The principal autoantibodies identified in AITD and the methods for detecting them are listed in Table 7-3. Antibodies to the TSH receptor are discussed in detail in Chapter 10, but, in brief, observation of a factor in serum of patients with Graves' disease causing long acting stimulation of thyroid hormone release from an animal's thyroid, in contrast to the short acting stimulation produced by TSH, led directly to our knowledge of anti-TSH-R antibodies. We summarize here a huge amount of clinical and laboratory research. The antibodies directed to the TSH-R are currently separated into three types. Some antibodies bind to an important epitope in TSH-R and activate the receptor, producing the same effects as TSH, in particular causing generation of cyclic AMP. These antibodies may be referred to as TSI or TSAb -- thyroid stimulating immunoglobulins or thyroid stimulating antibodies. Other antibodies bind to different, or the same epitopes and interfere with radiolabelled TSH binding in certain assays -- thus they are known as thyrotrophin binding inhibitory immunoglobulins or TBII. Still others bind and prevent the action of TSH -- thus blocking antibodies. These may either interfere directly with TSH binding or have less well characterised inhibitory effects. Numerous other names are used.

TSI cause non-TSH dependent (often called "autonomous") stimulation of thyroid function, which, if of sufficient intensity, is hyperthyroidism. TBII comprise the mixture of TSI and TSH blocking antibodies, and therefore function cannot be predicted from the TBII level. Predominance of TSI characterizes Graves' disease, and TSH blocking antibodies are often present in Hashimoto's disease and primary myxedema. Probably a combination is present in most patients with AITD. Recent work indicates that both types of TSH-R antibody are present in Graves’ sera at low concentration with high affinity and similar (but nonetheless subtly distinct) binding epitopes (84a). TSI directly cause thyroid overactivity, their level correlates roughly with disease intensity, and a drop in levels correlates loosely with disease remission. The intact TSH-R epitopes recognized by B cells remain uncertain, in large part because the epitopes are probably conformational, and made up of discontinuous but continguous portions of the extracellular domain of the receptor (85). Unlike TG and TPO antibodies which are polyclonal and not restricted by immunoglobulin subclass (reviewed in 66, 86), there is evidence that some TSH-R are restricted to particular heavy and light chain subclasses, which may indicate an oligoclonal origin (87, 88), and TSH-R stimulating antibodies are present at much lower concentration than TG and TPO antibodies (85). Recent work has shown that normal subjects can have TSH-R antibodies that bind to but do not activate the TSH-R and that generally have low affinity (88a). These natural autoantibodies may be the precursors of the TSI that cause Graves’ disease and it is possible that affinity maturation, with class switching of immunoglobulin isotype, is critical in determining the clinical consequences of TSH-R antibody production.

Precipitating antibodies to TG were first detected by mixing antibody and antigen in equivalent concentrations, or by agar gel diffusion, as in the Ouchterlony plate technique. Subsequently, much more sensitive methods were developed, such as solid phase ELISA (89) and RIA (90), and the tanned red cell hemagglutination test (TGHA) (91). In the latter, TG is adsorbed on the surface of red cells that have been treated with dilute tannic acid. Agglutination of these treated cells occurs in the presence of antibody to TG. By this method, antibody can be detected at a concentration that is 1/40,000th of that required for a positive precipitin reaction. Current RIAs for TGAb are as, or more sensitive, than TGHA. Immunoradiometric assays (IRMA) used currently involve binding of serum Ab to solid phase antigen, and secondary quantitation of antibody by binding labelled monoclonal anti-human Ig antibody. These tests are also reported to be very sensitive and specific. Hemagglutination titers of up to 1 in 5 million have been obtained with sera from patients with chronic thyroiditis. A high anti-TG titer (1/1,250 or more) is strong evidence of AITD and helps to distinguish it from multinodular goiter and thyroid carcinoma. In some instances, sufficient TG is released into the circulation to form circulating antigen-antibody complexes that prevent the detection of free antibodies, unless a special technique is used (90).

