Genetic evidence for distinct roles of COX-1 and COX-2 in the immediate and delayed phases of prostaglandin synthesis in mast cells.

Activation of mast cells by aggregation of their high-affinity IgE receptors stimulates prostaglandin (PG) D(2) synthesis and secretion. An immediate phase of PGD(2) synthesis, complete within 30 min, is followed by a delayed, second phase of PGD(2) production that reaches a maximum 4 to 8 h after activation. Activation of mast cells from COX-2 (-/-) mice stimulates the release of PGD(2) during the first 30 min, whereas activation of mast cells from COX-1 (-/-) mice does not generate any PGD(2) in the first 2 h. On the other hand, COX-2 (-/-) cells do not participate in delayed phase of PGD(2) synthesis, while COX-1 (-/-) cells secrete low levels of PGD(2) between 2 and 4 h after activation. These data demonstrate that (i) the first phase of PG synthesis is COX-1 dependent and (ii) the second, delayed phase of PG synthesis is dependent on activation-induced synthesis and activity of COX-2.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

Regulation of prostaglandin endoperoxide synthase-2 and IL-6 expression in mouse bone marrow-derived mast cells by exogenous but not endogenous prostanoids.

Mouse bone marrow-derived mast cells (BMMC), stimulated with stem cell factor, IL-1beta, and IL-10, secrete IL-6 and demonstrate a delayed phase of PGD(2) generation that is dependent upon the induced expression of PG endoperoxide synthase (PGHS)-2. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate PGHS-2 induction and IL-6 secretion in mouse BMMC. Exogenous PGE(2), which acts through G protein-coupled receptors, and 15-deoxy-Delta(12,14)-PGJ(2), which is a ligand for peroxisome proliferator-activated receptor (PPAR)gamma, elicited a 2- to 3-fold amplification of PGHS-2 induction, delayed-phase PGD(2) generation, and IL-6 secretion in response to stem cell factor, IL-1beta, and IL-10. The effect of PGE(2) was reproduced by the E prostanoid (EP)1 receptor agonist 17-trinor-PGE(2), and the EP1/EP3 agonist, sulprostone, but not the EP2 receptor agonist, butaprost. Although BMMC express PPARgamma, the effects of 15-deoxy-Delta(12,14)-PGJ(2) were not reproduced by the PPARgamma agonists, troglitazone and ciglitazone. PGHS-2 induction, but not IL-6 secretion, was impaired in cPLA(2)-deficient BMMC. However, there was no impairment of PGHS-2 induction in BMMC deficient in hematopoietic PGD synthase or PGHS-1 in the presence or absence of the PGHS-2 inhibitor, NS-398. Thus, although exogenous prostanoids may contribute to amplification of the inflammatory response by augmenting PGD(2) generation and IL-6 secretion from mast cells, endogenous prostanoids do not play a role.     

Regulation of type V phospholipase A2 expression and function by proinflammatory stimuli.

Types IIA and V secretory phospholipase A2 (sPLA2) are structurally related to each other and their genes are tightly linked to the same chromosome locus. An emerging body of evidence suggests that sPLA2-IIA plays an augmentative role in long-term prostaglandin (PG) generation in cells activated by proinflammatory stimuli; however, the mechanism underlying the functional regulation of sPLA2-V remains largely unknown. Here we show that sPLA2-V is more widely expressed than sPLA2-IIA in the mouse, in which its expression is elevated by proinflammatory stimuli such as lipopolysaccharide. In contrast, proinflammatory stimuli induced sPLA2-IIA in marked preference to sPLA2-V in the rat. Cotransfection of sPLA2-V with cyclooxygenase (COX)-2, but not with COX-1, into human embryonic kidney 293 cells dramatically increased the interleukin-1-dependent PGE2 generation occurring over a 24 h of culture period. Rat mastocytoma RBL-2H3 cells overexpressing sPLA2-V exhibited increased IgE-dependent PGD2 generation and accelerated beta-hexosaminidase exocytosis. These results suggest that sPLA2-V acts as a regulator of inflammation-associated cellular responses. This possible compensation of sPLA2-V for sPLA2-IIA in many, if not all, tissues may also explain why some mouse strains with natural disruption of the sPLA2-IIA gene exhibit few abnormalities during their life-spans.     

A negative regulator of delayed prostaglandin D2 production in mouse mast cells

We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.     

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.     

