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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 ϩ 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 ϩ T cells were also investigated after IFN-␥ 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 ϩ T cells cultivated in conditioned medium derived from human corneal fibroblasts showed decreased cell proliferation and increased apoptosis. IFN-␥ 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. (Invest Ophthalmol Vis Sci. 2007;48:4148 -4152) DOI:10.1167/iovs.05-1336 C orneal 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 hemecontaining enzyme that catalyzes the opening of the pyrrole ring of L-tryptophan to yield N-formylkynurenine, which rapidly degrades to give kynurenine. 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 ...

† These authors contributed equally to this work.Abstract. The present study was designed to determine the effects of leptin on lipid metabolism and gene expression during differentiation and maturation of the 3T3-L1 murine preadipocyte. The preadipocytes were induced to differentiate in a growth medium containing 10% calf serum and a hormonal cocktail for 2 days. The cells were next allowed to maturate for 14 days in the growth medium supplemented with 10 μg/ml insulin or 500 ng/ml insulin-like growth factor (IGF)-I in the absence or presence of supplemented leptin. Leptin, at a dose of 5 to 500 ng/ml, had no effect on proliferation of undifferentiated 3T3-L1 cells. However, leptin suppressed the insulin-or IGF-I-stimulated lipid accumulation and enhanced the release of glycerol, a measure of lipolysis, in a dose-dependent manner during and after the maturation of the cell. Moreover, leptin at a dose of 50 ng/ml inhibited IGF-I gene expression during the entire differentiation and maturation and also peroxisome proliferator activated receptor (PPAR)-γ expression during late maturation as monitored by semi-quantitative reverse transcription-polymerase chain reaction. However, leptin exerted no effect on the expression of transforming growth factor-β, CCAT/enhancer binding protein-α and PPAR-δ. Taken together, results suggest the anti-lipogenic and lipolytic effects of leptin in differentiating and mature adipocytes may have been partly mediated by suppressing the expression of PPAR-γ and IGF-I genes.Key words: Leptin, IGF-I, PPAR, Adipocyte, Differentiation, Lipid (Endocrine Journal 55: 827-837, 2008) ADIPOCYTES are highly specialized cells which play an important role in energy homeostasis by harboring energy reservoirs as lipid droplets consisting of triglycerides [1,2]. These reservoirs, however, have been implicated in a host of major human health problems, because an excessive or insufficient energy reserve results in a metabolic disorder known as obesity or lipodystrophy, respectively [3,4]. The cellular development and subsequent metabolic processes controlling the energy reserve of adipocdytes are regulated by a number of transcription factors and autocrine/paracrine as well as endocrine agents. Insulinlike growth factor (IGF)-I stimulates the proliferation and differentiation including lipid synthesis and also inhibits lipolysis in the adipose cell lines in a fashion similar to that of insulin [5][6][7]. Transforming growth factor-β also has a stimulatory effect on proliferation of preadipocytes [8], but, unlike IGF-I, this peptide inhibits differentiation of preadipocyte cell lines [9,

PurposeTo establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM).MethodsPrimary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively.ResultsSuccessful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15±10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV.ConclusionsIHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

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