IV-induced IFN-β release and subsequent TRAIL expression depend on activation of PKR and NF-κB in alveolar macrophages.

<p>(A) Murine AM were A/PR8 infected <i>ex vivo</i> and expression of phosphorylated PKR (p-PKR) and total PKR were assessed by western blot. Wt or <i>pkr^−/</i>⁻ AM were A/PR8-infected <i>ex vivo</i> and NF-κB p65 activation was comparatively analysed by TransAM assay (quantifying p65 binding to a consensus-binding site oligo by a colorimetric method) and depicted as fold activation of mock-infected control (B) or phosphorylated IRF-3 in relation to total IRF-3 protein expression or total IRF-7 expression were determined by western blot at the given time points pi and densitometry data is depicted as relative expression (C). (D) Wt vs. <i>pkr^−/</i>⁻ AM or IKK inhibitor- vs. DMSO-treated wt AM were A/PR8-infected <i>ex vivo</i> and IFN-β concentrations in supernatants were analysed 24 h pi. (E) Wt or <i>pkr^−/</i>⁻ AM were either A/PR8-infected or stimulated with 180 U/ml rIFN-β, respectively, and additionally treated with IKK inhibitor vs. DMSO or anti-IFN-β vs. isotype Ab and TRAIL mRNA expression was quantified 16 h pi. (A) shows a representative western blot of 3 independent experiments. Bar graphs represent means ± SD of 3 (B, C) or 4 (D, E) independent experiments. * p<0.05; ** p<0.01; ***p<0.005; ctrl, control; iso, isotype; pi, post infection; Ab, antibody.