25
SCIENCE & PRACTICE
Thyroid-associated eye disease: pathophysiology
complications associated most typically with hyperthyroidism caused by Graves’ disease range from discomfort and lid retraction to disfiguring proptosis, diplopia, and sight loss. Usually called Graves’ ophthalmopathy, this condition is better termed thyroidassociated eye disease (or ophthalmopathy), emphasising that a spectrum of thyroid disorders may occur in these patients. It is one of the most difficult autoimmune disorders to investigate. Severe eye disease in uncommon, there is no animal model, and the target tissues are relatively inaccessible. Any hypothesis about the aetiology and pathogenesis must explain the recondite relation of ophthalmopathy to thyroid disease and the often asymmetric changes. I will concentrate on recent insights into the development of this condition; earlier work has been reviewed extensively.’1
Predisposing factors
The eye
Relation to autoimmune thyroid disease
hyperthyroidism occurs in about 90% of patients thyroid-associated eye disease. The remaining patients are said to have ophthalmic Graves’ disease, which in about Graves’
with
half of these
cases is associated with autoimmune whereas the others have no overt thyroid hypothyroidism, abnormalities biochemically. However, more detailed analysis has shown that most, if not all, of these patients have autoimmune thyroid disease, and this becomes more obvious with prolonged follow-up.1 About one-third of those who are euthyroid have circulating thyroid-stimulating hormone (TSH) receptor antibodies, whereas two-thirds have
thyroglobulin
or
thyroid
peroxidase
(microsomal)
antibodies.2 In a recent series of 22 such patients,3various novel immunological markers, based on detection of antibodies against thyroid membranes and the presence of thyroiditis on biopsy, were also assessed, and some evidence of thyroid autoimmunity was found in all cases. Not only do almost all patients with ophthalmopathy have an autoimmune thyroid disorder but also in most patients with Graves’ hyperthyroidism the eye is affected to some extent. This may often be clinically inapparent but can be detected with ultrasonography, computed tomography, or evaluation of saccadic eye movement.1 There is also a close temporal relation between onset of the two disorders; in more than two-thirds of patients, both hyperthyroidism and ophthalmopathy develop within two years.1 These increasingly technologically advanced clinical studies show that Graves’ hyperthyroidism and ophthalmopathy must share some pathogenetic mechanisms, which are probably related to cross-reactivity between autoantigens in the retrobulbar tissues and the thyroid. However, this leaves unexplained the infrequent development of eye disease in autoimmune hypothyroidism, in which presumably similar cross-reactivity is rare. Antigenic cross-reactivity also does not readily account for the development of severe (congestive) ophthalmopathy in only 3-5% of patients with Graves’ hyperthyroidism.1
approach to address these points has been the analysis of immunogenetic and environmental factors associated with severe ophthalmopathy. Several studies have compared human leucocyte antigen (HLA) alleles in patients who have Graves’ disease with and without severe eye disease. Generally these studies have failed to reveal any consistent important differences between the two groups. The prevalence of HLA-DR4 and HLA-DRw6 was higher in USA black patients with thyroid-associated eye disease than in control patients, but the importance of this finding is uncertain since there was no clear difference from patients with Graves’ thyrotoxicosis when the results were corrected for the number of alleles tested.4 The well recognised association of HLA-DR3 with Graves’ thyrotoxicosis in Caucasians may be slightly stronger in patients with eye disease in certain areas (eg, Hungary); this has not been found in the UK.5,6 Other candidate susceptibility genes, encoding immunoglobulin allotypes and T-cell receptor polymorphisms, have also been unrevealing,5 and the weak association with blood group P found in a survey of 18 diverse genetic markers did not survive statistical correction for the number of variables tested.6 One possibility not yet examined is that genetic susceptibility to Hashimoto’s thyroiditis confers moderate protection against the development of eye disease. The sex distribution is more nearly equal in patients with overt ophthalmopathy than in those with Graves’ hyperthyroidism.1 One explanation may lie in the strong association between smoking and ophthalmopathy. Both the presence and the severity of eye signs are associated with increasing tobacco consumption, until recently a habit more pronounced in men. The reasons for the association are unknown but could relate to immunomodulatory effects of smoking or to the enhanced release of cross-reactive autoantigens from the thyroid; smoking is goitrogenic and increases thyroglobulin release from the gland. Clearly there are other contributory factors, because the aetiological fraction produced by smoking is less than 0-5 (the maximum value for a disease-associated factor is 1 -0). Thyroid status and treatment may also be important. In a retrospective study of 90 patients referred for evaluation of ophthalmopathy, euthyroidism was less frequent in those with more severe eye signs.8 However, it is impossible to be sure that this effect is merely related to thyroid hormones, since the duration and previous treatment of thyroid dysfunction were not stated. For example, radioiodine therapy for Graves’ disease can lead to deterioration of pre-existing ophthalmopathy, although it does not initiate clinical disease in patients without previous eye One
involvement.9 Similar observations have been made after ADDRESS: Department of Medicine, University of Sheffield Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7A, UK (Prof A. P. Weetman, FRCP).
