Interferon-γ Induces Apoptosis and Expression of Inflammation-Related Proteins in Chang Conjunctival Cells

PURPOSE. The purpose of this study was to investigate the effect of interferon (IFN)γ on cell viability, cell growth, and apoptosis and on expression of apoptotic and inflammation-related proteins in epithelial conjunctival cells in vitro. Some aspects of transduction pathways of IFNγ-induced alterations were also investigated, especially the role of protein kinase C (PKC) and IFNγ transcriptional factor STATI. METHODS. A human conjunctival cell line was treated with different concentrations (30 and 300 U/ml) of human recombinant IFNγ. 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 was sought by phase-contrast microscopy, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and transmission electron microscopy and was confirmed by DNA electrophoresis and immunoblotting of poly(ADP-ribose) polymerase (PARP). The cell cycle and expression of apoptotic proteins Fas, bax, and p53; of inflammation-related proteins HLA-DR and intercellular adhesion molecule (ICAM)-1; and of IFNγ signal-transducing factor STATI were evaluated by flow cytometry and/or western blot analysis. To investigate PKC-related transduction pathways, two PKC modulators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and staurosporine, were applied for 3 hours, followed by IFNγ treatment for 72 hours. Moreover, the effects of PKC depletion were studied after a 24-hour application of TPA, also followed by IFNγ treatment for 72 hours. Then, Fas, ICAM-1, and HLA-DR expressions were studied by flow cytometry. RESULTS. IFNγ at 30 U/ml induced no change in cell cycle and in cell viability. Cell viability significantly decreased after 48 hours of treatment with 300 U/ml IFNγ, 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 of PARP. Moreover, IFNγ-treated cells overexpressed plasma membrane Fas, HLA-DR, and ICAM-1 in a dose- and time-dependent manner, and STATI in both nuclear and cytosolic cell fractions. Only 300 U/ml IFNγ-treated cells overexpressed bax, whereas Bcl-2 and p53 proteins were not modified. HLA-DR and Fas were upregulated after addition of staurosporine or after PKC-depleting treatment and repressed with TPA. Staurosporine, PKC depletion, and TPA all enhanced ICAM-1 expression. CONCLUSIONS. In our model, IFNγ induced expression of inflammatory molecules and apoptotic mediators, cell growth arrest, and apoptosis of Chang conjunctival cells. Moreover, our results suggest that activation of PKC is not involved in some IFNγ cellular effects that possibly imply the upregulation and nuclear translocation of STATI. IFNγ-induced apoptosis could explain in part the recently reported coexistence of inflammation and programmed cell death in ocular surface inflammatory disorders such as Sjogren's syndrome.