J. R. Wall

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.

Significance of Cytotoxic Eye Muscle Antibodies in Patients with Thyroid-Associated Ophthalmopathy

We have studied the clinical significance of cytotoxic antibodies against human eye muscle cells in patients with thyroid-associated ophthalmopathy (TAO). Eye muscle reactive antibodies were measured in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. A positive test was defined as % specific lysis greater than the upper limit of normal, taken as the mean plus two standard deviations for normal subjects tested concurrently. As parameters of the severity of the ophthalmopathy we measured the degree of proptosis (mm), level of intraocular pressure (IOP) (mmHg) and American Thyroid Association classes (0-6). ADCC tests were positive in 21 out of 42 patients with TAO and in 8 out of 14 patients with Graves disease without evident eye disease but in none of 12 normal subjects tested. In patients with TAO mean (+/- SE) IOP was significantly greater than that in patients with Graves disease without apparent eye involvement for the primary position and for all gaze positions. There were significant positive correlations between levels of eye muscle reactive cytotoxic antibodies and the severity of the eye disease quantitated as American Thyroid Association classes 0-6, the IOP in the primary position and on downgaze, but not with the degree of proptosis. These results suggest that cytotoxic antibodies, as detected in ADCC, may play a role in the eye muscle damage of TAO and that their measurement may provide a useful clinical test.

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 Hashimotos 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)

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.

Antibodies reactive with an intracellular epitope of a recombinant 64 kDa thyroid and eye muscle protein in patients with thyroid autoimmunity and ophthalmopathy.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies reactive with a 98 amino acid fragment, called D1, of a recombinant thyroid and eye muscle membrane protein corresponding to a MW of 64 kDa (called 1D) in the serum of patients with thyroid autoimmunity with and without ophthalmopathy. Antibodies against the D1 fragment expressed as a fusion protein with β galactosidase, were detected in 29% of patients with thyroid-associated ophthalmopathy (TAO) of <1 yr duration, in 33% of those with disease of >3 yr duration, in 40% of patients with Graves’ hyperthyroidism (GH) without evident eye disease, in 31% of patients with lid lag and retraction but no other signs of progressive ophthalmopathy, in 25% of patients with euthyroid Graves’ disease and in 43% of patients with untreated Hashimoto’s thyroiditis (HT), but in none of 14 patients with other (non-immunological) thyroid disorders. Although tests were positive in 6 out of the 15 patients with ophthalmopathy and no overt thyroid autoimmunity overall there was no close association of the antibodies with clinical features of the eye disease or its course. In those sera in which Western blotting for antibodies reactive with a 64 kDa eye muscle membrane protein and ELISA were both carried out there was no close correlation between the two tests. In order to better understand the significance of antibodies reactive with 64 kDa eye muscle and thyroid membrane autoantigens and their possible relationship to ophthalmopathy it will be necessary to perform prospective studies of patients with untreated Graves’ hyperthyroidism beginning before the onset of eye signs, using antibody assays incorporating full length recombinant proteins.

Expression of major histocompatibility complex class II antigen in nod mouse thyroid

Non obese diabetic (NOD) mice spontaneously develop thyroiditis in addition to diabetes. Mononuclear cells begin to infiltrate the thyroid of these animals in the first month of life. The expression of major histocompatibility complex (MHC) class II (Ia) antigens by cells in the thyroid from NOD mice of various ages with and without thyroiditis was examined. We found that only 1 of the 9 infiltrated thyroids from 18 8-33 day old NOD mice surveyed expressed MHC class II antigens. Therefore Ia antigen expression appears to be secondary to infiltration and does not initiate the autoimmune process. Fourteen of 17 (82.2%) infiltrated and 7 of 11 (63.6%) uninfiltrated thyroids from NOD mice aged 51-73 days contained cells expressing Ia antigens. Sixteen of 18 (88.9%) infiltrated and all 7 of the uninfiltrated thyroids from mice aged > 89 days contained Ia positive cells. These MHC class II expressing cells included thyroid epithelial cells (TEC), as well as interstitial cells such as macrophages. Ia positive cells in the thyroid have the potential of presenting thyroid specific antigen to infiltrating T cells and thereby maintaining or potentiating thyroid autoimmune destruction. Macrophages were observed in thyroid tissue from 9 of 11 (81.8%) infiltrated and 12 of 15 (80%) uninfiltrated 8-33 day old NOD mice, thyroids from 11 of 16 (68.7%) infiltrated and 6 of 9 (66.7%) uninfiltrated 51-73 day old NOD mice, as well as 28 of 29 (96.5%) uninfiltrated and all 9 of the uninfiltrated thyroid from NOD mice aged > 89 days. Thyroids from control age matched non autoimmune BALB/c mice were consistently Ia antigen negative while macrophages were seen in some of the animals aged > 60 days.

