Serological reactivity of recombinant 1D autoantigen and its expression in human thyroid and eye muscle tissue: a possible autoantigenic link in Graves' patients.
Thyroid-associated ophthalmopathy (TAO) is a potentially severe autoimmune disease, in and around the orbit, usually accompanied by Graves' disease. It was the goal of this study to develop a serological indicator for TAO and to characterize its expression in human thyroid and eye muscle tissue. Thus, we have recloned the full-length 1D-complementary DNA and assessed its expression levels in 90 healthy and diseased human thyroids. Only Graves' patients suffering from TAO (n = 29) displayed a significant, 2.1-fold increase of 1D expression levels (P = 0.029), compared with normal controls (n = 9), as assessed using the Mann-Whitney U-test for paired, nonnormally distributed samples. In contrast, a decrease of 1D expression (to 40% of control normal values) was confined to thyroid autonomy (n = 19, P = 0.032). In all other diseased human thyroids, including Graves' thyroids from patients not suffering from clinically overt TAO (n = 9), 1D expression levels were not different from the healthy controls. 1D gene expression was demonstrated in both healthy (n = 10) and diseased (n = 10) eye muscle tissues. Furthermore, a recombinant protein derived from baculovirus-infected Sf9 insect cells was purified under both nondenaturing and denaturing conditions. While under nondenaturing conditions, the molecular mass of recombinant 1D was determined to be 85 kDa; denaturing isolation yielded the expected 64-kDa protein. Autoantibodies against denatured 1D protein were not detectable in sera of diseased or healthy subjects. Immunoreactivity against the 85-kDa, nondenatured protein, evaluated in a panel of 222 different human sera, showed that 82% of Graves' patients suffering from TAO had autoantibodies against recombinant 1D, whereas only 5% of the healthy controls were positive for antibodies against 1D. Taken together, our results demonstrate a high disease sensitivity and specificity of recombinant, nondenatured 1D, to distinguish Graves' disease with or without TAO from other forms of thyroid and/or eye disease. Prospective studies will have to show whether autoantibodies against 1D can also be used as a prognosticator of TAO.
Institute for Immunology, Pathology and Molecular Biology, Hamburg, Germany. kromminga@labor-keeser-arndt.de
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Kromminga
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Hagel
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Arndt
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Schuppert
D000818:Animals; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D005123:Eye / Q000276:immunology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D009132:Muscles / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D012313:RNA / Q000032:analysis; D011994:Recombinant Proteins / Q000276:immunology; D018411:Spodoptera / Q000378:metabolism; D013961:Thyroid Gland / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens; D003598:Cytoskeletal Proteins; C103926:LMOD1 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D012313:RNA