ANTISENSE INHIBITION OF 85-KDA CPLA(2) BLOCKS ARACHIDONIC-ACID RELEASE FROM AIRWAY EPITHELIAL-CELLS

Inflammatory cytokines play a critical role in the initiation and perp etuation of inflammation. Several cytokines are known to increase the production of arachidonic acid (AA) metabolites, which may mediate cyt okine-induced acute and chronic inflammation. Although cytokines upreg ulate phospholipase A(2) (PLA(2)) in several target cells, the contrib ution of individual PLA(2) to cytokine-induced AA release and eicosano id production remains unclear because of the existence of various form s of cellular PLA(2). To examine the role of 85-kDa cytosolic PLA(2) ( cPLA(2)) in cytokine-induced AA release, a system was developed to inh ibit the expression of cPLA(2) in a human bronchial epithelial cell li ne (BEAS-2B cells) by antisense RNA. Cells stably expressing antisense cPLA(2) exhibited decreased cPLA(2) protein levels as well as decreas ed cPLA(2) activity assayed in vitro. The effects of cytokines interfe ron-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and in terleukin-1 alpha (IL-1 alpha) on the release of prelabeled [H-3]AA we re then tested in cells stably transfected with vector alone as well a s cells transfected with cPLA(2) antisense plasmid. IFN-gamma (300 U/m l), TNF-alpha (20 ng/ml), and IL-1 alpha (20 ng/ml) all induced a sign ificantly increased release of prelabeled [H-3]AA after 15 min to 2 h of treatment in control cells, and their effects were significantly re duced in cells transfected with cPLA(2) antisense vector. These result s demonstrate a critical role of cPLA(2) in inflammatory cytokine-indu ced AA metabolism.