26
irradiation to the neck for non-thyroid-associated disorders and, occasionally, after surgery.1 Such findings are compatible with the existence of a cross-reactive autoantigen, the thyroidal release of which is increased by such treatment. However, other possibilities should also be considered-eg, non-specific effects of radiation on circulating T-cell populations.
generally failed to identify any eye-muscle-specific proteins (human or porcine) that are recognised exclusively by sera from patients with thyroid-associated eye disease. 14-17 However, Zhang et ap8 have found that up to 50% of such sera react
with certain components of relative molecular
weights 95, 64, and 55 kDa. The 55 kDa antigen seemed to be confined to eye muscle, whereas those at 64 and 95 kDa shared with skeletal muscle and thyroid tissue. These studies remain unconfirmed so far. Indeed, other investigators have suggested that antibodies against the 64 kDa protein have no pathological importance,17 and a 64 kDa antigen recognised by normal sera has been found in retro-ocular fibroblasts.19 were
Extraocular
changes
Some clues about
pathogenesis have come from histological examination, although material is rare. The extraocular muscles are the principal targets of the autoimmune response and the main changes are in the interstitium. There is both a diffuse and a focal mononuclear cell infiltration primarily by activated T cells, with some B cells and occasional macrophages and mast cells (fig 1).10 This infiltration is accompanied by activation of the fibroblasts (which, in contrast to the muscle cells, express la or MHC class II molecules); interstitial oedema (due to increased glycosaminoglycan and water content), and, later, fat cell infiltration, fibrosis, and atrophy. Lymphocytes may infiltrate the lacrimal gland but the orbital fat and connective tissue are hardly affected. Apart from subsarcolemmal deposits of lipid and glycogen, the extraocular muscle fibres appear strikingly normal, with no histological evidence of active destruction as a cause of the disease." This observation is consistent with normal electromyographic studies of the extraocular muscles in most patients with severe ophthalmopathy; the restriction in movement is accounted for by the fibrosis and the increased volume of orbital contents.1 A separate change occurs in the levator palpebrae superioris muscle: there is negligible lymphocytic infiltration, extracellular volume is not increased, and the muscle fibres become greatly enlarged, leading to hypertrophy of the levator muscle and upper eyelid retraction."
Retrobulbar autoantibodies
autoantibodies, Circulating thyroid against thyroglobulin, thyroid peroxidase, and the TSH receptor, are not related to the development or severity of ophthalmopathy ; slightly higher concentrations may be present in eye disease patients, but there is a very wide range .2 The search for retrobulbar autoantibodies has had two goals-namely, to provide a tool for establishing the frequency and severity of ophthalmopathy and to provide information on the initiating autoantigen. Many approaches and antigen sources have been used, with the consequence that no consistent results have yet emerged. High concentrations of circulating IgG in human extraocular tissue can make detection of exogenous antibody binding very difficult. Therefore, ultracentrifuged membranes from porcine eye muscle were used to develop an enzyme-linked immunosorbent assay, initial results were promising.12 Antibodies against this source of antigen were found in 64% of patients with clinically overt thyroid-associated eye disease but were undetectable in controls (or in those with only Graves’ hyperthyroidism, a surprising observation in view of the very high frequency of covert eye disease). Furthermore, these antibodies seemed to be tissue specific, failing to react with porcine skeletal muscle, liver, or fat cell membranes. The sensitivity and specificity of this assay have proved far less impressive subsequently.13-17 In most studies there is a very close correlation between eye and skeletal muscle binding, as might be expected from the definition of actin, tubulin, and acetylcholine receptor as some of the important antigens.13 Immunoblotting studies have
Cross-reactive retrobulbar autoantigens The hypothetical autoantigen shared between retrobulbar tissue and the thyroid has eluded identification. Immunoreactive thyroglobulin-like material found in eye muscle membranes pointed to one candidate1 and interest was renewed recently with the discovery that thyroglobulin shares an impressive sequence homology and hydropathy profile with acetylcholinesterase. Could thyroglobulin antibodies bind to eye muscle acetylcholinesterase and thereby produce eye disease? A shared epitope on the two molecules (that was probably sequential rather than conformational), which bound antibodies in sera from patients with thyroid-associated eye disease, has been identified. Only one of eight Hashimoto sera reacted, despite the high titres of thyroglobulin antibodies in these patients.20 If indeed this is a sequential epitope present only on the unfolded proteins (known as a cryptotope or unfoldon), it is difficult to understand how antibodies against it could have a primary role in the disease.21 The reasons why recognition of such a cryptotope should be restricted to eye muscle and only occur in Graves’ disease (when thyroglobulin antibodies are more frequent in Hashimoto’s thyroiditis) are also unclear. A second candidate, initially recognised by screening an expressed human thyroid cDNA library with Hashimoto sera, is a novel 64 kDa autoantigen.22 This antigen is probably membrane bound and has no sequence homology with known thyroid antigens. The mRNA for this protein can be detected in human thyroid tissue and eye muscle (but not skeletal muscle), whereas in the dog the canine homologue was present in all organs tested. It is unclear whether this molecule is related to the 64 kDa antigen referred to earlier, 18 since autoantibodies that recognise the recombinant protein may not correlate with the presence of ophthalmopathy and have even been detected in apparently healthy controls.22 Whether the widespread distribution of the protein in dogs occurs in man is also not yet known.
