Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells

In murine P388D1 macrophages, the generation of prostaglandin E2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A2 (PLA2), Group IV cytosolic PLA2 (cPLA2), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA2 that induces expression of Group V PLA2, which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E2 generation. Because Group V PLA2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA2, blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA2 previously associated with the outer cellular surface. Localization of Group V PLA2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA2. Collectively, these results suggest that encapsulation of Group V PLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA2 and COX-2 for efficient prostaglandin synthesis.     

Phosphorylation of cytosolic group IV phospholipase A(2) is necessary but not sufficient for Arachidonic acid release in P388D(1) macrophages

Activation of the cytosolic Group IV phospholipase A(2) (cPLA(2)) by agonists has been correlated with the direct phosphorylation of the enzyme by members of the mitogen-activated protein kinase (MAPK) cascade. Phosphorylation of the cPLA(2) increases the specific activity of the enzyme, thereby stimulating the arachidonic acid release. We show here, however, that conditions that lead to full phosphorylation of the cPLA(2) do not lead to enhanced AA release. As the above observations were made under both Ca(2+)-dependent and Ca(2+)-independent conditions, they emphasize that the current paradigm for activation of the cPLA(2) in cells involving both phosphorylation and Ca(2+) is incomplete and that other factors should be taken into account.     

Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by /=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.     

Localization and functional interrelationships among cytosolic Group IV, secreted Group V, and Ca2+-independent Group VI phospholipase A2s in P388D1 macrophages using GFP/RFP constructs

P388D(1) cells exposed to bacterial lipopolysaccharide (LPS) mobilize arachidonic acid (AA) for prostaglandin synthesis in two temporally distinct pathways. The "immediate pathway" is triggered within minutes by receptor agonists such as platelet-activating factor (PAF) but only if the cells have previously been primed with LPS for 1 h. The "delayed pathway" occurs in response to LPS alone over the course of several hours. We have now investigated the subcellular localization of both the Group IV cytosolic phospholipase A(2) (cPLA(2)) and the Group V secreted PLA(2) (sPLA(2)) during these two temporally distinct routes of AA release. We have prepared cells overexpressing fusion proteins of sPLA(2)-GFP and cPLA(2)-RFP. In the resting cells, cPLA(2)-RFP was uniformly located throughout the cytoplasm, and short-term treatment with LPS did not induce translocation to perinuclear and/or Golgi membranes. However, such a translocation occurred almost immediately after the addition of PAF to the cells. Long-term exposure of the cells to LPS led to the translocation of cPLA(2)-RFP to intracellular membranes after 3 h, and correlates with a significant release of AA in a cPLA(2)-dependent manner. At the same time period that the delayed association of cPLA(2) with perinuclear membranes is detected, an intense fluorescence arising from the sPLA(2)-GFP was found around the nucleus in the sPLA(2)-GFP stably transfected cells. In parallel with these changes, significant AA release was detected from the sPLA(2)-GFP transfectants in a cPLA(2)-dependent manner, which may reflect cross-talk between sPLA(2) and cPLA(2). The subcellular localization of the Group VIA Ca(2+)-independent PLA(2) (iPLA(2)) was also investigated. Cells overexpressing iPLA(2)-GFP showed no fluorescence changes under any activation condition. However, the iPLA(2)-GFP-expressing cells showed relatively high basal AA release, confirming a role for iPLA(2) in basal deacylation reactions. These new data illustrate the subcellular localization changes that accompany the distinct roles that each of the three kinds of PLA(2) present in P388D(1) macrophages play in AA mobilization.     

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.     

Group V phospholipase A(2)-mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D(1) macrophages

P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.     

Phytosterols decrease prostaglandin release in cultured P388D1/MAB macrophages

Cardiovascular disease (CVD) remains the leading cause of death in Western societies. Atherosclerosis is a major cardiovascular related disorder that is responsible for 50% of all mortality in the United States. Several epidemiological studies suggest that consumption of a plant-based diet is associated with a decreased incidence of cardiovascular abnormalities. Phytosterols, especially beta-sitosterol, are plant sterols that have been shown to exert protective effects against cardiovascular diseases as well as many types of cancer. Monocyte/macrophage cells are involved with the inflammatory process. Accumulation of these cells in arteries is one of the initial events leading to atherosclerosis. Macrophages are capable of supplying the atherosclerotic vessel with substantial amounts of prostaglandins. Prostaglandins have been shown by numerous studies to play a key role in the atherosclerosis process. They can affect platelet aggregation, vasodilation or constriction of blood vessels, and the adherence of monocytes to the vessel walls. The purpose of this study was to examine the effect of phytosterols on the release of PGE(2) and PGI(2) from lipopolysaccharide (LPS)-stimulated P388D(1)/MAB macrophage cells. P388D(1)/MAB cells were supplemented with 16 microM cholesterol, beta-sitosterol or campesterol using cyclodextrin as a vehicle. Phytosterol supplementation led to a significant decrease in cellular growth at various time points throughout a 7-day treatment period, especially after 3 days of treatment. Macrophages incorporated the supplemented phytosterols into their membranes which accounted for 26% of total membrane sterols. Cholesterol supplementation at 16 microM however, had no effect on membrane sterols. Supplementation with 16 microM concentration of beta-sitosterol or campesterol resulted in a significant inhibition of PGE(2) and PGI(2) release from macrophage cells as compared to the vehicle control. Of the two phytosterols, beta-sitosterol supplementation exhibited a greater inhibitory effect. PGE(2) release was decreased 68% by beta-sitosterol and 55% by campesterol, while cholesterol supplementation was not as effective, as it led to a 37% decrease. Similarly, release of PGI(2) from macrophages was inhibited 67% by beta-sitosterol and 52% by campesterol treatment, while enrichment of the cells with cholesterol, led to a 35% decrease in PGI(2) release. The decrease in prostaglandin release was not due to alteration in the expression of cPLA(2) and COX-2 enzymes which suggests that alterations in the activities of these enzymes may be responsible for the observed changes in prostaglandin release. It was concluded that phytosterol incorporation into macrophages may offer protection from atherosclerosis by reducing their prostaglandin release and thus slowing down the atheroma development.     

