Antigen-initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 ± 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/10⁶ cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [³H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A₂, C and D and not by lipase A₁. The antigen-initiated release of PAF-acether, leukotriene C₄ (LTC₄), and leukotriene B₄ (LTB₄), and the secretion of the granule marker β-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of β-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-0-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min. The capacity of mouse bone marrow-derived mast cells sensitized with IgE and stimulated with specific antigen to form PAF-acether, LTC₄, and LTB₄ is an initial example of a homogeneous cell population responding to a biologically relevant activation/secretion process by the generation and release of two structurally unrelated major classes of lipid mediators orignating from membrane phospholipids.