Establishment And Regulation Of The B Lymphocytes And Orbital Fibroblasts Co-culture In Thyroid Associated Ophthalmopathy

Objective1. To establish and identify an in vitro experimental model of autoimmune thyroid-associated ophthalmopathy (TAO) by co-culture the orbital fibroblasts and peripheral B lymphocytes.2. To investigate the expression of Insulin-like growth factor-1 receptor (IGF-1R) on OFs, observe the inflammatory responses in the model, and explore the important role of B cells in TAO.3. Pathogenesis and therapeutic approaches for TAO were explored.Material and Method1. Orbital fibroblasts obtained from 15 patients with TAO and from 15 control subjects were used to set up primary cultures, and were identified by immunohistochemistry. B lymphocytes were isolated from peripheral blood obtained from 10 patients with TAO and 10 controls. B cells were enriched, purified by using immunomagnetic beads separation techniques and analyzed by flow cytometry.2. The IGF-1R of OFs were evaluated by flow cytometry and observed under confocal microscopy. The expression of interleukin-6 and chemoattractants RANTES were quantified by Enzyme-linked immunosorbent assay (ELISA) at 24hours,48 hours and 72 hours respectively within the co-culture models.3. The depleting effects of Rituximab (RTX) on B cells at several concentrations in different time were measured by MTS. The inhibition on the expression of IL-6 and RANTES by using RTX and IGF-1 binding protein on the co-culture model were analyzed by ELISA. Result1. The co-culture of orbital fibroblasts and B lymphocytes was established.2. The expression of IGF-1R on OFsinpatients with TAO was significantly higher than normal's.3. The expression of IL-6 and RANTES in each co-culture group was increased at 24 hours, while the T+T group (B lymphocytes and orbital fibroblasts all obtained from TAO patients) represented a highest expression.4. RTX and IGF-1 binding protein significantly inhibited the expression of IL-6 and RANTES in the T+T co-culture group at 48 hours.Conclusion1. An in vitro model that partially represents TAO, as an autoimmune inflammatory disease, was established by co-culture of orbital fibroblasts and peripheral B lymphocytes. The interactions between those two cells may play a role in the pathogenesis of TAO.2. IGF-1R may be the pathway in the interactions between OFs and B cells. It may mediate the process of TAO. Inflammatory responses may be depressed by blocking the receptor.3. B cell depleting agents RTX showed a well anti-inflammatory effect in the vitro model. Monoclonal anti-CD20 antibody therapy may be a novel treatment option in the treatment of TAO.