@phdthesis{SoutoCarneiro2000,
  author    = {Souto-Carneiro, Maria Margarida},
  title     = {Molecular and functional analyses of human synovial B-lymphocytes in rheumatoid arthritis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-2308},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2000},
  abstract  = {B-cells of the rheumatoid synovial tissue are a constant part of and, in some histopathological subtypes, the dominant population of the inflammatory infiltrate, located in the region of tissue destruction. The pattern of B-cell distribution and the relationship to the corresponding antigen-presenting cells (follicular dendritic reticulum cells: FDCs) show a great variety. B-cells may exhibit (i) a follicular organization forming secondary follicles; (ii) follicle-like patterns with irregularly formed FDC networks, and (iii) a diffuse pattern of isolated FDCs. Molecular analysis of immunoglobulin VH and VL genes from human synovial B-cell hybridomas and synovial tissue demonstrates somatic mutations due to antigen activation. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation, which is involved in the generation of autoantibodies. An autoantibody is defined as "pathogenic" if it fulfills the Witebsky-Rose-Koch criteria for classical autoimmune diseases: definition of the autoantibody; induction of the disease by transfer of the autoantibody; and isolation of the autoantibody from the disease-specific lesion. B-cells from rheumatoid synovial tissue show specificity for FcIgG, type II collagen, COMP, sDNA, tetanus toxoid, mitochondrial antigens (M2), filaggrin and bacterial HSPs. The contributions of these antigens to the pathogenesis of RA are still hypothetical. A possible contribution could derive from crossreactivity and epitope mimicry: due to crossreaction, an antibody directed originally against a foreign infectious agent could react with epitopes from articular tissues, perpetuating the local inflammatory process. The characteristic distribution pattern, the localisation within the area of tissue destruction, the hypermutated IgVH and IgVL genes, and their exclusive function to recognize conformation-dependent antigens suggest a central role for B-cells in the inflammatory process of rheumatoid arthritis. Therefore, the analysis of synovial B-cell hybridomas and experimental expression of synovial IgVH and IgVL genes will help to characterise the antigens responsible for the pathogenesis of rheumatoid arthritis. In the present study 55 IgVH genes amplified from 3 different anatomical regions of a RA patient were analysed adding further information on synovial B-cell maturation and recirculation in RA. This analysis demonstrated somatically mutated IgVh genes in all different regions with amino acid deletions and mixed IgVh molecules, suggesting the existence of a novel pathway to generate (auto)antibody specificities. The comparison of amino acid sequences of amplified genes belonging to the VH1 family (with predominantly the same germline counterpart) exhibited a strong homology, indicating an apparently conserved mutational pattern. This suggests that the number of antigens activating B-cells in the different locations is restricted. The most striking result was the finding of clonally related sequences in different anatomical regions indicating a recirculation of activated B-cells between the different affected joints. Also in the present study a synovial B-cell hybridoma was analyzed for its specific recognition of cartilage antigens. A heptameric peptide of cartilage oligomeric protein (COMP) could be defined as the target structure. The IgVH-gene (IgHV4-59*01) of the IgG2l hybridoma has somatically mutated genes with high R/S values in the CDR regions (9:2). Thus, indicating that this hybridoma originates from a synovial B-cell which has been antigen activated/selected for its affinity. To analyse the presence of the clonotypic IgHV4-59*01 sequences in other cases of RA and osteoarthritis (OA) synovitis, primers specific for the CDR3 rearrangement of this hybridoma were used. The clonotypic and clone related sequences (98 per cent ± 1 per cent homology) could only be detected in synovitis of RA cases but not in OA cases indicating that this B-cell is specific to RA synovitis. The identified heptameric peptide of COMP was used in a peptide ELISA to analyse whether there is a specific binding in RA serum samples. Serum samples (IgG) from RA patients (n=22) showed a significant higher efficiency to the COMP heptamer than the OA sera (n=24) and the age matched healthy controls (n=20) (for both p<1x10-4, Students t-test). The specificity of this B-cell hybridoma may therefore be defined as RA specific. Since COMP is restricted to cartilage and tendons which are organs specifically affected in RA this COMP specific autoantibody represents the first organ specific autoantibody in RA. The IgG2 COMP specific autoantibody with somatically mutated IgVH genes is different from germline encoded, antigen clearing IgM autoantibodies and may therefore be directly involved as an "arthritogenic autoantibody" in cartilage and tendons destruction by complement activation.},
  subject      = {Rheumatoide Arthritis},
  language  = {en}
}