September 2007
Volume 48, Issue 9
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Immunology and Microbiology  |   September 2007
Expression of Indoleamine 2,3-Dioxygenase in Human Corneal Cells as a Local Immunosuppressive Factor
Author Affiliations
  • Yang-Hwan Ryu
    From the Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul, Korea.
  • Jae-Chan Kim
    From the Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul, Korea.
Investigative Ophthalmology & Visual Science September 2007, Vol.48, 4148-4152. doi:https://doi.org/10.1167/iovs.05-1336
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      Yang-Hwan Ryu, Jae-Chan Kim; Expression of Indoleamine 2,3-Dioxygenase in Human Corneal Cells as a Local Immunosuppressive Factor. Invest. Ophthalmol. Vis. Sci. 2007;48(9):4148-4152. https://doi.org/10.1167/iovs.05-1336.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its functional ability as a local immunosuppressive factor.

methods. The expression profile of IDO was identified in primary cultures of human corneal cells (fibroblasts, epithelial cells, endothelial cells) by RT-PCR and Western blot analysis. The immunosuppressive function of IDO was assessed by culturing human CD4+ T cells with conditioned medium derived from the three types of human corneal cells, and changes in proliferation and apoptosis were determined. IDO expression and its apoptotic effects on CD4+ T cells were also investigated after IFN-γ treatment.

results. Among the three types of human corneal cells, IDO mRNA and protein expression were observed in human corneal fibroblasts and epithelial cells, with higher levels in the human corneal fibroblasts. Human CD4+ T cells cultivated in conditioned medium derived from human corneal fibroblasts showed decreased cell proliferation and increased apoptosis. IFN-γ treatment significantly induced IDO expression and showed apoptotic effects on immune cells.

conclusions. These results suggest that human corneal fibroblasts are relatively immunoresistant and that the IDO expression can act as one of the factors for the maintenance of immune privilege in the cornea.