Antibodies directed against TG are rarely present in children without evidence of thyroid disease. The prevalence in “well" persons increases with age, and low levels are frequently present in normal adults (91). The greatest frequency occurs in women aged 40-60 years. The frequency of antibodies in well persons correlates with the incidence of lymphocytic infiltration found on microscopic examination of "normal" thyroids (92), and antibody levels correlate well with the presence of lymphocyte infiltration in the thyroid (93). Over 90% of patients with Hashimoto's thyroiditis have these antibodies. Low to moderate titers (< 1/2500) are found in half of patients with Graves' disease. High antibody levels in this disease are often found in patients who become hypothyroid after thyroidectomy (94) and ¹³¹I treatment (95). Almost all patients with idiopathic hypothyroidism have high titers. Antibody levels are either absent or low in patients with subacute (De Quervain's) thyroiditis, who may present clinically like patients with Hashimoto's thyroiditis. In general human TG and its autoantibody bind complement weakly due to the widely scattered epitopes which are unable to allow antibody cross-linking (66).

The second important antigen-antibody system was originally recognized by antibodies which, by immunofluorescence, were observed to bind to non-denatured thyroid cytoplasm, to fix complement in the presence of human thyroid membranes ("microsomes"), or to bind to microsome-coated red cells (the MCHA assay). We now know this antigen is TPO (see previous Section 3), but will in discussions refer to it both as microsomal antigen, since this conforms to many original reports, and as TPO, the designation used in recent studies (Fig. 7-8). Almost all patients with Hashimoto's thyroiditis have TPO/microsomal antibodies. They also occur in the normal population in the absence of clinically significant thyroid disease: in a recent survey of a population followed for 20 years, 26% of adult women and 9% of adult men had microsomal and/or TG levels (91). However, the presence of such antibodies was shown to be associated with an increased risk of future hypothyroidism, especially if the TSH was also raised (subclinical hypothyroidism). Few sera from AITD contain TG antibodies in the absence of microsomal antibodies, but the converse is not true, so that screening for AITD could be undertaken initially with assays for microsomal/TPO antibodies (96). This is particularly the case if the hemagglutination assay is used for TG antibodies; sensitive RIAs may detect a very high frequency of TG antibodies in individuals with autoimmune thyroid disease, even more than TPO antibodies (97). Current assay techniques include MCHA, RIA, ELISA, and IRMA, and use of purified TPO (98) or recombinant human TPO. Although all antibodies reacting with "microsomes" may not be directed to TPO, it is likely that TPO antibodies constitute the very major portion of "antimicrosomal" activity (99). These antibodies also bind to and inhibit the enzymatic function of TPO, as shown by Okamoto et al (100). This effect is probably limited in vivo by inability of the antibodies to penetrate the thyroid and reach TPO on the surface of the cells facing the colloid space.

Antibodies detected by these techniques are believed to be similar to antibodies that fix complement in the presence of extracts from a thyrotoxic gland (101), and to a cytotoxic antibody found in patients with Hashimoto's thyroiditis (71, 102). Sera from patients with Hashimoto's thyroiditis usually have high cytotoxic activity (103). Complement-mediated sublethal injury probably occurs in vivo since complement containing complexes have been identified in thyroid tissue of patients with GD and HT (104). Thyroid cell expression of membrane proteins, especially CD59, helps prevent complement-mediated lysis (105), and this protein is upregulated by IL-1 and IFN-g.

With the sera of certain patients with Hashimoto's thyroiditis investigated by the fluorescent antibody technique, a reaction is localized to the nuclei of the thyroid slice (106). This is a coincidental antinuclear factor (antibody) and DNA antibodies are found in some AITD sera but have unknown significance (83, 107).

In early studies, TG antibodies were demonstrated by the passive cutaneous anaphylaxis technique and by a skin test in which an extract of human thyroid gland was injected intradermally (108). Positive skin reactions were found in patients with Hashimoto's thyroiditis and primary myxedema, and these conditions were closely correlated with the presence of circulating thyroid precipitins. The tissue change at the reaction site was that of an Arthus response. This type of study obviously carries a risk of viral transmission, and thus is not now acceptable (or necessary). The frequency of autoantibodies detected by the TGHA and MCHA tests combined in Hashimoto's thyroiditis was 95%: the figures in diffuse toxic goiter (Graves’ disease) and nontoxic nodular goiter were 87% and 25%, respectively (109). The highest antibody titers are found in Hashimoto's thyroiditis and diffuse toxic goiter.