T-cell responses Function studies have obviously been confined to circulating T cells, which will reflect imprecisely (if at all) the autoimmune responses of the cells infiltrating the retro-ocular muscles. T-cell recognition of antigens in eye muscle has been shown in patients with overt ophthalmopathy by assays for T-cell proliferation and production of migration inhibition factor, although the latter method has been difficult to reproduce.15.23 Like autoantibodies, the responsible antigens seem to be shared with skeletal muscle (and lacrimal gland) and T-cell reactivity is not confined to patients with thyroid-associated eye disease. Some efforts have been directed to identifying immunologically mediated cytotoxicity against eye muscle
27
cells in vitro. Presently there is no evidence for cytotoxic T cells specific for retrobulbar tissues. However, half of ophthalmopathy patients have antibodies that can mediate antibody-dependent cell-mediated cytotoxicity.18 This type of tissue injury is produced by natural killer cells that bind via their Fc receptors to antibodies on a target cell. The autoantibodies described do not fix complement, are undetectable in normal sera, and correlate with severity of eye disease (as judged by the intraocular pressure in the primary position). The antigen they recognise is unknown but is shared with thyroid cells. These findings are difficult to reconcile with the absence of muscle-cell destruction in
ophthalmopathy. The extraocular muscle fibroblast The histological evidence points to the retrobulbar fibroblast rather than myocyte as the primary target for the autoimmune process; a similar response to skin fibroblasts could account for Graves’ dermopathy, which is strongly associated with the presence of severe eye disease. Early experiments showed that glycosaminoglycan production by cultured normal retro-ocular fibroblasts was increased by co-incubation with lymphocytes from healthy donors, and this is further enhanced by mitogen stimulation of the T cells.1 This is entirely in keeping with the now recognised effects on fibroblasts of a variety of cytokines derived from T cells and macrophages: Cytokine (main source) Interleukin-I (macrophage) Tumour necrosis factor-a
(macrophage)
Effects on fibroblast Stimulates proliferation Variable effects on proliferation and collagen
synthesis Fibroblast growth factors*
Stimulate proliferation
(macrophage) Platelet derived growth factor (macrophage, platelet)
Stimulates proliferation and collagen synthesis
Transforming growth factors-p (macrophage, T cell) Interferon-&bgr; (macrophage, T cell) Lymphotoxin (T cell) Interferon-y (T cell)
Diverse; generally fibrogenic
Fibroblast activating factors
Enhance connective tissue
Inhibits
stimulate collagen synthesis by fibroblasts have been described, and a close correlation was reported between such immunoglobulins in Graves’ patients and severity of ophthalmopathy.24 Other investigations2S.26 have not reproduced these findings and a primary role for such antibodies does not accord with the presence of clinically significant ophthalmopathy in some patients without
TSH receptor antibodies. Patients with dermopathy have a subgroup of TSH receptor antibodies that are unusually potent stimulators of thyroid cell growth and protein synthesis 26 One product of TSH-stimulated thyroid cells can act as a cofactor to potentiate the mitogenic effect of insulin-like growth factor-1 on fibroblasts.27 That fibroblast growth factors could simulate the effect of this unknown cofactor suggests that excessive release of such thyroid-derived cytokines, stimulated by a subset of TSH receptor antibodies or by other intrathyroidal autoimmune mechanisms, could have a role in ophthalmopathy (and dermopathy). It is noteworthy that no study has yet addressed T-cell recognition of retro-ocular fibroblast antigens; it is entirely possible that local fibroblast activation could be due to cytokine release from infiltrating autoreactive T cells, unaccompanied by any antibody formation. Finally, the anatomical complexity of the extraocular muscles may explain the specificity of muscle involvement in ophthalmopathy.1,11 By comparison with skeletal muscle, the extraocular muscles have more spindles, more abundant interstitial connective tissue and nerve fibres, and a much greater blood flow. Pattern of blood supply varies both between and within individuals; this may be a factor in the asymmetry of clinical signs so frequently seen. There may be less difference between the six extraocular muscles in their pathological involvement than is apparent clinically; the characteristic limitation of elevation is due not only to fibrosis of the inferior rectus but also to changes in the fibrous connective tissue septa within the orbit. detectable
Conclusions
proliferation There is
increasing evidence that thyroid-associated ophthalmopathy is intrinsically linked to thyroid
Stimulates proliferation Inhibits proliferation and
collagen synthesis synthesis
(T cell) *Regulation
of the
important in
vanous
bioavailability
of fibroblast
growth factors
may be
diseases
the intramuscular inflammatory infiltrate could fibroblast activation and produce the clinical signs of ophthalmopathy as an end result, the reasons why the response should be so restricted to retro-ocular muscles and associated with thyroid autoimmunity are unclear. One explanation for localisation is that retrobulbar fibroblasts may be antigenically unique. To address this possibility, extracts of cultured fibroblasts from different sites have been used to detect specific antibodies in sera from patients with ophthalmopathy. Although an initial report suggested that there was diversity in antigen expression by fibroblasts, a follow-up study, in which immunoblotting was used to detect autoantibodies, did not fmd any antigenic differences between fibroblasts from different sites.19 About half the sera from patients with Graves’ disease recognised a 23 kDa protein found in all fibroblast preparations; the presence of these antibodies did not correlate with clinically obvious eye disease (or dermopathy). The association with thyroid autoimmunity may be due to the production of certain fibroblast stimulating factors in these disorders. Monoclonal TSH receptor antibodies that
Although cause
Fig 1-Section of extraocular muscle
in
thyroid-associated
eye
disease. Diffuse and focal infiltration
by T cells (red) and substantial interstitial
oedema; muscle fibres intact (original magnification x 400).
