Amplification Mechanisms of Inflammation: Paracrine Stimulation of Arachidonic Acid Mobilization by Secreted Phospholipase A2 Is Regulated by Cytosolic Phospholipase A2-Derived Hydroperoxyeicosatetraenoic Acid

Abstract

In macrophages and other major immunoinflammatory cells, two phospholipase A2 (PLA2) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A2 (cPLA2) and a secreted phospholipase A2 (sPLA2). In this study, the molecular mechanism underlying cross-talk between the two PLA2s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA2-dependent manner, and addition of exogenous group V sPLA2 to the activated cells increases the release. This sPLA2 effect is abolished if the cells are pretreated with cPLA2 inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA2. In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA2 activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA2 response to levels found in cells displaying normal cPLA2 activity. Moreover, 12- and 15-HPETE are able to enhance sPLA2 activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA2-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA2 with cPLA2 for a full AA mobilization response.