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.     

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.     

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 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.     

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.     

Clinical significance of a new autoantibody against a human eye muscle soluble antigen, detected by immunofluorescence.

The clinical significance of a circulating autoantibody against a recently identified soluble human eye muscle-derived antigen was studied in patients with Graves' ophthalmopathy and autoimmune thyroid disorders. Tests were positive in 73% of patients with Graves' ophthalmopathy, including six of seven with no associated thyroid disease (euthyroid Graves' disease). Tests were also positive in 27% of patients with hyperthyroidism but no clinically apparent eye disease, in 13% of patients with Hashimoto's thyroiditis without eye disease, in two of 12 patients with subacute thyroiditis, in one of 20 patients with nonimmunological thyroid disorders but in none of 39 normal subjects. There were significant positive correlations between serum levels of the antibody (expressed as a titre) and the severity of the eye muscle component quantified as an index as well as the duration of the eye disease. Antibodies were detected in three of five patients with only lid lag and state who subsequently developed active ophthalmopathy, in six of nine patients who developed eye disease after treatment of their hyperthyroidism and in one of eight first degree relatives of patients with Graves' ophthalmopathy. In addition three of the 12 patients with autoimmune thyroid disease without apparent eye involvement, but positive antibody tests, have developed ophthalmopathy since the time of testing. These findings suggest that tests for antibodies against a soluble human eye muscle antigen may be useful clinically as a diagnostic test and to predict the onset of eye disease in predisposed patients and subjects.     

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.     

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.     

Cell-mediated immunity to orbital tissue antigens in thyroid-associated ophthalmopathy determined using the leukocyte procoagulant activity assay.

While it is generally accepted that thyroid-associated ophthalmopathy (TAO), a progressive inflammatory disorder of the extraocular muscle and orbital connective tissue (OCT), is immunologically mediated the nature of the underlying abnormalities is poorly understood. Although there is considerable evidence for antibody-mediated immunity against both eye muscle (EM) and OCT antigens in TAO a role of cell-mediated immunity (CMI) has not been studied in detail. We have used a new sensitive test for CMI, the leukocyte procoagulant activity (LPCA) assay and tested blood leukocytes from patients with TAO and autoimmune thyroid disease (ATD) without evident ophthalmopathy for reactivity against pig eye muscle (PEM) and human thyroid and OCT membrane and soluble fractions. In some cases human EM fractions were also tested. Preparations of PEM membrane (PEMM) and human thyroid membrane induced a significant LPCA response in both groups of patients. PEM cytosol, human OCT membrane and cytosol and human spleen membrane did not evoke a significant response in either group of patients. There were significant positive correlations in patients with TAO between (i) LPCA in response to PEMM and that to human thyroid membrane and (ii) LPCA in response to human thyroid membrane and that to human EM membrane. In patients with TAO there were no significant associations between LPCA response to PEMM and the detection of serum antibodies to a 64 kDa EM membrane protein in immunoblotting, or between LPCA response to PEMM and the duration or severity of the ophthalmopathy or clinical evidence for eye muscle involvement. These findings confirm a role of cell-mediated immunity against eye muscle antigens in thyroid-associated ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Interactions of fibroblasts, adipocytes and immunocompeent cells in the pathogenesis of endocrine ophthalmopathy

Graves' ophthalmopathy is thought to result from a complex interplay of genetic and environmental factors. Various genes including those coding for HLA may determine a patient's susceptibility to the disease and its severity, but in addition numerous and often unknown environmental factors may determine its course. The orbital immune process is thought to be initiated, on the background of a permissive immunogenetic milieu, by circulating T cells directed against certain antigens on thyroid follicular cells that also recognize antigenic epitopes which are shared by tissues contained in the orbital space. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active Graves' ophthalmopathy has revealed limited variability of TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur during the early stages of Graves' ophthalmopathy. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which attract T cells by stimulating the expression of several adhesion molecules (e.g. ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. Adhesion molecules are known to be important for a variety of interactions between immunocompetent cells, preadipocyte fibroblasts and adipocytes. In addition, these molecules play a central role in lymphocyte activation and localization, facilitating antigen recognition, T cell costimulation, and various effector-target cell functions at the inflammatory sites, which result in amplification of the cellular immune process in active Graves' ophthalmopathy. T cells and macrophages populate the orbital space and release a number of cytokines (most likely a Th-1-type spectrum) into the surrounding tissues. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in Graves' ophthalmopathy, may aggravate tissue hypoxia and exert important immunomodulatory and pro-oxidant effects. Differentiation of orbital preadipocyte fibroblasts into mature adipocytes expressing increased levels of TSHR may also be driven by stimulation with circulating or locally produced cytokines or effectors. TSHR-directed autoantibodies or T cells may thus play a direct role promoting adipogenesis, glycosaminoglycan synthesis and expression of immunomodulatory proteins within the orbits. Once the net effect of these changes has come to increase the volume of the fatty connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and periorbital congestion will ensue.     

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.     

Antibodies against 1D,a Recombinant 64-kDa Membrane Protein,Are Associated with Ophthalmopathy in Patients with Thyroid Autoimmunity

We have tested for serum antibodies reactive with ₁D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to ₁D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-₁D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with ₁ D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against ₁D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both ₁D and the 64-kDa eye muscle protein were incubated with CHO (₁D) cell membrane, reactivity against ₁D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of ₁D.     