28
heterogeneity in black American patients with Graves’ disease. Arch
Fig
2-Possible
pathogenic mechanisms.
Predominant T-cell infiltration of the retro-ocular muscles may arise from recognition of antigens shared with the thyroid. These T cells, in addition to macrophages, release cytokines (see text) that stimulate fibroblasts, producing oedema and fibrosis.
the number of patients with ophthalmic Graves’ disease without any evidence of thyroid involvement continues to diminish as increasingly sophisticated measures of thyroiditis are used and follow-up is continued. The evidence for a separate immunogenetic predisposition to ophthalmopathy is inconsistent, and environmental factors such as smoking may be more important in the development of overt eye disease. In view of the long natural history of autoimmune thyroid disorders, it is impossible to be sure whether eye disease ever precedes or always follows the thyroid lesion. The most likely explanation for the association is the sharing of an autoantigen between the thyroid and the extraocular muscles: one candidate is a novel 64 kDa thyroid and eye-muscle antigen, although the cellular localisation of this is as yet unknown. The primary pathogenic mechanism in ophthalmopathy seems to be fibroblast stimulation, probably as a result of cytokine release by the activated T cells that accumulate in the muscles (fig 2). This localisation could be directed by T-cell recognition of cross-reactive antigens and by the anatomical features of the extraocular muscles. By contrast, there is little histological evidence for muscle cell destruction by the inflammatory infiltrate. Attempts to detect eye muscle autoantibodies have failed to prove their consistent presence in ophthalmopathy patients or their eye muscle specificity. Production of eye muscle reactive antibodies is probably a secondary event, that many of the antigens recognised are cytoskeletal components accounts for the broad specificity of these antibodies, which have an uncertain pathogenic role. Whether there are other antibodies that can stimulate fibroblasts directly or indirectly also remains unclear. Further knowledge about the pathogenic mechanisms should lead to safe and specific immunological treatment for this enigmatic condition.
autoimmunity;
REFERENCES 1. Jacobson DH, Gorman CA. Endocrine ophthalmopathy: current ideas concerning etiology, pathogenesis and treatment. Endocr Rev 1984; 5: 200-20. 2. Bech K. Thyroid antibodies in endocrine ophthalmopathy. A review. Acta Endocrinol 1989; 121 (suppl 2): 117-22. 3. Salvi M, Zhang Z-G, Haegert D, et al. Patients with endocrine ophthalmopathy not associated with overt thyroid disease have multiple thyroid immunological abnormalities. J Clin Endocrinol Metab 1990; 70: 89-94. 4. Sridama V, Hara Y, Fauchet R, De Groot LJ. HLA immunogenetic
Intern Med 1987; 147: 229-31. 5. Weetman AP, So AK, Warner CA, Foroni L, Fells P, Shine B. Immunogenetic markers in Graves’ ophthalmopathy. Clin Endocrinol 1988; 28: 619-28. 6. Kendall-Taylor P, Stephenson A, Stratton A, Papiha SS, Perros P, Roberts DF. Differentiation of autoimmune ophthalmopathy from Graves’ hyperthyroidism by analysis of genetic markers. Clin Endocrinol 1988; 28: 601-10. 7. Shine B, Fells P, Edwards OM, Weetman AP. Association between Graves’ ophthalmopathy and smoking. Lancet 1990; 335: 1261-64. 8. Prummel MF, Wilmar MD, Wiersinga M, Koornneef L, Berghout A, van der Gaag R. Effects of abnormal thyroid function on the severity of Graves’ ophthalmopathy. Arch Intern Med 1990; 150: 1098-1101. 9. Bartalena L, Marcocci C, Bogazzi F, Panicucci M, Lepri A, Pinchera A. Use of corticosteroids to prevent progression of Graves’ ophthalmopathy after radioiodine therapy for hyperthyroidism. N Engl J Med 1989; 321: 1350-59. 10. Weetman AP, Cohen S, Gatter KC, Fells P, Shine B. Immunohistochemical analysis of the retrobulbar tissues in Graves’ ophthalmopathy. Clin Exp Immunol 1989; 75: 222-27. 11. Campbell RJ. Immunology of Graves’ ophthalmopathy: retrobulbar histology and histochemistry. Acta Endocrinol 1989; 121 (suppl 2): 9-16. 12. Atkinson S, Holcombe M, Kendall-Taylor P. Ophthalmopathic immunoglobulin in patients with Graves’ ophthalmopathy. Lancet 1984; ii: 374-76. 13. Kadlubowski M, Irvine WJ, Rowland AC. Anti-muscle antibodies in Graves’ ophthalmopathy. J Clin Lab Immunol 1987; 24: 105-11. 14. Ahmann A, Baker JR, Weetman AP, Wartofsky L, Nutman TB, Burman KD. Antibodies to porcine eye muscle in patients with Graves’ ophthalmopathy; identification of serum immunoglobulins directed against unique determinants by immunoblotting and enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 1987; 64: 454-60. 15. Weetman AP, Fells P, Shine B. T and B cell reactivity to extraocular and skeletal muscle in Graves’ ophthalmopathy. Br J Ophthalmol 1989; 73: 323-27. 16. Schifferdecker E, Ketzler-Sasse U, Boehm BO, Ronsheimer HB, Scherbaum WA, Schoffling K. Re-evaluation of eye muscle autoantibody determination in Graves’ ophthalmopathy: failure to detect a specific antigen by use of enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting techniques. Acta Endocrinol 1989; 121: 643-50. 17. Weightman D, Kendall-Taylor P. Cross-reaction of eye muscle antibodies with thyroid tissue in thyroid-associated ophthalmopathy. J Endocrinol 1989; 122: 201-06. 