Abstract
Human monocytes possess several acylhydrolase activities and are capable of producing both prostanoids (PG) and leukotriene (LT) products upon acute stimulation with calcium ionophore, A23187 or phagocytosis of zymosan particles. The cytosolic 85-kDa phospholipase (PLA) A2 co-exists with the 14-kDa PLA2 in the human monocyte, but their respective roles in LT production are not well understood. Reduction in 85-kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85-kDa PLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocyte prostanoid formation. In contrast, neither treatment altered stimulated LTC4 production. This confirmed the important role of the 85-kDa PLA2 in prostanoid formation but suggests that it has less of a role in LT biosynthesis. Alternatively, treatment of monocytes with the selective, active site-directed 14-kDa PLA2 inhibitor, SB 203347, prior to stimulation had no effect on prostanoid formation at concentrations that totally inhibited LT formation. Addition of 20 microM exogenous arachidonic acid to monocytes exposed to SK 7111 or SB 203347 did not alter A23187-induced PGE2 or LTC4 generation, respectively, indicating that these agents had no effect on downstream arachidonic acid-metabolizing enzymes in this setting. Taken together, these results provide evidence that the 85-kDa PLA2 may play a more significant role in the formation of PG than LT. Further, utilization of SB 203347 provides intriguing data to form the hypothesis that a non-85-kDa PLA2 sn-2 acyl hydrolase, possibly the 14-kDa PLA2, may provide substrate for LT formation.
Highlights
Much work has been directed toward understanding the liberation of arachidonic acid (AA)1 from human monocyte phospholipids (PL) and its subsequent metabolism to a number of cyclooxygenase (COX) and 5-lipoxygenase (5-LO) products (1– 4)
Western Analysis of the Effect of Antisense on Unstimulated Monocytes—Human monocytes (5 ϫ 106/ml) from one donor were treated over 18 h with 3 M 85-kDa PLA2 initiation site-directed antisense, SK7111, or sense oligonucleotide, SK9030 (3 M) and cytosols were evaluated for 85-kDa PLA2 protein
We have provided further support for the co-existence of both the 85-kDa PLA2 and the type II 14-kDa PLA2 in human monocyte as cell-associated enzymes
Summary
Measurement of Stimuli-induced Eicosanoid Release—Monocytes (5 ϫ 106/ml) isolated as described previously (21) were incubated in RPMI 1640 medium (Life Technologies, Inc.) containing the treatment and/or the relevant vehicle after which the stimulus was added. Quantitation of Type II 14-kDa PLA2—To quantitate 14-kDa PLA2, human monocyte 100,000 ϫ g particulates from 4 donors were treated with 0.36 N H2SO4 for 1 h at 4 °C, brought back to pH 7.4 by addition of 2 M Tris buffer This sample was directly assessed for 14-kDa PLA2 mass using an enzyme-linked immunosorbent assay developed for rh type II 14-kDa PLA2 as described previously (21, 48). Human monocytes from 3–5 donors were treated with Lipofectin (5 g/ml) or 3 M SK 7111 for 18 h as described under “Experimental Procedures,” prior to challenge with either vehicle, A23187 (1 M, 7 min, 37 °C) or opsonized zymosan (5 mg/ml, 2 h, 37 °C). LTC4 and PGE2 measured in the cell free supernatants is expressed as ng/ml, mean Ϯ S.D. (n ϭ 3). *, significantly different from control at p Ͻ 0.01 using ANOVA. **, unstimulated control levels for LTC4 ranged from 27 to 145 pg/ml in both systems; unstimulated LT values are rounded to 0.1 for comparison
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