Antibodies in the serum of patients with autoimmune thyroid disorders react with a recombinant 98 amino acid fragment of a full length 64 kDa eye muscle membrane protein which is also expressed in the thyroid.

We have tested sera from patients with autoimmune thyroid disorders with or without ophthalmopathy for immunoreactivity, in a dot blot assay, against a recombinant 98 amino acid fragment of a cloned 64 kDa protein, D1, which is expressed in human eye muscle and thyroid, in the form of a Lac Z fusion protein. Tests were positive in 19 out of 40 patients with established thyroid-associated ophthalmopathy (TAO), in 12 out of 21 patients with Graves' hyperthyroidism (GH) without clinically evident ophthalmopathy, in 5 out of 10 patients with thyroid autoimmunity and lid retraction but no other signs of ophthalmopathy, in 4 out of 23 patients with Hashimoto's thyroiditis (HT) without evident ophthalmopathy and in 2 out of 18 patients with benign adenoma or multinodular goitre, but in only 2 out of 37 normal subjects tested. SDS-polyacrylamide gel electrophoresis and Western blotting for an antibody reactive with a 64 kDa antigen in pig eye muscle membranes was also carried out on sera from patients with TAO and GH. While immunoblotting for antibodies reactive with a 64 kDa protein was more often positive in patients with TAO, in whom 58% had serum antibodies which reacted with a 64 kDa protein, this was not the case in patients with GH without eye signs in whom the prevalence of positive immunoblot tests was 35%. Overall there was a fairly close correlation between the two tests although there were many exceptions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies targeting the calcium binding skeletal muscle protein calsequestrin are specific markers of ophthalmopathy and sensitive indicators of ocular myopathy in patients with Graves' disease

We have identified several eye muscle antigens and studied the significance of the corresponding serum autoantibodies in patients with Graves' disease. Of these antigens, only calsequestrin is expressed more in eye muscle than other skeletal muscles, which could explain at least partly the specific involvement of eye muscle in patients with Graves' disease. Earlier, we found a modest relationship between anti-calsequestrin antibodies and ophthalmopathy, but in that study we used calsequestrin prepared from rabbit heart muscle and measured antibodies by immunoblotting. We have reinvestigated the prevalences of anti-calsequestrin antibodies in larger groups of well-characterized patients with thyroid autoimmunity with and without ophthalmopathy and control patients and healthy subjects, using standard enzyme-linked immunosorbent assay incorporating highly purified rabbit skeletal muscle calsequestrin, which has a 97% homology with human calsequestrin, as antigen. Anti-calsequestrin antibodies were detected in 78% of patients with active congestive ophthalmopathy, in 92% of those with active inflammation and eye muscle involvement, but in only 22% of patients with chronic, 'burnt out' disease. Tests were also positive in 5% of patients with Graves' hyperthyroidism without evident ophthalmopathy (two patients) and one patient with 'watery eyes' but no other clear signs of congestive ophthalmopathy and IgA nephropathy and no known thyroid disease, but in no patient with Hashimoto's thyroiditis, toxic nodular goitre, non-toxic multi-nodular goitre or diabetes, or age- and sex-matched healthy subjects. In serial studies of all 11 patients with Graves' hyperthyroidism who had active ophthalmopathy at the time of the first clinic visit, or developed eye signs during the first 6 months, and positive anti-calsequestrin antibodies in at least one sample, anti-calsequestrin antibodies correlated with the onset of ocular myopathy in six patients. Antibodies targeting calsequestrin appear to be specific markers for ophthalmopathy and sensitive indicators of the ocular myopathy subtype of ophthalmopathy in patients with thyroid autoimmunity. However, these results must be considered preliminary until a large prospective study of patients with newly diagnosed Graves' hyperthyroidism, in which serum levels of calsequestrin antibodies are correlated with clinical changes and orbital eye muscle and connective tissue/fat volumes, has been carried out.     

