Abstract
Studies were conducted to characterize a human monocyte model where the role of the 85-kDa phospholipase A2 (PLA2) in prostanoid formation could be evaluated. The presence of an immunologically related 85-kDa PLA2 and type II 14-kDa PLA2 was demonstrated in human monocytes and their roles examined in lipopolysaccharide (LPS)-induced monocyte prostaglandin E2 (PGE2) formation. Exposure of human monocytes to LPS over 18 h resulted in the up-regulation of the mitogen-inducible cyclooxygenase-2 and was accompanied by production and release of prostaglandin E2 but not leukotriene C4. This coincided with a 2-fold increase in the 85-kDa PLA2 protein and activity levels. In contrast, there was no effect on the type II 14-kDa-like PLA2 activity measured in the 100,000 x g particulate fraction nor did LPS induce the release of type II 14-kDa PLA2 into the medium. Treatment with cycloheximide over 18 h resulted in a time-dependent decrease in cytosolic 85-kDa PLA2 protein and activity (half-life = 4 h), but there was no change in the particulate type II 14-kDa-like PLA2 activity. Monocytes were therefore exposed to an 85-kDa PLA2 initiation site-directed antisense oligonucleotide which specifically decreased the cytosolic 85-kDa PLA2 protein levels and activity in a concentration-dependent manner. This had no effect on the cyclooxygenase-2 (protein mass or the ability to convert arachidonic acid to PGE2) or the particulate fraction sn-2 acylhydrolytic activity but was associated with a decrease in LPS-induced PGE2 production. Taken together, these data support a role for the cytosolic 85-kDa PLA2 in LPS-induced monocyte PGE2 formation.
Highlights
Studies wereconducted to characterize a human Marshall, 1993; Dennis, 1983).The mammalian typeI1 16kDa monocyte modelwhere the role of the 85-kDaphospho- PLA, has been well characterized andis known to exisitn both lipaseA, (PLA,)in prostanoid formation could be evalu-an extracellular form in inflammatory fluids
PLA, initiation site-directed antisense oligonucleotide We have previously described the coexistence of a cytosolic which decreasedthe cytosolic 85-kDaP w 85-kDa-like PLA, and a cell-associated type I1 14-kDa-likeP&
Phospho- or 18 h and the cell-free media was examined for PGE, and rothioate oligonucleotides were useddue to the reported enhanced cell LTC, levels
Summary
Studies wereconducted to characterize a human Marshall, 1993; Dennis, 1983).The mammalian typeI1 16kDa monocyte modelwhere the role of the 85-kDaphospho- PLA, has been well characterized andis known to exisitn both lipaseA, (PLA,)in prostanoid formation could be evalu-an extracellular form in inflammatory fluids A cytosolic 85-kDa PLA, has been isolated and cloned from the human monocytic cell line, U937 (Clark et al, 1990; Kramer et al, 1991).This structurally distinct P& exhibits a preference for AA in the sn-2 position of PL and is regulatbeyd intracellular Ca” concentrakotriene C, This coincidedwith a 2-foldincrease in the tions and phosphorylation 1991; Diez and Mong, 1990; Svensson et al, 1993; Lin et al, there was no effect on the type I1 14-kDa-likePLA, ac- 1993; de Carvalho, et al, 1993) This enzyme was originally tivity measured in the 100,000 x g particulate fraction described inhumanandmurine monocyte/macrophage cell nor did LPS induce the release of type I1 14-kDa PLA, lines (Channon andLeslie, 1990;Clark et al, 1990) and canbe into the medium. Thishad no effect otnhe cyclooxygenase-2(pro- the role of the two enzymes in the regulationof monocyte AA tein mass or the ability to convert arachidonic acid to mobilization and lipid mediator formationhas yet t o be clearly
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