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Journal of Clinical Immunology

, Volume 27, Issue 2, pp 172–180 | Cite as

Immunohistochemical Studies Using Immunized Guinea Pig Sera with Features of Anti-Human Thyroid, Eye and Skeletal Antibody and Graves’ Sera

  • IldikÓ MolnÁr
  • Zita Szombathy
  • Ilona KovÁcs
  • A. JÓZsef SzentmiklÓsi
Article

Type 2 5′ deiodinase enzyme was observed in both thyroid and eye muscle tissues, highlighting its possible role as a common antigen in thyroid-associated ophthalmopathy. Sera of 105 Graves’ patients and 40 controls, and immunized guinea pig sera against TCSS peptide, showing homology to the amino acid sequence from 132 to 152 of type 2 5′ deiodinase, were investigated to demonstrate the binding effects to human thyroid, eye and skeletal muscle tissues.

Twenty-two Graves’ patients were positive for anti-TCSS peptide antibodies, of whom 18 cases had ophthalmopathy. The levels of anti-TCSS peptide antibodies were higher not only in Graves’ patients with (P<0.0001) and without (P<0.036) eye symptoms compared to controls but also the difference was significant between patients with and without ophthalmopathy (P<0.049). In Western blot, immunized sera showed binding reactions to the supernatant fractions of human thyroid, eye and skeletal muscle tissues at the range of 29 kDa. Patient sera with Graves’ ophthalmopathy resulted in positive reactions directed to membrane areas in thyroid follicular cells, and to fibers in eye and skeletal muscles using immunohistochemical method, while no positive staining was present after adding control sera. The binding features of immunized guinea pig sera exhibited similar staining in all human tissues but could be blocked with Graves’ sera. Our results suggest that type 2 5′ deiodinase enzyme protein could play a role as an antigen in Graves’ disease. Immunized guinea pig sera against TCSS peptide exhibited similar binding reactions and stainings to human thyroid, eye and skeletal muscle tissues as patient sera with Graves’ ophthalmopathy.

KEY WORDS

Type 2 5′ deiodinase Graves’ ophthalmopathy antibodies eye and skeletal muscle thyroid 