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2.

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2-4 h) of arachidonic acid (AA) and prostaglandin (PG) D2 in rat liver macrophages. Group IV cytosolic phospholipase A2 (cPLA2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA2 shows an enhanced in vitro activity. The Ca2+ dependence of cPLA2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA2 mRNA (after 2-4 h) and an enhanced expression of cPLA2 protein (after 8 h). The cellular cPLA2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA2 (sPLA2). LPS has no effect on the levels of Groups V and IIA sPLA2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA2 protein and sPLA2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA2.     

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.     

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.     

Regulation of cytosolic phospholipase A2 phosphorylation by proteolytic cleavage of annexin A1 in activated mast cells

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.     

Identification of a third pathway for arachidonic acid mobilization and prostaglandin production in activated P388D1 macrophage-like cells

Previous studies have demonstrated that P388D(1) macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A(2) (cPLA(2)), the secretory group V phospholipase A(2), and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D(1) macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A(2) nor requires the cells to be primed by LPS. The group VI Ca(2+)-independent phospholipase A(2) is also not involved. Zymosan appears to signal exclusively through activation of the cPLA(2), which is coupled to the cyclooxygenase-2. These results define a secretory PLA(2)-independent pathway for AA mobilization in the P388D(1) macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA(2) is sufficient to generate a prostaglandin biosynthetic response in the P388D(1) macrophages.     

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Regulation of arachidonic acid mobilization in lipopolysaccharide-activated P388D(1) macrophages by adenosine triphosphate

Murine P388D(1) macrophages exhibit a delayed prostaglandin biosynthetic response when exposed to bacterial lipopolysaccharide (LPS) for prolonged periods of time that is dependent on induction of the genes coding for Group V secretory phospholipase A(2) and cyclooxygenase-2. We herein report that LPS-induced arachidonic acid (AA) metabolite release in P388D(1) macrophages is strongly attenuated by the P2X(7) purinergic receptor antagonists periodate-oxidized ATP and pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid, and this is accompanied by suppression of the expression of both Group V secretory phospholipase A(2) and cyclooxygenase-2. The effect appears to be specific for LPS, because the P2 purinergic receptor antagonists do not affect P388D(1) cell stimulation by other stimuli such as platelet-activating factor or the Ca(2+) ionophore A23187. Moreover, extracellular nucleotides are found to stimulate macrophage AA mobilization with a pharmacological profile that implicates the participation of the P2X(7) receptor and that is inhibited by periodate-oxidized ATP. Collectively these results demonstrate coupling of the P2X(7) receptor to the AA cascade in P388D(1) macrophages and implicate the participation of this type of receptor in LPS-induced AA mobilization.     

Alpha-fetoprotein stimulates leukotriene synthesis in P388D1 macrophages

Alpha-fetoprotein (AFP) is able to bind specifically polyunsaturated fatty acids, especially arachidonic acid, the major precursor for prostaglandin and leukotriene synthesis. In P388D1 macrophages, AFP was found to reduce prostaglandin synthesis. This reduced synthesis was counter-balanced by a higher release of unmetabolized arachidonic acid and an enhanced production of leukotrienes. The same results were obtained with unactivated and activated cells irrespective of the activator used: lipopolysaccharide, Ca2+ ionophore A23187, phorbol myristate acetate, interferon-gamma, silica, or zymozan particles. The stimulation of leukotriene synthesis by AFP in macrophages thus appears to be a possible mechanism for the in vitro immunosuppressive effects of this oncofetal protein.     

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.     

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.     

Selective purine release from P388D1 macrophages injured by silica

Silica particles are toxic to primary cultures of macrophages or the P388D1 cell line in vitro. Loss of viability in these model systems is accompanied by depletion of ATP content within 3 to 6 hours. The mechanisms responsible for ATP depletion will be explored in this paper. After prelabeling for 1 hour with 3H-adenine, silica-treated cells released 60-80% of their labeled acid-soluble pool into the culture medium. This release did not occur after phagocytosis of nontoxic titanium dioxide particles and was specific for purines. ATP depletion was accompanied by purine catabolism: inosine, hypoxanthine, xanthine, and uric acid were detected in the culture medium using thin layer or high-performance liquid chromatography. The final xanthine oxidase step in purine catabolism generates reactive oxygen metabolites. Silica toxicity was not prevented by the xanthine oxidase inhibitor allopurinol nor exogenous purines. It is concluded that adenine nucleotide depletion and purine catabolism are not solely responsible for irreversible injury in silica toxicity. It is hypothesized that purine catabolism and release from injured macrophages may lead to generation of reactive oxygen species, injury to surrounding tissue, and fibrosis.