Clinical Endocrinology (1992)36, 307-323

Review

Autoimmune thyroiditis: predisposition and pathogenesis A. P. Weetman Department of Medicine, University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7A0, UK (Received 8 October 1991; returned for revision 5 December 1991; finally revised 19 December 1991; accepted 7 January 1992)

Introduction

Autoimmune thyroid disease is common, affecting approximately 1 % of the population while subclinical, focal thyroiditis and/or circulating thyroid autoantibodies can be found in about 15% of otherwise healthy subjects who are euthyroid. This frequency, combined with historical precedence, ready h e s s to the target organ and well established animal models, has led to a considerable research effort aimed at understanding the initiation and pathogenesis of thyroid autoimmunity, often with the hope that the lessons learned may be applicable to more serious but less accessible autoimmune diseases. In this review, I shall summarize recent developments in autoimmune hypothyroidism (Hashimoto’s thyroiditis and primary myxoedema), only touching on the important insights gained from experimental autoimmune thyroiditis (EAT) and other animal models, detailed elsewhere (Weetman, 1991). Similar autoimmune processes occur in Graves’ disease, which is uniquely distinguished by the presence of thyroid stimulating antibodies. These and other types of TSH receptor antibody are discussed in a separate review (Munro, 1992) and therefore will not be considered further here. Predisposition

As in many autoimmune disorders, it is the interplay of genetics and environment which determines the initiation of autoimmune thyroiditis, with a further modifying influence provided by endogenous factors such as age and sex hormones. lmmunogenetic factors

HLA-linked genes The major histocompatibility complex (MHC) of genes, called HLA in man, controls (in part) immune responsiveness to a variety of foreign and self antigens. In many autoimmune conditions, associations have been reported with certain M H C alleles, particularly

those encoded by the class I (HLA-A,B,C) and class I1 (HLA-D) regions. O n the whole, such associations tend to be stronger with class I1 than class I region genes. Many of the alleles within and between these regions are in linkage disequilibrium, that is, closely linked genes occur together more frequently than expected with random distribution. It is therefore difficult to determine from population-based surveys whether associations of a n autoimmune disease with a particular allele are truly an effect of this allele rather than one in linkage disequilibrium with it. To complicate matters, HLA genes have been renamed recently. A clear exposition of the old and new terminology can be found elsewhere (Tait &Harrison, 1991). As most analyses of HLA in thyroiditis to date have used serologically defined specificities, this nomenclature will be preserved below. Initial studies showed a n association of primary myxoedema with HLA-B8 in Caucasians, but none between class I region genes and Hashimoto’s thyroiditis (Irvine, 1978; Farid et al., 1981). With the availability of serological reagents for HLA-DR typing, this dichotomy was further emphasized, with reports of a n HLA-DR3 or DR5 association with atrophic thyroiditis (primary myxoedema) and Hashimoto’s thyroiditis respectively (Weissel et al., 1980; Farid et al., 1981). As D R 3 is in linkage disequilibrium with B8 in Caucasians, these results in primary myxoedema were consistent with previous observations, but subsequent reports have failed to confirm a simple subdivision of autoimmune thyroiditis (Table 1). Hashimoto’s thyroiditis was associated with D R 4 in one small study from Newfoundland, with D R 5 in patients from Toronto and Denmark and with DR3 in Hungary. In all cases, the relative risks conferred by these alleles were rather small. Differences could have been due in part to variations in the specificity of typing reagents, as up to 25% of serological HLA-DR typings may be incorrect (Mytilineos et al., 1990). The DNA-restriction fragment length polymorphism (RFLP) method of D R typing is less prone to error and by this method D R 3 (but not D R 4 or DR5) was more frequent in English patients with Hashimoto’s thyroiditis (Tandon et ul., 1991a). This method together with allele-specific oligonucleotide probing of amplified D N A sequences also allows typing of other class I1 alleles, particularly D Q A l , DQBl and DPBl, and such techniques have provided considerable insight into the immunogenetics of type 1 diabetes mellitus. One group has found that the strongest relative risk for Hashimoto’s thyroiditis is conferred by DQw7 (previously termed DQw3.1) rather than by D R 4 or D R 5 (Badenhoop et ul., 1990), suggesting that the primary disease susceptibility 307

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A. P. Weetman

Clinical Endocrinology (1992) 36

Table 1 Association of HLA-DR

Study Hashimoto’sthyroiditis Weissel el a/. (1980) Farid et al. (1981) Thomsen et af. (1983) Thompson and Farid (1985) Stenzsky er al. (1987) Vargas el al. (1988) Badenhoop et al. (1990)t

Region

Austria Newfoundland Denmark Newfoundland Eastern Hungary Toronto Toronto/London (UK)

Tandon er al. (1991a)t United Kingdom Mangklabruks et al. (1991)t Chicago Primary myxoedema Farid et al. (1 98 1)

Newfoundland

Number of Relative patients Association risk

39 40 41 21 68

60 64 86 63

50

DR5 DR5 Dw5* DR4 DR3 DR5 DR4 DR5 DR3

None

3.2 3.1 2.2 5.0 3.3 4.2 2.9 3.8 2.2 None

DR3

5.7

specificities with autoimmune hypothyroidism in Caucasians

* T cell-defined specificity equivalent to most of the serologically-defined DR5 specificity. t Defined by RFLP analysis. locus is closer to the DQAl and DQBl genes that encode the DQw7 specificity than to those of the DR locus. However, only DQw2 was associated with Hashimoto’s thyroiditis in another study (Tandon et al., 1991a),consistent with the strong linkage disequilibrium between DQw2 and DR3 seen in the normal Caucasian population. A further RFLP study from the USA found no HLA-DR or DQ associations with Hashimoto’s thyroiditis (Mangklabruks et al., 1991). Transracial studies have been useful in determining susceptibility genes in some autoimmune disorders but have not yet been conducted in sufficient detail to clarify the HLA associations in autoimmune thyroiditis. HLA-Bw46 and DR9 are increased in Chinese patients with Hashimoto’s thyroiditis (Hawkins et al., 1987; Wang et al., 1988), whereas in Japanese, B16 is increased and DR2 is decreased (Nakao et al., 1978; Sakurami et al., 1982): DR3 is unknown in healthy Japanese subjects. In a more recent report from Japan, the most impressive association was with a supertypic allele, HLA-DRw53, encoded by the DRB4 gene present only on a restricted number of haplotypes (DR4,7 and 9),the association with DR9 being much weaker (Honda et al., 1989). Again, a significant decrease in DR2 as well as in DRl, DRw6 and DRwl2 was noted in this group of Japanese Hashimoto patients; it is not clear whether these are nonspecific decreases due to the positive association with other alleles or represent a true protective effort. The former explanation seems more likely as no consistent protective alleles have been defined in other populations. Subdivision of these Japanese patients revealed that 30% had neither

thyroglobulin (Tg) nor microsomal/thyroid peroxidase (TPO) antibodies (this is an unusually high frequency, as TPO antibodies are usually found in over 90% of patients). HLA-DQwl (now known to include two specificities,WSand w6) was decreased and DQw4 increased in the seronegative patients. Thus, the HLA associations with autoimmune thyroiditis are relatively weak and inconsistent between populations. Case ascertainment, geographical influences or even a temporal change in susceptibility could account for the differences observed. There is reasonable evidence that Hashimoto’s thyroiditis and primary myxoedema differ immunogenetically; even those Caucasian Hashimoto patients with an increase in DR3 may differ from individuals with atrophic thyroiditis as only the latter is associated with the extended haplotype HLA-A1, B8, DR3, DQw2 (DPwl). It is possible that two or more loci within the MHC may determine predisposition to autoimmune thyroid disease. One o f these (DR3 or a gene close to it) may have a nonspecific effect in enhancing the likelihood of autoreactivity. This is suggested by the variety of immunological differences between DR3-positive and DR3-negative healthy individuals (Hashimoto et al., 1990) and would explain the frequency with which DR3 is associated with many autoimmune disorders, including Graves’ disease. Other MHC loci may determine which type of thyroid disease the patient develops; for instance, an unidentified gene in linkage disequilibrium with the haplotype Al, B8, DR3 may confer susceptibility to develop atrophic thyroiditis, whereas other genes, in linkage disequilibrium with DR4 or DR5 in some Caucasians or with DRw53 in Japanese, may be important in

