Involvement of group VIA calcium-independent phospholipase A2 in macrophage engulfment of hydrogen peroxide-treated U937 cells

Hydrogen peroxide-induced apoptosis of U937 cells results in substantial hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A(2) (iPLA(2)-VIA). However, abrogation of cellular iPLA(2)-VIA neither delays nor decreases apoptosis, suggesting that, beyond a mere destructive role, iPLA(2)-VIA may serve other specific roles. In this study, we report that phagocytosis of apoptosing U937 cells by macrophages is blunted if the cells are depleted of iPLA(2)-VIA by treatment with an inhibitor or an antisense oligonucleotide, and it is augmented by overexpression of iPLA(2)-VIA in the dying cells. Thus, the magnitude of macrophage phagocytosis correlates with the level of iPLA(2)-VIA activity of the dying cells. Eliminating by antisense oligonucleotide technology of cytosolic group IVA phospholipase A(2) does not attenuate phagocytosis of U937 dying cells by macrophages. Incubation of U937 cells with different fatty acids has no effect on either the extent of hydrogen peroxide-induced apoptosis or the degree of phagocytosis of the dying cells by macrophages. However, preincubation of the macrophages with lysophosphatidylcholine before exposing them to the dying cells blocks phagocytosis of the latter. These results indicate that formation of lysophosphatidylcholine by iPLA(2)-VIA in hydrogen peroxide-treated U937 cells to induce apoptosis directly contributes to their efficient clearance by macrophages.     

Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.     

Calcium-independent phospholipase A2 mediates proliferation of human promonocytic U937 cells

We have investigated the possible involvement of two intracellular phospholipases A(2), namely group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) and group IVA cytosolic phospholipase A(2) (cPLA(2)alpha), in the regulation of human promonocytic U937 cell proliferation. Inhibition of iPLA(2)-VIA activity by either pharmacological inhibitors such as bromoenol lactone or methyl arachidonyl fluorophosphonate or using specific antisense technology strongly blunted U937 cell proliferation. In contrast, inhibition of cPLA(2)alpha had no significant effect on U937 proliferation. Evaluation of iPLA(2)-VIA activity in cell cycle-synchronized cells revealed highest activity at G(2)/M and late S phases, and lowest at G(1). Phosphatidylcholine levels showed the opposite trend, peaking at G(1) and lowest at G(2)/M and late S phase. Reduction of U937 cell proliferation by inhibition of iPLA(2)-VIA activity was associated with arrest in G(2)/M and S phases. The iPLA(2)-VIA effects were found to be independent of the generation of free arachidonic acid or one of its oxygenated metabolites, and may work through regulation of the cellular level of phosphatidylcholine, a structural lipid that is required for cell growth/membrane expansion.     

Involvement of hydrogen peroxide in mistletoe lectin-II-induced apoptosis of myeloleukemic U937 cells

Mistletoe lectin-II, a major component of Korean mistletoe (Viscum album var. coloratum) induces apoptotic death in cancer cells. In this study, we demonstrated that lectin-II induced the generation of pro-oxidants and thus resulted in the apoptotic death of human myeloleukemic U937 cells. We observed that lectin-II-induced apoptotic death was inhibited by antioxidants including reduced glutathione (GSH), N-acetylcysteine (NAC), ebselen, mnTBP, catalase and pyrrolidine dithiocarbamate (PDTC). GSH and NAC also abolished the apoptotic DNA ladder pattern fragmentation of U937 cells after lectin-II stimulation. Obviously, lectin-II treatment of cells resulted in a remarkable generation of intracellular hydrogen peroxide (H2O2) as an early event, which was monitored fluorimetrically using scopoletin-horse radish peroxidase (HRP) assay and peroxide-sensitive fluorescent probe, DCF-DA. In addition, antioxidants inhibited the activation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) as well as cytosolic release of cytochrome c by mistletoe lectin-II. Moreover, lectin-II-induced activation of caspase-9 and 3-like protease and cleavage of poly(ADP-ribose) polymerase (PARP) were inhibited by pretreatment of cells with thiol antioxidants, GSH and NAC. Taken together, these results suggest that Korean mistletoe lectin-II is a strong inducer of pro-oxidant generation such as H2O2, which mediates the JNK/SAPK activation, cytochrome c release, activation of caspase-9 and caspase 3-like protease, and PARP cleavage in human myeloleukemic U937 cells.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Involvement of calcium-independent phospholipase A2 in uterine stromal cell phospholipid remodelling.