Corneal transplantation is generally understood to be highly successful because of the immunologic privilege of the cornea and anterior chamber. However, in clinical processes, transplanted corneas are not grafted into normal recipient beds. In patients with corneal disease, immunologic privilege is compromised, which leaves the graft prone to allograft rejection, the most frequent cause of corneal graft failure. Accordingly, systemic immunosuppression with a calcineurin blocker (cyclosporine or FK506) is widely used in corneal transplantation, but it can lead to adverse effects such as infection. This emphasizes the importance of maintaining the immune privilege on ocular surface. 
Indoleamine 2,3-dioxygenase (IDO) is a monomeric heme-containing enzyme that catalyzes the opening of the pyrrole ring of l-tryptophan to yield N-formylkynurenine, which rapidly degrades to give kynurenine. 1 Interferon (IFN)-γ is a strong inducer of IDO expression in cultured fibroblasts, 2 macrophages, 3 dendritic cells, 4 and many cancer cell lines. 5 However, IDO is poorly induced by lipopolysaccharide (LPS), 6 IFN-α, and IFN-β. 7  
The role of IDO in the survival of fetal allograft during pregnancy has been explored, and researchers have found that the expression of IDO in the placenta is crucial to the prevention of immunologic rejection of the fetal allograft. 8 In addition, T-cell proliferation in vitro was inhibited by human macrophage tryptophan catabolism by IDO. 9 Subsequently, it was reported that the expression of IDO suppresses T-cell responses by limiting the availability of tryptophan in local tissue microenvironments 10 and that human dendritic cells were shown to express IDO, which suppressed T-cell proliferation in vitro under certain culture conditions. 4 These results have suggested that the proliferation of infiltrated T cells is inhibited by IDO because it generates a tryptophan-deficient environment in local tissues. Recent research demonstrated that the expression of IDO by dermal fibroblasts mediates immune cell damage. 11  
Other studies have shown a role for IDO expression in cancer biology as a suppressor of antitumor immunity. Uyttenhove et al. 12 explored the role of IDO in tumors by using the P815 mouse mastocytoma cell line 12 and found that transfecting IDO into P815 tumor cells prevented their rejection by preimmunized hosts, enabling the tumor to circumvent a normally protective immune response. This finding is consistent with observations that the transfection of IDO renders tumor cell lines immunosuppressive in vitro 13 and that 1-methyl-tryptophan significantly delays tumor outgrowth in a model of Lewis lung carcinoma. 14 Despite the numerous studies suggesting the immunosuppressive functions of IDO in fields such as cancer biology, there have been no similar reports in human corneal cells. Therefore, this study was conducted to identify the expression profile of IDO in three types of human corneal cells (corneal epithelial cells, fibroblasts, endothelial cells) and to estimate its local immunosuppressive function on the ocular surface. 
Materials and Methods
Isolation and Primary Culture of Human Corneal Cells
Human eyes were obtained in accordance with the tenets of the Declaration of Helsinki and proper informed consent. Three types of human corneal cells (corneal epithelial cells, fibroblasts, endothelial cells) were isolated by tissue explant from a donated human cornea following procedures outlined in a previous report. 15 Briefly, the corneal tissue was incised into three layers (epithelium, stroma, endothelium) and cut into 2-mm2 explants. Each piece of explants was placed directly on a culture dish, with the epithelial, stromal, or endothelial side down. The cultures were submerged in the appropriate media for 1 week, and the cells were harvested and subcultured by trypsin digestion. 
Primary human corneal epithelial (PHCEp) cells were maintained in medium (EpiLife; Cascade Biologics, Portland, OR) containing 0.06 M CaCl2, and human corneal growth supplement (HCGS; Cascade Biologics); primary human fibroblasts (PHCFs) were grown in DMEM (WelGene Biopharmaceuticals, Daegu, Korea) supplemented with 10% FBS (WelGene Biopharmaceuticals) and 1% antibiotic (WelGene Biopharmaceuticals). Primary human corneal endothelial cells (PHCEn) were grown in reduced serum medium (Opti-MEM; (Gibco BRL, Invitrogen, Grand Island, NY) containing 5 ng/mL EGF (Sigma Chemical, St. Louis, MO), 20 ng/mL NGF (R&D Systems, Minneapolis, MN), 20 μg/mL ascorbic acid (Sigma Chemical), 0.005% insect lipid (Sigma Chemical), 0.2 μg/mL CaCl2, 0.02% chondroitin sulfate (Sigma Chemical), 1% RPMI 1640 vitamin mixture (Sigma Chemical), 8% FBS, and 1% antibiotic. Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C, subcultured using 0.25% trypsin-EDTA every 3 to 4 days, and used for experiments (no more than three passages). 
Isolation and Culture of Human CD4+ T Cells
Human CD4+ T cells were isolated through a two-step purification procedure. To obtain CD4+ T cells, peripheral blood mononuclear cells were purified using Ficoll (Sigma Chemical), and CD4+ T cells were positively selected (Human CD4 Cell Recovery Column Kit; Cedarlane Inc., Burlington, ON, Canada). Cells were grown in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C and subcultured using 0.25% trypsin-EDTA every 3 days. 
RT-PCR
