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Abstract
Purpose
To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its ability to act as a local immunosuppressive factor.
Methods
The expression profile of IDO was obtained with RT-PCR and Western blot of in a primary culture of human corneal cells (fibroblasts, epithelial cells and endothelial cells). In order to investigate the immunosuppressive function of IDO, immune cells were cultured in a human corneal cell-conditioned medium, and their prolifleration was identified by the MIT assay. Moreover, apoptotic effects of IDO in immune cells treated with IFN-Y were also investigated with apoptosis ELISA.
Results
Among the three different types of human corneal cells analyzed, mRNA and protein expression of IDO was observed only in human comeal fibroblasts. Immune cells cultured in a human comeal fibroblast-conditioned medium showed inhibited proliferation Moreover, IFN-Y-induced expression of IDO significantly enhanced apoptotic ability in a dose-depandant manner.
References
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Figure 1.
Expression of IDO in human corneal cells. RT-PCR was performed to identify the expression of IDO in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEn did not express IDO. IDO expression was observed in PHCEp and PHCF. PHCF showed significantly higher expression.
Figure 2.
Expression of IDO in human corneal cells. Western blotting performed to identify the expression of IDO in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEn did not express IDO. PHCF showed significantly higher IDO expression, and PHCEp also showed IDO expression.
Figure 3.
Proliferation of immune cells cultured in human corneal cells conditioned medium. In order to investigate the effects of IDO expressed by human corneal cells on the proliferation of immune cells, an MTT cell proliferation assay was performed. Human immune cells, THP-1 and Jurkat cells, were cultured in three different types of human corneal cells conditioned medium. To remove the effects of the medium, immune cells were cultured in human corneal cell-defined medium. (A) Although the human monocytes, THP-1, showed decreased proliferation compared with the control (cultured in RPMI1640), there were no effects of the conditioned medium. (B) Human T-lymphocytes (Jurkat cells) cultured in defined medium for human corneal cells showed slightly decreased proliferation. PHCEn-conditioned medium did not show any effects on proliferation of the Jurkat cells. However, PHCF and PHCEp-conditioned medium showed decreased proliferation of Jurkat cells (*P>0.05). Data represent the mean(SD of three separate experiments.
Figure 4.
Apoptosis of immune cells cultured in IFN-V -treated PHCF-conditioned medium. Immune cells were cultured in IFN-v-treated PHCF-conditioned medium for 24 hrs, and cellular apoptosis was measured by ELISA. The THP-1 culture was not affected by IFN-V treatment. However, Jurkat cells showed increased apoptosis in 1.0 and 5.0 ng/ml IFN-Y-treated conditioned medium. IDO neutralizer, 1-MT, treatment showed recovered apoptosis.
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