Internalization and degradation of type IIA phospholipase A(2) in mast cells

Whereas exogenous types IB and X secretory phospholipase A(2) (sPLA(2)) elicited prostaglandin D(2) (PGD(2)) production in mouse bone marrow-derived mast cells (BMMC), sPLA(2)-IIA was unable to do so. In search of a mechanism underlying this cellular refractoriness to exogenous sPLA(2)-IIA, we now report that this isozyme is promptly associated with cell surfaces, internalized, and then degraded in BMMC. Adsorption of sPLA(2)-IIA to BMMC was prevented by addition of heparin to the medium. Moreover, a heparin-nonbinding sPLA(2)-IIA mutant did not bind to BMMC. These results indicate that this sPLA(2)-IIA inactivation process depends on its rapid binding to heparan sulfate proteoglycan (HSPG) on BMMC surfaces. Thus, the present observations represent a particular situation in which cell surface HSPG exhibit a negative regulatory effect on cellular function of sPLA(2)-IIA, and argue that HSPG does not always act as a functional adapter for heparin-binding sPLA(2)s in mammalian cells as has been demonstrated before.     

Regulation of delayed prostaglandin production in activated P388D1 macrophages by group IV cytosolic and group V secretory phospholipase A2s

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.     

Group V secretory phospholipase A2 translocates to the phagosome after zymosan stimulation of mouse peritoneal macrophages and regulates phagocytosis

We have previously reported that group V secretory phospholipase A2 (sPLA2) amplifies the action of cytosolic phospholipase A2(cPLA2) alpha in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA2, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA2 on phagocytosis of zymosan. We report that group V sPLA2 is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA2 is recruited to the phagosome. There it co-localizes with cPLA2alpha, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA2-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA2 but IIA not group sPLA2. These data demonstrate that group V sPLA2 contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.     

Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

Profiling of eicosanoid production in the rat hippocampus during kainic acid-induced seizure: dual phase regulation and differential involvement of COX-1 and COX-2

Kainic acid (KA)-induced seizure in rat involves eicosanoid production in the brain, but their production mechanism and biological functions are poorly understood. We profiled the eicosanoid production during KA-induced seizure by a comprehensive lipidomics analysis using liquid chromatography-tandem mass spectrometry. Systemic KA administration caused production of large amounts of prostaglandin (PG) F(2alpha) and PGD(2) in the hippocampus, with smaller amounts of other PGs and hydroxyeicosatetraenoic acids. The production was biphasic, which consisted of an initial burst in the first 30 min and a sustained late phase production. The initial phase was specific to the hippocampus and was blocked by intracerebroventricular administration of KA receptor antagonists. A selective cyclooxygenase (COX)-2 inhibitor, NS398, completely inhibited the initial phase productions, except for PGD(2) and thromboxane B(2), whose productions were also dependent on COX-1. These results suggest that KA signals directly stimulate the arachidonic acid cascade in the initial phase and that COX-1 and COX-2, both constitutively expressed at low levels, differentially contribute to PG productions. In the late phase, a sustained PG production in hippocampus appears due to the increased COX-2 levels even with a limited arachidonic acid supply. The present study demonstrates a dual phase regulatory mechanism of eicosanoid production during KA-induced seizure, providing a biochemical basis for understanding the biosynthesis and roles of eicosanoids in the brain.     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.     

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.     

Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption

Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated approximately 50% in peritoneal macrophages from group V sPLA2-null mice compared with macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.     

Ceramide-induced enhancement of secretory phospholipase A2 expression via generation of reactive oxygen species in tumor necrosis factor-alpha-stimulated mesangial cells

Since prostanoids such as prostaglandin E2 play a pivotal role in modulating renal function, we investigated the involvement of ceramide in expression of secretory phospholipase A2 (sPLA2) and cyclooxygenase-2 (COX-2) in tumor necrosis factor-alpha (TNF-alpha)-stimulated mesangial cells. TNF-alpha stimulation increased ceramide generation in parallel with a decrease in sphingomyelin. Pretreatment with exogenous sphingomyelinase (SMase) dose-dependently enhanced TNF-alpha-stimulated increases in COX-2 protein and sPLA) activity. SMase also augmented TNF-alpha-mediated nuclear factor kappaB (NF-kappaB) activation. N-acetylcysteine (NAC), an antioxidant, completely inhibited the SMase-induced increase in sPLA2 activity, whereas NAC inhibited partially the activity stimulated with TNF-alpha alone. Under the conditions, NAC completely inhibited reactive oxygen species (ROS) production induced by SMase followed by TNF-alpha. These results suggest that ceramide elicits up-regulation of NF-kappaB through ROS production, which, in turn, leads to stimulation of COX-2 and sPLA2 expression. Therefore, ceramide may be implicated in the pathogenesis of renal abnormalities.