18. Zhang S-G, Medeiros-Neto G, Iacona A, et al. Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroidassociated ophthalmopathy. Acta Endocrinol 1989; 121 (suppl 2): 23-30. 19. Bahn RS, Gorman CA, Johnson CM, Smith TJ. Presence of antibodies in the sera of patients with Graves’ disease recognizing a 23 kilodalton fibroblast protein. J Clin Endocrinol Metab 1989; 69: 622-28. 20. Ludgate M, Dong Q, Dreyfus PA, et al. Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves’ ophthalmopathy. Autoimmunity 1989; 3: 167-76. 21. Laver WG, Air GM, Webster RG, Smith-Gill SJ. Epitopes on protein antigens: misconceptions and realities. Cell 1990; 61: 553-56. 22. Dong Q, Ludgate M, Vassart G. Cloning and sequencing of a novel 64 Kd autoantigen recognised by patients with autoimmune thyroid disease. J Clin Endocrinol Metab (in press). 23. Munro RE, Lamki L, Row VV, Volpé R. Cell-mediated immunity in the exophthalmos of Graves’ disease as demonstrated by the migration inhibition factor (MIF) test. J Clin Endocrinol Metab 1973; 37: 286-92. 24. Rotella CM, Zonefrati R, Toccafondi R, Valente WA, Kohn LD. Ability of monoclonal antibodies to the thyrotropin receptor to increase collagen synthesis in human fibroblasts: an assay which appears to measure exophthalmogenic immunoglobulins in Graves’ sera. J Clin Endocrinol Metab 1986; 62: 357-67. 25. Westermark K, Lilja K, Karlsson FA. Effects of sera and immunoglobulin preparations from patients with endocrine ophthalmopathy on the production of hyaluronate and the incorporation of tritiated thymidine in fibroblasts. Acta Endocrinol 1989; 121 (suppl 2): 85-89. 26. Tao T-W, Leu S-L, Kriss JP. Biological activity of autoantibodies associated with Graves’ dermopathy. J Clin Endocrinol Metab 1989; 69: 90-99. 27. Takahashi S-I, Conti M, Van Wyk JJ. Thyrotropin potentiation of insulin-like growth factor-I dependent deoxribonucleic acid synthesis in FRTL-5 cells: mediation by an autocrine amplification factor(s). Endocrinology 1990; 126: 736-45.
SCIENCE & PRACTICE
Thyroid-associated eye disease: pathophysiology
complications associated most typically with hyperthyroidism caused by Graves’ disease range from discomfort and lid retraction to disfiguring proptosis, diplopia, and sight loss. Usually called Graves’ ophthalmopathy, this condition is better termed thyroidassociated eye disease (or ophthalmopathy), emphasising that a spectrum of thyroid disorders may occur in these patients. It is one of the most difficult autoimmune disorders to investigate. Severe eye disease in uncommon, there is no animal model, and the target tissues are relatively inaccessible. Any hypothesis about the aetiology and pathogenesis must explain the recondite relation of ophthalmopathy to thyroid disease and the often asymmetric changes. I will concentrate on recent insights into the development of this condition; earlier work has been reviewed extensively.’1
Predisposing factors
The eye
Relation to autoimmune thyroid disease
hyperthyroidism occurs in about 90% of patients thyroid-associated eye disease. The remaining patients are said to have ophthalmic Graves’ disease, which in about Graves’
with
half of these
cases is associated with autoimmune whereas the others have no overt thyroid hypothyroidism, abnormalities biochemically. However, more detailed analysis has shown that most, if not all, of these patients have autoimmune thyroid disease, and this becomes more obvious with prolonged follow-up.1 About one-third of those who are euthyroid have circulating thyroid-stimulating hormone (TSH) receptor antibodies, whereas two-thirds have
thyroglobulin
or
thyroid
peroxidase
(microsomal)
antibodies.2 In a recent series of 22 such patients,3various novel immunological markers, based on detection of antibodies against thyroid membranes and the presence of thyroiditis on biopsy, were also assessed, and some evidence of thyroid autoimmunity was found in all cases. Not only do almost all patients with ophthalmopathy have an autoimmune thyroid disorder but also in most patients with Graves’ hyperthyroidism the eye is affected to some extent. This may often be clinically inapparent but can be detected with ultrasonography, computed tomography, or evaluation of saccadic eye movement.1 There is also a close temporal relation between onset of the two disorders; in more than two-thirds of patients, both hyperthyroidism and ophthalmopathy develop within two years.1 These increasingly technologically advanced clinical studies show that Graves’ hyperthyroidism and ophthalmopathy must share some pathogenetic mechanisms, which are probably related to cross-reactivity between autoantigens in the retrobulbar tissues and the thyroid. However, this leaves unexplained the infrequent development of eye disease in autoimmune hypothyroidism, in which presumably similar cross-reactivity is rare. Antigenic cross-reactivity also does not readily account for the development of severe (congestive) ophthalmopathy in only 3-5% of patients with Graves’ hyperthyroidism.1
