Interferon-gamma induces apoptosis and expression of inflammation-related proteins in Chang conjunctival cells

PURPOSE. The purpose of this study was to investigate the effect of interfe ron (IFN)gamma on cell viability, cell growth, and apoptosis and on express ion of apoptotic and inflammation-related proteins in epithelial conjunctiv al cells in vitro. Some aspects of transduction pathways of IFN gamma-induc ed alterations were also investigated, especially the role of protein kinas e C (PKC) and IFN gamma transcriptional factor STAT1. METHODS. A human conjunctival cell line was treated with different concentr ations (30 and 300 U/ml) of human recombinant IFN gamma. After 24, 48, and 72 hours of treatment, cell viability and relative cell number were studied with 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl tetrazolium bromide (MTT) and crystal violet colorimetric assays. The apoptotic process cr as sought by phase-contrast microscopy, 4',6'diamidino-2-phenylindole dihydrochloride (D API) staining, and transmission electron microscopy and was confirmed by DN A electrophoresis and immunoblotting of poly(ADP-ribose) polymerase (PARP). The cell cycle and expression of apoptotic proteins Fas, bar, and p53; of inflammation-related proteins HLA-DR and intercellular adhesion molecule (I CAM)-1; and of IFN gamma signal-transducing factor STAT1 were evaluated by flow cytometry and/or western blot analysis. To investigate PKC-related tra nsduction pathways, two PKC modulators, 12-O-tetradecanoyl-phorbol-13-aceta te (TPA) and staurosporine, were applied for 3 hours, followed by IFN gamma treatment for 72 hours. Moreover, the effects of PE;C depletion were studi ed after a 24-hour application of TPA, also followed by IFN gamma treatment for 72 hours. Then, Fas, ICAM-1, and HLA-DR expressions were studied by no w cytometry. RESULTS. IFN gamma at 30 U/ml induced no change in cell cycle and in cell v iability. Cell viability significantly decreased after 48 hours of treatmen t with 300 U/ml IFN gamma, associated with cell cycle alterations (decrease in number of cells in the S-M phase), apoptotic chromatin condensation and fragmentation, ladder pattern on DNA electrophoresis assay, and cleavage o f PARP. Moreover, IFN gamma-treated cells overexpressed plasma membrane Fas , HLA-DR, and ICAM-1 in a dose- and time-dependent manner, and STAT1 in bot h nuclear and cytosolic cell fractions. Only 300 U/ml IFN gamma-treated cel ls overexpressed bar, whereas Bcl-2 and p53 proteins were not modified. HLA -DR and Fas were upregulated after addition of staurosporine or after PKC-d epleting treatment and repressed with TPA. Staurosporine, PKC depletion, an d TPA all enhanced ICAM-1 expression. CONCLUSIONS. in our. model, IFN gamma induced expression of inflammatory mo lecules and apoptotic mediators, cell growth arrest, and apoptosis of Chang conjunctival cells. Moreover, our results suggest that activation of PRC i s not involved in some IFN gamma cellular effects that possibly imply the u pregulation and nuclear translocation of STAT1. IFN gamma-induced apoptosis could explain in part the recently reported coexistence of inflammation an d programmed cell death in ocular surface inflammatory disorders such as Sj ogren's syndrome.