The role of Ca2+-independent phospholipase A2 (iPLA2) in arachidonic (AA) and docosahexaenoic (DHA) acid incorporation and phospholipid remodelling in rat uterine stromal cells (UIII cells) was studied. Incorporation of AA and DHA into UIII cell phospholipids was Ca2+-independent. Bromoenollactone (BEL), a potent inhibitor of iPLA2, reduced lysophosphatidylcholine level and AA incorporation into phospholipids by approximately 20%. DHA incorporation was not affected by BEL, indicating that the pathways for AA and DHA incorporation are partially different. In control cells, the transfer of AA occurred mainly from diacyl-glycerophosphocholine (GroPCho) to alkenylacyl-glycerophosphoethanolamine (GroPEtn) and to a lesser extent from diacyl-GroPCho to diacyl-GroPEtn. [3H]DHA was redistributed from diacyl-GroPCho and alkylacyl-GroPEtn to alkenylacyl-GroPEtn. BEL treatment inhibited completely the redistributrion of AA within diacyl-GroPCho and diacyl -GroPEtn and reduced the [3H]DHA content of diacyl-GroPEtn, indicating that a BEL-sensitive iPLA2 controls the redistribution of polyunsaturated fatty acids to diacyl-GroPEtn. In contrast the redistribution of radioactive AA and DHA to alkenylacyl-GroPEtn was almost insensitive to BEL. The analysis of substrate specificity and BEL sensitivity of iPLA2 activity indicates that UIII cells exhibit at least two isoforms of iPLA2, one of which is BEL-sensitive and quite selective of diacyl species, and another one that is insensitive to BEL and selective for alkenylacyl-GroPEtn. Taken together, these results suggest that several iPLA2 participate independently in the remodelling of UIII cell phospholipids.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.     

Effects of saturated fatty acids on n-6 fatty acid metabolism in cultured human monocyte-like cells (U937)

Effects of supplementation of saturated fatty acids (16:0 and 18:0) on metabolism of the cytotoxic n-6 fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 16:0 and 18:0. Supplementation of either 16:0 or 18:0 has no significant effect on the uptake of 18:2n-6 and 18:3n-6. However, addition of 16:0 to the medium increased whereas 18:0 suppressed the cytotoxic effects of 18:2n-6 and 18:3n-6. In addition, 16:0 supplementation reduced the incorporation of n-6 fatty acids in cellular phospholipid fraction, and enhanced the metabolism of n-6 fatty acids, particularly the conversion of 20:3n-6 to 20: 4n-6 in U937 cells. Results with microsomes prepared from U937 cells also showed that 16:0 supplementation increased the 5 desaturase activity. This may be related in part to an increase in the availability of 20:3n-6, since results obtained in a separate study have shown that 16:0 competed with 20:3n-6 for incorporaton into the phospholipid molecule at sn-2 position. Increasing the availability and formation of long chain n-6 fatty acids, which are cytotoxic, might also be responsible for increasing cytotoxicity of 16:0 supplementation.     

Intracellular accumulation of free fatty acids in isolated white adipose cells

A simple, rapid, and accurate method was developed for measuring intracellular FFA levels in isolated white adipose cells using sucrose-(14)C or inulin carboxyl-(14)C as nontransportable, nonutilizable markers of the extracellular space. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose-(14)C or inulin carboxyl-(14)C retained in the floating packed adipose cells. In this way the FFA content of the adipose cells could be corrected for contamination by FFA bound to extracellular albumin. With this technique the initial events in hormone-activated lipolysis were studied under conditions of maximal and constant rates of triglyceride hydrolysis. The FFA content of isolated adipocytes of fed rats was 0.5 micro mole/g cell lipid. On addition of norepinephrine in the presence of medium albumin, the concentration of intracellular FFA rapidly increased and reached a plateau at a concentration of 2-2.5 micro moles/g cell lipid. In the presence of medium albumin an initial lag in glycerol release occurred and this was attributed to partial hydrolysis of triglyceride with retention of lower glycerides. After 5 min of incubation FFA and glycerol output was constant. In the absence of medium albumin norepinephrine-stimulated lipolysis was reduced more than 90% and extracellular FFA release was not detected. Nevertheless, intracellular FFA accumulation was identical to that seen in the presence of albumin. The data suggest that most of this intracellular pool of FFA is bound to cytoplasmic constituents.     

Activation of phospholipase Cepsilon by free fatty acids and cross talk with phospholipase D and phospholipase A2

This paper uses phospholipase Cepsilon as a model to demonstrate that lipids can act as ligands to bind to specific motifs and regulate protein activity via allosteric effects. Phospholipids such as phosphatidic acid and free fatty acids such as arachidonate are potent activators of PLCepsilon, increasing the rate of PI hydrolysis by 8-fold and 50-fold, respectively. The mechanism appears to be a reduction of K(m), as the substrate dependence curve is shifted to the left and K(m) is reduced 10-fold. The regulation of PLCepsilon by lipids appears to be physiologic, as reconstitution or cotransfection of either cPLA(2) or PLD with PLCepsilon leads to activation of phosphodiesterase activity. Additionally, TSA-201 cells transfected with PLCepsilon and fed arachidonic acid complexed with BSA had increased (4-5-fold) hydrolysis of polyphosphoinositides. This study demonstrates the ability of lipids to act as potent and direct mediators of protein function and identifies cross talk between different classes of phospholipase (PLD and PLA(2) with PLC) mediated via lipid products.     

