Prostaglandin E sub 2 Production by Endotoxin-Stimulated Alveolar Macrophages Is Regulated by Phospholipase C Pathways

Abstract

Background Eicosanoids play an important role in many aspects of systemic inflammatory responses and host defense. Although the synthesis of eicosanoids by different enzymes has been elucidated, the regulatory mechanism of eicosanoid production is not clear. We designed this study to investigate the hypothesis that PGE₂ production by endotoxin (lipopolysaccharide; LPS)-stimulated macrophages (MO) is dependent on phospholipase C (PLC) signaling pathways. Methods Rabbit alveolar macrophages (MO) were obtained by bronchoalveolar lavage. MO were suspended in RPMI-1640 medium at 1 X 10⁶ /mL and were exposed to Escherichia coli LPS (10 ng/mL) +/- various agonists and antagonists of PLC and its secondary messengers. After 24 hours of incubation, prostaglandin E₂ (PGE₂) production was measured by ELISA. Results LPS-activated MO produced four times as much PGE₂ as did control unstimulated MO. The increase in PGE₂ production was inhibited by PLC inhibitors (U73122 or D609) and a low-molecular-weight PLA₂ inhibitor, manoalide. An increase in intracellular calcium and activation of both the calmodulin and protein kinase C kinase pathways increase PGE sub 2 production. Conclusions PGE₂ production is intimately dependent on several phospholipases. Production is not only dependent on low-molecular-weight PLA₂ cleavage of arachidonic acid from membrane phospholipids, but also by-products of PLC activation. PLC-dependent intracellular Ca-calmodulin signaling and protein kinase C activation provide significant modulation of PGE₂ production.