Clinical Endocrinology (1992) 36

Hashimoto’s thyroiditis. Several candidates for these unknown MHC genes exist, including the class 111 loci which regulate production of certain complement components and the genes encoding two immunologically important cytokines, tumour necrosis factor (TNF) and lymphotoxin. Non-HLA genes There are no conclusive family studies of autoimmune thyroiditis which allow estimation of the genetic contribution made by HLA to the disease but, by analogy with Graves’ disease (in which similar relative risks for HLA associations have been reported), this is unlikely to be extensive. The risk of developing Graves’ disease in an HLA-identical sibling is 7- I6%, whereas the concordance rate for Graves’ disease in monozygotic twins is around 50% (Stenszky et al., 1985). Even allowing for ascertainment bias in the latter figure, these results suggest an important contribution from non-HLA genes. Population-based studies in Newfoundland have suggested an association with IgG heavy chain allotypes, especially in atrophic thyroiditis (Nakao et af., 1982). Subsequently, an interaction between a particular Gm3 allotype, g, and DR3 was observed which enhanced the risk for goitrous autoimmune thyroiditis in Hungary (Stenszky eta/,, 1987). Neither study has yet been confirmed and no significant difference was found in Japan in the distribution of Gm allotypes between normals and a small group of patients with Hashimoto’s thyroiditis (Tamai et al., 1985).However, in 10 families selected because two or more first degree relatives had Graves’ disease, those family members with Hashimoto’s thyroiditis had the same HLA and Gm haplotypes as individuals with Graves’ disease (albeit with separate haplotypes conferring disease susceptibility in the different families). This suggests the operation of common genetic factors (including Gm) in the two conditions, but how closely these highly selected patients reflect sporadic cases of Hashimoto’s thyroiditis is not clear. Family studies have also been helpful in elucidating the pattern of inheritance of thyroid autoantibodies. Burek and colleagues (1 982) reasoned that autoimmune disease in older patients may reflect a dominant environmental effect (see below), while a similar disease in children probably represents a greater genetic contribution. They therefore analysed the families of a group of children and adolescents with H ashimoto’s thyroiditis, to test whether this genetic predisposition might be mirrored by a high frequency of autoantibodies in siblings and parents. This was indeed the case. Furthermore, there was an additive relationship between the number of progeny with thyroid antibodies and the presence of these antibodies in neither, one or both parents: the sex of the parent did not matter. Using more sensitive assays, these results have been

Autoimmune thyroiditis

309

confirmed, and segregation analysis of I6 families with thyroiditis in at least two members suggested that the inheritance of thyroid autoantibodies is a Mendelian dominant trait in women, with reduced penetrance in men (Philips et al., 1990). Linkage analysis was uninformative for Gm allotypes but linkage with H LA markers could be excluded. To rule out the potential bias resulting from family selection, a second study examined the distribution of incidentally occurring thyroid autoantibodies in 49 families with neurological or psychiatric disease (Philips et al., 1991). Again, a dominant pattern of inheritance was found in women, with reduced penetrance in young women as well as in men of all ages. Results from animals with spontaneously occurring EAT strongly suggest that genes involved in T cell regulation and in target organ responsiveness are important determinants of disease (Weetman, 1991). In man, there is no evidence so far that genetic variation in thyroid autoantigens or thyrocyte behaviour contributes to susceptibility, although it is only recently that techniques to test these possibilities have become available. A RFLP of the T cell receptor a-chain variable (V) region has been associated with autoimmune hypothyroidism (Weetman et ul., 1987) and other T cell receptor polymorphisms have been linked to Graves’ disease and the ability to produce Tg antibodies (Demaine et al., 1989). As with HLA associations, these findings have not been reproduced in a study from the USA (Mangklabruks et ul., 1991) and how these germ-Iine polymorphisms could relate to disease susceptibility is not yet clear. Finally, a very high prevalence of thyroid autoimmunity has been reported in kindreds with familial Alzheimers’ disease as well as in Down’s syndrome, suggesting the influence of an unknown genetic factor on chromosome 21 (Ewins et al., 1991). In summary, it seems certain that other loci besides those within the MHC operate in determining the susceptibility to autoimmune thyroiditis. The propensity to form thyroid antibodies seems to be inherited as a Mendelian dominant trait (which is not MHC-encoded) but is modified by the age and sex of the individual, in turn presumably reflecting the effect of environmental factors and sex hormones. Other loci influence which of these antibody-positive individuals will go on to develop overt disease: possible candidates may lie within or outside the MHC. Non-genetic factors

The existence of these has already been hinted at. Two endogenous effects are important in modifying disease susceptibility. Ageing produces increasing exposure to environmental agents as well as changes in immunoregulation which may help initiate thyroiditis (Fong et al., 1981). Sex

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A. P. Weetman

hormones have a variety of effects on the immune system but it is apparent that oestrogens exacerbate EAT, whereas testosterone ameliorates it (Weetman, 1991). Prolactin may also have a role because asymptomatic autoimmune thyroiditis is more common in hyperprolactinaemic disorders (Ferrari et al., 1983). Altered levels of these hormones and the other immunological changes which accompany pregnancy presumably account for the remission seen in the last trimester and the subsequent exacerbation of subclinical thyroiditis and rise in thyroid autoantibody levels which constitute the entity of post-partum thyroiditis (Lazarus & Othman, 1991). It is not yet clear how many women who have had post-partum thyroiditis will later develop permanent autoimmune hypothyroidism, or whether repeated episodes of post-partum thyroiditis increase the likelihood of this happening. However, it is intriguing that the same HLA alleles, DR3, DR4 or DRS, have been associated with the two conditions in different Caucasian populations, suggesting a similar although heterogeneous genetic predisposition. Environmental factors also play a significant role. Epidemiological data support an enhancing effect of increasing iodide intake on thyroid autoimmunity. For instance, the prevalence of thyroid lymphocytic infiltration in the nontumorous portion of thyroidectomy specimens (removed for carcinoma) rose from 8 to 31 YO in the 5 years after the introduction of iodine prophylaxis in Argentina (Harach et al., 1985), and an increase in thyroid autoantibodies was found 3-6 months after the acute administration of iodized oil to otherwise healthy people in Corfu, an area of endemic goitre (Boukis et al., 1983). However, the clearest evidence for an effect of iodide has come from observations in EAT: a high iodine diet exacerbates thyroiditis, whereas a low iodine diet causes amelioration (Bagchi et al., 1985; Allen et al., 1986; Cohen & Weetman, 1988). Indeed, in the Obese strain of chicken, the uptake and metabolism of iodine is crucial to the development of thyroiditis; administration of perchlorate and mononitrotyrosine (to inhibit iodine transport and promote thyroidal iodine loss respectively) in ovo virtually prevents thyroiditis in these birds (Brown et al., 1991). This study is important as it implies a role for physiological concentrations of iodide, whereas some of the earlier studies in EAT had used fairly massive concentrations of iodide to supplement diets. Several mechanisms may explain this effect of iodide. Tg is more immunogenic in normal animals if it is highly iodized (Sundick et al., 1987) and the response of murine T cell hybridomas to Tg is also directly related to its iodine content (Champion et al., 1987). This may be because critical epitopes for T cell recognition of Tg contain T4 and consist of sequences flanking one of the hormonogenic sites; in iodine deficiency, an alternative site for hormonogenesis is