approach to address these points has been the analysis of immunogenetic and environmental factors associated with severe ophthalmopathy. Several studies have compared human leucocyte antigen (HLA) alleles in patients who have Graves’ disease with and without severe eye disease. Generally these studies have failed to reveal any consistent important differences between the two groups. The prevalence of HLA-DR4 and HLA-DRw6 was higher in USA black patients with thyroid-associated eye disease than in control patients, but the importance of this finding is uncertain since there was no clear difference from patients with Graves’ thyrotoxicosis when the results were corrected for the number of alleles tested.4 The well recognised association of HLA-DR3 with Graves’ thyrotoxicosis in Caucasians may be slightly stronger in patients with eye disease in certain areas (eg, Hungary); this has not been found in the UK.5,6 Other candidate susceptibility genes, encoding immunoglobulin allotypes and T-cell receptor polymorphisms, have also been unrevealing,5 and the weak association with blood group P found in a survey of 18 diverse genetic markers did not survive statistical correction for the number of variables tested.6 One possibility not yet examined is that genetic susceptibility to Hashimoto’s thyroiditis confers moderate protection against the development of eye disease. The sex distribution is more nearly equal in patients with overt ophthalmopathy than in those with Graves’ hyperthyroidism.1 One explanation may lie in the strong association between smoking and ophthalmopathy. Both the presence and the severity of eye signs are associated with increasing tobacco consumption, until recently a habit more pronounced in men. The reasons for the association are unknown but could relate to immunomodulatory effects of smoking or to the enhanced release of cross-reactive autoantigens from the thyroid; smoking is goitrogenic and increases thyroglobulin release from the gland. Clearly there are other contributory factors, because the aetiological fraction produced by smoking is less than 0-5 (the maximum value for a disease-associated factor is 1 -0). Thyroid status and treatment may also be important. In a retrospective study of 90 patients referred for evaluation of ophthalmopathy, euthyroidism was less frequent in those with more severe eye signs.8 However, it is impossible to be sure that this effect is merely related to thyroid hormones, since the duration and previous treatment of thyroid dysfunction were not stated. For example, radioiodine therapy for Graves’ disease can lead to deterioration of pre-existing ophthalmopathy, although it does not initiate clinical disease in patients without previous eye One
involvement.9 Similar observations have been made after ADDRESS: Department of Medicine, University of Sheffield Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7A, UK (Prof A. P. Weetman, FRCP).
26
irradiation to the neck for non-thyroid-associated disorders and, occasionally, after surgery.1 Such findings are compatible with the existence of a cross-reactive autoantigen, the thyroidal release of which is increased by such treatment. However, other possibilities should also be considered-eg, non-specific effects of radiation on circulating T-cell populations.
generally failed to identify any eye-muscle-specific proteins (human or porcine) that are recognised exclusively by sera from patients with thyroid-associated eye disease. 14-17 However, Zhang et ap8 have found that up to 50% of such sera react
with certain components of relative molecular
weights 95, 64, and 55 kDa. The 55 kDa antigen seemed to be confined to eye muscle, whereas those at 64 and 95 kDa shared with skeletal muscle and thyroid tissue. These studies remain unconfirmed so far. Indeed, other investigators have suggested that antibodies against the 64 kDa protein have no pathological importance,17 and a 64 kDa antigen recognised by normal sera has been found in retro-ocular fibroblasts.19 were
Extraocular
changes
Some clues about
pathogenesis have come from histological examination, although material is rare. The extraocular muscles are the principal targets of the autoimmune response and the main changes are in the interstitium. There is both a diffuse and a focal mononuclear cell infiltration primarily by activated T cells, with some B cells and occasional macrophages and mast cells (fig 1).10 This infiltration is accompanied by activation of the fibroblasts (which, in contrast to the muscle cells, express la or MHC class II molecules); interstitial oedema (due to increased glycosaminoglycan and water content), and, later, fat cell infiltration, fibrosis, and atrophy. Lymphocytes may infiltrate the lacrimal gland but the orbital fat and connective tissue are hardly affected. Apart from subsarcolemmal deposits of lipid and glycogen, the extraocular muscle fibres appear strikingly normal, with no histological evidence of active destruction as a cause of the disease." This observation is consistent with normal electromyographic studies of the extraocular muscles in most patients with severe ophthalmopathy; the restriction in movement is accounted for by the fibrosis and the increased volume of orbital contents.1 A separate change occurs in the levator palpebrae superioris muscle: there is negligible lymphocytic infiltration, extracellular volume is not increased, and the muscle fibres become greatly enlarged, leading to hypertrophy of the levator muscle and upper eyelid retraction."
