The kinetics and magnitude of the synergistic activation of the serum amyloid A promoter by IL-1 beta and IL-6 is determined by the order of cytokineaddition

Human serum amyloid A protein (A-SAA) is a major hepatic acute-phase protei n, the concentration of which increases by up to 1000-fold during inflammat ion. This induction is primarily due to synergistic transcriptional up-regu lation by pro-inflammatory cytokines, principally interleukin (IL)-1 and IL -6. Using HepG2 hepatoma cells transfected with pGL2-SAA2pt, a cytokine-res ponsive human SAA2 promoter/luciferase reporter gene construct, we show tha t stimulation with IL-1 beta prior to IL-6 is essential for maximal synergi stic transcriptional induction of the SAA2 gene. The reciprocal treatment, i.e. stimulation of the promoter with IL-6 before IL-IP, results in signifi cantly less synergistic activation of the SAA2 promoter. These findings str ongly suggest that in vitro studies of acute-phase-protein induction using combinations of cytokines should be designed to reflect the chronology of t heir participation in the cytokine cascade.