Clinical Endocrinology (1992) 36

used preferentially and thus these key epitopes will not be generated (Champion et al., 1991). However, iodide may well have other immunological effects, as a central pathogenic role for Tg autoreactivity seems much less important in human thyroiditis than in EAT. For instance, excess iodide exacerbates post-partum thyroiditis, yet this is a condition in which Tg antibodies are rarely found (Jansson et al., 1985). Highly reactive oxidized forms of iodine may be produced by reaction with oxygen radicals generated within the thyroid (considered further below) and these could have inflammatory potential or iodine could have direct or indirect immunological effects yet to be defined (Brown et al., 1991). Infection, particularly by viruses, is an environmental insult which seems an obvious candidate for initiating autoimmunity, although compelling evidence for this exists in very few human diseases. In fact, subacute thyroiditis, assumed to have a viral aetiology, typically does not result in thyroid autoantibody formation or chronic thyroiditis. Epstein-Barr virus (EBV) infection has recently been implicated in the development of autoimmune thyroiditis in three patients (Coyle et al., 1989) but much more work is required to consolidate this; obviously it is possible that many separate viruses may be relevant and that infection may precede the onset of thyroid disease by sufficient time to obscure any relationship. Again, the clearest evidence of an important role for infection has come from EAT. Certain strains of rat develop thyroiditis and Tg antibodies after thymectomy at weaning and sublethal irradiation (Penhale et al., 1973).This can be prevented by raising the animals under germ-free conditions; conversely, disease can be initiated in such pathogen-free animals by administering the gut microbial contents from normal rats (Penhale & Young, 1988). EAT in these animals depends on a major perturbation in the T cell repertoire and considerable caution is therefore required in extrapolating from the results to human thyroiditis, but this work does indicate a possible role for commensal rather than pathogenic micro-organisms. Finally, exogenous cytokines used as novel therapeutic agents seem unusually prone to cause thyroiditis. The precipitation of thyroid autoimmunity by leucocyte-derived a-interferon (a-IFN) was originally ascribed to contamination with y-IFN (Burman et al., 1986) but the same effect occurs with recombinant a-IFN, which may also induce other autoimmune disorders, such as pernicious anaemia and vasculitis (Ronnblom et al., 1991). Moreover, y-IFN administration in man does not precipitate thyroid autoimmunity (Kung et af., 1990), although murine thyroid isografts exposed to y-IFN in vitro are destroyed by T cells in transplated recipients (Frohman et al., 1991).Whether this is a species, dosage or technical difference is currently unknown. Infusions of interleukin-2 (IL-2) or granulocyte-

Clinical Endocrinology (1992) 36

Autoimmune thyroiditis

macrophage colony-stimulating factor can also cause the formation of thyroid autoantibodies and hypothyroidism (Van Liessum et al., 1989; Hoekman et al., 1991). Thus the broad range of immunological effects produced by these diverse cytokines (at very high concentrations) results in the common development of autoimmune thyroiditis, presumably in individuals who are already predisposed to develop this prevalent disorder. Whilst such effects may have little to do with the pathophysiology of spontaneous thyroid autoimmunity, they emphasize that several diverse, nongenetic factors could separately result in the production of autoimmune thyroid disease. From the foregoing, it seems likely that a combination of genetic, constitutional and environmental factors initiate autoimmune thyroiditis but the relative contribution of each of these, and almost certainly of undiscovered factors, is unknown. For instance, the potential role of environmental toxins, affecting the thyroid or the immune system, has not been examined in detail, yet substances such as anthracene derivatives are potent inducers of EAT in certain genetically susceptible strains of rat (Weetman, 1991). Infectious agents may well play a more important role than currently appreciated. Although there are no striking homologies of thyroid antigens with known viral or bacterial sequences, only a fraction of potential sequences are known. Therefore, infection resulting in molecular mimicry cannot yet be discounted as a triggering event, particularly taking into account the experimental evidence provided by germ-free animals (Penhale & Young, 1988). Direct infection of thyroid cells, leading to expression of neoantigens or release of autoantigens, seems a less likely route whereby pathogens may produce autoimmune thyroiditis, based on the outcome in most cases of subacute thyroiditis. Finally, the genetic and constitutional factors described above probably influence a variety of immunological interactions to be discussed next,

311

from antigen presentation, through T cell activation, to B cell production of thyroid autoantibodies. Only a proportion of the disordered immunoregulation which results may be thyroid specific, but non-specific effects may nevertheless result in thyroiditis when combined with particular environmental insults such as excess iodine or infectious agents which target thyrocytes. Pathogenesis

The archetypal immune response consists of antigen presentation by a 'professional' antigen presenting cell (APC), such as a macrophage, to a helper, CD4+ T cell which can then activate B cells (Fig. 1) (Weetman, 1991). For thyroiditis to occur, the autoimmune process must complete each of these steps to generate the effector mechanisms which result in tissue injury. In this section, therefore, antigen presentation, T cell responses and B cell responses are considered sequentially before reviewing the proximal causes of thyroid destruction. Before doing so, it is worth emphasizing that the role of the APC is to convert intact antigen into peptides of around 12-20 amino acids in size which can serve as T cell epitopes, binding to a groove in the MHC class I1 (or Ia) molecule expressed by the APC (MHC class I molecules bind peptides of around nine amino acids). The first stage in this process is uptake of antigen by the APC, generally via receptors (e.g. for mannose, complement or the Fc portion of immunoglobulins compIexed with antigen), allowing preferential handling of foreign rather than self antigens. The APC then processes antigen, either within lysosomes for phagocytosed antigen or in endosomes containing proteases which degrade antigen taken up by receptor endocytosis. Finally, the peptide which constitutes the epitope binds to a class I1 molecule within the cell and this bimolecular complex is expressed on the cell surface where it can interact

0-&J 0

08

f

APC e.g. mocrophoge

Fig. 1 Two types of antigen presentation.

In the upper half a conventional antigen presenting cell (APC), able to express a costimulatory signal, stimulates a helper T cell; in the lower half, antigen presentation in the absence of a costimulator leads to T cell tolerance (reproduced from Weetman, 1991, with permission of the publisher). fi Ia (or MHC class 11) molecule+peptide, t costimulatory signal.

