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.     

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.     

Group V secretory phospholipase A2 amplifies the induction of cyclooxygenase 2 and delayed prostaglandin D2 generation in mouse bone marrow culture-derived mast cells in a strain-dependent manner

Activation of mouse bone marrow-derived mast cells (BMMC) with stem cell factor (SCF) or IgE and antigen elicits exocytosis and an immediate phase of prostaglandin (PG) D(2) and leukotriene (LT) C(4) generation. Activation of BMMC by SCF, IL-1beta and IL-10 elicits a delayed phase of PGD(2) generation dependent on cyclooxygenase (COX) 2 induction. Cytosolic phospholipase A(2) alpha provides arachidonic acid in both phases and amplifies COX-2 induction. Pharmacological experiments implicate an amplifying role for secretory (s) PLA(2). We used mice lacking the gene encoding group V sPLA(2) (Pla2g5-/-) to definitively test its role in eicosanoid generation by BMMC. Pla2g5-/- BMMC on a C57BL/6 genetic background showed a modest reduction in exocytosis and immediate PGD(2) generation after activation with SCF or with IgE and antigen, while LTC(4) generation was not modified. Delayed-phase PGD(2) generation and COX-2 induction were reduced approximately 35% in C57BL/6 Pla2g5-/- BMMC and were restored by exogenous PGE(2). There was no deficit in either phase of eicosanoid generation by Pla2g5-/- BMMC on a BALB/c background. Thus, group V sPLA(2) amplifies COX-2 expression and delayed phase PGD(2) generation in a strain-dependent manner; it has at best a limited role in immediate eicosanoid generation by BMMC.     

Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.     

Role of group IIa and group V secretory phospholipases A(2) in the metabolism of lipoproteins. Substrate specificities of the enzymes and the regulation of their activities by sphingomyelin

Although many isoforms of secretory phospholipases A(2) (sPLA(2)) are known to be secreted by various inflammatory cells, and are present in plasma, their role in lipoprotein metabolism is unknown. We studied the in vitro hydrolysis of lipoprotein phospholipids by group IIa and group V sPLA(2), two structurally related enzymes with differing phospholipid specificities. The group V sPLA(2) was about 30 times more efficient than the group IIa enzyme in the hydrolysis of lipoprotein phosphatidylcholine (PC), and both enzymes were more active on high density liporotein (HDL) than on low density lipoprotein (LDL). The lower activity on LDL appears to be due to the higher sphingomyelin (SPH) concentration in this lipoprotein. PC hydrolysis in lipoproteins was stimulated significantly by enzymatic depletion of their SPH. The hydrolysis of PC in liposomes was inhibited by the incorporation of SPH, and this inhibition was reversed by treatment with sphingomyelinase. The incorporation of ceramide, on the other hand, stimulated the sPLA(2) activity significantly. Unlike most sPLA(2), which show no fatty acid preference, group V sPLA(2) released disproportionately more linoleate, and less arachidonate from lipoproteins. These studies show that group V sPLA(2) is physiologically more important than group IIa enzyme in lipoprotein metabolism, that the sPLA(2) activities are regulated by sphingomyelin and ceramide, and that the pathological effects of sPLA(2) may not be mediated through stimulation of eicosanoid synthesis.     

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.     

Groups IV, V, and X phospholipases A2s in human neutrophils: role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis.

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.     

Cellular arachidonate-releasing function and inflammation-associated expression of group IIF secretory phospholipase A2

Here we report the cellular arachidonate (AA)-releasing function of group IIF secretory phospholipase A(2) (sPLA(2)-IIF), a sPLA(2) enzyme uniquely containing a longer C-terminal extension. sPLA(2)-IIF increased spontaneous and stimulus-dependent release of AA, which was supplied to downstream cyclooxygenases and 5-lipoxygenase for eicosanoid production. sPLA(2)-IIF also enhanced interleukin 1-stimulated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. AA release by sPLA(2)-IIF was facilitated by oxidative modification of cellular membranes. Cellular actions of sPLA(2)-IIF occurred independently of the heparan sulfate proteoglycan glypican, which acts as a functional adaptor for other group II subfamily sPLA(2)s. Confocal microscopy revealed the location of sPLA(2)-IIF on the plasma membrane. The unique C-terminal extension was crucial for its plasma membrane localization and optimal cellular functions. sPLA(2)-IIF expression was increased in various tissues from lipopolysaccharide-treated mice and in ears of mice with experimental atopic dermatitis. In human rheumatoid arthritic joints, sPLA(2)-IIF was detected in synovial lining cells, capillary endothelial cells, and plasma cells. These results suggest that sPLA(2)-IIF is a potent regulator of AA metabolism and participates in the inflammatory process under certain conditions.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Secreted phospholipase A2 involvement in neurodegeneration: differential testing of prosurvival and anti-inflammatory effects of enzyme inhibition

