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Rheumatoid synovial fibroblasts activate endothelial cells and promote neutrophil recruitment in a flow based multicellular model of the rheumatoid pannus

In rheumatoid arthritis, leukocytes are recruited from the blood by vascular endothelial cells (EC) and accumulate in stromal tissue forming ectopic leukocyte aggregates that contribute to soft tissue remodelling and bone destruction. The signals that regulate the process of chronic leukocyte recruitment into the diseased joint are poorly defined; however, it is probable that cells of the tissue stroma that have undergone a disease-specific transition in phenotype contribute to inflammation by the inappropriate production of proinflammatory cytokines and chemokines [1, 2]. Here we have used a novel co-culture model [3] to monitor the role of stromal cells in regulating the quality and quantity of leucocytes that enter the synovium. We tested the hypothesis that fibroblasts from the rheumatoid joint, but not fibroblasts from normal tissue (skin), have the ability to drive inflammation by activating EC and promoting the recruitment of neutrophils.

EC were established in co-cultured with fibroblasts from the synovium (SF) or skin (DF) of RA patients on the opposite sides of porous transwell membranes. After 24 hours of EC conditioning, co-cultures were incorporated into a parallel plate leukocyte adhesion assay and the ability of EC to recruit flowing neutrophils determined. Neutrophils adhered efficiently to EC co-cultured with 5 × 105 SF (88 ± 12/mm2/106 cells perfused) but not to EC co-cultured with DF (12 ± 9/mm2/106 cells perfused). Anti-P-selectin and anti-E-selectin antibodies markedly decreased neutrophil adhesion, and an anti-CD18 antibody (the β2 integrin) abolished neutrophil recruitment. The antibody blockade of the neutrophil chemokine receptor CXCR2 but not CXCR1 also abolished neutrophil recruitment. Supernatants collected from cocultures of EC/SF, but not from SF cultured alone, activated EC to support neutrophil adhesion, indicating the presence of a soluble activating factor(s) generated exclusively in co-culture. EC/SF co-culture supernatants also contained a high concentration of IL-6 (2925.2 ± 521.2 pg/ml), and both IL-6 levels and neutrophil adhesion were reduced in a dose-dependent manner by the inclusion of hydrocortisone in the co-culture medium. An anti-IL-6 antibody also abolished neutrophil adhesion.

We propose that in the rheumatoid synovium, a previously unsuspected process of cross-talk involving IL-6 signalling occurs between synovial fibroblasts and vascular EC resulting in the upregulation of adhesion molecules and chemokines that support neutrophil recruitment. The observation that neutrophil adhesion in this co-culture system can be inhibited by anti-IL-6 neutralising antibodies or hydrocortisone suggests that this novel in vitro model of the synovium faithfully mimics important in vivo processes involved in leukocyte accumulation within the synovium. It also suggests that this new model can act as a useful screen for new anti-inflammatory therapies in rheumatoid arthritis.


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Smith, E., Lally, F., Filer, A. et al. Rheumatoid synovial fibroblasts activate endothelial cells and promote neutrophil recruitment in a flow based multicellular model of the rheumatoid pannus. Arthritis Res Ther 7 (Suppl 1), P77 (2005).

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