Activation by cytokines

\

+

I a castimulatorexpression and ontigen presentation

4 __ 'U Ia- non- APC e.g thyrocyte

Tcell stimulation

Tcel' @

I a expression and antigen presentation

-

T cell paralysis: peripheral tole ronce

312

Clinical Endocrinology (1992) 36

A. P. Weetman

Antigen presenting cell

b3-n-d ICbM-ILFA-3

Ia molecule Antigenic peptide

CD4

Tcell receptw

CD2

LFA-I

T cell

Fig. 2 Molecules involved in the interaction between an antigen presenting cell (such as a macrophage) and a helper T cell. (Reproduced from Weetman, 1991, with permission of the publisher.)

with an appropriate T cell receptor, leading to T cell activation (Fig. 2). The T cell receptor is composed of two chains, o! and /I each , with a distal V region and a proximal constant (C) region. There are up to 100 separate V gene segments for each chain, and this genomic diversity is increased by the multiple rearrangement combinations possible as a result of junctional and diversity gene segments which encode short sequences lying between the V and C regions. This results in a vast repertoire of T cell receptors which the individual can use to recognize the wide array of peptide/class I1 bimolecular complexes likely to be encountered. The interaction between class II/peptide and T cell receptor is stabilized by the CD4 molecule, which is expressed by a subset of T cells capable of stimulation by peptide plus MHC class I1 molecule. Although these CD4+ T cells are generally regarded as helper cells, in some circumstances they may be cytotoxic, killing target cells bearing class I1 molecules and appropriate antigen. A reciprocal subset of T cells express CD8 which stabilizes their interaction with cells presenting antigen combined with a class I molecule: the usual situation in which this occurs is recognition of a virally infected target cell by a cytotoxic T cell. An ill-defined proportion of CD8+ T cells can also display suppressor cell function, but this property too may be shared by CD4+ T cells. Antigen presentation in autoimmune thyroiditis

The observation that thyrocytes express MHC class I1 molecules in Graves’ disease and Hashimoto’s thyroiditis (Hanafusa et al., 1983) led to the hypothesis that such epithelial cells could become APCs, capable of stimulating autoreactive T cells by presentation of endogenous autoantigen (Bottazzo et al., 1983). T cell lines and clones were obtained from the thyroid lymphocytic infiltrate i n these

conditions which responded by proliferation when stimulated by autologous MHC class 11+ thyrocytes, providing good initial evidence for this suggestion (Londei et al., 1985; Weetman et al., 1986). However, the idea that aberrant class I1 expression by thyrocytes is the initiating event in autoimmune thyroiditis now seems unlikely, as the phenomenon is dependent on local T cell infiltration. Three lines of evidence support this. Firstly, thyroid cells from patients with or without autoimmune thyroiditis express class I1 molecules equally well in vitro after culture with the T cell-derived cytokine y-IFN, which is by far the most potent stimulator tested (Todd et al., 1985; Weetman et al., 1985a): T N F may augment this effect of y-IFN on thyroid cells (Weetman & Rees, 1988: Bucsema etal., 1989).Secondly, the distribution ofclass 11+ thyrocytes in autoimmune thyroid disease is strongly correlated with the presence nearby of lymphocytes containing y-IFN, suggesting a direct relationship between the two (Hamilton et al., 1991). TNF is also produced by the mononuclear cell infiltrate in autoimmune thyroiditis (Turner et al., 1987). Finally, in sequential studies of EAT in rats, the appearance of the lymphocytic infiltrate always precedes that of class I1 molecules on thyroid cells (Cohen et al., 1988). This still leaves a possible role for class 11-expressing thyrocytes in perpetuating the autoimmune response, but the evidence for this is currently equivocal. Primary cultures of mouse thyroid follicular cells, stimulated with y-IFN to induce class I1 molecules, failed to present Tg, foreign antigens or alloantigens to appropriate T cells, and this was not improved by adding IL-1 or phorbol esters which enhance the function of other types of APC (Ebner et a!., 1987; Minami et a[., 1987). The cells used in these experiments were positive for cytokeratin and secreted Tg but were tested after 3 weeks of culture, which may have altered potential antigen presenting properties. On the other hand, rat and mouse thyroid-derived epithelial lines have been established which can present antigens, although supplementary phorbol ester was required in one case (Stein & Stadecker, 1987; Kimura & Davies, 1991). Such thyroid cell lines are divergent from primary thyroid cultures in many respects and these observations may therefore reflect a newly acquired capacity for APC function by the lines with little relevance to disease pathogenesis. The initial results previously mentioned, showing stimulation of T cells by class I I + thyrocytes, may have reflected contamination by professional APCs such as macrophages and dendritic cells. The latter are particularly potent and their intrathyroidal number is increased in Graves’ disease and Hashimoto’s thyroiditis (Kabel et al., 1988). There seems no doubt that primary thyroid cell cultures are contaminated by a small proportion of mononuclear cells, as

Clinical Endocrinology (1992) 36

it is stimulation of this population to release y-IFN which accounts for the indirect enhancement of class I1 expression seen when thyroid cells are cultured with T cell mitogens such as phytohaemagglutinin (Weetman et al., 1985a). The addition of monocytes to class 11+ thyroid cells enhances their ability to stimulate T cells (Eguchi et al., 1988); whether this reflects secretion of some important cytokine or mere bolstering of limiting numbers of professional APCs in the thyrocyte cultures is at present unclear. In short, results with primary thyrocyte cultures may be questioned on the basis of contamination by other cells, whereas pure thyroid cells may no longer reflect the antigen-presenting capacity of their native parents. One way out of this impasse has been described in the analogous situation of type 1 diabetes mellitus, in which the pancreatic beta cells can also express MHC class I1 molecules, although even this is contentious as in animal models of diabetes, class 11+ insulin-containing cells in the islets are probably phagocytic leucocytes (In’t Veld & Pipeleers, 1988; Signore et ul., 1989). Transgenic mice were made which express class I1 molecules on their beta cells, using the insulin gene promoter to ensure targeting of the transgene. These animals developed diabetes, but without any evidence of insulitis (Lo el uf., 1988). It appears that this abnormal expression of M H C class I1 (and, in other mice, class I) molecules is diabetogenic by itself, possibly due to the massive amounts of these glycoproteins which are synthesized. However, autoimmune beta cell destruction does not occur and this is therefore not a model for human type 1 diabetes. Furthermore, T cells can actually become tolerized rather than be stimulated by the aberrantly expressed class I1 molecules, although this is not an inevitable feature of these transgenic mice (Lo et al., 1988; Gotz et al., 1990). The likely explanation for these rather paradoxical findings lies in the recent recognition of a costimulator or second signal which is required in addition to class I1 expression for antigen presenting function and which is produced by macrophages, dendritic cells and similar professional APCs (Mueller et al., 1989). In its absence, antigen presented by a class I1 molecule may have no effect or may induce tolerance in the T cell by some unknown mechanism (Fig. 1). This phenomenon has been demonstrated in a number of human systems, including class I1 keratinocytes (Gaspari et ul., 1988). A transgenic model also exists for this; mice whose beta cells hyperexpress y-IFN develop diabetes caused by cytotoxic lymphocytes specific for islet antigens, possibly due to y-IFN-mediated activation of anergic T cells (Sarvetnick et af., 1990). Thus y-IFN is one possible costimulator but is not known to be produced by normal endocrine cells. Other molecules, besides y-IFN, are involved in mediating this function for conventional APC and appear to be membrane +