REFERENCES

  1. 1.
    Bahn RS: Clinical Review 157. Pathophysiology of Graves’ ophthalmopathy. The cycle of disease. J Clin Endocrinol Metab 88:1939–1946, 2003PubMedCrossRefGoogle Scholar
  2. 2.
    Smith TJ, Koumas L, Gagnon A, Bell A, Sempowski GD, Phipps RP, Sorisky A: Orbital fibroblast heterogeneity may determine the clinical presentation of thyroid-associated ophthalmopathy. J Clin Endocrinol Metab 87:385–392, 2002PubMedCrossRefGoogle Scholar
  3. 3.
    Takashi Y, Akira S, Ichiro K, Takeo N, Yoshiharu I, Akira T, Sumiya E, Yoshiya T: An elevation of serum immunoglobulin E provides a new aspect of hyperthyroid Graves’ disease. J Clin Endocrinol Metab 85:2775–2778, 2000CrossRefGoogle Scholar
  4. 4.
    Kumar S, Coenen MJ, Scherer PE, Bahn RS: Evidence for enhanced adipogenesis in the orbits of patients with Graves’ ophthalmopathy. J Clin Endocrinol Metab. 89:930–935, 2004PubMedCrossRefGoogle Scholar
  5. 5.
    Mizokami T, Salvi M, Wall JR: Eye muscle antibodies in Graves’ ophthalmopathy: Pathogenic or secondary epiphenomenon? J Endocrinol Invest 27:221–229, 2004PubMedGoogle Scholar
  6. 6.
    Kromminga A, Hagel C, Arndt R, Schuppert F: Serological reactivity of recombinant 1D autoantigen and its expression in human thyroid and eye muscle tissue: A possible autoantigenic link in Graves’ patients. J Clin Endocrinol Metab 83:2817–2823, 1998PubMedCrossRefGoogle Scholar
  7. 7.
    Gunji K, De Bellis A, Kubota S, Swanson J, Wengrowicz S, Cochran B, Ackrell BAC, Salvi M, Bellastella A, Bizzarro A, Sinisi AA, Wall JR: Serum antibodies against the flavoprotein subunit of eye muscle autoimmunity in patients with Graves’ hyperthyroidism. J Clin Endocrinol Metab 84:1255–1262, 1999PubMedCrossRefGoogle Scholar
  8. 8.
    Gunji K, De Bellis A, Li AW, Yamada M, Kubota S, Ackrell B, Wengrowicz S, Bellastella A, Bizzarro A, Sinisi A, Wall JR: Cloning and characterization of the novel thyroid and eye muscle shared protein G2s: Autoantibodies against G2s are closely associated with ophthalmopathy in patients with Graves’ hyperthyroidism. J Clin Endocrinol Metab 85:1641–1647, 2000PubMedCrossRefGoogle Scholar
  9. 9.
    De Bellis A, Sansone D, Coronella C, Conte M, Iorio S, Perrino S, Battaglia M, Bellastella G, Wall JR, Bellastella A, Bizzarro A: Serum anibodies to collagen XIII: A futher good marker of active Graves’ ophthalmopathy. Clin Endocrinol 62:24–29, 2005CrossRefGoogle Scholar
  10. 10.
    Gopinath B, Musselman R, Adams CL, Tani J, Beard N, Wall JR: Study of serum antibodies against three eye muscle antigens and the connective tissue antigen collagen XIII in patients with Graves’ disease with and without ophthalmopathy: Correlation with clinical features. Thyroid 16:967–974, 2006.PubMedCrossRefGoogle Scholar
  11. 11.
    Tani J, Wall JR: Autoimmunity against eye-muscle antigens may explain thyroid-associated ophthalmopathy. CMAJ 175:239–241, 2006PubMedGoogle Scholar
  12. 12.
    Salvatore D, Tu H, Harney JW, Larsen PR: Type 2 iodothyronine deiodinase is highly expressed in human thyroid. J Clin Invest 98:962–968, 1996PubMedCrossRefGoogle Scholar
  13. 13.
    Hosoi Y, Murakami M, Mizuma H, Ogiwara T, Imamura M, Mori M: Expression and regulation of type II iodothyronine deiodinase in cultured human skeletal muscle cells. J Clin Endocrinol Metab 84:3293–3300, 1999PubMedCrossRefGoogle Scholar
  14. 14.
    Croteau W, Davey JC, Galton VA, Germain LSt: Cloning of the mammalian type II iodothyronine deiodinase. J Clin Invest 98:405–417, 1996PubMedGoogle Scholar
  15. 15.
    Merrifield RB: The synthesis of a tetrapeptide. Solid phase peptide synthesis. J Am Chem Soc 85:2149–2154, 1963CrossRefGoogle Scholar
  16. 16.
    Matsueda GR, Stewart JM: A p-ethylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides. Peptides 2:45–50, 1981PubMedCrossRefGoogle Scholar
  17. 17.
    Sakakibara S, Shimonishim Y, Kishida Y, Okada M, Sugihara K: Use of anhydrous hydrogen fluoride in peptide synthesis. I. Behavior of various protective groups in anhydrous hydrogen fluoride. Bull Chem Soc Jpn 40:2164–2167, 1967PubMedCrossRefGoogle Scholar
  18. 18.
