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DJ Park, D Chang, V Nguyen, K Chamie, RP Phipps, SE Feldon; MHC II and CD40 Ligand (L)-Dependent Induction of Cell Proliferation in Orbital Fibroblasts of Graves’ Patients During Co-culture with Autologous T-cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2226.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Orbital fibroblasts have been shown to express MHC class II and CD40. It has been postulated that the interaction between orbital fibroblasts and infiltrating autologous T-cells via these markers plays an important role in the pathogenesis of Graves’ ophthalmopathy. To determine if autologous T-cells induce proliferation of orbital fibroblasts in Graves’ patients, autologous mixed cell reactions were performed, and cell proliferation was measured by 3-H-Thymidine incorporation. Methods: Orbital fibroblasts were harvested from biopsies of orbital connective tissue in Graves’ patients, and autologous T-cells were purified from peripheral blood by nylon wool column separation. 5x103 orbital fibroblasts were plated on 96-well plates in RPMI1640 with 10% FBS and allowed to incubate for 24 h, after which time 1x105 irradiated autologous T-cells were added in co-culture for another 48 h. 3-H-Thymidine uptake during the last 24 hours of co-culture was measured by liquid scintillation. Parallel trials in which antibodies to MHC class II (at 0.2 µg/well and 1 µg/well) and CD40L (at 0.2 µg/well and 2 µg/well) were added to co-culture were performed to determine if T-cell dependent induction of 3-H-thymidine incorporation by orbital fibroblasts was dependent on MHC class II or CD40L. 3-H-Thymidine incorporation in gamma-irradiated T-cells and in orbital fibroblasts without co-culture were measured as negative controls. Results: Orbital fibroblasts exhibited significantly increased 3-H-Thymidine uptake when co-cultured with autologous T-cells compared to orbital fibroblasts cultured alone. Irradiated T-cells showed almost no incorporation of 3-H-Thymidine. Addition of MHC class II or CD40L neutralizing antibodies suppressed the T-cell-induced increase in 3-H-Thymidine incorporation of orbital fibroblasts. Conclusion: Autologous T-cells induce cell proliferation of orbital fibroblasts in patients with Graves’ ophthalmopathy in a manner that appears to be dependent on MHC class II and CD40 ligand. Thus, T-cells that infiltrate the orbit may be responsible for the increases in fibroblastic response characteristic of Graves’ ophthalmopathy.
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