmed that the presence of ant-TPO and anti-Tg antibodies more than doubled the risk of developing clinical AITD (117). There are also data that produce the propensity to produce thyroid autoantibodies is an autosomal dominant trait (118), and recent data have linked this propensity to the CTLA-4 gene, which codes for a modulator of the second signal for T-cell activation (119). Lastly, there is a large literature on the presence of TSHR-Ab as a predictor of recurrence of Graves' disease (27).
This may take the form of a direct insult to the thyroid by an infection or another external influence, including trauma (108,120). The mechanism for such initiation is most likely to be the activation and attraction of host immune cells and a potent bystander effect on thyroid-specific T cells. Clinical examples of this effect have been most impressive in pretibial myxedema in which recurrence is common after plastic surgery or injury (121).
The second case of thyrotoxicosis originally described by Parry in 1825 (122) was a 21-year-old woman who became symptomatic after she had fallen down the stairs in a wheelchair. Since that time, a major stress has often been associated with the onset of Graves' disease, including data on the high incidence of thyrotoxicosis among refugees from German concentration camps (123). Both acute and chronic stress induces an overall state of immune suppression by nonantigen-specific mechanisms, perhaps secondary to the effects of cortisol and corticotropin-releasing hormone at the level of the immune cell. In several studies, more patients with Graves' disease had a history of major stresses than control subjects (124,125,126), and women may be more susceptible to such stress (127). Acute stress-induced immune suppression may be followed by immune system hyperactivity, which could precipitate AITD, as in the postpartum period (see later in the chapter).
Far more women than men have Graves' disease (~ 10:1), and some evidence suggests that sex steroids may contribute to this difference in susceptibility. Evidence in favor of such an influence includes the presentation of disease onset at the time of the menopause and the fact that Graves' disease is uncommon before puberty. Although estrogen may influence the immune system, particularly the B-cell repertoire, this influence of estrogen has been one of B-cell suppression rather than stimulation (128). Progesterone, however, favors the production of Th2-type cytokines, which would result in enhanced autoantibody production, an important component in the development of Graves' disease (129). In animals, androgen protected against estrogen enhanced thyroiditis after Tg immunization. These results provide evidence for an influence of sex steroids on the development of AITD. It remains unclear how important their influence is (130).
The X Chromosome
Women have two X chromosomes, thus potentially receiving a double dose of any susceptibility genes. Evidence suggesting a possible susceptibility gene locus on the X chromosome (131,132) has not been substantiated in larger studies (133). Interest in the role of X-chromosome inactivation in the etiology of autoimmune disease is also relevant to this discussion (134). Since women may use different X chromosomes in different tissues, it is possible that an immune response may occur from undeleted T cells interacting with antigens from a different X chromosome to the one they were tolerant to.
The Postpartum Period
The onset, or recurrence, of Graves' disease in the postpartum period offers a unique opportunity to identify those factors that herald its onset. Unfortunately, only data relating to autoantibodies are available, and the role of antigen-specific T cells has not been explored. Graves' disease sometimes appears to be aggravated in early pregnancy (135), but most patients show a falling level of TSHR-Ab at this time with a decreasing need for antithyroid drugs, in just the same way as TPO and Tg antibodies fall (135,136). This decline in TSHR-Ab is clinically important because of the potential transfer of stimulating TSHR-Ab to the fetus (21,137). When the antibody level falls insufficiently, the fetus may be born with transient neonatal thyrotoxicosis. In addition, if the mother transfers TSHR-blocking antibodies, then transient neonatal hypothyroidism may ensue (20,138). Following delivery, and the end of the placental-driven immunosuppression, there is usually a rebound increase in the quantity of thyroid autoantibodies, including TSHR-Ab, in the serum of the mother with a peak 3 to 6 months later. The onset of new or recurrent thyrotoxicosis correlates with this rise in TSHR-Ab. Hence, the recurrence, or the new onset, of Graves' disease is seen with a similar timing as for postpartum thyroiditis and is caused by the rising levels of TSHR-Ab.
