Binding of Thyroglobulin and Thyroglobulin-Antithyroglobulin Immune Complex to Extraocular Muscle Membrane

Abstract

To study the possible role of a thyroid antigen and its immune complex in the pathogenesis of Graves' ophthalmopathy, the binding of human thyroglobulin (Tg) and Tg-anti-Tg immune complex (Tg-A) to human and bovine extraocular muscle membrane fractions were studied. Membrane fractions were prepared from homogenates by sucrose density gradient centrifugation and verified by electron microscopy. Tg-A was made by mixing Tg with human anti-Tg under conditions of antigen excess. In the optimal system, membrane fractions were incubated with 125I-Tg or 125I-Tg-A for 80 min at 37 C, in Tris buffer pH 7.4, in the presence of 0.8 mM EDTA and 13% dialyzed human serum. About 4% of 125I-Tg bound to human and bovine extraocular muscle membrane fractions of density <1.14 while binding of Tg-A was several-fold higher (up to 27%). Binding was detectable at 4 µg/ml of membrane fraction, and it increased progressively with fraction amount up to about 50 µg/ml; it was abolished by cooling to 4 C and diminished by heating to 56 C or by increasing buffer ionic strength. Binding of I25I-Tg was inhibited in the presence of added non-radioactive Tg. Binding of Tg-A to membrane fractions is believed to be secondary to prior binding of Tg. Tg and Tg-A binding was less to membrane fractions of cheek, thoracic or heart muscle, and of kidney than to extraocular muscle. Neither substance bound to membrane fractions obtained from liver or spleen. Muscle membrane fractions showed no affinity for labeled human anti-Tg, normal IgG globulin, H-TSH, growth hormone or immune complexes of TSH or growth hormone. An immune complex of Tg prepared with the (Fab')2 fragment of anti-Tg bound as well as Tg-A, but a complex prepared with the corresponding Fab fragment bound less well to membrane preparations. Binding of Tg and Tg-A to membrane fractions did not occur when incubations were carried out in the absence of serum. Subsequently, the obligatory factor in human serum was found to be gamma globulin. Individual sera varied considerably in efficacy in promoting binding, male sera being more potent than female. Species differences in sera were also noted; monkey and rabbit sera were potent but those of a number of other species were inactive. The data suggest that attachment of Tg or Tg-A to skeletal muscle, especially extraocular muscle, could play a role in triggering immunological events in the orbit leading to the development of Graves′ ophthalmopathy. (Endocrinology95: 434, 1974)