Total RNA was isolated by using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA (2 μg) was subjected to reverse transcription (RT) into cDNA (AccuPower RT Premix; Bioneer Co., Daejon, South Korea). Equal amounts of samples were used for PCR amplification of cDNA, with specific primers for human corneal cell markers and IDO. Human IDO primers were 5′-GGC AAA GGT CAT GGA GAT GT-3′ (sense) and 5′-CAG GAC GTC AAA GCA CTG AA-3′ (antisense; GenBank accession no. BC027882); they yielded a 552-bp fragment. PCR amplification was performed (AccuPower PCR Premix; Bioneer Co.) with 5 μL cDNA product in a total volume of 20 μL containing 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl2, 1 U DNA polymerase, 1 mM dNTP, and 20 pmol each specific primer. PCR cycling conditions were as follows: denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute. Forty cycles were performed. Amplified products were verified by electrophoresis in 2% agarose gel, stained with ethidium bromide, and photographed under ultraviolet (UV) transillumination. 
Western Blot Analysis
Total proteins of human corneal cells were extracted with a solution (Pro-Prep Protein Extraction Solution; Intron Biotechnology, Gyeoggi-Do, Korea) according to the manufacturer's instructions. Total proteins (10 μg/sample) were boiled for 5 minutes, separated on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was blocked in 5% skim milk in 1× TBST (50 mM NaCl, 20 mM Tris, and 0.05% Tween 20) for 1 hour at room temperature and was incubated with 1:200 diluted goat anti–human IDO monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by incubation with the horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Santa Cruz Biotechnology) for 2 hours at room temperature. The peroxidase activity was developed with enhanced chemiluminescence (ECL Western Blotting Detection Reagent; (Amersham Bioscience, Piscataway, NJ) and was visualized with exposure to film. 
IFN-γ Treatment and Preparation of Conditioned Medium Derived from Human Corneal Cells
After human corneal cells were attached, 1 ng/mL IFN-γ (R&D Systems) was added and further incubated for 24 hours. In some experiments, 1-methyl tryptophan (1-MT; Sigma Chemical) was used to neutralize IDO activity. After various treatments, conditioned media were harvested from human corneal cells. 
MTT Assay
We used 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT; Sigma Chemical) to measure changes in cell proliferation. Human CD4+ T cells were seeded in a 96-well plate at a concentration of 0.4 × 104 cells/100 μL medium/well (Falcon Co., Franklin Lakes, NJ), and the plates were kept at 37°C for 24 hours in a humidified atmosphere containing 5% CO2. Cells were refed with RPMI 1640 containing conditioned medium (CM) derived from human corneal cells and were further incubated for 24 hours. In some experiments, 1-MT (Sigma Chemical) was also added to neutralize IDO activity. Cells were incubated with MTT working solution (2 mg/mL) for 2 hours. After the MTT-treated medium was removed, 150 μL dimethyl sulfoxide (DMSO; Sigma Chemical) was added to each well to dissolve the formazan dye, which was quantified at 595 nm using an ELISA plate reader. 
Apoptosis Detection ELISA
ELISA (Cell Death Detection ELISAPLUS; Roche, Mannheim, Germany) was used to detect apoptosis in CD4+ T cells according to the manufacturer's instructions. Human CD4+ T cells were cultured in RPMI 1640 containing CM derived from three different types of human corneal cells with or without 1 ng/mL of 1-MT for 24 hours CD4+ T cells were harvested and lysed with lysis buffer and incubated for 30 minutes at RT, then centrifuged at 200g for 10 minutes. Supernatant (12 μL) was carefully removed and placed in the streptavidin-coated multiwell plate, and 80 μL immunoreagent was added. The reactions were induced for 2 hours at RT and were then rinsed 3 times with incubation buffer. ABTS solution (100 μL/well) was added after the incubation buffer was removed, and optical density at 405 nm was measured against the ABTS solution, which was used as a blank. 
Statistical Analysis
The effects of CM derived from human corneal cells on the proliferation of human CD4+ T cells were analyzed by Duncan's one-way ANOVA procedure using a statistical analysis system (SAS Institute, Cary, NC). 
Results
Expression of IDO in Human Corneal Cells
Through the use of RT-PCR analysis, each type of primary cultured human corneal cells was identified by its inherent markers. PHCEp cells expressed CK3, PHCFs expressed keratocan and lumican, and PHCEn cells expressed Na+/K+ ATPase. None of the three types of cells expressed the dendritic cell marker CD83 (Fig. 1)
Expression of IDO in all three types of cells was assessed by RT-PCR (Fig. 2)and Western blot analysis (Fig. 3) . IDO was expressed in PHCEp and PHCF, but not in PHCEn. IDO expression level in PHCF was significantly higher than in PHCEp. 
Proliferation of Immune Cells Cultured with or without Conditioned Medium
To test the proliferative effects of IDO expressed by human corneal cells on human CD4+ T cells, an MTT cell proliferation assay was performed with human CD4+ T cells cultivated in CM derived from PHCEp, PHCF, or PHCEn. A specific antagonist, 1-methyl tryptophan (1-MT), was added to neutralize IDO activity. To exclude the effects of culture medium itself, CD4+ T cells were also cultured in a different kind of defined medium (DM) for human corneal cells and tryptophan supplier 1-MT. Human CD4+ T cells, derived from human peripheral blood, showed slightly decreased proliferation when cultured in DM for human corneal cells. PHCEn-CM did not affect the proliferation of CD4+ T cells. However, PHCF- and PHCEp-CM inhibited the proliferation of CD4+ T cells, and PHCF-CM significantly decreased the proliferation of CD4+ T cells (P < 0.05 Fig. 4 ). The decreased proliferation of human CD4+ T cells by PHCF-CM was reversed to the similar level of defined medium for human corneal cells with 1-MT treatment. 
Strongly Induced Expression of IDO in Human Corneal Cells by IFN-γ Treatment
