Urinary leukotriene E4 (LTE4) concentrations have been measured in six asthmatic patients with aspirin sensitivity and in five asthmatic subjects tolerant of aspirin, before and after provocation with aspirin or placebo. Aspirin-sensitive subjects showed an average 21% fall in FEV1 after aspirin challenge whereas control individuals had a 2% fall in FEV1 after ingestion of 100 mg aspirin. The resting urinary LTE4 concentrations in asthmatic subjects sensitive to aspirin were 243 pg/mg creatinine (range 50 to 1,041), and these were on average sixfold greater than those in control asthmatic subjects. Further, there was a mean fourfold increase in urinary LTE4 levels at 3 to 6 h after aspirin, but not placebo, challenge in aspirin-sensitive asthmatic subjects that was not seen in the control asthmatic individuals. Leukotriene release may play a central role in the mechanisms of asthmatic attacks produced by aspirin ingestion.

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Conclusions regarding the contribution of low molecular weight secretory phospholipase A 2 (sPLA 2 ) 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 sPLA 2 enzymes. To elucidate the role of group V sPLA 2 , we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C 4 and prostaglandin E 2 was attenuated ϳ50% in peritoneal macrophages from group V sPLA 2 -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 sPLA 2 -null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA 2 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.The first step in the biosynthesis of eicosanoids is the release of arachidonic acid from cell membrane phospholipids by phospholipase A 2 . Several classes of phospholipase A 2 have been described in mammals (1, 2). Cytosolic phospholipase A 2 (cPLA 2 ) 1 ␣ is an 85-kDa cytosolic enzyme that uses a catalytic serine residue and preferentially cleaves arachidonic acid from cell membrane phospholipids (3). The Ca 2ϩ -dependent translocation of cPLA 2 -␣ from the cytosol to the nuclear envelope (4), a prominent site of eicosanoid biosynthesis, is dependent on a Ca 2ϩ -dependent lipid binding (C-2) domain. Paralogues of cPLA 2 -␣ (cPLA 2 -␤ and cPLA 2 -␥) have been described previously (5, 6). cPLA 2 -␤ has a M r of 110,000 and shares 30% identity with cPLA 2 -␣, including a functional C-2 domain. cPLA 2 -␥ has a M r of 61,000, shares 29% sequence identity with cPLA 2 -␣, lacks a C-2 domain, and is Ca 2ϩ -independent. Mammalian low molecular weight secretory phospholipase A 2 (sPLA 2 ) enzymes, which are now 10 in number, are characterized by a conserved motif containing a catalytic histidine residue, by their relatively small size of ϳ14 kDa, and by their highly disulfide-linked tertiary structures (7-13). They are distinguished from one another by their structures, their biochemical properties, and their tissue distribution. Calcium-independent phospholipase A 2 enzymes have been described in myocardium and in leukocytes (14, 15). They have been implicated in membrane remodeling, regulation of store operated calcium channels, apoptosis, and release of arachidonic acid. The fourth group of phospholipase A 2 enzymes comprises the acetyl hydrolases of platelet activating factor (16).Given the complexity and size of the phospholipase A 2 family, targeted gene disruption is a suitable approach to elucidating the role(s) of individual enzymes and proved fruitful in determining the role of cPLA 2 -␣ in regulating eicosan...

We have used mice in which the gene for cytosolic phospholipase A 2 (cPLA 2 ) has been disrupted to demonstrate the absolute requirement for cPLA 2 in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA 2 prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D 2 generation. These defects occurred with two distinct stimuli, stem cell factor and IgE͞antigen, which were, however, sufficient for signal transduction defined by exocytosis of ␤-hexosaminidase. Whereas cPLA 2 is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD 2 generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD 2 .

Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.

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We have previously reported that group V secretory phospholipase A 2 (sPLA 2 ) amplifies the action of cytosolic phospholipase A 2 (cPLA 2 ) ␣ 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 sPLA 2 , 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 sPLA 2 on phagocytosis of zymosan. We report that group V sPLA 2 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 sPLA 2 is recruited to the phagosome. There it co-localizes with cPLA 2 ␣, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C 4 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 sPLA 2 -null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA 2 but not group IIA sPLA 2 . These data demonstrate that group V sPLA 2 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.Phospholipase A 2 (PLA 2 ) 2 hydrolyzes the sn-2 position of cell membrane phospholipids to release free fatty acids and lysophospholipids. Where the fatty acid is arachidonic acid, it serves as the precursor for biosynthesis of leukotrienes and prostaglandins (1). Free fatty acids also may signal through peroxisome proliferator-activated receptors (2), and lysophospholipids activate cell surface G protein-coupled receptors (3). The family of PLA 2 enzymes includes the cytosolic PLA 2 (cPLA 2 ) isoforms that utilize a catalytic serine (4, 5). cPLA 2 ␣ is critical for the release of arachidonic acid for leukotriene and prostaglandin generation (6, 7). The calcium-independent isoforms of PLA 2 , which also use a catalytic serine, have been implicated in cell membrane remodeling (8), spermatogenesis (9), and regulation of store-operated calcium channels (10). There are 10 mammalian secretory PLA 2 enzymes (11-14) that use a catalytic histidine and that have been implicated in amplifying cPLA 2 ␣-dependent eicosanoid generation (15-17) and in regulating secretory granule exocytosis (18). In certain circumstances, sPLA 2 enzymes may regulate eicosanoid generation independently from cPLA 2 ␣ (19). Certain sPLA 2 isoforms may act as ligands for cell surface receptors (20). However, the roles of the sPLA 2 enzymes have largely been inferred from their biochemical properties, the effects of their overexpression in transfected c...

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.