    Werner SC: Modification of classification of eye changes of Graves’ disease: Recommendations of the Ad Hoc Committee of American Thyroid Association. J Clin Endocrinol Metab 44:2034, 1977CrossRefGoogle Scholar
  19. 19.
    Mourits PM, Prummel ME, Wiersinga WM, Kornneef L: Clinical activity score as a guide in the management of patients with Graves’ ophthalmopathy. Clin Endocrinol (Oxford) 47:9–14, 1997CrossRefGoogle Scholar
  20. 20.
    Bradford MM: A rapid and sensitive method for the qualification of microgram quantites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976PubMedCrossRefGoogle Scholar
  21. 21.
    Molnar I., Kaczur V., Boros A., Krajczar G., Balzs C.: IgA autoantibodies against human eye muscle antigen detected by Western blotting and immunohistochemical methods in Graves’ disease. J Endocrinol Invest 18:408–414, 1995PubMedGoogle Scholar
  22. 22.
    Darras VM, Hume R, Visser TJ: Regulation of thyroid hormone metabolism during fetal development. Mol Cell Endocrinol 151:37–47, 1999PubMedCrossRefGoogle Scholar
  23. 23.
    Köhrle J: Local activation and inactivation of thyroid hormones: The deiodinase family. Mol Cell Endocrinol 151:103–119, 1999PubMedCrossRefGoogle Scholar
  24. 24.
    Molnar I, Csathy L: Autoantibodies against the type 2 5′-deiodinase are involved in the development of Graves’ ophthalmopathy. In Program of 12th international Thyroid Congress, Kyoto, Japan, p 176 (Abstract), 2000Google Scholar
  25. 25.
    Nakahara R, Tsunekawa K, Yabe S, Nara M, Seki K, Kasahara T, Ogiwara T, Nishino M, Kobayashi I, Murakami M: Association of antipitiutary antibody and type 2 iodothyronine deiodinase antibody in patients with autoimmune thyroid disease. Endocrinol J 52:691–699, 2005Google Scholar
  26. 26.
    Stadtman TC: Selenoproteins—tracing the role of a trace element in protein function. PloS Biol 3:2077–2079, 2005CrossRefGoogle Scholar
  27. 27.
    Gereben B, Goncalves C, Harney JW, Larsen PR, Bianco AC: Selective proteolysis of human type 2 deiodinase: A novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation. Mol Endocrinol 14:1697–1708, 2000PubMedCrossRefGoogle Scholar
  28. 28.
    Zeöld A, Pormüller L, Dentice M, Harney JW, Curcio-Morelli C, Tente SM, Bianco AC, Gereben B: Metabolic instability of type 2 deiodinase is transferable to stable proteins independently of subcellular localization. J Biol Chem 281:31538–31543, 2006PubMedCrossRefGoogle Scholar
  29. 29.
    Burmeister LA, Pachucki J, St. Germain DL: Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138:5231–5237, 1997PubMedCrossRefGoogle Scholar
  30. 30.
    Steinsapir J, Bianco AC, Buettner C, Harney J, Larsen PR: Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme’s active center. Endocrinology 141:1127–1135, 2000PubMedCrossRefGoogle Scholar
  31. 31.
    Kaminski HJ, Li Z, Richmonds C, Ruff RL, Kusner L: Susceptibility of ocular tissues to autoimmune diseases. Ann NY Acad Sci 998:362–374, 2003PubMedCrossRefGoogle Scholar
  32. 32.
    Andrikoula M, Tsatsoulis A: The role of FAS-mediated apoptosis in thyroid disease. Eur J Endocrinol 144:561–568, 2001PubMedCrossRefGoogle Scholar
  33. 33.
    Pittoni V, Valesini G: The clearence of apoptotic cells: Implications for autoimmunity. Autoimmunity Rev 1:154–161, 2002CrossRefGoogle Scholar
  34. 34.
    Salvatore D, Bartha T, Harney JW, Larsen PR: Molecular biological and biochemical characterization of the human type 2 selenodeiodinase. Endocrinology 137:3308–3315, 1996PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • IldikÓ MolnÁr
    • 1
    • 4
  • Zita Szombathy
    • 2
  • Ilona KovÁcs
    • 2
  • A. JÓZsef SzentmiklÓsi
    • 3
  1. 1.3rd Department of Internal MedicineKenézy County and Teaching HospitalDebrecenHungary
  2. 2.PathologyKenézy County and Teaching HospitalH-4043 DebrecenHungary
  3. 3.Department of Pharmacology and PharmacotherapyUniversity Debrecen, Medical and Health Science CenterDebrecenHungary
  4. 4.3rd Department of Internal MedicineKenézy County and Teaching HospitalDebrecenHungary

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