The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells

Human monoblast U937 cells contain a soluble phospholipase A2 (PLA2) that is activated over the range of 150-600 nM Ca2+ and is stable only at neutral pH. We have purified this PLA2 over 34,000-fold to near homogeneity using sequential ion exchange, hydrophobic interaction, and gel filtration chromatography steps. The protein has a Mr of approximately 100,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 5.1. Four lines of evidence indicate that this 100-kDa polypeptide represents the PLA2. (i) The intensity of staining of the 100-kDa protein was proportional to the degree of purification of PLA2 activity, (ii) the relative staining intensity of the 100-kDa protein precisely paralleled the elution profile of PLA2 activity during chromatography steps, (iii) the PLA2 activity recovered from a nondenaturing gel (greater than 60% of the total activity applied) coincided exactly with the major high molecular weight protein detected by silver staining, and (iv) monoclonal antibodies against the 100-kDa protein immunoprecipitated the PLA2. We conclude that the cytosolic PLA2 isolated from U937 cells represents a novel, high molecular weight PLA2 responding to physiological (intracellular) changes in Ca2+ concentration and therefore may play a critical role in cellular signal transduction processes and the biosynthesis of lipid mediators.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E₂, profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFα, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H₂O₂), either exogenous or endogenously generated by TNFα, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H₂O₂ that are involved in TNFα signaling and the pathways through which H₂O₂ modulates Cox-2 expression. Results indicate that H₂O₂-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H₂O₂ generation. Signaling triggered by H₂O₂ for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H₂O₂ plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H(2)O(2) can induce such an adaptive response without elevating the cellular capacity to degrade H(2)O(2), and were able to confer the cells a cross-resistance to an H(2)O(2)-independent lethal stimulus, C(2)-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-kappa B to the nuclei. This event was promoted or abolished when either IKK alpha or a dominant negative mutant of I kappa B, respectively, was overexpressed. The overexpression of IKK alpha also resulted in the suppression of H(2)O(2)-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the I kappa B mutant. The protective effect of IKK alpha was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H(2)O(2). Moreover, the overexpression of IKK alpha resulted in an enhancement of H(2)O(2)-induced resistance to C(2)-ceramide. The overall data suggest that NF-kappa B mediates the H(2)O(2) adaptation induced in a manner independent of H(2)O(2)-degrading activity.     

Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes

As a part of their surveillance functions in the immune system, monocytes/macrophages secrete large amounts of the bactericidal enzyme lysozyme to the extracellular medium. We report here that lysozyme secretion in activated U937 promonocytes depends on a functional calcium-independent phospholipase A(2) (iPLA(2)). Inhibition of the enzyme by bromoenol lactone or by treatment with a specific antisense oligonucleotide results in a diminished capacity of the cells to secrete lysozyme to the extracellular medium. Calcium-independent PLA(2) is largely responsible for the maintenance of the steady state of lysophosphatidylcholine (lysoPC) levels within the cells, as manifested by the marked decrease in the levels of this metabolite in cells deficient in iPLA(2) activity. Reconstitution experiments reveal that lysoPC efficiently restores lysozyme secretion in iPLA(2)-deficient cells, whereas other lysophospholipids, including lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylethanolamine, are without effect. Arachidonic acid mobilization in activated U937 cells is under control of cytosolic phospholipase A(2) (cPLA(2)). Selective inhibition of cPLA(2) results in a complete abrogation of the arachidonate mobilization response, but has no effect on lysozyme secretion. These results identify iPLA(2)-mediated lysoPC production as a necessary component of the molecular machinery leading to lysozyme secretion in U937 cells and rule out a role for cPLA(2) in the response. Collectively, the results demonstrate distinct roles in inflammatory cell signaling for these two intracellular phospholipases.     

Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.     

Differential effects of unsaturated fatty acids on modulation of endotoxin-induced tissue factor activation in cultured human leukemia U937 cells.

We examined the direct effects of unsaturated fatty acids, oleic (18:1 n-9), linoleic (18:2 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) on tissue factor (TF) activity in the human leukemia monocytic U937 cell line. After exposing cells to fatty acids for 16 h, there were no significant effects on either TF activity or its activation induced by bacterial endotoxin (LPS). When the cells were primed with fatty acids for 24 h, 48 h or 72 h, the TF activity remained essentially unchanged. However, the extent of TF-activation induced by LPS depended on the length of priming, and the dose and the degree of unsaturation of the fatty acids to which cells were exposed. After a 72-h priming, 18:1 produced 40-60 per cent elevation in LPS-challenge. In contrast, approximately 20-50 per cent reduction in LPS-challenge was achieved by 18:2, 20:5 and 22:6 at high concentrations. The results suggest that chronic exposure of U937 cells to unsaturated fatty acids leads to modulation of the TF-activation in response to LPS.