During pregnancy, both T-cell and B-cell function is diminished, and the rebound from this immunosuppression is thought to contribute to the development of the postpartum thyroid syndromes. Fetal progenitor cells persist in some maternal circulations for 20 or more years after childbirth (139,140). Although it appears that this observation is not true after all pregnancies, as documented so well in experimental animals (141), when it does occur the maternal T cells may or may not have become tolerant to paternal alloantigens. Tolerance certainly appears to have occurred during a successful pregnancy but, presumably, may also be lost in the postpartum period in some women (142). However, the more different the paternal HLA class II genes (DR and DQ) when compared with the mother, and, therefore, the more different the haplotype of the fetus, the stronger the immune suppression in the resulting pregnancy. This causes a greater relief from autoimmune diseases, such as rheumatoid arthritis, during pregnancy (143). Could microchimerism explain these HLA data and contribute to the rebound and to the female preponderance in autoimmune disease, including that of the thyroid gland? Recent data from a number of groups have confirmed the presence of intrathyroidal microchimerism in patients and animals with AITD (144,145) (Fig. 23A.11). Their persistence in the postpartum period, when the immune suppression of pregnancy is no longer active, may cause them to be a major thyroid autoimmune stimulant unless a permanent state of tolerance is induced. Failure to develop such tolerance may have serious consequences. Support for this hypothesis comes from evidence that in some women these fetal cells may initiate an intradermal “graft versus host” reaction, leading to scleroderma and long-standing immune stimulation (146).
FIGURE 23A.11. Intrathyroidal fetal microchimerism in the gland of a mouse with experimental thyroiditis. By following the mating of a fluorescent green mouse with a normal female, it was possible to detect green fluorescent male fetal cells in the thyroid tissue of the pregnant female mouse who had been previously immunized with thyroglobulin and developed experimental autoimmune thyroiditis. Such observations confirmed the presence of not just male DNA within the thyroid gland but intact fetal cells, which may have pluripotential properties. Only mice with thyroiditis had significant accumulation of fetal cells within their thyroid glands. (From Ando T, Imaizumi M, Graves PN, et al. Intrathyroidal fetal microchimerism in Graves' disease. J Clin Endocrinol Metab 2002;87:3315–3320, with permission.)
As well as being a substrate for the production of thyroid hormones, there is evidence that iodine can act as an immune stimulant precipitating the onset of AITD and may also be directly toxic to the thyroid epithelial cell. There is strong support for increased iodination of Tg being involved in the onset of autoimmune thyroiditis (147), and polymorphisms in the Tg gene have been associated with AITD. Similarly, iodine-induced thyrotoxicosis becomes common when iodine-deficient individuals are supplemented with dietary iodide (148). This takes the form of either toxic nodular goiter or Graves' disease. In addition, patients with Graves' thyrotoxicosis become more difficult to control with antithyroid drugs if their dietary iodine intake is too high, probably because iodine increases the stores of preformed hormone. In areas of iodine sufficiency, dietary iodine probably has little influence on the onset of Graves' disease. Nevertheless, iodine-containing drugs, such as amiodarone, frequently precipitate Graves' disease in susceptible patients, particularly in iodine-deficient areas (149), while hypothyroidism is more common in iodine-replete parts of the world. This is due to the onset of either a direct toxic action on the thyroid cells or the precipitation of hypothyroidism in patients with autoimmune thyroiditis.
Irradiation has differential effects on T-cell subsets, setting the stage for immune dysregulation. The most impressive data for irradiation-induced thyroid autoimmunity has come from the areas downwind of the Chernobyl nuclear accident, where many of the children developed thyroid antibodies when compared with unexposed populations (150). However, there are no data yet on the precipitation of Graves' disease in these populations. Radioiodine treatment has been reported to exacerbate or precipitate ophthalmic Graves' disease, most likely by the same effects on different T-cell subsets in a susceptible population (151).
The intrathyroidal lymphocytic infiltrate is the initial abnormality in AITD and can be correlated with the levels of thyroid autoantibodies. In susceptible persons, mostly women, thyroid-specific T cells and B cells capable of developing Graves' disease become activated (152,153,154,155). The two major theories of autoimmune disease etiology at this time are molecular mimicry and bystander effects. Molecular mimicry relies on crossover specificity of an immune response (perhaps an extrathyroidal infection), with an intrathyroidal antigen leading to the presence of activated T cells in the gland. In the bystander hypothesis, an intrathyroidal insult such as a virus would coincidentally activate T cells present in the gland. These activated T cells would then induce the expression of HLA class I and class II antigens by the thyroid epithelial cells, which, together with their second signal potential and that of other APCs present, would provide an efficient means of clonally expanding locally infiltrated thyroid antigen-specific cells capable of initiating disease. Once activated, the thyroid-specific T cells would induce B-cell proliferation and secretion of TSHR-Ab, and hyperthyroidism would ensue.
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Editors: Braverman, Lewis E.; Utiger, Robert D.