To investigate the inducing effects of IFN-γ on IDO expression, RT-PCR was performed in IFN-γ–treated human corneal cells. IFN-γ treatment significantly induced expression of IDO in all three types of human corneal cells, including PHCEn, which did not express IDO in the absence of IFN-γ (Fig. 5)
CD4+ T-Cell Apoptosis Induced by Combination of IFN-γ and Conditioned Medium Derived from Human Corneal Cells
Human CD4+ T cells were cultured with different doses of IFN-γ and CM derived from PHCEp (Fig. 6A) , PHCF (Fig. 6B) , or PHCEn (Fig. 6C) . CD4+ T cells treated with CM from each type of cells and 0 to 5.0 ng/mL IFN-γ showed a dose-dependent increase in apoptosis as measured by ELISA. Supplementation of the human CD4+ T-cell cultures in the presence of CM and 0 to 5.0 ng/mL IFN-γ with IDO antagonist, 1-MT, led to no apoptotic effects of each combination on the CD4+ T cells, except that the CD4+ T cells cultured in the combination of 5.0 ng/mL IFN-γ and CM showed slightly increased apoptosis. 
Discussion
It is generally believed that corneal transplantation is highly successful because the ocular surface is composed of immunologically privileged sites. However, some external stimuli and corneal diseases lead to allograft rejection followed by graft failure. Therefore, maintaining the immune privilege of the ocular surface is important for corneal surgery and the inhibition of corneal inflammation. 
The key factor explored in this study is IDO, which is a monomeric heme-containing enzyme found primarily in cancer cell lines, 5 human placenta, 8 macrophages, 9 and dendritic cells. 4 In this study, the RT-PCR for IDO mRNA and Western blotting for IDO protein in corneal cells revealed that the expression of IDO was observed only in PHCEp and PHCF but not in PHCEn cells. PHCFs showed a significantly higher expression of IDO than did other types of corneal cells (Figs. 2 3) . Furthermore, these cultured cells only showed the positive expression of their inherent markers, keratocan and lumican (Fig. 1) , not dendritic cell marker CD83, suggesting that PHCFs, which were not contaminated by other cells, can highly express IDO. The expression of IDO generates a tryptophan-deficient local environment that inhibits the proliferation of T lymphocytes. 4 Therefore, these results suggest that the significantly higher expression of IDO in PHCFs can possibly cause a tryptophan-deficient environment in the corneal stroma. This explains that IDO can act as one of the factors for the maintenance of immune privilege on the ocular surface. In addition, it is hypothesized that the expression of IDO by PHCEp and PHCFs helps to maintain peripheral tolerance through regulation of the allogeneic T-cell response. Human CD4+ T cells showed a significant decrease in cell proliferation in PHCF-conditioned medium (Fig. 4) , which may be explained by the higher mRNA and protein expression of IDO in PHCF. Jager et al. 15 showed that cultured human corneas obtained from normal eyes secrete unidentified factors by adding the supernatant of cultured cornea to a mixed lymphocyte. Our data demonstrated that IDO can be one of the previously reported unidentified factors. 
Moreover, reports have shown that tryptophan deprivation causes activated T-cell apoptosis, induced by expression of the Fas ligand, 16 and that a tryptophan metabolite, 3-hydroxyanthranilic acid, induces apoptosis of THP-1 and U937 cells. 17 These results indicate that immune cell damage induced by IDO may be a key contributor to IDO-mediated immune tolerance. Therefore, IDO expressed by human corneal cells is biologically active and immunosuppressive because of its inhibition of the proliferation of T lymphocytes. 
IFN-γ treatment significantly induced IDO expression in all three types of human corneal cells, even PHCEn, the only cell type that did not express IDO without IFN-γ treatment (Fig. 5) . Therefore, IFN-γ acts as a strong inducer of IDO expression not only in PHCF but also in PHCEn cells. 
The dose-dependent enhancement of IFN-γ and the mediating role of IDO in immune cell apoptosis were further investigated by ELISA. The results of ELISA on the CD4+ T cells cultured in IFN-γ–treated PHCF-conditioned medium revealed increased optical density, which is a hallmark of apoptosis. Moreover, treatment with IFN-γ, which is a known strong inducer of IDO expression, significantly increased the apoptosis of CD4+ T cells in a dose-dependent manner (Fig. 6) . These results demonstrate that the expression of IDO may be synergistically enhanced by treatment with an inducer such as IFN-γ and that the inhibited CD4+ T-cell proliferation results from the apoptotic effects of IDO induced by IFN-γ–stimulated PHCFs. 
In conclusion, the expression of IDO in PHCF and PHCEp (but not in PHCEn) cells, resulting in the cellular damage of CD4+ T cells, was demonstrated using conditioned medium of PHCFs and PHCEp. In addition, the immune cell damage caused by IDO resulted from apoptosis. IFN-γ was a strong inducer of IDO and acted synergistically with PHCF to enhance apoptotic death of CD4+ T cells by inducing IDO expression. This immune cell damage caused by human corneal cell-induced IDO explains the immune resistance of PHCFs and suggests that IDO can act as one of the factors in the maintenance of immune privilege on the ocular surface. 
Accordingly, these results may be used for the maintenance of immune privilege, improvement of corneal surgery, and inhibition of corneal inflammation. Therefore, the findings of this study support the feasibility and benefits of using a local immunosuppressive factor such as IDO to prevent rejection in corneal transplantation. 
 
