Diseased synovium in severe, active rheumatoid arthritis contains lymphocyte germinal centers able to produce immunoglobulin at splenic levels. Studies of these immunoglobulins have focused on rheumatoid factor (RF) content and have shown that, in seropositive RA, 5-10% of the repertoire consists of both 1gM and 1gG anti-1gG Fc. There is strong circumstantial evidence that the resultant immune complexes are involved in the pathogenesis and progression of the disease. However, the three idiotypes which are most commonly expressed by 1gM RF paraproteins, e.g., Wa, Po and Bla, appear to be a minor component of synovia RF. Thus, the genetic composition of the RA synovial antibody repertoire - both non-RF and RF - remains an open question. During the course of this project, we will analyze the antibody repertoire expressed by synovial mononuclear cells in order to test the hypothesis that synovial immunoglobulin production is antigen-driven and, therefore, involved in the pathogenesis of the disease. Considerable support for this approach derives from murine models. Sequence analysis of antibody repertoires in the MRL/lpr mouse suggests that autoantibodies generated during nonspecific stimulation by LPS or secondary immunization appear to be polyclonal and reflect the germline repertoire; whereas in autoimmune states, highly self-reactive antibodies are clonally related and demonstrate all the characteristics of antigen-driven immunoglobulin production. Murine models also suggest that the physico-chemical properties of specific V germline elements may influence the manifestations of autoimmune disease, hence polymorphism or heteromorphism in the genomic repertoire may be an additional, independent factor in disease susceptibility. cDNA libraries from rheumatoid synovial cells will be generated and the antibody repertoire characterized at the nucleotide sequence level. Evidence of restriction in V region utilization in Cmu- and Ckappa-containing transcripts will be sought. Comparisons will be made between RF paraproteins, the germline compartment, EBV-transformed synovial cell lines from RA patients, and synovial repertoires at different stages of disease. Evidence of antigen-specific clonal expansion will be sought by analyzing the Vh-Dh-Jh joins of representative Cgamma- and Calpha-containing transcripts as well as the Vkappa-Jkappa cDNAs. Analysis of the extent and distribution of somatic mutation among these clones will provide additional evidence for or against antigen-driven selection. Combinatorial expression libraries will be generated to test for possible antigen specificity. Candidate germline autoreactive elements will be identified, their extent of polymorphism in the human population established, and their correlation, if any, to selected disease manifestations established. These studies should not only shed significant light into a poorly understood manifestation of rheumatic disease, but also serve as reference for future studies of abnormal immune responses.
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