USE OF THE ACUTE-PHASE SERUM AMYLOID A2 (SAA2) GENE PROMOTER IN THE ANALYSIS OF PROINFLAMMATORY AND ANTIINFLAMMATORY MEDIATORS - DIFFERENTIAL KINETICS OF SAA2 PROMOTER INDUCTION BY IL-1-BETA AND TNF-ALPHA COMPARED TO IL-6

A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammator y mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that clos ely mimics the response of the endogenous SAA2 gene to such stimuli: i .e. single treatments induce transcriptional activation by IL-1 beta a nd ThrF-alpha to a greater extent than by IL-6 at 12-24 h. However, ti mecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a mu ch greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL- 6 to give a 10-fold increase in transcriptional readout over single cy tokine treatments, The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore , in synergistic cytokine combinations, it blocks only the IL-1 beta d riven component indicating that the effect is biological and not attri butable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory sig nalling in a cytokine specific manner, and far the evaluation of the p ro- and anti-inflammatory properties of novel natural and synthetic mo lecules.