The two-component C3-tat/HIV promoter is a broadly responsive sensor of chronic inflammation. (a and b) Induction by the principal proinflammatory cytokines in vitro. Primary synovial fibroblasts were infected in vitro by incubating overnight with the indicated Ad vectors at a multiplicity of infection of 100. Twenty-four hours later, cells were starved in 0.5% FBS medium for 24–48 hours, followed by stimulation for 6 hours with the indicated cytokines (20 ng/ml) or LPS (10 μg/ml). Luciferase activity in cell lysates was normalized to total protein. (a) The levels of inflammation-inducible C3-tat/HIV–driven expression are comparable to those attainable with the CMV promoter. Data are presented as mean of duplicate measurements within an individual experiment; error bars show SD. (b) The C3-tat/HIV promoter provides a large dynamic range of response. The values of fold induction were calculated as the average of readings in independent experiments performed in duplicate (two experiments with IL-1 and IL-6 and three experiments with TNF-α and LPS). (c) In vivo induction following reactivation of SCW arthritis. On day –42, animals were primed by IA injection of PG-APS in both ankle joints. The same joints were injected with the indicated Ad vectors (10⁷ PFU in 10 μl) on day –2. On day 0, arthritis was reactivated by an intravenous injection of PG-APS. Luciferase activity was measured in extracts of pulverized joints (each bar shows the mean ± SD of two joints). In three independent experiments, luciferase activity peaked on day 2–4 and remained elevated through day 4. Unstim., unstimulated; IV, intravenous; rlu, relative luciferase units.