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). h TSHR 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 ma y serve as a common antigen in the thyroidal and extrathyroidal manife stations of GD. Using techniques other than RT-PCR, we examined whethe r RNA encoding hTSHR and hTSHR-specific immunoreactivity can be detect ed and localized in cultured orbital fibroblasts (OF), orbital connect ive tissue, extraocular muscle and Various extraorbital tissues derive d from both patients with Graves' ophthalmopathy (GO) and normal indiv iduals. Using in situ hybridization with a digoxigenin-labeled antisen se oligonucleotide probe specific for the extracellular domain of hTSH R, specific perinuclear and cytoplasmic hTSHR gene expression was dete cted in OF of patients with GO and, to a lesser degree, normal individ uals. Using a highly sensitive immunostaining technique and a panel of monoclonal and polyclonal antibodies directed against different epito pes of recombinant hTSHR, distinct cytoplasmic hTSHR-like immunoreacti vity was detected in methanol-fixed OF and orbital connective tissue, which was absent in abdominal fibroblasts, or when using isotype-match ed non-immune immunoglobulins. Mouse monoclonal and pig polyclonal hTS HR antibodies detected cytoplasmic hTSHR-like immunoreactivity in peri mysial fibroblasts within extraocular muscle, but not in extraocular m uscle fibers. Immunocytochemical staining with rabbit polyclonal hTSHR antibody revealed, in addition, distinct cell surface-associated immu noreactivity in paraformaldehyde-fixed OF but not in abdominal fibrobl asts. Taken together, our results suggest that RNA encoding hTSHR is p resent and actively processed to immunoreactive hTSHR-like protein in OF residing within orbital connective tissue and extraocular muscle. T hese data support the concept that OF expressing intact or variant hTS HR may act as extrathyroidal targets for sensitized T-cells and immuno globulins in GD.