Abstract
Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.
Highlights
Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content
We demonstrate that adipose TG lipase (ATGL) is expressed in human MCs and that siRNA-mediated silencing of ATGL greatly reduces the amounts of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) released upon activation of MCs
The current investigation into AA metabolism in MCs is based on our key observation that silencing of ATGL expression leads to a marked accumulation of neutral lipids in cytoplasmic LDs and, concomitantly, to a reduced production of eicosanoids by these cells
Summary
Mature human MCs were generated exactly as described previously [9]. CD34+ progenitor cells were isolated from fresh buffy coats prepared from the peripheral blood of healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). The isolated CD34+ cells were cultured for at least 6 weeks at 37°C under serum-free conditions in Iscove’s modified Dulbecco’s medium supplemented with BIT 9500 serum substitute (StemCell Technologies), L-glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and recombinant human stem cell factor ( termed KITL, KIT ligand) (100 ng/ml; PeproTech, Rocky Hill, NJ). All experiments were carried out with mature MCs (i.e., with cells that had been allowed to differentiate for 6 to 8 weeks) [17]
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