IL-1-β, TNF-, and IFN-γ, alone and in combination, upregulate Fas expression in cultured TEC. TEC subcultured after 10 to 13 days of primary culture were incubated with the cytokines. At 24, 48, or 72 hours, TEC were collected by trypsin treatment and labeled with anti–HLA-DR or anti-Fas antibody. (A) A representative experiment shows that, after 48 hours of incubation, IL-1-β, TNF-, and IFN-γ, both alone and together, increased Fas MFI. Vertical bars on Fas staining histograms indicate the Fas MFI level in TEC cultured in control conditions, ie, in medium. (B) mRNA was extracted from TEC cultured without (−) or with (+) 1 ng/mL IL-1-β, 10 ng/mL TNF-, and 500 U/mL IFN-γ. Lane 1 corresponds to the molecular weight (MW) marker (pUC18 DNA Marker Hae III digest; Sigma). mRNA was submitted to RT-PCR. In three independent experiments, cytokine-activated TEC showed a strong increase in Fas mRNA levels in comparison to TEC cultured in control conditions, whereas β-actin expression was not modified. (C) The MFI ratio is the ratio between Fas MFI measured in the presence of one or several cytokines and Fas MFI measured in control conditions. MFI ratios are expressed as a function of time. Results are means ± SEM of four independent experiments. (D) The proportion of HLA-DR–positive cells was analyzed in the same experiments.