Abstract
The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.
Highlights
Mast cells play a vital role in acute and chronic allergic inflammation by virtue of their ability to release inflammatory mediators and cytokines upon antigen activation [1,2,3,4,5]
Apoptosis of bone marrow-derived mast cells (BMMC) in Cytokine-depleted Media—Our previous studies showed that BMMC cultured in IL-3/stem cell factor (SCF) maintained low intracellular levels of arachidonic acid (AA) [36]
This protease activity was reduced to control levels (4.4 Ϯ 0.1 CPP32 units) by a caspase inhibitor. These initial data suggested that a marker of apoptosis was increased when BMMC were placed in cytokine-depleted media for 24 h
Summary
Mast cells play a vital role in acute and chronic allergic inflammation by virtue of their ability to release inflammatory mediators and cytokines upon antigen activation [1,2,3,4,5]. AA is rapidly released and utilized for the formation of eicosanoids [7]. This can be accomplished by one or more of several PLA2 enzymes with different molecular characteristics that have been described in mast cells (8 –10). The mole quantities of AA, but not other unsaturated fatty acids, correlated with the number of apoptotic cells and were inversely proportional to the number of live cells This accumulation of AA can be explained by a decrease in CoA-IT activity, which results in a diminished capacity of mast cells to remodel AA from phosphatidylcholine (PC) to phosphatidylethanolamine (PE). BMMC, murine bone marrow-derived mast cell(s); PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; HPLC, high pressure liquid chromatography; SCF, stem cell factor; IL, interleukin; FITC, fluorescein isothiocyanate; MDA, malondialdehyde; NL, neutral lipids; CoA-IT, CoA-independent transacylase; TXB2, thromboxane B2
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