A Heparin-sensitive Phospholipase A2 and Prostaglandin Endoperoxide Synthase-2 Are Functionally Linked in the Delayed Phase of Prostaglandin D2 Generation in Mouse Bone Marrow-derived Mast Cells

Abstract

BALB/cJ mouse bone marrow-derived mast cells (BMMC) developed with interleukin (IL)-3 can be stimulated by c-kit ligand (KL) in the presence of IL-10 and IL-1beta for sequential immediate and delayed generation of prostaglandin (PG) D2 through utilization of constitutive prostaglandin endoperoxide synthase (PGHS) -1 and induced PGHS-2, respectively (Murakami, M., Matsumoto, R., Austen, K. F., and Arm, J. P. (1994) J. Biol. Chem. 269, 22269-22275). We now report that BALB/cJ BMMC stimulated with KL + IL-10 + IL-1beta also exhibit the biphasic release of [3H]arachidonic acid with an immediate phase over the first 10 min followed by a delayed phase from 2 to 7 h. The delayed phase of arachidonic acid release and of PGD2 generation was inhibited by heparin, which concomitantly released a phospholipase (PL) A2 from the cells into the supernatant. Both dexamethasone and a type II PLA2 inhibitor, 12-epi-scalaradial, suppressed delayed-phase PGD2 generation at concentrations that did not affect immediate eicosanoid generation. Transcripts for type IIA PLA2, as assessed by reverse transcription-polymerase chain reaction, were progressively induced in BALB/cJ BMMC treated for 2 to 7 h with KL + IL-10 + IL-1beta; the induction of these transcripts was down-regulated by 10(-6) M dexamethasone. The expression of steady-state transcripts and protein for cytosolic PLA2 (cPLA2) did not change. PGHS-2-dependent delayed-phase PGD2 generation elicited by IgE-dependent activation of BALB/cJ BMMC primed with KL + IL-10 was also accompanied by the induction of type IIA PLA2 transcripts and was suppressed by heparin, with concomitant release of PLA2 into the supernatant. However, both the direct, cytokine-stimulated and the cytokine-primed, IgE-dependent, delayed-phase PGD2 generation occurred in BMMC from C57BL/6J mice, which have a natural disruption of the type IIA PLA2 gene. Thus, kinetic, pharmacologic, and genetic analyses suggest that an inducible, heparin-sensitive PLA2, rather than cPLA2, provides arachidonic acid to concomitantly induced PGHS-2 for delayed-phase PGD2 biosynthesis in activated BMMC. Furthermore, this heparin-sensitive PLA2 likely represents a novel PLA2 or a new function for a known low molecular weight PLA2.