Identification of autoantigen recognized by autoimmune ophthalmopathy sera with immunoblotting correlated with orbital computed tomography.

We have demonstrated that there is an antibody related to extraocular muscle enlargement in autoimmune ophthalmopathy (Graves' ophthalmopathy, thyroid-associated correlated with orbital computed tomography (CT). This study was designed to identify the autoantigen and to determine whether there are common antigens among the extraocular muscle, the lacrimal gland, and the thyroid. We prepared a 100,000g sediment fraction of porcine extraocular muscle, lacrimal gland, thyroid, and human thyroid, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with sera from patients with Graves' disease, with or without ophthalmopathy, classified by symptoms and signs combined with orbital CT and normal controls. The results showed there was an approximately 55-kDa protein band which was recognized by the sera in 32.1% (9/28) of patients with autoimmune ophthalmopathy and in 47.3% (9/19) of patients with extraocular muscle enlargement demonstrated by orbital CT. It was significantly higher than the positive rates in patients without autoimmune ophthalmopathy and normal controls (15.8 and 11.1%, respectively, P < 0.025). However, there was no common antigen among the extraocular muscles, the lacrimal gland, and the thyroid. To further confirm this eye muscle-specific antigen, the approximately 55-kDa protein band was cut and solubilized from the nitrocellulose paper after SDS-PAGE, and electrophoretically transferred and used as an antigen in enzyme-linked immunosorbent assay. The absorbance was significantly higher in patients with autoimmune ophthalmopathy than patients without ophthalmopathy (P < 0.005), and normal controls (P < 0.01). Our findings suggest that an approximately 55-kDa protein may be a possible antigen in the eye muscle related to autoimmune ophthalmopathy.     

Antithyroglobulin monoclonal and autoantibodies cross-react with an orbital connective tissue membrane antigen: a possible mechanism for the association of ophthalmopathy with autoimmune thyroid disorders.

The possibility that Graves' ophthalmopathy and autoimmune thyroid disorders may be associated because of autoimmune reactions against antigens shared between human orbital and thyroid tissues was investigated using anti-thyroglobulin (Tg) monoclonal and autoantibodies. Eleven of 16 mouse monoclonal antibodies (MCAB) tested reacted, in an enzyme-linked immunosorbent assay (ELISA), with an antigen in human orbital connective tissue membranes (OCTmem), but not with the OCT soluble fraction, or with membrane or soluble fractions of human eye muscle, lacrimal gland or skin connective tissue. The anti-OCTmem activity was absorbed by OCTmem and Tg, but not by liver membranes or bovine serum albumin (BSA). In preliminary studies four out of 113 human MCAB against thyroid or orbital tissue antigens showed reactivity restricted to Tg and OCTmem. Sera from approximately 50% of patients with autoimmune thyroid disorders, with or without ophthalmopathy, also reacted with OCTmem. The autoantibody activity correlated closely with serum titres of antithyroglobulin but not with the presence, duration, or severity of the eye disease. The OCTmem reactivity was absorbed by Tg, thyroid membranes, and OCTmem but not liver membranes, membranes prepared from other orbital tissues, or BSA. The OCTmem-Tg shared antigen site appeared not to be native thyroglobulin since, (i) MCAB and serum autoantibodies did not react with the cytosol fraction of OCT, and (ii) because the membrane antigen was not solubilizable. Because not all patients with ophthalmopathy have detectable anti-Tg antibodies and, conversely, because not all patients with detectable anti-Tg antibodies develop ophthalmopathy it is unlikely that autoimmunity against a OCTmem-Tg shared antigen is the primary mechanism of Graves' ophthalmopathy, although this possibility has not been excluded. On the other hand the reaction of anti-Tg autoantibodies with OCT membranes may be a model for other autoimmune reactions against other thyroid-orbital tissue-shared antigens. While the pathogenesis of Graves' ophthalmopathy is likely to be multifactorial, humoral and cellular reactions against primary orbital antigens, thyroid-orbitol tissue shared antigens, or both, are likely to play important roles.     

