PROSTAGLANDIN E(2) PRODUCTION BY ENDOTOXIN-STIMULATED ALVEOLAR MACROPHAGES IS REGULATED BY PHOSPHOLIPASE-C PATHWAYS

Background: Eicosanoids play an important role in many aspects of syst emic inflammatory responses and host defense. Although the synthesis o f eicosanoids by different enzymes has been elucidated, the regulatory mechanism of eicosanoid production is not clear. We designed this stu dy to investigate the hypothesis that PGE(2) production by endotoxin ( lipopolysaccharide; LPS)-stimulated macrophages (MO) is dependent on p hospholipase C (PLC) signaling pathways. Methods: Rabbit alveolar macr ophages (MO) were obtained by bronchoalveolar lavage. MO were suspende d in RPMI-1640 medium at 1 x 10(6)/mL and were exposed to Escherichia coli LPS (10 ng/mL) +/- various agonists and antagonists of PLC and it s secondary messengers. After 24 hours of incubation, prostaglandin E( 2) (PGE(2)) production was measured by ELISA. Results: LPS-activated M O produced four times as much PGE(2) as did control unstimulated MO. T he increase in PGE(2) production was inhibited by PLC inhibitors (U731 22 or D609) and a low-molecular-weight PLA(2) inhibitor, manoalide. An increase in intracellular calcium and activation of both the calmodul in and protein kinase C kinase pathways increase PGE(2) production. Co nclusions: PGE(2) production is intimately dependent on several phosph olipases. Production is not only dependent on low-molecular-weight PLA (2) cleavage of arachidonic acid from membrane phospholipids, but also by-products of PLC activation. PLC-dependent intracellular Ca-calmodu lin signaling and protein kinase C activation provide significant modu lation of PGE(2) production.