Autoimmune thyroiditis 313

bound, as soluble factors released from mononuclear cells are unable to promote APC function in cells which d o not possess the unknown costimulator. Phorbol esters induce an activity which mimics this second signal and we have recently found a consistent enhancing effect of phorbol ester pretreatment of the ability of primary thyrocyte cultures to stimulate T Celt alloreactivity (unpublished observations). Thus, class I1 expression by thyroid cells seems unlikely to initiate thyroid autoimmunity, but could perpetuate it by increasing autoantigen presentation to neighbouring T cells. However, recent evidence suggests that if a costimulatory signal is not supplied by a class 11+cell presenting antigen, T cells will be refractory to stimulation and could even become tolerant. It is unknown whether thyroid or other endocrine cells can make this second signal. Molecular characterization of the costimulator and delineation of its cellular distribution will provide crucial insights into this question. At a more basic level, it seems worth recalling that y-IFN infusions d o not induce thyroiditis, at least in man (Kung et al., 1990) and that the intrathyroidal infiltrate contains abundant dendritic cells, as well as B cells which can act as APCs for thyroid autoantigens (Hutchings et al., 1987). Very small numbers (lo5) of murine dendritic cells pulsed with Tg, or derived from mice with EAT, can induce thyroiditis in recipients (Knight etal., 1988). I t is thereforequestionable whether any additional antigen-presenting capacity supplied by thyrocytes would have much impact on the evolution of autoimmune thyroid disease, particularly as the stimulation of T cells produced by thyrocytes, in those experiments in which it has been observed, seems to be relatively weak. T Cell responses in autoimmune thyroiditis

Two major autoantigens, Tg and TPO, provoke an immune reponse in autoimmune thyroiditis. Autoreactivity against the TSH receptor, at the level of B cell at least, occurs infrequently in autoimmune hypothyroidism, although it is central to the aetiology of Graves’ disease, and this is reviewed elsewhere (Munro, 1992). Other autoantigens may also be recognized in autoimmune thyroiditis, as demonstrated by the cloning of two novel antigens, ATRA-I, recognized by about 27% of Hashimoto sera (Hirayu et al., 1987), and a 64 kDa antigen present in thyroid and eye muscle and also recognized by a small proportion (21 %) of Hashimoto sera (Dong et al., 1991), but so far little further information is available on the importance of these in pathogenesis. While most attention has been paid to autoantibodies, there is no doubt that thyroid antigen recognition by T cells is central to the development of autoimmune thyroiditis: this is an essential prelude to B cell stimulation and autoreactive T cells may have important

314 A. P. Weetman

inflammatory effects, such as cytotoxicity and release of cytokines, which are independent of autoantibody synthesis. It is therefore important to establish why such T cells arise, as this is likely to be a major factor in the pathogenesis of thyroid autoimmunity. T cell tolerance Several mechanisms, which are not mutually exclusive, prevent autoimmune responses in general:

(i) Sequestered autoantigen, that is, hidden from recognition by the immune system; (ii) T cell tolerance, due to clonal deletion or anergy (in the latter, T cells continue to exist but are unable to respond to antigen; the peripheral tolerance induced by class 11+ epithelial cells described above is one form of anergy). Most T cells become tolerant in the thymus during ontogeny (central tolerance); (iii) B cell tolerance, due to deletion or anergy (which depends on antigenic valency); (iv) Active suppression of autoreactivity; (v) Idiotype-anti-idiotype regulation (which may in part be involved in mediating suppressor T cell function). Tg is certainly not sequestered from the immune system as it is present in the circulation, nor is it likely that TPO is completely hidden, as intraepitheliai lymphocytes are present in thyroid follicles and low levels of TPO can be detected in the blood. Furthermore, release of all thyroid antigens presumably occurs after thyroid injury, such as trauma, surgery or infection, yet does not result in autoimmune disease. It is likely that T cell tolerance induced in the neonatal thymus is a major mechanism which prevents thyroid autoimmunity; disturbing the process in animals by thymectomy leads, in certain strains, to EAT (Penhale et al., 1973; Kojima et al., 1976).Central tolerance may be one way in which the MHC affects the development of autoreactivity as T cells are positively as well as negatively selected within the thymus by their recognition of antigen presented by MHC class I1 molecules, and it is now apparent that the particular MHC molecules expressed by an animal can thus influence its T cell repertoire. Experiments with transgenic mice have shown that while central T cell tolerance to autoantigens is of considerable importance in removing autoreactive T cells, the process is incomplete (Kisielow et af., 1988). The autoreactive cells which remain untolerized are presumably held in check either by the active mechanisms of T cell suppression or antiidiotypic regulation, or by the imposition of peripheral tolerance. Active mechanisms may not be necessary as susceptible T cells can be accepted, provided they do not encounter autoantigen. If a self antigen is presented to

Clinical Endocrinology (1992) 36

susceptible T cells by an endocrine cell expressing class I1 molecules (e.g. following a viral infection of a gland, resulting in local y-IFN production), the relative efficiency of peripheral tolerance described above is obvious as the class 11-positive endocrine cells, unable to provide a second signal, could avert autoimmunity by inducing anergy. In contrast, active suppression appears to require considerable effort by the immune system to maintain defences for an event that may rarely, if ever, occur in an individual. As an aside, susceptible autoreactive B cells may also be permitted because they are harmless in the absence of helper T cells specific for the same autoantigen. It is therefore not surprising that autoantibodies against Tg (and TPO to a lesser extent) are often found in otherwise healthy individuals, particularly with ageing or after EBV infection (Fong et al., 1981). Exposure of these susceptible B cells to polyclonal activators such as EBV (more likely with age) may bypass the need for T cell help, or in some cases the active mechanisms to prevent T cell autoreactivity may be imperfect. As discussed below, other autoimmune processes seem to be required for progression of thyroid autoantibody formation to thyroiditis and clinical disease. There is considerable evidence from EAT to show that T cells reactive to Tg are incompletely tolerant in normal animals (ElRehewy et al., 1981) and, given the high frequency of autoimmune thyroiditis in the population, the same is almost certainly true in man. However, it is likely that a hierarchy exists, with fewer susceptible T cells being permitted which are reactive to TPO or the TSH receptor (as these have greater pathogenicity) than T cells directed against Tg. Direct quantitation of susceptible T cells specific for thyroid antigens in otherwise healthy subjects has not been possible, as the numbers are too small for current techniques. Instead, most attention has focused on the role of thyroid-specific suppressor T cells in preventing thyroid autoimmunity, the importance of which has been argued most cogently by Volpe (1988).

T cell-mediated suppression Antigen-specific T suppressor cells have been characterized by their inhibitory effects on a functionally defined cytokine, migration inhibition factor (MIF), produced by T cells sensitized to thyroid antigens in patients with autoimmune thyroiditis (Okita et al., 1980). The production of MIF in such experiments was abolished by the addition of normal, allogeneic T cells which were therefore postulated to have suppressor function, the absence of such cells in thyroiditis resulting in unrestrained MIF synthesis (Okita et al., 1981). Antigen specificity was demonstrated by the ability of T cells from diabetic patients (which produce MIF when stimulated by pancreatic antigens) to inhibit MIF production by Hashimoto T cells in

