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. 1989 Apr;134(4):865–877.

Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in myasthenia gravis.

A Marx 1, T Kirchner 1, F Hoppe 1, R O'Connor 1, B Schalke 1, S Tzartos 1, H K Müller-Hermelink 1
PMCID: PMC1879786  PMID: 2468286

Abstract

Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients.

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  1. Berrih S., Arenzana-Seisdedos F., Cohen S., Devos R., Charron D., Virelizier J. L. Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells. J Immunol. 1985 Aug;135(2):1165–1171. [PubMed] [Google Scholar]
  2. Dardenne M., Savino W., Bach J. F. Thymomatous epithelial cells and skeletal muscle share a common epitope defined by a monoclonal antibody. Am J Pathol. 1987 Jan;126(1):194–198. [PMC free article] [PubMed] [Google Scholar]
  3. Engel E. K., Trotter J. L., McFarlin D. E., McIntosh C. L. Thymic epithelial cell contains acetylcholine receptor. Lancet. 1977 Jun 18;1(8025):1310–1311. doi: 10.1016/s0140-6736(77)91343-5. [DOI] [PubMed] [Google Scholar]
  4. Gilhus N. E., Aarli J. A., Christensson B., Matre R. Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients. J Neuroimmunol. 1984 Nov;7(1):55–64. doi: 10.1016/s0165-5728(84)80006-5. [DOI] [PubMed] [Google Scholar]
  5. Gotti C., Conti-Tronconi B. M., Raftery M. A. Mammalian muscle acetylcholine receptor purification and characterization. Biochemistry. 1982 Jun 22;21(13):3148–3154. doi: 10.1021/bi00256a018. [DOI] [PubMed] [Google Scholar]
  6. Gown A. M., Vogel A. M. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. J Cell Biol. 1982 Nov;95(2 Pt 1):414–424. doi: 10.1083/jcb.95.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  8. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundelfinger E. D., Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature. 1987 Jul 16;328(6127):215–220. doi: 10.1038/328215a0. [DOI] [PubMed] [Google Scholar]
  9. Hohlfeld R., Toyka K. V., Miner L. L., Walgrave S. L., Conti-Tronconi B. M. Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis. J Clin Invest. 1988 Mar;81(3):657–660. doi: 10.1172/JCI113369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kao I., Drachman D. B. Thymic muscle cells bear acetylcholine receptors: possible relation to myasthenia gravis. Science. 1977 Jan 7;195(4273):74–75. doi: 10.1126/science.831257. [DOI] [PubMed] [Google Scholar]
  11. Kawanami S., Kamei H., Oita J., Kurokawa M., Uchida Y., Hayashi K. Acetylcholine-receptor-like protein from human thymoma associated with myasthenia gravis. J Neurol. 1987 May;234(4):207–210. doi: 10.1007/BF00618251. [DOI] [PubMed] [Google Scholar]
  12. Kirchner T., Hoppe F., Müller-Hermelink H. K., Schalke B., Tzartos S. Acetylcholine receptor epitopes on epithelial cells of thymoma in myasthenia gravis. Lancet. 1987 Jan 24;1(8526):218–218. doi: 10.1016/s0140-6736(87)90032-8. [DOI] [PubMed] [Google Scholar]
  13. Kirchner T., Schalke B., Melms A., von Kügelgen T., Müller-Hermelink H. K. Immunohistological patterns of non-neoplastic changes in the thymus in Myasthenia gravis. Virchows Arch B Cell Pathol Incl Mol Pathol. 1986;52(3):237–257. doi: 10.1007/BF02889966. [DOI] [PubMed] [Google Scholar]
  14. Kirchner T., Tzartos S., Hoppe F., Schalke B., Wekerle H., Müller-Hermelink H. K. Pathogenesis of myasthenia gravis. Acetylcholine receptor-related antigenic determinants in tumor-free thymuses and thymic epithelial tumors. Am J Pathol. 1988 Feb;130(2):268–280. [PMC free article] [PubMed] [Google Scholar]
  15. Kubalek E., Ralston S., Lindstrom J., Unwin N. Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata. J Cell Biol. 1987 Jul;105(1):9–18. doi: 10.1083/jcb.105.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lindstrom J., Criado M., Ratnam M., Whiting P., Ralston S., Rivier J., Sarin V., Sargent P. Using monoclonal antibodies to determine the structures of acetylcholine receptors from electric organs, muscles, and neurons. Ann N Y Acad Sci. 1987;505:208–225. doi: 10.1111/j.1749-6632.1987.tb51293.x. [DOI] [PubMed] [Google Scholar]
  18. Lindstrom J., Gullick W., Conti-Tronconi B., Ellisman M. Proteolytic nicking of the acetylcholine receptor. Biochemistry. 1980 Oct 14;19(21):4791–4795. doi: 10.1021/bi00562a012. [DOI] [PubMed] [Google Scholar]
  19. Lindstrom J., Schoepfer R., Whiting P. Molecular studies of the neuronal nicotinic acetylcholine receptor family. Mol Neurobiol. 1987 Winter;1(4):281–337. doi: 10.1007/BF02935740. [DOI] [PubMed] [Google Scholar]
  20. Matsumoto Y., Furuya A., Kobayashi T., Tsukagoshi H. Primary cultures of human myasthenia gravis thymus and normal thymus. Studies of cell morphology, cell proliferative pattern and localization of alpha-bungarotoxin binding sites on cultured thymic cells. J Neurol Sci. 1986 Sep;75(2):121–133. doi: 10.1016/0022-510x(86)90088-2. [DOI] [PubMed] [Google Scholar]