The putative role of fibroblasts in the pathogenesis of Graves' disease: evidence for the involvement of the insulin-like growth factor-1 receptor in fibroblast activation

Graves' disease when fully expressed affects the thyroid gland and connective tissues of the orbit and pretibium. While the glandular disease is relatively well-characterized, the pathogenesis of the orbital and dermal components remains enigmatic. In the following article, we review some of the evidence suggesting that fibroblast activation in Graves' disease might play an integral role in the tissue remodeling associated with ophthalmopathy. The thyrotropin receptor (TSHR) is expressed at low levels in several connective tissue depots and by their derivative fibroblasts, including those from the orbit. Little direct evidence currently links extra-thyroidal TSHR expression with Graves' disease. Very recent observations now implicate the insulin-like growth factor-1 receptor (IGF-1R) as a fibroblast activating antigen. When immunoglobulins from patients with the disease, with or without clinical ophthalmopathy, bind IGF-1R on the surface of fibroblasts, the receptor becomes activated and upregulates the expression of two T lymphocyte chemoattractants, IL-16 and RANTES. Thus, IGF-1R may represent a second self-antigen with a pathogenic role in extra-thyroidal Graves' disease.     

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)     

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.     

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.     

Peripheral parameters of oxidative stress in patients with infiltrative Graves' ophthalmopathy treated with corticosteroids

Infiltrative ophthalmopathy, which may develop in patients with Graves' disease, is considered an inflammatory disorder of autoimmune background. There is growing evidence that changed reactive oxygen species (ROS) metabolism plays an important role in pathogenesis of autoimmune diseases. Corticotherapy is a principal method of ophthalmopathy treatment, and its therapeutic effect is partially connected with influence on ROS generation systems. This study was undertaken to investigate corticosteroids treatment influence on blood extracellular indices of ROS metabolism in Graves' ophthalmopathy patients. Plasma indices of free radical generation and scavenging were determined in 22 euthyroid patients with active infiltrative Graves' ophthalmopathy initially, after intensive corticotherapy and after completing of steroid treatment. Age- and sex-matched 24 healthy volunteers and 25 euthyroid Graves' patients without overt ophthalmopathy served as controls. In the ophthalmopathy patients hydrogen peroxide (H(2)O(2)), lipid hydroperoxides (ROOH), thiobarbituric acid-reacting substances (TBARS) and ceruloplasmin (CP) levels and superoxide dismutase (SOD) and catalase (CAT) activities were increased, whereas glutathione peroxidase (GPx) and glutathione reductase (GR) activities were reduced. Intensive corticotherapy resulted in normalization (partial for ROOH) of ROS metabolism peripheral markers. After the withdrawal of corticosteroids a reduction of ophthalmopathy clinical activity was present, yet a marked restoration of increased oxidative stress indices was observed, along with activation of antioxidant defence systems (not significant for CAT activity). These data demonstrate that corticosteroids are effective in reduction of peripheral oxidative stress present in infiltrative Graves' ophthalmopathy, but this effect tends to be transient.     

Treatment of graves' ophthalmopathy with cyclosporin a

Summary To evaluate the effect of Cyclosporin A in the management of Graves' ophthalmopathy, we treated 13 patients with severe euthyroid endocrine eye disease with the immunosuppressive drug. Follow up consisted of clinical investigation, laboratory evaluation with detailed thyroid function tests and complete ophthalmological assessments including A and B scan ultrasound and computerized tomography (CT) of the orbits. Cyclosporin A given over 4–7 months resulted in improvement of clinical signs and symptoms of the disease due to a positive effect of the drug on soft tissue changes. The more objective methods did not demonstrate any unequivocal change in proptosis, visual acuity, intraocular pressure and thickness of extraocular muscles. Cyclosporin A was not able to stop acute progression of ophthalmopathy in one patient and did not obviate surgical decompression of the orbits in two more patients. There was no measurable effect of treatment on the underlying immunological process with regard to thyroid disease.Abbreviations CT computerized tomography - CyA Cyclosporin A - TBG thyroxine binding globulin - TDA TSH displacing activity - T₃ triiodothyronine - T₄ thyroxine - TSH thyroid stimulating hormone Presented in part at the annual meeting of the Deutsche Gesellschaft für Endokrinologie (1984) Acta Endocrinol 105 Suppl 264:83     

Long-term pathologic changes of alpha1-acid glycoprotein (orosomucoid) glycoforms in autoimmune thyroid disease

OBJECTIVE: We measured alpha1-acid-glycoprotein (AGP) in patients with autoimmune thyroid disease to study a possible relationship between microheterogeneity of the naturally occurring glycoforms of AGP and autoimmune thyroid disease. DESIGN, PATIENTS, MEASUREMENTS: In a group of 12 fasting thyrotoxic patients (11 females, mean age: 43 years) with newly diagnosed Graves' disease (subgroup 1), we measured serum concentrations of total AGP and its 3 glycoforms (micromol/l, crossed affinity immunoelectrophoresis with con A in the first dimension gel) as well as total thyroxine, total triiodothyronine, thyrotropine, thyroid peroxidase antibodies (anti-TPO), antibodies against the TSH receptor (TRAb, TRAK), at baseline and after 12 months of antithyroid drug therapy (ATD). For comparison, 4 subgroups of thyroid patients (patients with Graves' disease and thyroid associated ophthalmopathy (TAO) (subgroup 2, n = 10), radioiodine treated Graves' patients (subgroup 3, n = 7), Graves' patients without TAO (subgroup 4, n = 13), patients with Hashimoto's thyroiditis (subgroup 5, n = 8)) and 25 normal controls (17 females, mean age: 38 years) were studied. RESULTS: In subgroups of TRAb positive Graves patients' serum levels of glycoform 1, 2 or 3 increased significantly (p < 0.005) after 12 months of ATD as compared to both baseline of that person or normal controls. No significant changes were found in the TRAb negative Hashimoto subgroup. CONCLUSION: Patients with autoimmune Graves' disease changed their relationship to AGP, and thus a role of AGP and its 3 glycoforms is suggested in the pathogenesis of Graves' disease.