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A8.36 Activated human B cells induce an inflammatory phenotype in synovial fibroblasts
  1. Theresa Tretter,
  2. Hannah Stoerch,
  3. Lars Tykocinski,
  4. Babak Moradi,
  5. Hanns-Martin Lorenz
  1. Department Internal Medicine V, Div. Rheumatology, University Hospital Heidelberg, Heidelberg, Germany

Abstract

Background and Objectives Synovial fibroblasts (SF) are key players in pathogenesis and progression of rheumatoid arthritis (RA). A cross-talk between immune cells and SF is suggested to contribute to chronification of the disease. RA-SF can be found in close proximity to B cells in vivo, with activated proliferating B cells often organised in germinal center- like pseudofollicles. While various reports deal with the influence of SF on B cells, our intention was to clarify the potential influence of B cells on SF

Materials and Methods Highly purified CD19+ B-cells were separated from PBMC by magnetic cell sorting. B-cells were polyclonaly stimulated by BCR-cross-linking with anti-IgM/IgG Antibody or SAC (Staphylococcus aureus Cowan I antigen) and cocultured with SF, that were isolated from synovial biopsies of patients with osteoarthritis (OA-SF). After 48-72h cytokine production was determined by ELISA of cell culture supernatants and phenotypical changes in both cell populations were determined by flow cytometry

Results SF and B cells cultured alone produced low amounts of IL-6 and IL-8. However, in B-SF cocultures the concentrations of both cytokines increased up to 100-fold suggesting that the observed values not just reflect additive but rather synergistic effects. Intracellular staining with fluorochrome-labelled anti-cytokine Abs revealed that the increased cytokine concentrations originated from SF. In contrast, B cell specific production of TNF-alpha and IL-1-beta decreased in cocultures with SF by more than 50%, suggesting an inhibitory feedback mechanism by activated SF towards the B cells. Of note, after removal of B cells SF-specific cytokine production remained constantly increased for several days. Separation of B cells from SFs by semipermeable cell culture inserts (transwell) did not abolish observed effects, suggesting that soluble factors were the main mediators. Addition of neutralizing Abs against TNF-alpha and IL-1-beta to the cocultures significantly reduced SF activation. All effects proved to be stable with little variation between individual donors, different B cell: SF ratios, cell culture media or B cell stimuli used. In addition to IL-6 and IL-8, the production of Matrix-Metalloproteinase-3 (MMP-3) increased in coculture with B cells as well, suggesting that OA-SF developed potentially tissue destructive properties.

Conclusions Activated B cells interact with SF in a reciprocal crosstalk resulting in conversion of OA-SF into SF with an inflammatory phenotype. This process is mediated by B cell derived TNF-alpha, IL-1 beta and other yet to be identified soluble factors.

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