Sera from some patients can contain antibodies that have T ₄and T ₃binding activity (81,110). This activity represents another antibody response to the TG antigen. The antibodies do not alter thyroid function significantly, but can cause confusion in diagnosis due to artifacts in the T ₄and T ₃RIA.

The cytotoxicity of circulating antibodies has also been explored using systems to detect antibody-dependent cell-mediated cytotoxicity (ADCC) in which nonimmunized lymphocytes (NK cells) or macrophages act as effector cells and kill antigen-coated target cells, following incubation of the targets with antibody (111, 112). This reaction does not require complement, instead depending on the interaction of antibody on the target cell with Fc receptors on the effector cells. The exact role of ADCC in the pathogenesis of autoimmune thyroid disease is unclear, as it has been investigated only as an in vitro phenomenon. Antibodies capable of mediating ADCC on target cells include those against TG and TPO, but other antigens may also be targets, and sera from patients with Hashimoto’s thyroiditis, primary myxedema and Graves’ disease cause ADCC, although the frequency is lower in Graves’ disease (86, 113).

Antibody titers for all types of autoantibody obviously increase during the process of development of AITD, but this is not clearly documented. It is possible that one critical step in the production of TG autoimmune responsiveness is the generation of immunoreactive C-terminal fragments during hormone synthesis (which results in oxidative stress); these fragments may also lead to preferential presentation of TG epitopes by thyroid cells (114). After first observation, they tend to be stable over months. Recent studies have shown that so-called natural autoantibodies against TG may be more important in pathogenesis than previously thought. These low affinity, mainly IgM antibodies, which are frequent in healthy individuals, can complex TG with complement and such opsonized complexes can be taken up by B cells and presented to CD4 ⁺T cells, most likely in some regulatory fashion (115).

Radioactive iodine therapy leads to a rise in thyroid antibody levels in general in Graves' disease (116), and acute viral infections, or exposure to high levels of IL-2 (117) or IFN-a (118) also does so. With treatment of Graves' disease, or replacement therapy in Hashimoto's thyroiditis or myxedema, there is characteristically a gradual reduction in antibody levels over months or years, and some patients with total destruction of thyroid tissue eventually lose detectable antibody titers.

Information is beginning to emerge on the specific B cell epitopes for TG and TPO. There are two major conformational epitopes on the TG molecule that are recognized differentially by sera from healthy subjects and those with AITD; linear epitopes are recognized by polyclonal antibodies from healthy individuals (119 - 121). Similar studies on TPO have indicated at least eight major domains for human autoantibodies which are probably conformational epitopes. Using recombinant proteins and synthetic peptides, human anti-TPO antibodies are found to recognize apparently linear epitopes in the area of amino acids 590-622 and 710-722 (122) but, again, the important epitopes are most likely to be conformational (57, 66). A full 3-dimensional model will be required to identify TPO epitopes fully (123).

Peripheral blood mononuclear cells (PBMC) and thyroid lymphocytes from patients with AITD have among them activated cells that spontaneously secrete anti-TG and anti-McAg/TPO antibodies (124, 125). B cell production of antibodies to McAg/TPO and TG is most easily shown using cells incubated with mitogens. Specific antibody secretion in response to PBMC stimulation by TG or purified TPO is more difficult to demonstrate (125, 126). In patients with AITD, approximately 50 B cells secreting anti-TG antibodies are found per 10 ₆PBMC (~2% of total Ig secreting cells) by using plaque-forming assays after stimulation of PBMC with pokeweed mitogen. B cells from AITD patients synthesize antibodies in response to insolubilized TG bound to Sepharose (127), which appears to function as an especially good antigen. There are reports of production of anti-TSH-R antibodies in vitro, but in general this response has been difficult to observe.

