ESSENTIAL REQUIREMENT OF CYTOSOLIC PHOSPHOLIPASE A(2) FOR ACTIVATION OF THE PHAGOCYTE NADPH OXIDASE

Arachidonic acid (AA) can trigger activation of the phagocyte NADPH ox idase in a cell-free assay. However, a role for AA in activation of th e oxidase in intact cells has not been established, nor has the AA gen erating enzyme critical to this process been identified, The human mye loid cell line PLB-985 was transfected to express p85 cytosolic phosph olipase A(2) (cPLA(2)) antisense mRNA and stable clones were selected that lack detectable cPLA(2). cPLA(2)-deficient PLB-985 cells differen tiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D-3, indicating that cPLA(2) is not involved in the differentiation process. Neither cPLA(2) nor stimulated [H-3]AA release were detectable in differentiated cPLA(2)-deficient PLB-985 c ells, demonstrating that cPLA(2) is the major type of PLA(2) activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxida se subunits during differentiation of cPLA(2)-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety o f soluble and particulate stimuli, but the addition of exogenous AA fu lly restores oxidase activity. This establishes an essential requireme nt of cPLA(2)-generated AA for activation of phagocyte NADPH oxidase.