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.     

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A₂ (cPLA₂). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA₂-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA₂ activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.     

Protein kinase C beta is required for human monocyte chemotaxis to MCP-1

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused approximately 80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKCalpha and PKCbeta. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKCbeta-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKCbeta-sense ODN and PKCalpha-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR2, indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKCbeta is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKCbeta isoform in this important process.     

PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration.

Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.     

Soluble fractalkine prevents monocyte chemoattractant protein-1-induced monocyte migration via inhibition of stress-activated protein kinase 2/p38 and matrix metalloproteinase activities

In this study, we address the question of the cross-talk between two chemokines that are cosecreted during inflammation, namely monocyte chemoattractant protein-1 (MCP-1) and soluble fractalkine (s-FKN), toward monocyte migration. We found that s-FKN fails to induce MonoMac6 cell migration per se. Interestingly, this chemokine antagonizes transendothelial migration and chemotaxis of MonoMac6 cells and freshly isolated human monocytes induced by MCP-1, indicating a direct effect of s-FKN on monocytic cells. In this study, we found that stress-activated protein kinase (SAPK)1/c-Jun N-terminal kinase 1 and SAPK2/p38 are involved in the control of MCP-1-induced MonoMac6 cell migration. We demonstrated that s-FKN abrogates the MCP-1-induced SAPK2/p38 activation as well as the upstream Pyk2 activity. Furthermore, we observed that s-FKN also inhibits the activity of a major matrix metalloproteinase (MMP), namely MMP-2. Taken collectively, our results indicate that the s-FKN antagonizes the chemoattractant effect of MCP-1 on monocytes, likely by inhibiting crucial signaling pathways, like SAPK2/p38 and MMP-2 activities.     

Toll-like receptor 9-stimulated monocyte chemoattractant protein-1 is mediated via JNK-cytosolic phospholipase A2-ROS signaling

Monocyte chemoattractant protein-1 (MCP-1) influences monocyte migration into sites of inflammation. This study highlights the importance of cytosolic phospholipase A2 (cPLA2)-mediated reactive oxygen species (ROS) signaling processes in the regulation of MCP-1 release as a result of toll-like receptor (TLR) activation. In macrophages, activation of TLR9 induced MCP-1 and cPLA2-phosphorylated arachidonic acid (AA) release. Inhibition of cPLA2 blocked CpG-induced MCP-1 and AA release. Although CpG stimulates phosphorylation of ERK, p38 and JNK, only inhibition of the JNK signaling pathways attenuated MCP-1 release, suggesting that the TLR9-mediated MCP-1 release was dependent upon the JNK pathway. TLR9 activation also stimulated ROS generation, while inhibition of NADPH oxidases (Noxs) blocked CpG-induced MCP-1 release. The CpG treatment increased macrophage Nox1 mRNA level, however it had no effect on macrophage Nox2 mRNA level. Overall, these results suggest that CpG enhances ROS generation through cPLA2-dependent pathways, which results in MCP-1 release.     

Differential participation of phospholipase A(2) isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800μM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxigenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.     

Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67(phox) and p47(phox)

The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A(2) (cPLA(2)) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA(2) regulation of NADPH oxidase activation by evaluating the effects of cPLA(2) on translocation and phosphorylation of p67(phox) and p47(phox). We report that translocation and phosphorylation of p67(phox), as well as p47(phox), occur upon activation of human monocytes and that decreased cPLA(2) protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA(2) mRNA, blocked the stimulation-induced translocation of p47(phox) and p67(phox) from the cytosol to the membrane fraction. Inhibition of translocation of both p47(phox) and p67(phox) by cPLA(2) AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA(2) enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA(2)-deficient human monocytes. These results represent the first report that cPLA(2) activity or AA is required for p67(phox) and p47(phox) translocation in human monocytes. Although cPLA(2) was required for translocation of p47(phox) and p67(phox), it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA(2) regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47(phox) and p67(phox). These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.     

Role of Ca2+-independent phospholipase A2 on arachidonic acid release induced by reactive oxygen species

Previous studies have shown that reactive oxygen species (ROS) enhance arachidonic acid (AA) release and the subsequent AA metabolism in macrophages. The purpose of this study was determined the implication of phospholipases A2 (PLA2s) in these events. Our results show that oxidative stress induced by exogenous adding of hydrogen peroxide or superoxide anion in macrophage RAW 264.7 and mouse peritoneal macrophage cultures caused a marked enhancement of calcium-independent PLA2 (iPLA2) activity,whereas the increment of secreted PLA2 (sPLA2) and calcium-dependent cytosolic PLA2 (cPLA2) activities were slight. This increase of iPLA2 activity by ROS was rapid and dose-dependent. ROS also induced a significant [3H] arachidonic acid (AA) release. The iPLA2 selective inhibitor, bromoenol lactone, almost completely suppressed the mobilization of [3H]AA induced by ROS whereas antisense oligonucleotide against cPLA2 did not have any appreciable effect. Thus, our data show that iPLA2 activity is involved in the mechanism by which ROS increases the availability of free AA in macrophages RAW 264.7. Moreover, the protein kinase C (PKC) inhibitor, calphostin C, and calcium chelators had no effect on the [3H]AA release induced by ROS, suggesting this is a regulatory role of iPLA2.     

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)β, to lipid mobilization in proximal mast cells and distal fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.     

LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid ligand present in oxidized low-density lipoprotein. The effects of LPA were investigated, first separately on endothelial cells (EC) and monocytes. Using Ki16425 (an LPA₁ and LPA₃ receptor antagonist), GW9662 [a peroxisome proliferator-activator receptor (PPAR) antagonist], and pertussis toxin (that inhibits G_i/o), we demonstrate that LPA enhances IL-8 and monocyte chemoattractant protein-1 expression through a LPA₁-, LPA₃-, G_i/o- and PPAR-dependent manner in the EAhy926 cells. The effect of LPA on chemokine overexpression was confirmed in human umbilical vein endothelial cells. LPA was able to enhance monocyte migration at concentrations <1 µM and to inhibit their migration at LPA concentrations >1 µM, as demonstrated by using a chemotaxis assay. We then investigated the effects of LPA on the cross-talk between EC and monocytes by evaluating the chemotactic activity in the supernatants of LPA-treated EC. At 1 µM LPA, both cell types respond cooperatively, favoring monocyte migration. At higher LPA concentration (25 µM), the chemotactic response varies as a function of time. After 4 h, the chemotactic effect of the cytokines secreted by the EC is counteracted by the direct inhibitory effect of LPA on monocytes. For longer periods of time (24 h), we observe a monocyte migration, probably due to lowered concentrations of bioactive LPA, given the induction of lipid phosphate phosphatase-2 in monocytes that may inactivate LPA. These results suggest that LPA activates EC to secrete chemokines that in combination with LPA itself might favor or not favor interactions between endothelium and circulating monocytes. lysophosphatidic acid; endothelial cells; monocytes; chemotaxis     

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.     

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.     

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.     

Attenuation of the pulmonary inflammatory response following butylated hydroxytoluene treatment of cytosolic phospholipase A2 null mice

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.