Retrobulbar autoantibodies
autoantibodies, Circulating thyroid against thyroglobulin, thyroid peroxidase, and the TSH receptor, are not related to the development or severity of ophthalmopathy ; slightly higher concentrations may be present in eye disease patients, but there is a very wide range .2 The search for retrobulbar autoantibodies has had two goals-namely, to provide a tool for establishing the frequency and severity of ophthalmopathy and to provide information on the initiating autoantigen. Many approaches and antigen sources have been used, with the consequence that no consistent results have yet emerged. High concentrations of circulating IgG in human extraocular tissue can make detection of exogenous antibody binding very difficult. Therefore, ultracentrifuged membranes from porcine eye muscle were used to develop an enzyme-linked immunosorbent assay, initial results were promising.12 Antibodies against this source of antigen were found in 64% of patients with clinically overt thyroid-associated eye disease but were undetectable in controls (or in those with only Graves’ hyperthyroidism, a surprising observation in view of the very high frequency of covert eye disease). Furthermore, these antibodies seemed to be tissue specific, failing to react with porcine skeletal muscle, liver, or fat cell membranes. The sensitivity and specificity of this assay have proved far less impressive subsequently.13-17 In most studies there is a very close correlation between eye and skeletal muscle binding, as might be expected from the definition of actin, tubulin, and acetylcholine receptor as some of the important antigens.13 Immunoblotting studies have
Cross-reactive retrobulbar autoantigens The hypothetical autoantigen shared between retrobulbar tissue and the thyroid has eluded identification. Immunoreactive thyroglobulin-like material found in eye muscle membranes pointed to one candidate1 and interest was renewed recently with the discovery that thyroglobulin shares an impressive sequence homology and hydropathy profile with acetylcholinesterase. Could thyroglobulin antibodies bind to eye muscle acetylcholinesterase and thereby produce eye disease? A shared epitope on the two molecules (that was probably sequential rather than conformational), which bound antibodies in sera from patients with thyroid-associated eye disease, has been identified. Only one of eight Hashimoto sera reacted, despite the high titres of thyroglobulin antibodies in these patients.20 If indeed this is a sequential epitope present only on the unfolded proteins (known as a cryptotope or unfoldon), it is difficult to understand how antibodies against it could have a primary role in the disease.21 The reasons why recognition of such a cryptotope should be restricted to eye muscle and only occur in Graves’ disease (when thyroglobulin antibodies are more frequent in Hashimoto’s thyroiditis) are also unclear. A second candidate, initially recognised by screening an expressed human thyroid cDNA library with Hashimoto sera, is a novel 64 kDa autoantigen.22 This antigen is probably membrane bound and has no sequence homology with known thyroid antigens. The mRNA for this protein can be detected in human thyroid tissue and eye muscle (but not skeletal muscle), whereas in the dog the canine homologue was present in all organs tested. It is unclear whether this molecule is related to the 64 kDa antigen referred to earlier, 18 since autoantibodies that recognise the recombinant protein may not correlate with the presence of ophthalmopathy and have even been detected in apparently healthy controls.22 Whether the widespread distribution of the protein in dogs occurs in man is also not yet known.
T-cell responses Function studies have obviously been confined to circulating T cells, which will reflect imprecisely (if at all) the autoimmune responses of the cells infiltrating the retro-ocular muscles. T-cell recognition of antigens in eye muscle has been shown in patients with overt ophthalmopathy by assays for T-cell proliferation and production of migration inhibition factor, although the latter method has been difficult to reproduce.15.23 Like autoantibodies, the responsible antigens seem to be shared with skeletal muscle (and lacrimal gland) and T-cell reactivity is not confined to patients with thyroid-associated eye disease. Some efforts have been directed to identifying immunologically mediated cytotoxicity against eye muscle
27
cells in vitro. Presently there is no evidence for cytotoxic T cells specific for retrobulbar tissues. However, half of ophthalmopathy patients have antibodies that can mediate antibody-dependent cell-mediated cytotoxicity.18 This type of tissue injury is produced by natural killer cells that bind via their Fc receptors to antibodies on a target cell. The autoantibodies described do not fix complement, are undetectable in normal sera, and correlate with severity of eye disease (as judged by the intraocular pressure in the primary position). The antigen they recognise is unknown but is shared with thyroid cells. These findings are difficult to reconcile with the absence of muscle-cell destruction in
ophthalmopathy. The extraocular muscle fibroblast The histological evidence points to the retrobulbar fibroblast rather than myocyte as the primary target for the autoimmune process; a similar response to skin fibroblasts could account for Graves’ dermopathy, which is strongly associated with the presence of severe eye disease. Early experiments showed that glycosaminoglycan production by cultured normal retro-ocular fibroblasts was increased by co-incubation with lymphocytes from healthy donors, and this is further enhanced by mitogen stimulation of the T cells.1 This is entirely in keeping with the now recognised effects on fibroblasts of a variety of cytokines derived from T cells and macrophages: Cytokine (main source) Interleukin-I (macrophage) Tumour necrosis factor-a
(macrophage)
Effects on fibroblast Stimulates proliferation Variable effects on proliferation and collagen
synthesis Fibroblast growth factors*
Stimulate proliferation
(macrophage) Platelet derived growth factor (macrophage, platelet)
Stimulates proliferation and collagen synthesis