Figure 1.
 
Messenger RNA expression of human corneal cell markers. Each type of primary cultured human corneal cell expressed its own inherent markers but did not express the dendritic cell marker CD83.
Figure 1.
 
Messenger RNA expression of human corneal cell markers. Each type of primary cultured human corneal cell expressed its own inherent markers but did not express the dendritic cell marker CD83.
Figure 2.
 
Expression of IDO mRNA in human corneal cells. RT-PCR was performed to identify the expression of IDO mRNA in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEp and PHCF expressed IDO, and PHCF showed more higher expressions than PHCEp. However, PHCEn did not express IDO.
Figure 2.
 
Expression of IDO mRNA in human corneal cells. RT-PCR was performed to identify the expression of IDO mRNA in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEp and PHCF expressed IDO, and PHCF showed more higher expressions than PHCEp. However, PHCEn did not express IDO.
Figure 3.
 
Expression of IDO protein in human corneal cells. Western blot analysis was performed to identify the expression of IDO in primary cultured human corneal cells at the protein level. Among three different types of human corneal cells, PHCEp and PHCFs expressed IDO protein, whereas PHCEn did not. IDO protein level was higher in PHCFs.
Figure 3.
 
Expression of IDO protein in human corneal cells. Western blot analysis was performed to identify the expression of IDO in primary cultured human corneal cells at the protein level. Among three different types of human corneal cells, PHCEp and PHCFs expressed IDO protein, whereas PHCEn did not. IDO protein level was higher in PHCFs.
Figure 4.
 
Proliferation of CD4+ T cells cultured with conditioned medium derived from human corneal cells with or without 1-methyl tryptophan. Effects of IDO expressed by human corneal cells on the proliferation of CD4+ T cells were assessed by an MTT cell proliferation assay. Human CD4+ T cells were cultured in RPMI 1640 medium mixed with CM derived from PHCEp, PHCFs, or PHCEn with or without 1-MT. To exclude the effects of each medium used for cell cultures, cells were also cultured in RPMI 1640 medium mixed with DM for each type of human corneal cells. Human CD4+ T cells cultured in DM for each type of human corneal cells showed slightly decreased proliferation. Although cells were cultured in PHCEp and PHCEn-CM–treated medium did not show any proliferative effects, PHCF-CM–treated CD4+ T cells showed significantly decreased proliferation (P < 0.05), which was reversed by the IDO neutralizer 1-MT. Data represent the mean ± SD of three separate experiments. *P < 0.05, compared with no 1-MT treatment.
Figure 4.
 
