Pathway-oriented profiling of lipid mediators in macrophages

Abstract

Macrophages produce various kinds of lipid mediators including eicosanoids and platelet-activating factor. Since they are produced from common precursors, arachidonic acid-containing phospholipids, regulations of metabolic pathways underlie the patterning of lipid mediator production. Here, we report a pathway-oriented profiling strategy of lipid mediators by a newly developed multiplex quantification system. We profiled mouse peritoneal macrophages in different activation states. The analysis of kinetics revealed the differences in the production time course of various lipid mediators, which also differed by the macrophage types. Scatterplot matrix analysis of the inhibitor study revealed correlations of lipid mediator species. The changes of these correlations provided estimates on the effects of lipopolysaccharide priming. We also found a highly linked production of 11-hydroxyeicosatetraenoic acid and prostaglandin E2, implying the in vivo property of cyclooxygenase-mediated 11-hydroxyeicosatetraenoic acid production. The present approach will serve as a strategy for understanding the regulatory mechanism of lipid mediator production.

Fig. 1

Metabolic pathway of lipid mediator production. All the lipid mediators quantified in the present study are the metabolites of arachidonic acid-containing phospholipids. Arachidonic acid produced by PLA₂ enzymes is further metabolized to PGs, LTs, and HETEs by the downstream enzymes such as COX, LO, and various terminal synthases. PLA₂ also produces lyso-PAF, which can be metabolized into PAF. 5-HpETE, 5-hydroperoxyeicosatetraenoic acid.

Fig. 2

Lipid mediator production profiles of mouse peritoneal macrophages. Resident (A) and TG-induced (B) macrophages with or without 12-h LPS priming were stimulated with vehicle (0.1% DMSO) or 2 μM A23187, and lipid mediator production within 30 min was quantified. Values are expressed as means ± SD.

Fig. 3

Kinetics of lipid mediator production. Non-primed resident- and TG-induced macrophages were stimulated with 2 μM A23187 for indicated periods, and lipid mediator production was quantified. Results for PGs (A), LTs (B), HETEs (C), and PAF (D) are shown.

Fig. 4

Effects of various inhibitors on lipid mediator production. Resident- (A) and TG-induced (B) macrophages with or without 12-h LPS priming were pretreated with indicated concentrations of pyrrolidine-1 (Pyr-1), SC560, NS398 or AA861 for 30 min. Lipid mediator production after 30 min stimulation with 2 μM A23187 in the presence of inhibitors was measured. Results for PGE₂ (left panels) and LTC₄ (right panels) are shown. The data are normalized with A23187-stimulated controls. Minus (−), non-stimulated control.

Fig. 5

Correlation analysis of lipid mediators. (A) Pairwise correlations of lipid mediators are visualized in a scatterplot matrix. Each panel in the matrix contains a scatterplot for a pair of lipid mediators, and each dot in the scatterplot corresponds to a single measurement. Results for a single set of inhibitor experiment in the resident macrophages are shown. Blue and red dots indicate results from non-primed and LPS-primed cells, respectively. Density ellipses, α = 0.95. (B,C) A close-up view of scatterplot matrix for 6-keto-PGF_1α, TxB₂, and PGE₂ for resident- (B) and TG-induced (C) macrophages. (D,E) Correlation of PGE₂ and 11-HETE in the resident- (D) and TG-induced (E) macrophages. Lines are linear fittings for non-primed (blue) and LPS-primed (red) cells.