Isotype and immunoglobulin subclass distribution of eye muscle membrane reactive antibodies in the serum of patients with thyroid-associated ophthalmopathy as detected in Western blotting.

We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of anti-eye muscle antibody in patients with thyroid-associated ophthalmopathy by quantitative western blot.

To investigate the prevalence of antibody against rat eye muscle membrane antigen, as determined from SDS-polyacrylamide gel electrophoresis and western blotting, in sera from patients with thyroid-associated ophthalmopathy (TAO), we quantitatively analyzed the binding activity with a rat eye muscle membrane 64 kDa protein using chromato-scanner. Eye muscle antibody activity was expressed as ratio of density of the 64 kDa band to that at 66 kDa found with all normal sera and phosphate buffered saline. The mean (+/- SD) eye muscle antibody activity was 2.7 +/- 2.7 in TAO (P < 0.01 v.s. normal), 1.5 +/- 1.7 in Graves' disease without evident eye disease, 1.6 +/- 2.5 in Hashimoto's thyroiditis and 0.45 +/- 0.26 in normal subjects. A positive band at 64 kDa was found in 71% of patients with TAO, 36% of those of Graves' disease without evident eye disease and in 35% of patients with Hashimoto's thyroiditis without eye disease. The prevalence of this antibody activity tended to correlate to the severity of ophthalmopathy. Furthermore, the level of eye muscle antibody activity decreased in parallel with the improvement of eye signs in two patients. Sera reactive with rat eye muscle membrane 64 kDa protein reacted also with a human eye muscle membrane 64 kDa protein but not with human thyroid, liver, spleen or pancreas membrane preparations. In conclusion, antibody to rat eye muscle membrane 64 kDa protein is present in TAO and may be a useful clinical marker of ophthalmopathy.     

Extraocular muscle autoimmunity and orbital fat inflammation in thyroid-associated ophthalmopathy

The immunological basis for the ophthalmopathy associated with Graves’ hyperthyroidism is both poorly understood and controversial. The mechanism for its link with thyroid autoimmunity is unknown but likely to be due to autoimmunity against some thyroid and orbital tissue-shared antigen, such as the thyroid-stimulating hormone receptor, which is expressed on the orbital pre-adipocyte and extraocular muscle cell, or the putative ‘eye muscle cell membrane antigen’. Chronic upper-eyelid retraction, which sometimes occurs as a dominant feature of ophthalmopathy or as an isolated abnormality, is a common and related orbital disorder. Recent evidence that antibodies targeting the calcium-binding protein calsequestrin are specific and sensitive markers of eye muscle and upper-eyelid muscle damage has highlighted the need for diagnostic antibody tests in ophthalmopathy. In the context of this confusion, this review will address the nature of the autoimmune reactions in thyroid-associated ophthalmopathy, focusing on the eye muscle.     

Peripheral blood lymphocyte transformation to G2s in patients with autoimmune thyroid disease with and without ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Cellular and humoral autoimmune responses against human eye muscle membrane antigen in Graves' disease

75 patients with Graves' disease (54 with ophthalmopathy) were investigated using the tests of leucocyte adherence inhibition and immune adsorption with 125I-labelled Staphylococcus Protein A, against human eye muscle "crude" membrane antigen. The results of positive leucocyte adherence inhibition (10 out of 26 vs. 1 out of 28, P less than 0.05) and anti-human eye muscle membrane antibody index (mean +/- S.D.) (1.89 +/- 1.20 vs. 0.84 +/- 0.38, P less than 0.001) showed a correlation with the patients with clinically active eye disease and the HLA-B8 antigen in Graves' ophthalmopathy (P less than 0.01). Positive leucocyte adherence inhibition was observed in 9 out of 21 cases of Graves' disease without ophthalmopathy, but its prognostic relevance has to be confirmed in the development of ophthalmopathy.     

