Participation of Cytosolic Phospholipase A2 in Eicosanoid Generation by Mouse Bone Marrow-Derived Mast Cells

c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.

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Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells

Biochemical Journal ◽

10.1042/bj3520311 ◽

2000 ◽

Vol 352 (2) ◽

pp. 311-317 ◽

Cited By ~ 23

Author(s):

Noriaki NAKATANI ◽

Naonori UOZUMI ◽

Kazuhiko KUME ◽

Makoto MURAKAMI ◽

Ichiro KUDO ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Phospholipase A2 ◽

Lipid Mediators ◽

Cytosolic Phospholipase A2 ◽

Prostaglandin D2 ◽

Wild Type ◽

Cytosolic Phospholipase

Cytosolic phospholipase A2 (cPLA2) plays a critical role in mast-cell-related allergic responses [Uozumi, Kume, Nagase, Nakatani, Ishii, Tashiro, Komagata, Maki, Ikuta, Ouchi et al. (1997) Nature (London) 390, 618–622]. Bone-marrow-derived mast cells from mice lacking cPLA2 (cPLA-/- mice) were used in order to better define the role of cPLA2 in the maturation and degranulation of such cells. Cross-linking of high-affinity receptors for IgE (FcεRI) on cells from cPLA-/-mice led to the release of negligible amounts of arachidonic acid or its metabolites, the cysteinyl leukotrienes and prostaglandin D2, indicating an essential role for cPLA2 in the production of these allergic and pro-inflammatory lipid mediators. In addition, the histamine content of the mast cells and its release from the cells were reduced to 60%. While these results are in agreement with a reduced anaphylactic phenotype of cPLA-/- mice, the ratios of release of histamine and β-hexosaminidase were, paradoxically, significantly higher for cells from cPLA-/- mice than for those from wild-type mice. Consistently, IgE-induced calcium influx in mast cells was greater and more prolonged in cells from cPLA-/- mice than in those from wild-type mice. Thus the loss of cPLA2 not only diminishes the release of lipid mediators, but also alters degranulation. While the overall effect is still a decrease in the release of mast cell mediators, explaining the in vivo findings, the present study proposes a novel link between cPLA2 and the degranulation machinery.

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Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.13.6107 ◽

1995 ◽

Vol 92 (13) ◽

pp. 6107-6111 ◽

Cited By ~ 30

Author(s):

M. Murakami ◽

J. F. Penrose ◽

Y. Urade ◽

K. F. Austen ◽

J. P. Arm

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Phospholipase A2 ◽

Interleukin 4 ◽

Mouse Bone Marrow ◽

Eicosanoid Synthesis ◽

Induced Expression ◽

Kit Ligand ◽

Cytosolic Phospholipase ◽

Prostaglandin Endoperoxide Synthase

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Phosphorylation and activation of Ca2+-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE

Biochemical Journal ◽

10.1042/bj3040923 ◽

1994 ◽

Vol 304 (3) ◽

pp. 923-928 ◽

Cited By ~ 18

Author(s):

S Currie ◽

E F Roberts ◽

S M Spaethe ◽

N W Roehm ◽

R M Kramer

Keyword(s):

Enzyme Activity ◽

Mast Cells ◽

Phospholipase A2 ◽

Electrophoretic Mobility ◽

Tyrosine Kinases ◽

Cytosolic Phospholipase A2 ◽

Cross Linking ◽

High Affinity ◽

Cytosolic Phospholipase ◽

Murine Mast Cell

In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.

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