Proliferation of CD4+ T cells cultured with conditioned medium derived from human corneal cells with or without 1-methyl tryptophan. Effects of IDO expressed by human corneal cells on the proliferation of CD4+ T cells were assessed by an MTT cell proliferation assay. Human CD4+ T cells were cultured in RPMI 1640 medium mixed with CM derived from PHCEp, PHCFs, or PHCEn with or without 1-MT. To exclude the effects of each medium used for cell cultures, cells were also cultured in RPMI 1640 medium mixed with DM for each type of human corneal cells. Human CD4+ T cells cultured in DM for each type of human corneal cells showed slightly decreased proliferation. Although cells were cultured in PHCEp and PHCEn-CM–treated medium did not show any proliferative effects, PHCF-CM–treated CD4+ T cells showed significantly decreased proliferation (P < 0.05), which was reversed by the IDO neutralizer 1-MT. Data represent the mean ± SD of three separate experiments. *P < 0.05, compared with no 1-MT treatment.
Figure 5.
 
Induction of IFN-γ on IDO expression. RT-PCR was performed in IFN-γ–treated human corneal cells. All three types of cells (PHCEp, PHCEn, and PHCF) showed significantly increased expression of IDO on IFN-γ treatment. PHCEn, the only type of cell that did not express IDO without IFN-γ treatment (Cont.), showed strongly induced IDO expression.
Figure 5.
 
Induction of IFN-γ on IDO expression. RT-PCR was performed in IFN-γ–treated human corneal cells. All three types of cells (PHCEp, PHCEn, and PHCF) showed significantly increased expression of IDO on IFN-γ treatment. PHCEn, the only type of cell that did not express IDO without IFN-γ treatment (Cont.), showed strongly induced IDO expression.
Figure 6.
 
Apoptosis of CD4+ T cells cultured in IFN-γ–treated CM. Human CD4+ T cells were cultured in IFN-γ–treated human corneal cell CM with or without 1-MT for 24 hours, and cellular apoptosis was measured by ELISA. Each type of cell showed increased apoptosis from 1.0 to 5.0 ng/mL IFN-γ–treated CM. CD4+ T-cell apoptosis induced by the IFN-γ–treated CM was reversed by IDO neutralizer 1-MT.
Figure 6.
 
Apoptosis of CD4+ T cells cultured in IFN-γ–treated CM. Human CD4+ T cells were cultured in IFN-γ–treated human corneal cell CM with or without 1-MT for 24 hours, and cellular apoptosis was measured by ELISA. Each type of cell showed increased apoptosis from 1.0 to 5.0 ng/mL IFN-γ–treated CM. CD4+ T-cell apoptosis induced by the IFN-γ–treated CM was reversed by IDO neutralizer 1-MT.
The authors thank IOVS volunteer editor Dongli Yang (University of Michigan, Ann Arbor) for editing the manuscript. 
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Figure 1.
 
Messenger RNA expression of human corneal cell markers. Each type of primary cultured human corneal cell expressed its own inherent markers but did not express the dendritic cell marker CD83.
Figure 1.
 
Messenger RNA expression of human corneal cell markers. Each type of primary cultured human corneal cell expressed its own inherent markers but did not express the dendritic cell marker CD83.
Figure 2.
 
Expression of IDO mRNA in human corneal cells. RT-PCR was performed to identify the expression of IDO mRNA in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEp and PHCF expressed IDO, and PHCF showed more higher expressions than PHCEp. However, PHCEn did not express IDO.
Figure 2.
 
Expression of IDO mRNA in human corneal cells. RT-PCR was performed to identify the expression of IDO mRNA in primary cultured human corneal cells. Among three different types of human corneal cells, PHCEp and PHCF expressed IDO, and PHCF showed more higher expressions than PHCEp. However, PHCEn did not express IDO.
Figure 3.
 
Expression of IDO protein in human corneal cells. Western blot analysis was performed to identify the expression of IDO in primary cultured human corneal cells at the protein level. Among three different types of human corneal cells, PHCEp and PHCFs expressed IDO protein, whereas PHCEn did not. IDO protein level was higher in PHCFs.
Figure 3.
 
