Studies on Monoclonal Antibodies to TSH Receptors

Heterogeneity and Pathophysiological Significance of Antibodies to TSH Receptor

Abstract

Recent studies revealed that anti-TSH receptor autoantibodies are involved in the pathogenesis of both Graves' disease and a part of hypothyroidism, but precise mechanism of action of these antibodies remained to be studied. In order to delineate the heterogeneity of these antibodies and their pathophysiological significance, we produced monoclonal antibodies to TSH receptor and studied their characteristics.
Mouse monoclonal antibodies to TSH receptor were derived from spleen cells of mice immunized with partially purified human TSH receptor, which was prepared by TSHcoupled affinity chromatography of thyroid membrane solubilized with Triton X-100. By fusing spleen cells and mouse myeloma cells in the presence of polyethylene glycol and selecting with limiting dilution method, 5 hybridomas were obtained. Among 3 antibodies, which inhibited TSH binding to thyroid membrane (TSH displacing activity, TDA), 2 inhibited TSH stimulation of thyroid adenylate cyclase (AC) (human thyroid adenylate cyclase inhibitor activity, HTACI), and one showed no bioactivity. Among other 2 antibodies without TDA, 1 stimulated AC (human thyroid adenylate cyclase stimulator activity, HTACS) and the other inhibited TSH stimulation (HTACI). All activities of these antibodies were dependent on IgG concentration and disappeared by treatment of anti-mouse IgG antibodies.
In addition, 4 human-human hybridomas were established by fusing human peripheral lymphocytes of patients with Graves' disease and nongoitrous hypothyroidism with human lymphoblastoid cell line. Among 2 antibodies with TDA, one antibody inhibited TSH stimulation of AC, inhibiting TSH binding competitively and another antibody stimulated AC, inhibiting TSH binding noncompetitively. Among the other 2 antibodies, which did not inhibit TSH binding but were shown to bind to TSH receptor by immunoprecipitation, one stimulated AC and the other inhibited TSH stimulation of AC. Among 2 antibodies with HTACI, one antibody with positive TDA inhibited stimulation of AC by stimulative antibodies with positive TDA, but the other without TDA inhibited stimulation of AC by both antibodies with or without positive TDA. These inhibitory antibodies did not inhibit stimulation of AC by Forskolin and Gpp (NH) p, which are known to affect other parts of receptor-AC system than receptor unit.
These data suggest that anti-TSH receptor antibodies are heterogenous in the mode of binding to the receptor and in their bioactivities, and may be involved in the pathogenesis of both Graves' disease and a part of idiopathic hypothyroidism.