Abstract
Mononuclear cells often form highly organized lymphoid structures in the chronically inflamed synovial tissue of patients with rheumatoid arthritis (RA) within which B-cells are activated and may differentiate into effector plasma cells. The analysis of those activated B-cells and the determination of their specificity is of great importance for the understanding of the pathogenesis of RA. Here, we describe a technique that combines histological analysis of synovial tissue with a molecular analysis of the Vgene repertoire at the level of the single B-cell.
Immunohistochemical staining of tissue sections allows us to identify the activated B-cells. Those cells are then isolated using a micromanipulator and the rearranged immunoglobulin (Ig) genes amplified, cloned and sequenced. The combination of the V(D)J gene segments and the pattern of somatic mutations in the V-region genes, allows us to identify clonal relationships between the isolated B cells. Once Ig genes for a heavy and a light chain have been isolated from individual B-cells, they can be used to generate recombinant antibodies. These antibodies can be used to determine the antigens which support the activation of B-cells in the inflamed synovial tissue
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References
Wernick, R. M., Lipsky, P. E., Marban-Arcos, E., Maliakkal, J. J., Edelbaum, D., and Ziff, M. (1985) IgG and IgM rheumatoid factor synthesis in rheumatoid synovial membrane cell cultures. Arthritis Rheum. 28, 742–752.
Natvig, J. B., Randen, I., Thompson, K., Forre, O., and Munthe, E. (1989) The B cell system in the rheumatoid inflammation. New insights into the pathogenesis of rheumatoid arthritis using synovial B cell hybridoma clones. Springer Semin Immunopathol 11, 301–313.
Trentham, D. E., Townes, A. S., and Kang, A. H. (1977) Autoimmune to type II collagen an experimental model of arthritis. J. Exp. Med. 146, 857–868.
Sebbag, M., Simon, M., Vincent, C., Masson-Bessiere, C., Girbal, E., Durieux, J. J., and Serre, G. (1995) The antiperinuclear factor and the co-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 95, 2672–2679.
Schellekens, G. A., de Jong, B. A., van den Hoogen, F. H., van de Putte, L. B., and van Venrooij, W. J. (1998) Citrulline is an essential constituent of antigenic determinants recognised by rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 101, 273–281.
Despres, N., Boire, G., Lopez-Longo, F. J., and Menard, H. A. (1994) The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis. J. Rheumatol. 21, 1027–1033.
Zvaifler, N. J. (1973) The immunopathology of joint inflammation in rheumatoid arthritis. Adv. Immunol. 16, 265–336.
Takemura, S., Braun, A., Crowson, C., Kurtin, P. J., Cofield, R. H., O’Fallon, W. M., Goronzy, J. J., and Weyand, C. M. (2001) Lymphoid neogenesis in rheumatoid synovitis. J. Immunol. 167, 1072–1080.
Randen, I., Mellbye, O. J., Forre, O., and Natvig, J. B. (1995) The identification of germinal centres and follicular dendritic cell networks in rheumatoid synovial tissue. Scand. J. Immunol. 41, 481–486.
Schroder, A. E., Greiner, A., Seyfert, C., and Berek, C. (1996) Differentiation of B cells in the nonlymphoid tissue of the synovial membrane of patients with rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 93, 221–225.
Kim, H. J., and Berek, C. (2000) B cells in rheumatoid arthritis. Arthritis Res. 2, 126–131.
Berek, C., Berger, A., and Apel, M. (1991) Maturation of the immune response in germinal centres. Cell 67, 1121–1129.
Jacob, J., Kelsoe, G., Rajewsky, K., and Weiss, U. (1991) Intraclonal generation of antibody mutants in germinal centres. Nature 354, 389–392.
Kim, H. J., Krenn, V., Steinhauser, G., and Berek, C. (1999) Plasma cell development in synovial germinal centers in patients with rheumatoid and reactive arthritis. J. Immunol. 162, 3053–3062.
Matsuda, F., and Honjo, T. (1996) Organisation of the human immunoglobulin heavy-chain locus. Adv. Immunol. 62, 1–29.
Cook, G. P., and Tomlinson, I. M. (1995) The human immunoglobulin VH repertoire. Immunol. Today 16, 237–242.
Zachau. (1996) The Human Immunoglobulin k Genes. The Immunologist 4, 49–54
Frippiat, J. P., Williams, S. C., Tomlinson, I. M., Cook, G. P., Cherif, D., Le Paslier, D., Collins, J. E., Dunham, I., Winter, G., and Lefranc, M. P. (1995) Organisation of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4, 983–991.
Tonegawa, S. (1983) Somatic generation of antibody diversity Nature 302, 575–581
Berek, C. and Ziegner, M. (1993) The maturation of the immune response. Immunol. Today 14, 400–404.
Kuppers, R., Zhao, M., Hansmann, M. L., and Rajewsky, K. (1993) Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J, 12, 4955–4967.
Greiner, A., Neumann, M., Stingl, S., Wassink, S., Marx, A., Riechert, F., and Muller-Hermelink, H. K. (2000) Characterisation of Wue-1, a novel monoclonal antibody that stimulates the growth of plasacytoma cells lines. Virchows Arch. 437, 372–379.
Brezinschek, H. P., Brezinschek, R. I., and Lipsky, P. E. (1995) Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J. Immunol. 155, 190–202.
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Kim, HJ., Berek, C. (2007). Single Cell Analysis of Synovial Tissue B-Cells. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 136. Humana Press. https://doi.org/10.1007/978-1-59745-402-5_3
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DOI: https://doi.org/10.1007/978-1-59745-402-5_3
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