c-Kit Ligand Mediates Increased Expression of Cytosolic Phospholipase A2, Prostaglandin Endoperoxide Synthase-1, and Hematopoietic Prostaglandin D2 Synthase and Increased IgE-dependent Prostaglandin D2 Generation in Immature Mouse Mast Cells

Abstract

We have examined the cytokine regulation of IgE-dependent prostaglandin (PG) D2 generation in mouse mast cells by assessing the changes in the levels of the transcript, translated protein, and activity of the enzymes involved in the synthesis of PGD2 from endogenous arachidonic acid. When mouse mast cells, derived by culture of bone marrow cells with WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 (BMMC), were cultured in recombinant ckit ligand (KL), sensitized with IgE, and stimulated with antigen, PGD2 generation increased 3-fold; when KL was combined with IL-3, IL-9, or IL-10, PGD2 generation increased 6-8-fold above that produced by the cells cultured in IL-3 alone. The increased IgE-dependent PGD2 generation by BMMC was apparent after 1 day of culture, reached a maximum after 2-4 days of culture, and was dose-dependent for KL and for each of the accessory cytokines. IgE-dependent generation of leukotriene C4 increased 2-fold after the cells were cultured with KL and was not increased by the addition of IL-3, IL-9, or IL-10. Assays for steady-state transcripts by RNA blotting, for protein by SDS-PAGE/immunoblotting, and for function by enzymatic activities revealed that KL alone stimulated the increased expression of cytosolic phospholipase A2 (cPLA2), prostaglandin endoperoxide synthase (PGHS)-1, and the terminal enzyme, hematopoietic PGD2 synthase, without a change in expression of 5-lipoxygenase. IL-3, IL-9, and IL-10 each enhanced the KL-induced expression of PGHS-1. In contrast, transcripts for PGHS-2, which were detected transiently after the cells had been cultured for 5 h in KL+IL-3, were not expressed during the period of subsequent increase in IgE-dependent PGD2 generation. These findings demonstrate that KL up-regulates expression of cPLA2, PGHS-1, and hematopoietic PGD2 synthase, leading to a relatively selective increase in IgE-dependent production of PGD2 from endogenously released arachidonic acid in BMMC, and they provide the first example of cytokine regulation of hematopoietic PGD2 synthase.

Activation of Cytosolic Phospholipase A2 by Platelet-derived Growth Factor Is Essential for Cyclooxygenase-2-dependent Prostaglandin E2 Synthesis in Mouse Osteoblasts Cultured with Interleukin-1

“…These results suggest that arachidonic acid is produced and metabolized at the NE and ER. Murakami et al (41) reported that there is a linkage between cPLA 2 and cox-1 in c-kit ligand-primed, IgE-dependent PGD 2 synthesis in mouse mast cells. A linkage between cPLA 2 and cox-2 has also been suggested in monocytes and fibroblasts (42,43).…”

Cytosolic phospholipase A2is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells

“…Mouse recombinant IL-3, IL-9, and IL-10 were produced by expression in insect cells, and their concentrations were determined as described (10,20). William Smith (Michigan State University, East Lansing, MI) supplied rabbit polyclonal antiserum to PGHS-2, and Jim Clark (Genetics Institute, Boston, MA) supplied rabbit polyclonal antiserum to cPLA 2 .…”

Impaired Kit- but Not FcεRI-initiated Mast Cell Activation in the Absence of Phosphoinositide 3-Kinase p85α Gene Products

“…Reagents-Mouse recombinant KL and IL-3 were expressed by the infection of Sf9 insect cells with recombinant baculovirus (58). Rat mAb 2.4G2 anti-mouse Fc␥RIIB/III, mouse IgE mAb IgE-3 anti-trinitrophenyl (TNP), FITC-rat mAb R35-72 anti-mouse IgE, FITC-rat mAb 2B8 anti-mouse Kit, and FITC-rat IgG 2b were purchased from Pharmingen.…”