FIGURE 6.

Increased mRNA stability is not responsible for increased TNF-α production by AMφ stimulated with the NTHi OMP PCP and IFN-β. Resident AMφ from normal C57BL/6 mice were cultured for 3 h in the presence of medium alone (C′) (light gray bars), 100 ng/ml PCP (□), or a combination of PCP and IFN-β (1000 U/ml) (■). After 3 h, the RNA polymerase inhibitor actinomycin D (10 μg/ml, ActD) was added to a fraction of the samples and all the samples were incubated for 3 additional hours. Total cellular RNA was collected and processed for real-time PCR and samples were analyzed for TNF-α mRNA expression. The relative quantities of specific mRNA from treated AMφ are plotted compared with the calibrator mRNA (TNF-α mRNA in control AMφ, which is defined as a value of 1). dRn is baseline-corrected, reference dye-normalized fluorescence.*, p <0.05, unpaired Student t test. Data are mean ± SEM of triplicate wells in each of two independent experiments with similar results.