Clinical Endocrinology (1992)36

response to thyroid antigen and, conversely, Hashimoto T cells can inhibit MIF production by diabetic T cells elicited with pancreatic antigens (Topliss et al., 1983). However, the phenotype of these suppressor T cells has not been defined and it is unclear how they recognize HLA-mismatched target helper cells. The results of the MIF assay in thyroid autoimmunity have not been consistently reproduced (Ludgate et al., 1985)and trivial explanations such as cytotoxicity (due to alloreactivity) or consumption of lymphokines have proved responsible in other situations in which the operation of suppressor cells in vitro has been invoked (Gunther et al., 1982). If there is a defect in antigen-specific T suppressor cell function in thyroid autoimmunity, it is only partial and may reflect a broad range of suppressor dysfunction (Mori et al., 1985; Davies & Platzer, 1985). This is further supported by removal of the CD8+ T cell population (believed to contain a substantial part of the suppressor T cell pool) which enhances the proliferative response of Hashimoto CD4+ cells stimulated by either Tg or TPO (Weetman, 1988). In contrast, CD8+ T cell removal does not lead to a proliferative response by T cells from normal individuals. These results make it unlikely that CD8+ T cells have a major role in preventing thyroid autoreactivity in healthy subjects, although it is possible that CD4+ T cells could also act as suppressors. One problem with almost all of these studies is that although circulating T cells have been analysed, their relationship to intrathyroidal events is unknown. Moreover, it is impossible to be certain that any alteration in suppressor cell function is a primary rather than secondary event. Some have even questioned the very existence of suppressor cells (Moller, 1988), but this is an extreme view and the cloning of human antigen-specific T suppressor cells (Salgame et af., 1989) suggests new ways of examining such cells in thyroid autoimmunity, including the molecular mechanisms which mediate suppression. Modulation of Tg-specificT cell function by anti-idiotypic antibodies has been demonstrated in EAT, although the relative importance of this in preventing thyroiditis in vivo has not been addressed (Roubaty e al., 1990). There is no available information on the role of anti-idiotypic control of thyroid autoimmunity in man at the level of the T cell. However, there is clearly potential for this to occur and without the need to invoke antibodies; recognition of T cell receptor idiotypes by T cells bearing anti-idiotypic receptors could control autoreactivity in a way indistinguishable from T cell-mediated suppression. Such a system has already been proposed in experimental autoimmune encephalomyelitis (EAE) in which vaccination of animals with peptides from the antigen recognition site of the T cell receptor induces cells which prevent disease (Janeway, 1989).

Autoimmune thyroiditis 315

Clonality of the T cell response This work in EAE stemmed from the exciting observation that, in certain strains of rats and mice, recognition of autoantigen (myelin basic protein) was confined to lymphocytes bearing a restricted repertoire of T cell receptors (Wraith et al., 1989). If the same holds true in human autoimmunity, there is considerable scope for novel therapies, including vaccination with the receptor peptides just mentioned, which could prevent or cure disease without generalized immunosuppression. Limited evidence exists for restricted clonality of intrathyroidal T cells in Hashimoto’s thyroiditis. Studies on two patients using monoclonal antibodies against three T cell receptor Vg families (VD5, VP6 and VP12) showed no abnormal distribution (Teng et al., 1990) and RFLP analysis to detect T cell receptor gene rearrangements indicative of clonal expansion found evidence for this in only one of six patients (Katzin et al., 1989; Lipoldova et al., 1989). Similar results, showing a polyclonal response, have been obtained in Graves’ disease (Kaulfersch et al., 1988). In contrast, restriction of Va gene usage (to six of 18 possible V gene families) was found in one patient with Hashimoto’s thyroiditis studied by a more refined technique, namely reverse transcription of mRNA and DNA amplification using Va-specific oligonucleotide primers (Davies et al., 1991). Similar degrees of restriction were observed in four further patients with Graves’ disease, although the Va families used differed between patients. Clearly more work is required in this area, particularly addressing receptor usage by cloned, thyroid antigen-specific T cells, as the whole T cell population studies performed so far have examined multiple specificities, some of which may not even be related to thyroid antigen recognition. A second problem, more difficult to overcome, is that intrathyroidal T cells can be examined only once the disease is established; oligoclonality at the initiation of thyroiditis may be obscured subsequently by the effects of disease progression and treatment. Identification of the T cell epitopes of thyroid antigens is a related issue, as restricted T cell clonality in EAE has been demonstrated for some but not all of the epitopes on myelin basic protein. Studies previously described, analysing murine T cell epitopes on Tg, suggest that one of the iodinated hormonogenic sites is of critical importance (Champion et al., 1991), although it should be noted that these experiments used selected T cell hybridomas whose pattern of antigen recognition may not correspond to the primary T cell repertoire in intact animals. Tg epitopes for human T cells have not yet been defined. Weak, variable responses to synthetic TPO peptides containing motifs predicted to be T cell epitopes have been found in studies using lymphocytes from Hashimoto’s patients, suggesting a polyclonal T cell response to this

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autoantigen (Fukuma et uf., 1990; Tandon et al., 1991b). While some patients gave reasonable responses to one or more peptides, others responded equaly well to different sequences. T cells from control subjects were also occasionally stimulated by these peptides. It is not yet known whether such individuals possess an excess of susceptible T cells, perhaps putting them at risk of autoimmunity, or whether such responses are fortuitous cross-reactions. In one patient with Graves’ disease, T cell clones were found to recognize three different epitopes on TPO (two located between amino acid positions 535-551 and 632-645) and three Vg gene segments were used by separate clones reacting with a single TPO epitope (Dayan e l al., 1991). Further sets of T cell clones from two Graves’ patients did not react with these TPO epitopes. Taken together, it seems that there may be many T cell epitopes on TPO and the response to these is heterogeneous both between and within individuals. Such observations complement those discussed above, suggesting that a highly restricted repertoire of autoreactive T cells is not a feature of autoimmune thyroiditis. B cell responses in autoimmune thyroiditis

As already discussed, incomplete B cell tolerance exists for Tg and probably TPO. Tg antibodies with low affinity, low titre and of the IgM class are frequently detected in healthy individuals and belong to the group of so-called natural autoantibodies which may have a ‘housekeeping’ tole in removing effete cell products from the circulation (Grabar, 1975). Thyroid antibodies in autoimmune thyroiditis are predominantly IgG class and of high titre and affinity, and this change in their characteristics is likely to require provision of cytokines by antigen-specific T cells to the autoreactive B cells (Bresler et ul., 1990). As a result of this interaction within the thyroid, lymph nodes and bone marrow, thyroid-specific B cells appear in the bloodstream in varying states of activation, proliferation and differentiation (Weetman et af., 1985b). Whilst such circulating B cells have frequently been used to characterize the autoimmune response to thyroid antigens, this heterogeneity makes it difficult to interpret data concerning thyroid-specific B cell numbers and function. Even within the thyroid there are specific B cell compartments which make differing contributions to autoantibody synthesis (Atherton et al., 1985).

