Establishment of an in vitro test system to evaluate the down-regulatory activities of natural products on IL-4

Interleukin-4 (IL-4), a representative T(H)2 cytokine, plays a pathologic role in the onset of various allergic diseases including atopic dermatitis, atopic rhinitis, and asthma. Several drug candidates that down-regulate IL-4 expression have been studied for their possible use as antiallergic agents in clinical settings. Therefore, an in vitro test to evaluate IL-4 promoter activities might be useful for selecting candidates of novel natural therapeutics. The promoter region (741 to +56) of IL-4 was cloned upstream of a luciferase gene in the plasmid pGL4.14 with a hygromycin resistance gene as a selection marker to generate pGL4.14-IL-4. Treatment with PMA and A23187 highly increased luciferase activity by approximately 10-fold compared with the control in both EL-4 thymoma and RBL-2H3 cells transiently transfected with pGL4.14-IL-4, as well as in stable cell lines constantly expressing pGL4.14-IL-4. Cyclosporin A and dexamethasone, well-known anti-allergic agents, significantly down-regulated the activity in a dosedependent manner. The feasibility of this system was evaluated by measuring the down-regulatory activities of various extracts from the TBRC plant library on PMA- and A23187-induced luciferase activities of IL-4 promoter, and by measuring IL-4 production in cultured cells using ELISA assays. The results of this study suggest that this primary screening system is simple and time-saving, and might be suitable for the selection of natural therapeutic candidates for allergic disease by measuring the down-regulatory effects of natural products on the IL-4 promoter.