There is increased interest in the contribution of secreted phospholipase A2 (sPLA2) enzymes to neurodegenerative diseases. Systemic treatment with the nonapeptide CHEC-9, a broad spectrum uncompetitive inhibitor of sPLA2, has been shown previously to inhibit neuron death and aspects of the inflammatory response in several models of neurodegeneration. A persistent question in studies of sPLA2 inhibitors, as for several other anti-inflammatory and neuroprotective compounds, is whether the cell protection is direct or due to slowing of the toxic aspects of the inflammatory response. To further explore this issue, we developed assays using SY5Y (neuronal cells) and HL-60 (monocytes) cell lines and examined the effects of sPLA2 inhibition on these homogeneous cell types in vitro. We found that the peptide inhibited sPLA2 enzyme activity in both SY5Y and HL-60 cultures. This inhibition provided direct protection to SY5Y neuronal cells and their processes in response to several forms of stress including exposure to conditioned medium from HL-60 cells. In cultures of HL-60 cells, sPLA2 inhibition had no effect on survival of the cells but attenuated their differentiation into macrophages, with regard to process development, phagocytic ability, and the expression of differentiation marker CD36, as well as the secretion of proinflammatory cytokines TNF-α and IL-6. These results suggest that sPLA2 enzyme activity organizes a cascade of changes comprising both cell degeneration and inflammation, processes that could theoretically operate independently during neurodegenerative conditions. The effectiveness of sPLA2 inhibitor CHEC-9 may be due to its ability to affect both processes in isolation. Testing potential anti-inflammatory/neuroprotective compounds with these human cell lines and their conditioned media may provide a useful screening tool prior to in vivo therapeutic applications.     

Bacterial cell membrane hydrolysis by secreted phospholipases A(2): a major physiological role of human group IIa sPLA(2) involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A(2) (human sPLA(2)) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA(2)s. This specificity is due to selective penetration of the highly cationic human sPLA(2)50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA(2) activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA(2) as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA(2)s. Overall, the results highlight the potential of the human sPLA(2) as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.     

Group IIA and group V secretory phospholipase A(2): quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A(2) (sPLA(2)) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75-80% of the total amount of synthesized group IIA sPLA(2) was secreted. The synthesized group V sPLA(2), however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA(2). We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA(2) with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA(2) expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA(2) show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA(2) was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA(2) was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.     

Mechanisms by which elevated intracellular calcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2

Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A2 (sPLA2) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A2 (cPLA2). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA2. Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA2. Together these data suggest that lysolecithin induces susceptibility through both cPLA2-dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA2-independent pathway. This cPLA2-independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.     

Distinctiveness of secretory phospholipase A2 group IIA and V suggesting unique roles in atherosclerosis

Clinical observations strongly support an association of circulating levels of secretory phospholipases A(2) (sPLA(2)) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA(2)-IIA and sPLA(2)-V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.     

Transgenic expression of group V, but not group X, secreted phospholipase A2 in mice leads to neonatal lethality because of lung dysfunction

In an effort to elucidate the functions of secreted phospholipase A2 (sPLA2) enzymes in vivo, we generated transgenic (Tg) mice for group V sPLA2 (sPLA2-V) and group X sPLA2 (sPLA2-X), which act potently on phosphatidylcholine in vitro.We found that sPLA2-V Tg mice died in the neonatal period because of respiratory failure. The lungs of sPLA2-V Tg mice exhibited atelectasis with thickened alveolar walls and narrow air spaces, accompanied by infiltration of macrophages and only modest changes in eicosanoid levels. This severe pulmonary defect in sPLA2-V Tg mice was attributable to marked reduction of the lung surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol. Given that the expression of sPLA2-V is greatly elevated in human lungs with severe inflammation, our present results raise the intriguing possibility that this isozyme may contribute to ongoing surfactant hydrolysis often observed in the lungs of patients with respiratory distress syndrome. In contrast, sPLA2-X Tg neonates displayed minimal abnormality of the respiratory tract with normal alveolar architecture and surfactant composition. This unexpected result was likely because sPLA2-X protein existed as an inactive zymogen in most tissues. The active form of sPLA2-X was detected in tissues with inflammatory granulation in sPLA2-X Tg mice. These results suggest that sPLA2-X mostly remains inactive under physiological conditions and that its proteolytic activation occurs during inflammation or other as yet unidentified circumstances in vivo.     

Group IID heparin-binding secretory phospholipase A(2) is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues.

Group IID secretory phospholipase A(2) (sPLA(2)-IID), a heparin-binding sPLA(2) that is closely related to sPLA(2)-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA(2)-IIA. Here we identified the residues of sPLA(2)-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA(2)-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA(2)s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA(2)-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA(2)-IID and sPLA(2)-X constitutively, the former of which was negatively regulated by IL-1. sPLA(2)-IID, but not other sPLA(2) isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA(2)-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA(2)-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.     

Exogenously added human group X secreted phospholipase A(2) but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells

Mammalian secreted phospholipases A(2) (sPLA2s) comprise a group of at least eight enzymes, including the recently identified group X sPLA2. A bacterial expression system was developed to produce human group X sPLA2 (hGX). Inhibition studies show that the sPLA2 inhibitor LY311727 binds modestly more tightly to human group IIA sPLA2 than to hGX and that a pyrazole-based inhibitor of group IIA sPLA2 is much less active against hGX. The phospholipid head group preference of vesicle-bound hGX was determined. hGX binds tightly to phosphatidylcholine vesicles, which is thought to be required to act efficiently on cells. Tryptophan 67 hGX makes a significant contribution to interfacial binding to zwitterionic vesicles. As little as 10 ng/ml hGX releases arachidonic acid for cyclooxygenase-2- dependent prostaglandin E(2) generation when added exogenously to adherent mammalian cells. In contrast, human group IIA, rat group V, and mouse group IB sPLA2s are virtually inactive at releasing arachidonate when added exogenously to adherent cells. Dislodging cells from the growth surface enhances the ability of all the sPLA2s to release fatty acids. Studies with CHO-K1 cell mutants show that binding of sPLA2s to glycosaminoglycans is not the basis for poor plasma membrane hydrolysis by group IB, IIA, and V sPLA2s.     

A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions

We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naive or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50 to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.