Analysis of human thyrotropin receptor gene expression and immunoreactivity in human orbital tissue

The human thyrotropin receptor (hTSHR) represents an autoantigen that plays a central role in the hyperthyroidism of Graves' disease (GD). hTSHR transcripts have recently been detected by reverse transcription (RT)-PCR in various extrathyroidal tissues, suggesting that the hTSHR may be more widely distributed than previously thought, and that it may serve as a common antigen in the thyroidal and extrathyroidal manifestations of GD. Using techniques other RT-PCR, we examined whether RNA encoding hTSHR and hTSHR-specific immunoreactivity can be detected and localized in cultured orbital fibroblasts (OF), orbital connective tissue, extraocular muscle and various extraorbital tissues derived from both patients with Graves' ophthalmopathy (GO) and normal individuals. Using in situ hybridization with a digoxigenin-labeled antisense oligonucleotide probe specific for the extracellular domain of hTSHR, specific perinuclear and cytoplasmic hTSHR gene expression was detected in OF of patients with GO and, to a lesser degree, normal individuals. Using a highly sensitive immunostaining technique and a panel of monoclonal and polyclonal antibodies directed against different epitopes of recombinant hTSHR, distinct cytoplasmic hTSHR-like immunoreactivity was detected in methanol-fixed OF and orbital connective tissue, which was absent in abdominal fibroblasts, or when using isotype-matched non-immune immunoglobulins. Mouse monoclonal and pig polyclonal hTSHR antibodies detected cytoplasmic hTSHR-like immunoreactivity in perimysial fibroblasts within extraocular muscle, but not in extraocular muscle fibers. Immunocytochemical staining with rabbit polyclonal hTSHR antibody revealed, in addition, distinct cell surface-associated immunoreactivity in paraformaldehyde-fixed OF, but not in abdominal fibroblasts. Taken together, our results suggest that RNA encoding hTSHR is present and actively processed to immunoreactive hTSHR-like protein in OF residing within orbital connective tissue and extraocular muscle. These data support the concept that OF expressing intact or variant hTSHR may act as extrathyroidal targets for sensitized T-cells and immunoglobulins in GD.