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The exophthalmogenic activity of the beta subunit of bovine thyrotropin is only 10% to 20% that of the thyrotropin molecule or of an exophthalmogenic factor produced by partial pepsin digestion of purified thyrotropin preparations. The alpha subunit of thyrotropin, luteinizing hormone, and both subunits of luteinizing hormone have no exophthalmogenic activity.

The present report demonstrates that mouse tumor thyrotropin preprarations have exophthalmogenic activity. In addition it shows that the exophthalmogenic activity of mouse tumor thyrotropin can be increased by partial pepsin digestion, whereas the thyroid stimulating activity of mouse tumor thyrotropin is rapidly destroyed by such treatment. Thus, after 30 min of pepsin digestion, mouse tumor thyrotropin has 130% of its initial exophthalmogenic action but only 10% of its thyroid stimulating activity. Preparations of human thyrotropin are similarly sensitive to partial pepsin digestion, i.e., there is a rapid destruction of thyroid stimulating activity but a very much slower destruction of exophthalmogenic activity. Thus, after 30 min of pepsin digestion, preparations of human thyrotropin retain 80% of their exophthalmogenic activity but only 20-30% of their thyroid stimulating action. Since these results are analogous to those obtained in studies of the partial pepsin digestion of bovine thyrotropin (1), partial pepsin digestion of both purified human and purified mouse tumor thyrotropin preparations should yield an exophthalmogenic fragment of the TSH molecule devoid of thyroid stimulating action.

Patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity to antigens present in extracts of certain normal human tissue; namely,thyroid gland and retroorbital tissue. The delayed hypersensitivity can be assayed in vitro by quantitating the amount of a lymphokine, migration inhibition factor (MIF), which is produced when T lymphocytes of patients with Graves' disease and exophthalmos are exposed to these antigens. In the present report, a partial purification is described for the retro-orbital tissue antigen which is responsible for the positive leucocyte migration inhibition factor assay (MIF assay) exhibited by a sensitized lymphocytes of these patients. The purified retro-orbital tissue antigen preparation demonstrates a 50- to 150-fold higher specific activity over crude homogenates in its ability to act as an antigen in the MIF assay of exophthalmic patients. Immunodiffusion, ultracentrifugation, and disc electrophoretic data indicate that this purified antigen preparation, obtained from normal human, retro-orbital tissue, contains thyroglobulin or a derivative of thyroglobulin; immunofluorescence studies localize the anti-thyroglobulin reactive material to the plasma membranes of extraocular muscle fibers of normal individuals. On the basis of these data it is concluded that thyroglobulin or a derivative of the thyroglobulin molecule is present in the orbital muscle of normal individuals. Since thyroglobulin purified from normal human thyroid glands and the purified retro-orbital tissue preparation are nearly equivalent as antigens in the MIF assay of exophthalmic patients, we conclude that thyroglobulin or an antigenic component of the thyroglobulin molecule is one of the antigens to which patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity.

When grown in the presence of thyrotropin, dog thyroid cells in culture from follicle-like structures, take up labeled iodide, and iodinate macromolecular components in the cell. When grown in the absence of thyrotropin, dog thyroid cells in culture form a monolayer, take up only 6% of the iodide of follicular cells, and do not iodinate macromolelcular components in the cell. The iodide uptake in monolayer cells does, however, reflect an incorporation process unique to thyroid cells because hepatocytes and fibroblasts do not have the capacity of the monolayer cells to take up iodide. Thyrotropin stimulation of monolayer cells for a prolonged period (3-8 days) causes the cAMP levels of these cells to return to levels identical to those in follicular cells. The increased cAMP levels are not due to the induction of an adenylate cyclase enzyme, because homogenates of monolayer cells have a thyrotropin-stimulable adenylate cyclase activity. The low level of cAMP, thus, seems to be a problem of receptor coupling to the adenylate cyclase enzyme. The return of cAMP to normal levels is accompanied by an increase in iodide uptake and by macromolecular organification; the return of cAMP levels to normal values is not accompanied by follicular development. The majority (75%) of the iodinated macromolecular product accumulated by follicular thyroid cells, by monolayer thyroid cells stimulated with thyrotropin for a prolonged period, or by thyroid cells treated with dibutyryl cAMP from the onset of culture has the characteristics of 19 S thyroglobulin. The remainder appears to be low mol wt material which may be thyroglobulin-related i.e., be either precursor or biodegraded material.