Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity

BACKGROUND The role of calcium-independent phospholipase A(2) (iPLA(2)), a component of the three major PLA(2) families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA(2)-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA(2) is unidentified. We also demonstrated that the iPLA(2)-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA(2) may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA(2) (cPLA(2)), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA(2) regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A(2), iPLA(2), and cPLA(2), in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. METHODS Isolated human PMNs were incubated with the PLA(2) inhibitor selective for iPLA(2), iPLA(2), or cPLA(2) and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. RESULTS The iPLA(2)-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA(2)-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA(2), but not iPLA(2) or cPLA(2), completely inhibited the platelet activating factor/fMLP-induced chemotaxis. CONCLUSION iPLA(2) is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA(2) inhibitor suggests that AA may not be relevant to these cytotoxic functions.