Muscle and species reactivity of mouse monoclonal antibodies to human eye muscle membrane antigens

We studied the tissue and species reactivity of mouse monoclonal antibodies (MCAB) produced by immunizing mice with a 100,000g ultracentrifuged preparation of human eye muscle (HEM) membranes. Twenty-three MCABs, 20 of which reacted in an enzyme-linked immunosorbent assay (ELISA) with HEM membrane, 2 with human thyroid membrane, and 1 nonreactive negative control, were selected for the study. The muscle and species specificity of 6 of the most reactive and more restrictively reactive MCAB were studied in more detail. All reacted in ELISA with human skeletal muscle membrane and, to a lesser extent, with human cardiac muscle membrane, but not with human brain membrane. The 6 MCAB cross-reacted with eye muscle membrane prepared from pig but not rat, although reactivity with human tissue was greatest for all MCAB tested. When tested in immunoblotting with HEM and thyroid membranes, 3 of 6 MCAB reacted with a 64-kDa protein in HEM, 2 of which also reacted with an antigen of the same molecular weight in thyroid membrane. In a complement-mediated antibody-dependent cytotoxicity assay, 5 of 19 MCAB lysed HEM cells, 6 of 21 lysed human skeletal muscle cells, and 10 of 22 lysed human thyroid cells. These findings support results from earlier clinical studies which showed that eye muscle membrane reactive autoantibodies in the serum of patients with thyroid-associated ophthalmopathy cross-react with membrane prepared from other striated muscle. The significance of eye muscle, skeletal muscle, and thyroid cross-reactivity of MCAB is discussed in the context of autoimmune thyroid disease and ophthalmopathy.

Nature and significance of orbital autoantigens and their corresponding autoantibodies in thyroid-associated ophthalmopathy.

There is now considerable evidence that the pathogenesis of thyroid-associated ophthalmopathy is closely linked to the presence of a shared autoantigen(s) in the thyroid and the eye muscle, against which cytotoxic mechanisms are directed. Although the orbital connective tissue is certainly involved in the orbital inflammatory process, a 64 kDa membrane protein expressed by both the eye muscle and the thyroid and recognized consistently by antibodies in the sera of TAO patients, seems to be the most likely target candidate. While its presence in non ocular skeletal muscle is not as well established, more recent data tend to suggest the existence of a 64 kDa molecule in the three tissues. The availability of a cDNA encoding a 572 amino acid protein corresponding to a MW of 63-64 kDa, which may be the same molecule, will allow us to determine more clearly the structural characteristics of the different molecules proposed as targets. The role of the corresponding autoantibodies in the pathogenesis of the eye disease is far less well defined. Whether they play a role in the induction of the ophthalmopathy or only represent helpful markers remains to be clarified.

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 Hashimotos 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)

Cross-reactive antibodies in the serum of balb/c mice immunized with thyroid or eye muscle membranes

During the course of immunizing balb/c mice with eye muscle (EM) or thyroid (THY) membranes for monoclonal antibody (MCAB) production their sera frequently contain antibodies which react against both EM and THY membranes in enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In order to further study this phenomenon we have analyzed sera from 27 balb/c mice, including 10 that were studied serially, and their tissues examined histologically at sacrifice. Following immunization serum and, in some cases, the corresponding MCAB produced by fusion of the mouse spleen cells with a mouse myeloma cell line, were tested for EM and THY cross-reactivity in an ELISA and by immunoblotting. The number of antibodies demonstrated in Western blotting identified as bands of reactivity, and ELISA levels, expressed as optical density — increased with time, each peaking at around 10–12 weeks. THY and EM antibody cross-reactivity was demonstrated in the majority of mice, serum from mice immunized with THY membranes reacting with these membranes as well as with pig EM membranes in both ELISA and immunoblotting and, conversely, sera from mice immunized with pig EM membranes also reacting with THY membranes in the two tests. In Western blotting a variety of THY and EM-reactive antibodies were demonstrated including those directed against a 64 kDa protein, shown to be an important autoantigen in thyroid-associated ophthalmopathy. There was also some cross-reactivity with brain membranes, used as control antigen in both tests and in immunization, although to a lesser degree, but very little to liver and orbital connective tissue membrane. While there were several exceptions there was, overall, a fairly close correlation between serum reactivities and tissue specificities of the corresponding MCAB. No mouse demonstrated significant lymphocytic infiltration in THY or orbital tissues and none developed features of an ophthalmopathy although the experimental protocol was not optimised for the production of autoimmunity. This is the first report of the production of antibodies reactive against putative THY and EM shared epitopes in experimental animals and provides further support for the notion that the mechanism for the association of ophthalmopathy with autoimmune THY disorders in humans may be immunological cross-reactivity.