Differential Mobilization of the Phospholipid and Triacylglycerol Pools of Arachidonic Acid in Murine Macrophages

Abstract

Innate immune cells such as monocytes and macrophages contain high levels of arachidonic acid (AA), part of which can be mobilized during cellular activation for the formation of a vast array of bioactive oxygenated metabolites. Monocytes and macrophages present in inflammatory foci typically incorporate large amounts of AA, not only in membrane phospholipids, but also in neutral lipids such as triacylglycerol. Thus, it was of interest to investigate the metabolic fate of these two AA pools in macrophages. Utilizing a variety of radiolabeling techniques to distinguish the phospholipid and triacylglycerol pools, we show in this paper that during an acute stimulation of the macrophages with yeast-derived zymosan, the membrane phospholipid AA pool acts as the major, if not the only, source of releasable AA. On the contrary, the AA pool in triacylglycerol appears to be used at a later stage, when the zymosan-stimulated response has declined, as a source to replenish the phospholipid pools that were consumed during the activation process. Thus, phospholipids and triacylglycerol play different in roles AA metabolism and dynamics during macrophage activation.

Keywords: arachidonic acid; inflammation; membrane phospholipid; monocytes/macrophages; phospholipase A2; triacylglycerol.

Figure 1

AA distribution in murine macrophages. (A) AA incorporation into the lipids of mouse peritoneal macrophages. The cells were either untreated (open bars) or treated with 20 µM AA (orange bars). Afterward, the various lipid classes were isolated and their AA content was measured by GC-MS. (B) The cells, prelabeled with [³H]AA, were either untreated (open symbols) or treated with 0.5 mg/mL zymosan (black symbols) for the times indicated. Afterward, the extracellular media were removed and analyzed for ³H-radioactivity content. The data are expressed as mean values ± S.E.M. (n = 6).

Figure 2

AA mobilization by zymosan-stimulated peritoneal macrophages. The [³H]AA-labeled cells were either untreated (Ctrl) or stimulated with 0.5 mg/mL zymosan for 2 h in the absence (Zym) or presence of 1 µM pyrrophenone (Zym + Pyrr), or 20 µM atglistatin (Zym + AGS), or 10 µM bromoenol lactone (Zym + BEL). Afterward, the [³H]AA released to the supernatants (A) or remaining in phospholipids (B) or TAG (C) was quantified as described in Materials and Methods. The data are expressed as mean values ± S.E.M. (n = 6). * p < 0.05, significantly different from untreated cells.

Figure 3

Source of AA release in zymosan-stimulated macrophages. The cells, labeled with [³H]AA and [¹⁴C]AA were treated with 0.5 mg/mL zymosan for 1 h. Afterward, the ³H/¹⁴C ratios of extracellularly liberated free AA, PL and TAG were calculated. The data are expressed as mean values ± S.E.M. (n = 6).

Figure 4

Transfer of [³H]AA from TAG to phospholipids. Cells prelabeled with [³H]AA were either untreated (A) or stimulated with 0.5 mg/mL zymosan for 2 h (B). Afterward, they were washed and transferred to fresh media, and the distribution of [³H]AA content in phospholipids (maroon symbols) and TAG (open symbols) was determined at the times indicated. The data are expressed as mean values ± S.E.M. (n = 6).