Antisense inhibition of 85-kDa cPLA2 blocks arachidonic acid release from airway epithelial cells.

Inflammatory cytokines play a critical role in the initiation and perpetuation of inflammation. Several cytokines are known to increase the production of arachidonic acid (AA) metabolites, which may mediate cytokine-induced acute and chronic inflammation. Although cytokines upregulate phospholipase A2 (PLA2) in several target cells, the contribution of individual PLA2 to cytokine-induced AA release and eicosanoid production remains unclear because of the existence of various forms of cellular PLA2. To examine the role of 85-kDa cytosolic PLA2 (cPLA2) in cytokine-induced AA release, a system was developed to inhibit the expression of cPLA2 in a human bronchial epithelial cell line (BEAS-2B cells) by antisense RNA. Cells stably expressing antisense cPLA2 exhibited decreased cPLA2 protein levels as well as decreased cPLA2 activity assayed in vitro. The effects of cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha) on the release of prelabeled [3H]AA were then tested in cells stably transfected with vector alone as well as cells transfected with cPLA2 antisense plasmid. IFN-gamma (300 U/ml), TNF-alpha (20 ng/ml), and IL-1 alpha (20 ng/ml) all induced a significantly increased release of prelabeled [3H]AA after 15 min to 2 h of treatment in control cells, and their effects were significantly reduced in cells transfected with cPLA2 antisense vector. These results demonstrate a critical role of cPLA2 in inflammatory cytokine-induced AA metabolism.