Use of Monoclonal Antibodies to Investigate a Possible Role of Thyroglobulin in the Pathogenesis of Graves' Ophthalmopathy*

Abstract

One possible mechanism for Graves' ophthalmopathy is that the progressive orbital inflammation is initiated by formation of thyroglobulin (Tg)-anti-Tg immune complexes at sites of Tg binding to extraocular muscle membranes. In this study monoclonal antibodies (MCAB) against human Tg were used as probes (1) to identify Tg in eye muscle membranes prepared from normal subjects and (2) to measure binding of human Tg and Tg-anti-Tg immune complexes to eye muscle membranes. Reactivity of anti-Tg MCAB with Tg, thyroid, and eye muscle membranes was determined by binding of [125I]anti-Tg monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA), and the indirect immunofluorescence technique. Seven membrane fractions, prepared by differential sucrose gradient centrifugation, were used. Whereas [125I]anti-Tg MCAB bound to all thyroid membrane fractions tested, no [125I]anti-Tg bound to eye muscle membranes. Similarly, reactivity of anti-Tg MCAB with eye muscle membranes was not demonstrated in ELISA or immunofluorescence tests. Although Tg-anti-Tg immune complexes bound to thyroid membranes, such complexes did not bind to eye muscle membranes. Significant binding of [125I]human Tg to eye muscle or thyroid membranes was not demonstrated for any membrane preparation. On the other hand moderate, but significant, binding to skeletal muscle was shown. Similar results were found using an ELISA. Binding of [125I]anti-Tg-Tg complexes of [125I]Tg to thyroid and eye muscle membranes was not affected by the presence of normal human serum, phosphate ions, pH, or incubation temperature, conditions claimed by others to be critical for Tg and Tg-anti-Tg immune complex binding. Since Tg is not present in normal human eye muscle a major role of Tg, or Tg-anti-Tg immune complexes, in the pathogenesis of Graves' ophthalmopathy appears to have been excluded by these findings.

Identification of Thyroglobulin in Orbital Tissues of Patients with Thyroid-Associated Ophthalmopathy

“…In all these studies (14)(15)(16)(17)29) orbital tissues were obtained at autopsy from subjects with neither thyroid disease nor TAO. Because serum Tg levels in normal subjects and in patients without thyroid diseases are usually low or undetectable (30), it is likely that little, if any, deposition of Tg occurs in orbital tissues to detectable levels.…”

“…Based on these findings, it was postulated that Tg may be an antigen involved in the pathogenesis of TAO. However, Wall and associates (16,17) obtained conflicting results in separate studies. In particular, they failed to detect binding of monoclonal anti-TgAbs to eye muscles or to show Tg binding to extraocular muscle membranes (16).…”

“…However, Wall and associates (16,17) obtained conflicting results in separate studies. In particular, they failed to detect binding of monoclonal anti-TgAbs to eye muscles or to show Tg binding to extraocular muscle membranes (16). In the present study we show that intact Tg of thyroid origin is present in orbital tissues from three patients with TAO, but not in orbital tissues from subjects without thyroid or eye disease, supporting the possibility that this thyroid antigen may play a role in the pathogenesis of TAO.…”

Ocular Manifestations of Graves' Disease: A Review

“…A different approach to the question of orbital autoantibodies was recently made by raising monoclonal antibodies from mice immunized with potential orbital antigens (Kodama et al 1982). With these monoclonal antibodies, crude orbital homogenates could be fractionated into separate (xeno-) antigens and these purified antigens were then used to screen for serum autoantibodies in patients.…”

Ciclosporine and Graves’ Ophthalmopathy

“…However, neither the presence nor the extent of the ophthalmopathy has been shown to be correlated with the level of circulating antithyroglobulin antibody [7]. In addition, thyroglobulin cannot be de tected in normal human retro-orbital mus cles and there is no evidence of a specific binding site for thyroglobulin in this tissue [8].…”