Eye muscle membrane reactive antibodies are not detected in the serum or immunoglobulin fraction of patients with thyroid-associated ophthalmopathy using an ELISA and crude membranes

We tested sera and purified immunoglobulin (Ig) fractions from patients with autoimmune thyroid disorders (AITD), with and without ophthalmopathy, and normal subjects, for the presence of antibodies reactive with eye muscle membrane antigens in an optimized enzyme-linked immunosorbent assay (ELISA). We found no correlation between ELISA results and the presence or severity of ophthalmopathy in patients with AITD for either serum or Ig, and there were no significant differences between the mean values (+/- SE) for the three groups (AITD with ophthalmopathy, AITD without ophthalmopathy and normals) for either serum or Ig. In contrast Ig from 8 of 19 (45%) patients with thyroid-associated ophthalmopathy reacted with a 64 kDa eye muscle membrane antigen in SDS-polyacrylamide gel electrophoresis and Western blotting, while tests were positive in only one of the 8 patients with AITD without eye disease and in none of the 8 normal subjects. The presence of antibodies to a 64 kDa antigen in immunoblotting did not correlate with the levels of antibodies measured in ELISA. We conclude that the ELISA, incorporating a crude membrane fractions as antigen, is not useful as a clinical test for eye muscle autoantibodies.     

Time-dependent effect of cyclosporin-A on the TSH-receptor antibody synthesis in patients with Graves' disease

Two patients with hyperthyroidism and Graves' ophthalmopathy were treated with cyclosporin A (CyA), in addition to methimazole, after failure of steroid therapy. Eye disease showed favorable responses and TSH receptor antibody concentration showed precipitous decline in concentrations compared to a gradual linear decline in antibody concentrations observed in 10 patients not treated with CyA. These results prompted us to investigate the in vitro influence of CyA on the synthesis of TSH receptor antibody by a patient's lymphocytes (with highest antibody concentration) in response to thyroid membrane antigen. CyA caused a dose-dependent reduction of TSH receptor antibody synthesis compared to control cultures. The effect of CyA was more marked when added to lymphocyte culture at the same time rather than 24 h after addition of antigen, consistent with CyA's interference of early T cell triggering by antigen. This study emphasizes the importance of helper T cells in synthesis of TSH-receptor antibody by cells and suggests that the drug may be therapeutically beneficial in severe Graves' ophthalmopathy and/or Graves' hyperthyroidism resistant to conventional treatment.     

Immunologically mediated cytotoxicity against human eye muscle and thyroid cells in euthyroid and thyrotoxic Graves' ophthalmopathy

We have studied the role of eye muscle and thyroid autoimmunity in patients with Graves' hyperthyroidism with or without ophthalmopathy in an area of relatively low iodine intake. Antibody dependent cell mediated cytotoxicity (ADCC) and complement mediated antibody dependent cytotoxicity (CMAC) against thyroid and eye muscle cells, and levels of antibodies against TSH receptor antigen and the thyroid microsomal antigen (thyroid peroxidase) were determined in three groups of patients: (1) thyrotoxic with exophthalmos (TX, n = 28), (2) thyrotoxic without ophthalmopathy (GR, n = 10), and (3) euthyroid ophthalmopathy (EU, n = 12). The thyroid glandular mass of the EU group was significantly less (P less than 0.01) compared with TX or GR. Mean (+/- SD) TSH receptor antibody (TRAb) level was 27 +/- 14% in EU which was significantly lower compared with TX (52.4 +/- 20%) and GR (59 +/- 18%). The prevalence of microsomal antibodies were similar and not significantly different in the three groups. On the other hand the prevalence of positive ADCC and CMAC tests was significantly greater, and at higher levels, in EU (ADCC THY CELLS 10.9 +/- 8.9% SL, ADCC Eye muscle = 25.9 +/- 20% SL, CMAC = 70.2 +/- 43% SL) and TX (ADCC THY CELLS = 9.3 +/- 9.2% SL, ADCC Eye muscle = 20.1 +/- 19% SL, CMAC = 62.4 +/- 30% SL) compared to GR (ADCC THY CELLS = 4.4 +/- 9.5% SL, ADCC Eye muscle = 7.7 +/- 6.7% SL, CMAC = 24.7 +/- 23% SL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Assays of TSH-receptor antibodies in 576 patients with various thyroid disorders: their incidence, significance and clinical usefulness