Expression of IDO protein in human corneal cells. Western blot analysis was performed to identify the expression of IDO in primary cultured human corneal cells at the protein level. Among three different types of human corneal cells, PHCEp and PHCFs expressed IDO protein, whereas PHCEn did not. IDO protein level was higher in PHCFs.
Figure 4.
 
Proliferation of CD4+ T cells cultured with conditioned medium derived from human corneal cells with or without 1-methyl tryptophan. Effects of IDO expressed by human corneal cells on the proliferation of CD4+ T cells were assessed by an MTT cell proliferation assay. Human CD4+ T cells were cultured in RPMI 1640 medium mixed with CM derived from PHCEp, PHCFs, or PHCEn with or without 1-MT. To exclude the effects of each medium used for cell cultures, cells were also cultured in RPMI 1640 medium mixed with DM for each type of human corneal cells. Human CD4+ T cells cultured in DM for each type of human corneal cells showed slightly decreased proliferation. Although cells were cultured in PHCEp and PHCEn-CM–treated medium did not show any proliferative effects, PHCF-CM–treated CD4+ T cells showed significantly decreased proliferation (P < 0.05), which was reversed by the IDO neutralizer 1-MT. Data represent the mean ± SD of three separate experiments. *P < 0.05, compared with no 1-MT treatment.
Figure 4.
 
Proliferation of CD4+ T cells cultured with conditioned medium derived from human corneal cells with or without 1-methyl tryptophan. Effects of IDO expressed by human corneal cells on the proliferation of CD4+ T cells were assessed by an MTT cell proliferation assay. Human CD4+ T cells were cultured in RPMI 1640 medium mixed with CM derived from PHCEp, PHCFs, or PHCEn with or without 1-MT. To exclude the effects of each medium used for cell cultures, cells were also cultured in RPMI 1640 medium mixed with DM for each type of human corneal cells. Human CD4+ T cells cultured in DM for each type of human corneal cells showed slightly decreased proliferation. Although cells were cultured in PHCEp and PHCEn-CM–treated medium did not show any proliferative effects, PHCF-CM–treated CD4+ T cells showed significantly decreased proliferation (P < 0.05), which was reversed by the IDO neutralizer 1-MT. Data represent the mean ± SD of three separate experiments. *P < 0.05, compared with no 1-MT treatment.
Figure 5.
 
Induction of IFN-γ on IDO expression. RT-PCR was performed in IFN-γ–treated human corneal cells. All three types of cells (PHCEp, PHCEn, and PHCF) showed significantly increased expression of IDO on IFN-γ treatment. PHCEn, the only type of cell that did not express IDO without IFN-γ treatment (Cont.), showed strongly induced IDO expression.
Figure 5.
 
Induction of IFN-γ on IDO expression. RT-PCR was performed in IFN-γ–treated human corneal cells. All three types of cells (PHCEp, PHCEn, and PHCF) showed significantly increased expression of IDO on IFN-γ treatment. PHCEn, the only type of cell that did not express IDO without IFN-γ treatment (Cont.), showed strongly induced IDO expression.
Figure 6.
 
Apoptosis of CD4+ T cells cultured in IFN-γ–treated CM. Human CD4+ T cells were cultured in IFN-γ–treated human corneal cell CM with or without 1-MT for 24 hours, and cellular apoptosis was measured by ELISA. Each type of cell showed increased apoptosis from 1.0 to 5.0 ng/mL IFN-γ–treated CM. CD4+ T-cell apoptosis induced by the IFN-γ–treated CM was reversed by IDO neutralizer 1-MT.
Figure 6.
 
Apoptosis of CD4+ T cells cultured in IFN-γ–treated CM. Human CD4+ T cells were cultured in IFN-γ–treated human corneal cell CM with or without 1-MT for 24 hours, and cellular apoptosis was measured by ELISA. Each type of cell showed increased apoptosis from 1.0 to 5.0 ng/mL IFN-γ–treated CM. CD4+ T-cell apoptosis induced by the IFN-γ–treated CM was reversed by IDO neutralizer 1-MT.
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