B cell development Rapid progress has been made in delineating the mechanisms whereby helper T cells stimulate antibody production by B cells. Contrary to earlier expectations, T cell help is mediated by cytokines without antigen specificity. Furthermore, most B cell-stimulating cytokines act at multiple stages of B cell development, many have

Clinical Endocrinology (1992) 36

overlapping functions and all so far described have effect on other cell lineages. The apparent antigen specificity of B cell stimulation provided by T cells is the result of localized delivery of these cytokines which occurs when appropriate helper T cells form conjugates with B cells bearing antigen and MHC class I1 molecules. IL-6 is one of the most important cytokines affecting B cell development. Although produced by monocytes and T cells, many other types of cell can make IL-6, including thyrocytes (Grubeck-Loebenstein et al., 1989; Zheng et al., 1991). The production of IL-6 by thyrocytes is enhanced by TNF, IL-l and y-IFN (Weetman et al., 1990a). As thyroid cells are exposed to these cytokines in thyroiditis, it is likely that abundant IL-6 will be produced within the diseased gland, in turn stimulating plasma cell formation and leading to localized autoantibody formation. Thyroid cells also appear to synthesize IL-la in Hashimoto’s thyroiditis (Zheng et al., 1991) and this too could have effects on B cell activation and differentiation. In addition, both IL-1 and IL-6 activate T cells; their localized production by thyrocytes may therefore further promote the development of thyroiditis by stimulating cell-mediated effector mechanisms. Tg antibodies Antibodies to Tg in autoimmune thyroiditis are directed against a restricted number of determinants (Chan et al., 1987). The autoantibody responses in individuals with thyroiditis are generally against species-specific epitopes on Tg, whereas naturally occurring Tg antibodies from otherwise healthy individuals appear to recognize mainly thyroxine-containing determinants that are not species-specific (Bresler et a!., 1990). Perhaps through evolutionary pressure to conserve natural antibodies with housekeeping function, genes may have been selected to code for Tg antibodies directed against the most conserved part of the molecule. As discussed above, these hormonogenic sites may be modified by dietary iodine but this seems to affect only T cell epitopes; in EAT, naturally occurring autoantibodies react equally well with Tg of high and low iodine content (Sundick et al., 1987). It is likely that the ongoing autoimmune process leads to thyroid injury with autoantigen release in patients with thyroiditis, and this diversifies the Tg autoantibody response from the ‘natural’ state, in turn producing the stable polyclonal spectrotypes which characterize Hashimoto’s thyroiditis (Delves & Roitt, 1988). Tg antibodies do not fix complement, a property that seems related to the widely spaced epitopes on this large molecule which prevent IgG cross-linking (Adler et a!., 1984). Although some assays have suggested that restriction of the Tg antibody response to the non-complement fixing IgG4 subclass may be responsible (Parkes et al., 1984), this seems unlikely as affinity purification of the IgG subclasses

Clinical Endocrinology (1992) 36

shows that, although IgG4 is over-represented, it accounts for around only 30% of the Tg antibody contained in total IgG (Weetman et al., 1989a).Tg antibodies of both the IgGl and IgG4 subclasses can mediate antibody-dependent cellmediated cytotoxicity (ADCC), which may have pathogenic importance (see below). Anti-idiotypic antibodies against Tg antibodies have been described in EAT (Zanetti & Bigazzi, 1981) but their role in regulating autoantibody production in vivo is unknown. Thyroid peroxidase antibodies The immunological as well as molecular characteristics of TPO have been reviewed extensively elsewhere (Banga et d., 1991). There now seems little doubt that the thyroid microsomal/microvillar antigen and TPO are identical, and it is this antigen which is recognized by complement-fixing,cytotoxic antibodies in autoimmune thyroiditis (Khoury et al,, 1981). Despite its location on the apical surface of thyroid cells, antibodies can reach and bind to the antigen in viuo (Khoury et al., 1984). Antibodies against TPO in thyroiditis are polyclonal, being found in all four IgG subclasses, distributed between both light chains and recognizing at least six different epitopes, including one or both of the sites for catalytic peroxidation (Doble et a/., 1988; Weetman et al., 1989a).Whether patient subsets can be identified which correspond to the different serological reactivities to TPO is unclear. Antibodies binding to the catalytic sites may inhibit enzyme activity. which could contribute to the generation of hypothyroidism; TPO antibodies from otherwise healthy subjects in contrast do not block TPO activity (Kohno er a/., 1991). Although these results have recently been disputed on methodological grounds (Salter ef al., 1991), it is possible that a sufficiently pure microsomal preparation is required to demonstrate enzyme inhibition in uifro (Banga e f al., 1991). Another epitope which has provoked some controversy is one reportedly shared with Tg (Kohno et al., 1988). Sera recognizing this epitope were detected in a high proportion ofpatients with thyroiditis but not in euthyroid controls with thyroid antibodies (Naito et al., 1990).Others have found no evidence for cross-reactivity, again relating previous results to differences in TPO purity (Henry et al., 1991). The pathogenic role of TPO antibodies is considered in the next section.

Pathogenic mechanisms in autoimmune thyroiditis Both classical limbs of the immune response, humoral and cellular, participate in tissue injury. Several lines of evidence suggest a major role for complement-fixing antibodies, almost certainly against TPO, in mediating cell damage. Immune complexes have been demonstrated around the

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317

thyroid follicular basement membrane in Hashimoto’s thyroiditis, as have terminal complement complexes (TCCs), the fluid phase products released after formation of membrane attack complexes (MACs) (Weetman et al., 1989b). In addition, serum TCC concentrations are raised in autoimmune thyroid diseases and their level may reflect disease activity. A striking but often unappreciated feature of complementmediated injury is the resistance of nucleated cells to lysis by complement which is homologous (i.e. derived from the same species).Two mechanisms account for this: active removal of MACs from the cell surface, requiring energy expenditure, and expression of proteins which prevent assembly and insertion of MACs into the cell membrane. Human thyro? cytes in vitro are relatively resistant to lysis by homologous complement, and this seems to be due in part to the expression of two protective proteins, CD59 and MIP/HRF; both are upregulated by exposure of thyrocytes to cytokines (IL-I, TNF and y-IFN) which are known to be produced by the thyroid lymphocytic infiltrate (Weetman et al., 1990b, Tandon et a[., 1992). However, even sublethal complement attack impairs the response of thyroid cells to TSH and causes them to release prostaglandins and reactive oxygen metabolites, which have potent inflammatory potential. It is possible that scavenging of such oxygen radicals by antithyroid drugs (known to be concentrated by the thyrocyte) and by antioxidants administered experimentally explains the amelioration of thyroiditis observed with these agents (Weetman et al., 1984; Bagchi et al., 1990). Compelling evidence for the role of thyroid-specific cytotoxic T cells exists in murine EAT (Creemers et al., 1983) but such cells have been difficult to demonstrate in human thyroiditis. Expansion of the infiltrating lymphocytes by culture with IL-2 has shown that many of the T cells in Hashimoto’s thyroiditis (but not Graves’ disease) have nonspecific cytotoxic effects (Canonica et al., 1985; Del Prete et al., 1986; MacKenzie er al., 1987). Such lymphokineactivated killer (LAK) cells have been well described in nonautoimmune settings: their pathogenic relevance to thyroiditis is obscure, as is the killing of thyrocytes by NK cells, which also lacks target specificity. Only one thyroid-specific cytotoxic T cell clone, derived from a Hashimoto patient, has so far been described; the nature of the autoantigen recognized by these cells was not elucidated (MacKenzie et al., 1987). Despite this, indirect evidence suggests that thyrocytes themselves enhance their susceptibility to cell-mediated injury in thyroiditis by expressing adhesion molecules, which are central to the recognition of targets by cytotoxic T lymphocytes (Fig. 2). Thyroid cells in autoimmune thyroiditis express intercellular adhesion molecule- I (ICAM-I),