The incidence and the significance of TSH-receptor antibodies in Graves' disease and in various thyroid disorders have been evaluated. TSH-binding inhibiting antibodies (TBIAb) and thyroid stimulating antibodies (TSAb) were detected in a large proportion of Graves' disease patients (TBIAb in 68.8% and TSAb in 77.8%), in a small number of patients with idiopathic myxoedema or Hashimoto's thyroiditis, and were not detected in patients with endemic euthyroid goitre, differentiated thyroid carcinoma and toxic adenoma. Furthermore, TSH-receptor antibodies were present in some patients with toxic multinodular goitre (TBIAb in 12.7% and TSAb in 15.9%). When TSH-receptor and other thyroid autoantibodies were compared, it was found that 13 of the 15 Graves' patients with negative tests for thyroglobulin and thyroid microsomal antibodies were positive for TSH-receptor antibodies. On the other hand, 9 of the 11 patients with toxic multinodular goitre who had positive TSH-receptor antibody tests, also had serum thyroglobulin and/or thyroid microsomal antibodies. No significant differences in the prevalence of TSH-receptor antibodies were found in Graves' patients irrespective of the presence of ophthalmopathy or pretibial myxoedema. Elevated TBIAb activity at the end of anti-thyroid drug treatment was found in 52.9% of Graves' patients who subsequently relapsed, while in Graves' patients in remission TBIAb was always negative. TSH-receptor antibody results were not predictive of the outcome of radioiodine treatment in Graves' disease. Finally no correlation could be found between TBIAb and TSAb in Graves' disease and Hashimoto's thyroiditis. In conclusion: the high incidence of TSH-receptor antibodies in Graves' disease confirms their pathogenetic role in the development of hyperthyroidism; TSH-receptor antibodies in Graves' disease are not significantly associated with the presence of ophthalmopathy or pretibial myxoedema; TSH-receptor antibody assays may be useful for the diagnosis of Graves' disease in the absence of other signs of autoimmunity. TBIAb seems to be a good predictor of relapse in Graves' patients treated with anti-thyroid drugs; a fraction of toxic multinodular goitre could be a nodular variant of Graves' disease.     

The prevalence of anti-acetylcholinesterase antibodies in autoimmune disease

A robust and precise enzyme linked immunosorbent assay (ELISA) with proven sensitivity and specificity has been employed to detect human antibodies (allogenic/autogenic) to human acetylcholinesterase (AChE). The sensitivity of the method has been established using mouse monoclonal antibodies (0.8 ng/ml) and uniquely, human sera positive for anti-Yt(a) allogenic antibodies, to one phenotypic form (most common) of human AChE. The latter was also used as the positive human control to ensure functionality of the assay. The ELISA method was used to establish a normal distribution curve for absorbance values employing sera from healthy blood donors Subsequently, the ELISA was employed to investigate the prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease and patients with non-autoimmune thyroid disease (diseased control). The results indicate that there is not a high prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease. The lack of anti-AChE autoantibodies in patients' with clinically apparent Graves' ophthalmopathy, mitigates against there being a causal role of such antibodies in Graves' associated eye disease.     