  21. McAllister R. M., Isaacs H., Rongey R., Peer M., Au W., Soukup S. W., Gardner M. B. Establishment of a human medulloblastoma cell line. Int J Cancer. 1977 Aug 15;20(2):206–212. doi: 10.1002/ijc.2910200207. [DOI] [PubMed] [Google Scholar]
  22. Momoi M. Y., Lennon V. A. Purification and biochemical characterization of nicotinic acetylcholine receptors of human muscle. J Biol Chem. 1982 Nov 10;257(21):12757–12764. [PubMed] [Google Scholar]
  23. Müller-Hermelink H. K., Marino M., Palestro G. Pathology of thymic epithelial tumors. Curr Top Pathol. 1986;75:207–268. doi: 10.1007/978-3-642-82480-7_7. [DOI] [PubMed] [Google Scholar]
  24. Papiernik M., Nabarra B., Bach J. F. In vitro culture of functional human thymic epithelium. Clin Exp Immunol. 1975 Feb;19(2):281–287. [PMC free article] [PubMed] [Google Scholar]
  25. Patrick J., Stallcup W. B. Immunological distinction between acetylcholine receptor and the alpha-bungarotoxin-binding component on sympathetic neurons. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4689–4692. doi: 10.1073/pnas.74.10.4689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pyke K. W., Gelfand E. W. Morphological and functional maturation of human thymic epithelium in culture. Nature. 1974 Oct 4;251(5474):421–423. doi: 10.1038/251421a0. [DOI] [PubMed] [Google Scholar]
  27. Radzun H. J., Parwaresch M. R., Feller A. C., Hansmann M. L. Monocyte/macrophage-specific monoclonal antibody Ki-M1 recognizes interdigitating reticulum cells. Am J Pathol. 1984 Dec;117(3):441–450. [PMC free article] [PubMed] [Google Scholar]
  28. Ratnam M., Nguyen D. L., Rivier J., Sargent P. B., Lindstrom J. Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles. Biochemistry. 1986 May 6;25(9):2633–2643. doi: 10.1021/bi00357a052. [DOI] [PubMed] [Google Scholar]
  29. Ratnam M., Sargent P. B., Sarin V., Fox J. L., Nguyen D. L., Rivier J., Criado M., Lindstrom J. Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochemistry. 1986 May 6;25(9):2621–2632. doi: 10.1021/bi00357a051. [DOI] [PubMed] [Google Scholar]
  30. Schluep M., Willcox N., Vincent A., Dhoot G. K., Newsom-Davis J. Acetylcholine receptors in human thymic myoid cells in situ: an immunohistological study. Ann Neurol. 1987 Aug;22(2):212–222. doi: 10.1002/ana.410220205. [DOI] [PubMed] [Google Scholar]
  31. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  32. Sealock R. Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes. J Neurosci. 1982 Jul;2(7):918–923. doi: 10.1523/JNEUROSCI.02-07-00918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Syapin P. J., Salvaterra P. M., Engelhardt J. K. Neuronal-like features of TE671 cells: presence of a functional nicotinic cholinergic receptor. Brain Res. 1982 Jan 14;231(2):365–377. doi: 10.1016/0006-8993(82)90373-0. [DOI] [PubMed] [Google Scholar]
  34. Tzartos S., Langeberg L., Hochschwender S., Lindstrom J. Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. FEBS Lett. 1983 Jul 11;158(1):116–118. doi: 10.1016/0014-5793(83)80688-7. [DOI] [PubMed] [Google Scholar]
  35. Tzartos S., Langeberg L., Hochschwender S., Swanson L. W., Lindstrom J. Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity. J Neuroimmunol. 1986 Jan;10(3):235–253. doi: 10.1016/0165-5728(86)90105-0. [DOI] [PubMed] [Google Scholar]
  36. Wada K., Ballivet M., Boulter J., Connolly J., Wada E., Deneris E. S., Swanson L. W., Heinemann S., Patrick J. Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science. 1988 Apr 15;240(4850):330–334. doi: 10.1126/science.2832952. [DOI] [PubMed] [Google Scholar]
  37. Whiting P. J., Vincent A., Newsom-Davis J. Myasthenia gravis: monoclonal antihuman acetylcholine receptor antibodies used to analyze antibody specificities and responses to treatment. Neurology. 1986 May;36(5):612–617. doi: 10.1212/wnl.36.5.612. [DOI] [PubMed] [Google Scholar]
  38. Whiting P., Lindstrom J. Purification and characterization of a nicotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci U S A. 1987 Jan;84(2):595–599. doi: 10.1073/pnas.84.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Willcox N., Schluep M., Ritter M. A., Schuurman H. J., Newsom-Davis J., Christensson B. Myasthenic and nonmyasthenic thymoma. An expansion of a minor cortical epithelial cell subset? Am J Pathol. 1987 Jun;127(3):447–460. [PMC free article] [PubMed] [Google Scholar]
  40. Williams C. L., Lennon V. A. Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin. J Exp Med. 1986 Oct 1;164(4):1043–1059. doi: 10.1084/jem.164.4.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]

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