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which binds to lymphocytic function-associated molecule-1 (LFA-1) on T cells, and this expression can be enhanced in ritvo by treatment with y-IFN or TNF (Weetman et al., 1989~).Blocking ICAM-1 with an appropriate monoclonal antibody partially prevents both T cell adherence to thyrocytes and LAK cell-mediated lysis (Weetman et al., 1990~). While one group has suggested that this property of ICAM-1 expression by thyrocytes is unique to Hashimoto's thyroiditis (Bagnasco et al., 1991), the weight ofevidence is in favour of it being a non-specific effect, presumably mediated by cytokines released in all types of autoimmune thyroid disease (Weetman et al., 1989~;Martin et al., 1990; Zheng et al., 1990). Thyrocytes also express LFA-3 which binds to CD2 on T cells (Zheng et al., 1990). Like ICAM-I, LFA-3 is upregulated by cytokines and participates in T cell adherence to thyrocytes (N. Tandon et al., unpublished observations). Besides rendering thyrocytes more susceptible to T cellmediated lysis, ICAM-1 and LFA-3 expression could enhance the possible antigen presenting function of thyrocytes, as adhesion molecule binding precedes that between T cell receptor and the MHC class 11-antigen bimolecular complex. Conversely, class I1 expression by thyroid cells could lead to their destruction by CD4+ cytotoxic T cells. Both of these remain theoretical possibilities. A second form of specific cytotoxicity, ADCC, is mediated by NK cells via their interaction through immunoglobulin Fc receptors with antibody bound to a target cell, contrasting with the non-specific effector function of NK cells in the absence of antibody. Studies of NK cell numbers and ADCC function in autoimmune thyroiditis have been inconclusive in demonstrating abnormalities (Bogner et al., 1984; Sack et al., 1986). Recently it has been suggested that the thyroid antibodies which mediate ADCC in uitro are not directed against TPO, as previously supposed, but recognize instead a novel autoantigen (Bogner et a[., 1990). As mentioned above, several new thyroid autoantigens have been identified by molecular cloning and further definition of antigen/antibody systems with the potential to mediate ADCC should soon be possible. Whether Tg antibodies contribute in uiuo to ADCC is unknown, although they can mediate this function in uitro (Weetman et al., 1989a). Finally, cytokines released by the inflammatory infiltrate may have a role in altering thyroid cell function and even causing direct injury. Thyroid cell lines like FRTL-5 are prevented from dividing by culture with y-IFN and TNF (together but not alone), although primary thyrocyte cultures are far less susceptible to such effects (Weetman & Rees, 1988), incidentally reinforcing the caveats discussed previously regarding extrapolation from experiments with cell lines. Human thyrocyte cultures appear resistant to any

Clinical Endocrinology (1992) 36

cytotoxicity mediated by cytokines (McLachlan et al., 1990). Nonetheless, more subtle effects on thyroid cell function may be produced by TNF, y-IFN, IL-1 and IL-6 which impair the thyroid response to TSH (Nagayama et al., 1987; Sato et a/., 1990; Weetman et al., 1990a). Conclusions

Autoimmune thyroiditis is the result of an interaction between genetic predisposition and non-genetic factors; their relative contribution varies between patients. HLA genes partly determine susceptibility but the effects are weak and population dependent; immunoglobulin genes may also be important as associations with Gm allotypes have been reported and thyroid autoantibody production is established as an autosomal dominant trait. The impact of other genes remains to be defined. Sex hormones modify the penetrance of thyroid autoantibody production and thyroiditis, and pregnancy has a major effect on these parameters which may be due in part to alterations in sex hormones and prolactin. There is compelling evidence from EAT that infection and dietary iodide are two environmental factors which can initiate disease, and epidemiological studies support a role for iodide in human thyroiditis. It is probable that the great majority of thyroid-reactive T cells are deleted or become tolerant within the thymus during development; those that escape may be held in check by active suppression (perhaps via anti-idiotypic interactions with other T cells) or by peripheral tolerance, whereby exposure to thyroid antigens and class I1 MHC molecules on y-IFN-stimulated thyrocytes, in the absence of a costimulatory signal, leads to anergy. It remains possible that class I1 thyrocytes can present autoantigen in certain circumstances, perhaps acquiring the ability to deliver a second signal in established thyroiditis, but the contribution this might make seems unlikely to be very large. Thyrocytes display additional responses to cytokines released by the inflammatory infiltrate, synthesising IL-6, increasing ICAM- 1 and LFA-3 expression, producing oxygen radicals and prostaglandins and enhancing their resistance to complement-mediated injury by expression of CD59 and MIP/HRF. Given the complexity of control over autoreactive T and B cells, and the participation of the thyroid cells themselves in a number of immunological processes, it is perhaps not surprising that autoimmune thyroiditis is so common. What of the future? There is clearly a lot still to be learned about the immunogenetics of thyroiditis as the contribution from the MHC HLA-D region is small: family studies using a range of candidate gene probes will be required to advance our knowledge. The search for environmental triggering factors will intensify, probably through the use of animal +

Clinical Endocrinology (1992) 36

models, and the exact role ofdietary iodine should be defined soon. It is also reasonable to expect rapid progress in the areas of intrathyroidal cytokine effects and interactions, T and B cell epitopes on thyroid antigens, and restriction of T cell receptor V gene usage by infiltrating lymphocytes. However, developments in basic immunology, such as characterization of costimulatory signals and precise identification of T suppressor cells, will be necessary to answer the difficult questions regarding antigen presentation by thyrocytes and the importance of active peripheral control of thyroid-specific T cells in preventing autoimmune thyroiditis. From such developments, we may also expect an answer to the question that applies to many autoimmune diseases: if central tolerance is critical to preventing autoreactivity, how can the entire set of self antigens be presented to immature T cells within the thymus so that the appropriate cells are deleted? This is not simply a question of autoantigen number; the ontogeny of certain proteins and their presence firmly within or on specific cells seems intuitively to militate against their intrathymic expression. There is little doubt that thyroiditis will continue to serve as an excellent model for organ-specific autoimmunity and answers to these problems are likely to be applicable t o other conditions such as type 1 diabetes, in which difficulties of target organ access in particular preclude much of the work that is possible with the thyroid. Acknowledgement

The support of the Wellcome Trust is gratefully acknowledged. References Adler, T.R., Beall, G.N., Curd, J.G., Heiner, D.C. & Shabharwal, U.K. (1984) Studies of complement activation and IgG subclass restriction of anti-thyroglobulin. Clinical and Experimental Immunology, 56,383-389. Allen, E.M., Appel, M.C. & Braverman, L.E. (1986) The effect of iodine ingestion on the development of spontaneous lymphocytic thyroiditis in the diabetes prone BB/W rat. Endocrinology, 118, 1977-1981. Atherton, M.C., McLachlan, S.M., Pegg, C.A.S., Dickinson, S., Baylis, P., Young, E.T., Proctor, S.J. & Rees Smith, B. (1985) Thyroid autoantibody synthesis by lymphocytes from different lymphoid organs; fractionation of B cells on density gradients. Immunology, 55, 27 1-279. Badenhoop, K., Schwarz, G., Walfish, P.G., Drummond, V., Usadel, K.H. & Bottazzo, G.F. (1990) Susceptibility to thyroid autoimmune disease: molecular analysis of HLA-D region genes identifies new markers for goitrous Hashimoto’s thyroiditis. Journal of Clinical Endocrinology and Metabolism, 71, 1131. Bagchi, N., Brown, T.R., Hedergen, D.M., Dhar, A. & Sundick, R. (1990) Antioxidants delay the onset of thyroiditis in obese strain chickens. Endocrinology, 127, 1590- 1595.

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Autoimmune thyroiditis: predisposition and pathogenesis.

Clinical Endocrinology (1992)36, 307-323 Review Autoimmune thyroiditis: predisposition and pathogenesis A. P. Weetman Department of Medicine, Univer...
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