Soluble CTLA-4 in autoimmune thyroid diseases: relationship with clinical status and possible role in the immune response dysregulation

CTLA-4 molecule, expressed by activated T and B lymphocytes, transduces an inhibitory signal. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. The aim is to evaluate the augmented sCTLA-4 serum levels in different autoimmune thyroid diseases when compared with normal donors or with non-autoimmune hyperthyroidism and to investigate the functional activities and suggest the possible pathogenetic role of sCTLA-4. We demonstrate the presence of a soluble form of CTLA-4 in 59/90 sera from patients with autoimmune thyroid diseases (both Graves' disease and autoimmune thyroiditis). sCTLA-4 levels were not related to specific clinical manifestations, such as clinical thyroid status (hypo- or hyperthyroidism), circulating thyroid hormones, or other clinical features (ophthalmopathy). sCTLA-4 production does not seem to be affected by disease evolution during time. We showed that sCTLA-4 from sera of patients with thyroid autoimmunity is able to bind its physiological ligands CD80/CD86 and displays functional activities on different in vitro systems (T-cell proliferation induced by specific soluble antigens, bi-directional mixed lymphocyte reaction). In conclusion, we demonstrate an increment of sCTLA-4 in serum of patients with autoimmune thyroid diseases. Its possible pathogenetic role during autoimmune processes can be speculated: sCTLA-4 can specifically inhibit the early T-cell activation by blocking the interaction of CD80/CD86 with the co-stimulatory receptor CD28. Conversely, higher levels of sCTLA-4 could compete with membrane-bound CTLA-4 for CD80/CD86, in later T lymphocytes activation phase, causing a reduction of inhibitory signaling.     

Endocrine ophthalmopathy: a re-evaluation of the association with thyroid autoantibodies.

Autoantibodies to the three major thyroid autoantigens, the TSH-receptor (TSH-R), thyroid peroxidase (TPO) and thyroglobulin (TG), have been investigated in 63 Graves' patients with severe endocrine ophthalmopathy. In agreement with other studies, TSH-R antibodies were detectable in 88% of patients and dominated the autoantibody spectrum. TPO antibodies were detectable in 60% of patients and TG antibodies in only 25% of patients. The prevalences, as well as the amounts, of all three thyroid autoantibodies were not significantly different from the values in 51 Graves' patients without clinically significant ophthalmopathy. However, in the subgroup of patients with TG antibodies, the ophthalmopathy patients displayed a shift towards IgG4 TG antibodies. Furthermore, in the same TG antibody positive subgroup, the amount of TSH-R antibody was significantly higher in the ophthalmopathy patients than in Graves' patients without ophthalmopathy. These qualitative differences in thyroid autoantibodies in patients with and without ophthalmopathy raise the possibility that further qualitative differences, such as thyroid autoantibody epitopes, may exist in patients with ophthalmopathy. Our observations, combined with recent evidence for the presence of TSH-R specific mRNA in retro-orbital tissue, suggest that it may be premature to dismiss the potential pathogenetic or diagnostic value of thyroid autoantibodies, particularly TSH-R antibodies, in Graves' ophthalmopathy.     

Peripheral Blood Lymphocyte Transformation to G2s in Patients with Autoimmune Thyroid Disease with and without Ophthalmopathy

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.     

Peripheral blood T lymphocyte sensitisation against calsequestrin and flavoprotein in patients with Graves' ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune disorder of the extraocular and eyelid muscles and surrounding connective and adipose tissue. Although mononuclear cell infiltration of orbital tissue is a characteristic feature of TAO the likely role of T lymphocyte reactivity against eye muscle antigens in the initiation of eye muscle damage in TAO has not been explored in detail. Therefore, we tested for T lymphocyte sensitisation to three eye muscle antigens namely, calsequestrin, G2s and flavoprotein (Fp), in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects. T lymphocyte reactivity was determined in a proliferation assay, results being expressed as stimulation index (SI). Mean ( +/- SE) SI for patients with GO, but not GH without ophthalmopathy, were significantly greater than that for normal subjects for calsequestrin and Fp, but not G2s. Mean ( +/- SE) SI was also significantly increased in patients with active ophthalmopathy, but not chronic ophthalmopathy, compared to normal subjects, for calsequestrin and Fp, but not G2s. Overall, positive lymphocyte proliferation to calsequestrin was demonstrated in 59% of patients with GO and 33% of patients with GH, which was significantly greater than in normals for both groups. In conclusion, we have demonstrated significant T lymphocyte reactivity to calsequestrin and, to a lesser extent, Fp in patients with GO. Because calsequestrin is located in the cell membranes of the eye muscle cell during the myotubular stage of the cell cycle, its targeting might be the primary reaction which leads to extraocular muscle inflammation in patients with GH.     

