J Korean Ophthalmol Soc.  2007 Aug;48(8):1126-1133.

Expression of Local Immunosuppressive Factor, Indoleamine 2,3-dixygenase, in Human Coreal Cells

• Ryu YH
• Kim JC

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 MTT assay. Moreover, apoptotic effects of IDO in immune cells treated with IFN-gamma 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 corneal fibroblasts. Immune cells cultured in a human corneal fibroblast-conditioned medium showed inhibited proliferation. Moreover, IFN-gamma-induced expression of IDO significantly enhanced apoptotic ability in a dose-depandant manner.
CONCLUSIONS
Our results suggest that human corneal fibroblasts are relatively immuno-resistant and that expression of IDO may be one of the factors involved in the immune tolerance observed in corneal grafts.

Keyword

Indoleamine 2,3-dioxygenase; Corneal fibroblasts; Immune privilege

MeSH Terms

Apoptosis
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Epithelial Cells
Fibroblasts
Humans*
Immune Tolerance
Indoleamine-Pyrrole 2,3,-Dioxygenase
RNA, Messenger
Transplants
Indoleamine-Pyrrole 2,3,-Dioxygenase
RNA, Messenger

Figure

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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.

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  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.

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  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.

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  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.

Reference

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