During initiation of AITD, thyroid autoantibody formation presumably occurs in lymph nodes draining the thyroid. In fully developed AITD, the thyroid is clearly an important source of autoantibody. In fact, since there are relatively few circulating specific autoantibody-secreting B cells (128), it has been suggested that autoantibody formation occurs mainly or uniquely in the thyroid, where spontaneous autoantibody secretion by B cells are more easily demonstrated (129). This is supported by the histopathological features, including the demonstration of thyroid antigen-specific B cells and the occurrence of secondary immunoglobulin gene rearrangement in intrathyroidal lymphoid follicles, together with a congruent pattern of adhesion molecule and chemokine expression (130). However, lymph nodes, bone marrow and possibly other organs also contribute to autoantibody production (131) and this explains why patients with apparently destroyed thyroid tissue, or those with resected thyroids, continue to have circulating thyroid auto-antibodies

Techniques for identification of T lymphocyte reactivity to foreign or autologous antigens depend on culturing mixed peripheral leukocytes or semipurified thyroid or blood lymphocytes with an antigen to which the cells may have been pre-sensitized. Upon re-exposure to antigen, the sensitized cells change to a blast-like immature form, synthesize new protein, RNA, and DNA, and directly or through liberated effector molecules alter the function of target cells. Different endpoints characterize the various assays, including measurement of [ ³H]-thymidine uptake, assay of migration inhibition factor (MIF), or leukocyte migration inhibition (LMI) (132), assessment of the mobility of lymphocytes, and cytokine assay, all after stimulation with antigen in culture.

Numerous reports have shown that T cell immunity can be detected in Graves' disease, Hashimoto’s thyroiditis, and primary myxedema, although responsivity of T cells to thyroid antigens is much less than to exogenous antigens such as tetanus toxoid or tuberculin. PBMC, or thyroid T cells plus B cells and APCs, respond to TG and microsomal antigen with production of antibodies, although the response is usually weak and not present in many patients (127, 128). Peripheral blood T cells respond to incubation with TG or microsomal antigen by thymidine incorporation, the so-called proliferation assay (133, 134). Responses by separated lymphocytes are generally weak; better responses are seen by adding IL-2 to thyroid antigen-stimulated cultures of diluted whole blood (135). T cells secrete lymphotoxin in response to incubation with thyroid particulate membrane material, which presumably includes microsomal antigen (136). Thyroid T cells responding to TG are of the CD4 ⁺T helper type (137), or occasionally CD8 ⁺cells (138). T cells also respond to crude thyroid antigen, possibly "microsomal antigen", in LMI (132, 139). T cell lines and short term T cell clones (CD4 ⁺) are stimulated during coculture with TECs to incorporate [ ³H]-thymidine; DR+ TECs are especially effective stimulators (140 - 142). The identity of the antigen recognized on TECs is unknown but may well be TPO and/or TG.

The specific peptide epitope fragments of TPO recognized by lymphocytes of patients with HT were noted previously. T cell epitopes present within the extracellular domain of the TSH-R are also heterogeneous with peptides bearing sequences of aa 158-176, 237-252, and 248-263 and 343-362 being especially important (143) but other epitopes (aa 57-71, 142-161, 202-221, 247-266) have been identified by others using different assay parameters (144). HLA-DR3 molecules bind TSH-R peptides with high affinity, which may explain the genetic association of this HLA specificity with Graves’ disease (145).

T cell responses to an antigenic stimulus may use a wide variety of variable (V) TCR gene segments, or the response may involve (be "restricted" to) a few V segments. Restriction of autoreactive T cells to use of one or more V gene segments has been found in some experimental autoimmune models (4). Restricted Va and Vb usage has been reported by Davies and co-workers (146, 147) but not found by others in the whole intrathyroidal lymphocyte population (148, 149). However, CD8 ⁺do display a degree of restriction although their autoreactive potential is at present not known (150). Presumably at an early stage of disease, the T cell response is clonally restricted, but as it advances, spreading of the immune response occurs, involving many more epitopes, leading to an unrestricted response as demonstrated in an animal model of AITD (151).

While T cell immunization is conventionally recognized by a stimulatory effect of antigen, direct T cell cytotoxicity of thyroid cells has been recognized in a few studies. For example, Davies and co-workers developed a CD8 ⁺T cell clone which was cytotoxic to autologous TEC and was appropriately class I restricted (152). An interesting potential consequence of T lymphocytic adherence to thyroid cells, via ICAM-1/LFA-3 interaction, is the stimulation of thyroid cell proliferation, which could lead to goiter formation (153).