Comparative study on IgG and IgA antibodies against human thyroid and eye-muscle antigens in Graves' ophthalmopathy.

Circulating IgG and IgA anti-thyroid and anti-eye muscle antibodies were investigated in 87 patients with Graves' disease (60 cases with ophthalmopathy). The ELISA method was used. Both IgG and IgA antibodies were demonstrated against human thyroid and eye-muscle membrane or cytosol antigens. Anti-eye-muscle antibodies of the IgA type were observed more frequently than those of the IgG type (25 cases vs. 18 were demonstrated with membrane antigens and 37 cases vs. 23 with cytosol antigens). The respective distributions for thyroid antigens the cytosol fraction were 55 cases vs. 13 and 18 cases vs. 36. A significant difference was observed in the anti-thyroid IgG levels and the anti-eye-muscle membrane or cytosol levels between the patients with Graves' disease and those in control group (P less than 0.001). The difference in the IgA antibody to thyroid and eye-muscle antigens was significant between the patients with and without ophthalmopathy (P less than 0.002). The strong correlation between the levels of IgA antibodies to thyroid and those to the eye-muscle cytosol fractions might be connected with the theory of the common aetiology of the thyroid and eye diseases in Graves' ophthalmopathy (P less than 0.001). Circulating IgA anti-human thyroid and eye-muscle antibodies seemed to have a diagnostic relevance in the development of ophthalmopathy in Graves' ophthalmopathy.     

Restricted tissue reactivity of autoantibodies to a 64-kDa eye muscle membrane antigen in thyroid-associated ophthalmopathy.

We studied the tissue specificity of eye muscle (EM) membrane-reactive autoantibodies detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In preliminary studies, such antibodies were shown to react, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, with human thyroid (THY) and other human skeletal muscle (HSM) membrane antigens. We carried out absorption with human EM (HEM), THY, and HSM membranes of sera from patients with TAO and autoimmune thyroid disease without ophthalmopathy which reacted with one or more of 55-, 64-, and 95-kDa antigens in pig eye muscle (PEM) membrane in immunoblotting, the majority of which were also cytotoxic to HEM cells in an antibody-dependent cell-mediated cytotoxicity assay. In Western blotting, serum antibodies reactive with PEM membrane antigens of 55, 64, and 95 kDa were cross-absorbed by HEM, THY, and HSM but not by spleen or brain membranes and showed some species specificity, being absorbed by pig and human, but not bovine, EM membranes. When incubated with cultured HEM, THY, and HSM cells in vitro, autoantibodies in TAO sera immunoprecipitated a 64-kDa antigen from the first two tissues, but not from HSM, suggesting a specific binding to autoantigenic epitopes in HEM and THY. Sera from patients with TAO as well as those from patients with thyroid autoimmunity without ophthalmopathy immunoprecipitated a approximately 66-kDa protein, shown to be distinct from the 64-kDa antigen. The restricted immunological cross-reactivity of antibodies to a THY and HEM 64-kDa membrane antigen is discussed in the context of the association of ophthalmopathy with thyroid autoimmunity. Further experiments are needed to show whether autoantibodies to the 64-kDa eye muscle and thyroid shared antigen are cytotoxic, and thus likely to play a major role in the pathogenesis of the eye disease, or just markers of the orbital autoimmune process.