Transforming growth factors-p (macrophage, T cell) Interferon-&bgr; (macrophage, T cell) Lymphotoxin (T cell) Interferon-y (T cell)
Diverse; generally fibrogenic
Fibroblast activating factors
Enhance connective tissue
Inhibits
stimulate collagen synthesis by fibroblasts have been described, and a close correlation was reported between such immunoglobulins in Graves’ patients and severity of ophthalmopathy.24 Other investigations2S.26 have not reproduced these findings and a primary role for such antibodies does not accord with the presence of clinically significant ophthalmopathy in some patients without
TSH receptor antibodies. Patients with dermopathy have a subgroup of TSH receptor antibodies that are unusually potent stimulators of thyroid cell growth and protein synthesis 26 One product of TSH-stimulated thyroid cells can act as a cofactor to potentiate the mitogenic effect of insulin-like growth factor-1 on fibroblasts.27 That fibroblast growth factors could simulate the effect of this unknown cofactor suggests that excessive release of such thyroid-derived cytokines, stimulated by a subset of TSH receptor antibodies or by other intrathyroidal autoimmune mechanisms, could have a role in ophthalmopathy (and dermopathy). It is noteworthy that no study has yet addressed T-cell recognition of retro-ocular fibroblast antigens; it is entirely possible that local fibroblast activation could be due to cytokine release from infiltrating autoreactive T cells, unaccompanied by any antibody formation. Finally, the anatomical complexity of the extraocular muscles may explain the specificity of muscle involvement in ophthalmopathy.1,11 By comparison with skeletal muscle, the extraocular muscles have more spindles, more abundant interstitial connective tissue and nerve fibres, and a much greater blood flow. Pattern of blood supply varies both between and within individuals; this may be a factor in the asymmetry of clinical signs so frequently seen. There may be less difference between the six extraocular muscles in their pathological involvement than is apparent clinically; the characteristic limitation of elevation is due not only to fibrosis of the inferior rectus but also to changes in the fibrous connective tissue septa within the orbit. detectable
Conclusions
proliferation There is
increasing evidence that thyroid-associated ophthalmopathy is intrinsically linked to thyroid
Stimulates proliferation Inhibits proliferation and
collagen synthesis synthesis
(T cell) *Regulation
of the
important in
vanous
bioavailability
of fibroblast
growth factors
may be
diseases
the intramuscular inflammatory infiltrate could fibroblast activation and produce the clinical signs of ophthalmopathy as an end result, the reasons why the response should be so restricted to retro-ocular muscles and associated with thyroid autoimmunity are unclear. One explanation for localisation is that retrobulbar fibroblasts may be antigenically unique. To address this possibility, extracts of cultured fibroblasts from different sites have been used to detect specific antibodies in sera from patients with ophthalmopathy. Although an initial report suggested that there was diversity in antigen expression by fibroblasts, a follow-up study, in which immunoblotting was used to detect autoantibodies, did not fmd any antigenic differences between fibroblasts from different sites.19 About half the sera from patients with Graves’ disease recognised a 23 kDa protein found in all fibroblast preparations; the presence of these antibodies did not correlate with clinically obvious eye disease (or dermopathy). The association with thyroid autoimmunity may be due to the production of certain fibroblast stimulating factors in these disorders. Monoclonal TSH receptor antibodies that
Although cause
Fig 1-Section of extraocular muscle
in
thyroid-associated
eye
disease. Diffuse and focal infiltration
by T cells (red) and substantial interstitial
oedema; muscle fibres intact (original magnification x 400).
28
heterogeneity in black American patients with Graves’ disease. Arch
Fig
2-Possible
pathogenic mechanisms.
Predominant T-cell infiltration of the retro-ocular muscles may arise from recognition of antigens shared with the thyroid. These T cells, in addition to macrophages, release cytokines (see text) that stimulate fibroblasts, producing oedema and fibrosis.
the number of patients with ophthalmic Graves’ disease without any evidence of thyroid involvement continues to diminish as increasingly sophisticated measures of thyroiditis are used and follow-up is continued. The evidence for a separate immunogenetic predisposition to ophthalmopathy is inconsistent, and environmental factors such as smoking may be more important in the development of overt eye disease. In view of the long natural history of autoimmune thyroid disorders, it is impossible to be sure whether eye disease ever precedes or always follows the thyroid lesion. The most likely explanation for the association is the sharing of an autoantigen between the thyroid and the extraocular muscles: one candidate is a novel 64 kDa thyroid and eye-muscle antigen, although the cellular localisation of this is as yet unknown. The primary pathogenic mechanism in ophthalmopathy seems to be fibroblast stimulation, probably as a result of cytokine release by the activated T cells that accumulate in the muscles (fig 2). This localisation could be directed by T-cell recognition of cross-reactive antigens and by the anatomical features of the extraocular muscles. By contrast, there is little histological evidence for muscle cell destruction by the inflammatory infiltrate. Attempts to detect eye muscle autoantibodies have failed to prove their consistent presence in ophthalmopathy patients or their eye muscle specificity. Production of eye muscle reactive antibodies is probably a secondary event, that many of the antigens recognised are cytoskeletal components accounts for the broad specificity of these antibodies, which have an uncertain pathogenic role. Whether there are other antibodies that can stimulate fibroblasts directly or indirectly also remains unclear. Further knowledge about the pathogenic mechanisms should lead to safe and specific immunological treatment for this enigmatic condition.
autoimmunity;
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