Fas- and interferon γ-induced apoptosis Chang conjunctival cells: Further investigations

PURPOSE. Previously interferon (IFN)γ-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were demonstrated. The aim of this study was to further investigate the mechanisms of IFNγ-, Fas-, and cycloheximide (CHX-(induced programmed cell death, with special attention to the role of transcriptional factors NF-κB and STAT1. METHODS. In a human conjunctival cell line (Chang conjunctival cells) apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab) alone (24 or 48 hours) or, as previously reported, with 300 U/ml of human recombinant IFNγ alone (48 hours). To study role of IFNγ on Fas-induced apoptosis, cells were treated first with IFNγ at 30 U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied for 24 hour. Moreover, to study the influence of CHX on Fas- and IFNγ-induced apoptosis, cells were treated for 24 hours with 300 U/ml IFNγ together with a nontoxic concentration (1 μg/ml) of CHX, or with 500 ng/ml anti-Fas ab together with 1 μg/ml CHX (24 hours). After treatment, cell viability (neutral red assay), mitochondrial membrane potential (rhodamine 123 assay), chromatin condensation (Hoechst 33342 assay), and the index Hoechst/neutral red were studied by cold light microplate cytometry. The apoptotic process was sought for by contrast phase microscopy and DAPI staining and was confirmed by immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 were investigated by Western blot analysis. NF-κB and STAT DNA-binding activities were studied by electrophoretic mobility shrift assays (EMSA). RESULTS. After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20% and 30%, respectively, of apoptotic cells were observed. When anti-Fas sera were applied after IFNγ pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by an index Hoechst/neutral red greater than one. All these modifications were preceded by a decrease in mitochondrial membrane potential. EMSA revealed that NF-κB was activated after IFNγ and anti-Fas ab treatments and inhibited after CHX treatment. STAT1 was strongly activated after IFNγ treatment and only in a minor degree after anti-Fas ab treatment. STAT1-binding activity persisted after CHX treatment. CONCLUSIONS. The relative resistance of Chang cells toward Fas-induced apoptosis could be related to the activation of NF-κB. IFNγ-induced programmed cell death preferentially involves the activation of STAT1 that counterbalances NF-κB antiapoptotic effects. In fact, Fas-induced apoptosis was potentiated by IFNγ or CHX treatments. These results suggest NF-κB activation could maintain cell viability as well as participate in IFNγ-induced inflammatory modifications, whereas STAT1 activation could provide, in this model, a proapoptotic signal.