Abstract
Purpose:
1.To establish and to identify an in vitro experimental model of autoimmune TAO by co-culture the orbital fibroblasts and peripheral B lymphocytes. 2.To investigate the Insulin-like growth factor-1(IGF-1R) receptor expressing on OFs, observe the inflammatory responses in the model, and explore the important role of B cells in TAO. 3.Exploring pathogenesis and therapeutic approaches for TAO.
Methods:
1.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 using immunomagnetic beads separation techniques and analyzed by folw 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 of were quantified by ELISA at 24hours,48 hours and 72 hours after co-culture. 3.The depleting effects of Rituximab on B cells at several concentrations in different time were measured by MTS. The inhibition on the expression of IL-6 and RANTES after using RTX and IGF-1 binding protein on the co-culture model were analyzed by ELISA.
Results:
1.The co-culture of orbital fibroblasts and B lymphocytes was established. 2.The expression of IGF-1R on OFs in patients 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, especially in the T+T group (B lymphocytes and orbital fibroblasts all obtained from the TAO patients) is the highest. 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.
Conclusions:
1.An in vitro model that partially represents TAO, as an autoimmune inflammatory disease, was established by co-culture of orbital fibroblasts and peripheral B lymphoctes. The interactions between the two cells may play a role in TAO pathogenesis. 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 after blocking the recptor. 3.B cell depleting agents RTX have a good anti-inflammatory effects in the vitro model. Monoclonal anti-CD20 antibody therapy may be a novel treatment option in TAO in the future.
Keywords: 637 pathology: experimental •
432 autoimmune disease •
554 immunohistochemistry