In addition to the antibody and T cell responses, circulating immune complexes are found in patients with autoimmune thyroid disease as would be anticipated], although their pathogenic importance appears minimal. In a certain sense this is most fortuitous. Since many individuals have circulating TG antibodies and antigen, if the immune complexes caused serious disease, it would be a plague. Fortunately the immune complexes of TG and its antibody do not bind complement weakly and do not cause serious illness such as immune-complex nephritis, except in rare instances (154, 155). Immune complexes, including complement terminal components, can be recognized around the basement membrane of thyroid follicular cells (104) and may cause sublethal effects including release of proinflammatory mediators by TECs (156). Release of TG into the circulation can cause formation of immune complexes which are rapidly removed from the circulation, and the process could lead to depletion of circulating antibody levels. It is possible that this antigen-dependent antibody depletion contributes to the lower levels of anti-TG antibody found in Graves' disease, compared to Hashimoto's thyroiditis, since the thyroid of Graves' disease releases more TG than that of Hashimoto's thyroiditis.

Many studies have been reported on natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC); their conclusions vary. Endo et al (162) found NK cells were decreased in Graves' disease and Hashimoto's thyroiditis, and presented evidence that this was due to saturation of their Fc receptors by immune complexes. Normal NK effector function was found in Hashimoto's thyroiditis PBMC (158) in one study, although by phenotyping, decreased NK cells in Graves' disease, and increased NK cells in Hashimoto's thyroiditis were reported in another (159). ADCC of thyroid cells, mediated by normal PBMC, was induced by anti-McAg/TPO antibody positive sera (160) but other, unknown antibody-antigen systems also contribute (113). Effector cell activity in ADCC was increased in Hashimoto's thyroiditis and in post-partum thyroiditis, and thought to be related to thyroid cell destruction (161). Other data have indicted that ADCC may be more important in primary myxedema than Hashimoto’s thyroiditis explaining the difference in clinical presentation (162), but this has not been confirmed in two other studies showing equal ADCC activity in sera from both diseases (113, 163).

Cytokines lie at the heart of the autoimmune response and can have a number of direct and indirect effects (Fig. 7-9). For example, IFN-g is produced in the thyroid by infiltrating lymphocytes and causes HLA class I expression on the surface of TECs to increase and initiates class II expression. It also has a direct inhibitory function on TEC iodination and TG synthesis (164, 165). These effects are mediated by multiple, temporally distinct mechanisms, at least in part acting via effects on cAMP response element-regulated gene expression (166). IFN-g is not essential for the development of AITD in mice but exacerbates disease activity (167). IL-2 can activate lymphocytes to produce IFN-g, and activate NK cells. TNF is produced by infiltrating macrophages and is potentially cytotoxic to TEC. TEC can produce several cytokines, including IL-1, which may activate T cells, IL-6, which stimulates T and B cells and IL-8, a chemokine which attracts inflammatory cells (reviewed in 165). Dendritic cells are important sources of IL-1b and IL-6 in the thyroid and can inhibit thyroid follicular cell growth (168). In addition, IL-1a causes dissociation of junctional complexes between thyroid cells which could expose hidden autoantigens (169). Vascular endothelial growth factor expression is increased in AITD and is important in angiogenesis in autoimmune goiters (170). Cytokines also seem to play a major role in the pathogenesis of thyroid-associated ophthalmopathy through their stimulatory actions on orbital fibroblasts (167, 171). Exogenous cytokines given therapeutically can also precipitate autoimmune thyroid disease, probably in predisposed individuals. The best described such reaction is α interferon (118).

To summarize, augmented pools of activated and resting T and B cells reactive to thyroid antigen exist in patients with AITD. The time course of development of these reactive cells, before clinical disease is apparent, has not been established. The cells respond to biochemically normal antigen, and some reactive cells exist in otherwise healthy individuals. Immune complex formation appears to be of limited importance in the disease process. K and NK activity may be reduced in Graves' disease and increased in Hashimoto's thyroiditis and may contribute to the course of the disease - proliferative in Graves' disease and destructive in Hashimoto's thyroiditis. Cytokines have multiple actions in the thyroid in AITD and are likely to determine clinical manifestations such as ophthalmopathy. The role of the TEC in the autoimmune response is not simply passive and, as discussed below, the interaction between TECs and cells of the classical immune system may be critical in determining the outcome of an initially mild thyroiditis.