Establishment And Regulation Of The Co-culture Experimental Model Of Thyroid Associated Ophthalmopathy

PurposeTo establish and to identify an in vitro experimental model of autoimmune thyroid- associated ophthalmopathy (TAO) by co-culture technique. In this model, T-cell mediated inflammatory responses of orbital fibroblasts were regulated by using a selective cyclooxygenase-2 inhibitor(COX-2 inhibitor). Mechanistic and therapeutic approaches for TAO were explored.MethodsOrbital fibroblasts obtained from 10 patients with TAO and from 10 control subjects were used to set up primary cultures , and were identified by immunohistochemistry. T lymphocytes were isolated from peripheral blood (PBTL) obtained from 20 patients with TAO and 20 controls. PBTLs were enriched, purified and analyzed by flow cytometry. Co-cultured cells, OFs and PBTLs, were monitored by immunofluorescence microscopy and their ultrastructure was observed by electron microscopy. The expression of interleukin-1α, interleukin-1β, interleukin-2 and interleukin -6 were quantified by Enzyme-linked immunosorbent assay(ELISA) at 24 hours, 48 hours and 72 hours after co-cultured. Real-time PCR was used to measure the expression of each group's COX-2 mRNA. The effects of Celecoxib, a COX-2 inhibitor, at several concentrations on the co-culture cell models were tested by 3-(4, 5)-dimethylthiahiazo(-z-yl)-3, 5-di-phenytetrazoliumromide (MTT) .The inhibition of Celecoxib on the expression of inflammatory cytokines , interleukin-1α, interleukin-1β, interleukin-2 and interleukin-6, were analyzed by ELISA. The co-culture cell model, autologous or non-autologous T lymphocytes co-cultured with OFs, was studied by immunofluorescence microscopy, electron microscopy, to compare the levels of the inflammatory cytokines.ResultsThe co-culture of orbital fibroblasts and T-lymphocytes was established. There was no obvious difference between the co-cultured OFs with autologous and non-autologous T lymphocytes by immunofluores-cence microscopy and electron microscopy. The expression of IL-1α, IL-1β, IL-2 and IL-6 in each co-culture group was increased at 24h, especially to the T + T group(T lymphocytes and the orbital fibroblasts all obtained from the TAO patients). The significant differences between the T+T group and the others were observed (P = 0.001, 0.008, 0.012); The level of IL-6 in N+N group (T lymphocytes and the orbital fibroblasts obtained from the normal's)at 24 hours was the highest. The level of IL-6 in all groups continued to increase during the 72 hours, while the N+T group (T lymphocytes obtained from the normal's and the orbital fibroblasts obtained from the TAO patients) was the highest. The difference in expression between the different times were obvious (P =0.000, 0.004, 0.001) . There was no difference of inflammatory cytokines between the co-cultured OFs with autologous and non- autologous T lymphocytes. The expression of the COX-2 mRNA in patients with TAO was statistically higher than normal's, while the T+T group was the highest; Different concentrations of clecoxib, 1μmol/L, 2μmol/L, 5μmol/L and 10μmol/L, showed the dose- and time-dependent inhibitory effects on proliferation of OFs in each group. Celecoxib (2μmol/L for 12 hours) inhibited the expression of IL-1α, IL-1β, IL-2 and IL-6 all with time-dependent fashion. In T+T group, after 2μmol/L Celecoxib treatment, the levels of IL-1β, IL-2, IL-6 at 12 hours, 24 hours and 36 hours had the statistical difference from the other groups.ConclusionsAn in vitro model that represents TAO, an autoimmune inflammatory disorder, was established by co-culture of orbital fibroblasts and acti-vated T lymphocytes. There was no essentially morphologic difference in all co-culture groups at light microscopic level. Although the morphology of co-cultured cells remained unchanged, metabolic alterations of co-cultured OFs were observed by electron microscopy, specifically in the T+T group. The expression of the IL-1α, IL-1β, IL-2, IL-6 and COX-2 mRNA could be detected from all the co-culture groups. The T+T group showed the highest levels except for the IL-6.The enhanced metabolism and the upregulation of the inflammatory factors and pro-inflammatory gene such as COX-2 confirmed that the inflammatory reaction of OFs was stimulated by activated T-lymphocytes. The suppres-sed levels of IL-1α, IL-1β, IL-2 and IL-6, by COX-2 inhibitor, especially in T+T group, showed the importance of COX-2 pathways in regulation of autoimmune inflammatory reactions. For the first time, the co-culture model provides the direct mechanistic evidence of T lymphocyte-mediated inflammatory reaction of orbital fibroblasts. It also provides an in vitro model for testing a variety of therapeutic agents such as anti-inflammatory drugs, T-cell inhibitors, cytokine antagonists, etc. to regulate autoimmue inflammatory reactions of TAO.