Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage-like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.     

Intracellular calcium does not appear to be essential for arachidonic acid release from stimulated macrophages as shown by studies with Quin-2

The present investigation was undertaken to study the potential role of intracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages activated by inflammatory stimuli. The intracellular calcium concentration, as measured using fluorescent probe Quin-2, was 112 +/- 8.4 nM. The chelation of intracellular calcium with Quin-2 did not affect the release of arachidonic acid from macrophages upon stimulation with phorbol myristate acetate, opsonized zymosan or calcium ionophore A23187. However, the removal of calcium from the extracellular medium resulted in a 30-50% decrease in arachidonic acid release from phorbol myristate acetate- and zymosan-stimulated macrophages and also the stimulation of arachidonic acid release from calcium ionophore-stimulated cells were nullified. These studies indicated the existence of calcium-dependent and independent mechanisms modulating the release of arachidonic acid from macrophages subjected to inflammatory stimuli.     

Interactions of lectins with CHO cell surface membranes. I. Competition studies indicate concanavalin A and WGA bind to discrete populations of sites

Human platelet-derived growth factor (PDGF) stimulates release of arachidonic acid from cellular phospholipids, synthesis and release of prostaglandins from the cell, and initiation of DNA synthesis in cultures of 3T3 Swiss mouse fibroblasts at similar concentrations with four independent preparations representing a million-fold range of purification. Stimulation of archidonic acid and prostaglandin release is an early event (beginning within minutes) in the response to PDGF treatment. Incubating cells with PDGF at 4°C followed by washing leads to activation of archidonic acid release on warming the cells to 37°C, consistent with binding of the factor to the cell surface. PDGF-stimulated arachidonic acid release, prostaglandin release, and initiation of DNA synthesis are all inhibited by phenylglyoxal at similar concentrations. These results suggest that activation of arachidonic acid release from phospholipids plays an essential role in the mechanism by which PDGF stimulates the initiation of DNA synthesis in 3T3 cells. The stimulation of initiation of DNA synthesis by PDGF does not appear to be mediated by the synthesis of prostaglandins or other known arachidonic acid metabolites because neither indomethacin (a fatty acid cyclooxygenase inhibitor) nor phenidone (a lipoxygenase inhibitor) inhibit initiation of DNA synthesis at concentrations which inhibit arachidonic acid metabolism. Although the activation of arachidonic acid release by PDGF is a calcium-dependent process, a simple calcium flux appears unimportant to the mechanism of activation. Evidence was also obtained against an involvement of sodium fluxes or proteolytic activity in the mechanism of stimulating arachidonic acid release by PDGF or serum.     

Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx.

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2+ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Ca2+ was added to the incubation medium. When arachidonic acid release was examined in Ca(2+)-depleted cells, the response took place only in presence of external Ca2+. Verapamil, a voltage-dependent Ca2+ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2+ influx is functionally linked to arachidonic acid release and hence to phospholipase A2 activation in mouse peritoneal macrophages.     

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.     

Group X secretory phospholipase A(2) induces potent productions of various lipid mediators in mouse peritoneal macrophages

We have previously shown the expression of group X secretory phospholipase A(2) (sPLA(2)-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA(2)-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA(2)-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA(2)s. In resting macrophages, sPLA(2)-X elicited a modest production of prostaglandin E(2) and thromboxane A(2). After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA(2)-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA(2) inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA(2)-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA(2)-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.     

Activation of cytosolic phospholipase A2alpha in resident peritoneal macrophages by Listeria monocytogenes involves listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.     

Release of arachidonic acid and formation of oxygenated derivatives after complement attack on macrophages: role of channel formation

Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.     

1-14C]Arachidonic acid incorporation into glycerolipids and prostaglandin synthesis in peritoneal macrophages: effect of chloramphenicol

Peritoneal macrophages from normal mice were labelled with [1-14C]arachidonic acid after 2 h culture. The uptake of arachidonic acid into cellular lipids was rapid, time-dependent and can be represented within the limit of the studied times by a parabolic regression. Indomethacin decreased the kinetics of uptake; this inhibition is dose-dependent. Chloramphenicol had no effect on macrophage [1-14C]arachidonic acid uptake. After 3 h, the radioactivity was recovered in phosphatidylcholine (38.6%), phosphatidylserine-phosphatidylinositol (8.5%), phosphatidylethanolamine (22.1%), diacylglycerol (2.9%), triacyglycerol (2%) and cholesteryl ester (11.8%). Chloramphenicol and indomethacin inhibited the labelling of phospholipids and stimulated the labelling of neutral lipids and cholesteryl ester. Studies on arachidonic acid release from glycerolipids of prelabelled 2-h cultured macrophages showed that phosphatidylcholine and phosphatidylserine-phosphatidylinositol are the major source of arachidonic acid in prostaglandin synthesis in macrophages stimulated or not by zymosan. Chloramphenicol inhibited release of fatty acid from phosphatidylcholine and phosphatidylserine-phosphatidylinositol; indomethacin had no effect. Both drugs inhibited prostaglandin synthesis in stimulated or non-stimulated macrophages. In the culture medium, indomethacin increased the release of free arachidonic acid by stimulated macrophages. Possible explanations for the mechanisms underlying these effects are presented. It is concluded that indomethacin and chloramphenicol exert profound effects on the metabolism of phospholipids and its zymosan activation. Chloramphenicol appears to impair prostaglandin synthesis through several mechanisms and especially through phospholipase inhibition.     

Serum and plasma stimulate prostaglandin production by alveolar macrophages

Fetal bovine serum (FBS) stimulated rabbit alveolar macrophages to synthesize prostaglandins (PG) and release lysosomal enzymes. This stimulatory action was not entirely due to the effect of foreign protein in FBS, since rabbit serum and plasma, both homologous and autologous, also induced release of PGs and lysosomal enzymes. Rabbit serum and plasma are less effective than FBS as a stimulus for PG release, with rabbit serum being more potent than plasma at the same concentration. Bovine serum albumin elicited a dose-dependent increase of arachidonic acid release by macrophages, but not of PG production. Hence, the fatty acid "trapping" effect of albumin in serum and plasma is not responsible for the PG stimulation. The PG stimulating factors were stable at 56 degrees C for 30 min., but lost half the activity after heating at 100 degrees C for 10 min. Gel permeation chromatography of FBS showed several peaks of PG stimulating and arachidonic acid releasing activity. The molecular weight of the major one (150,000 daltons) is similar to that of immunoglobulin G. Rabbit IgG, when added to the macrophage culture, stimulated release of arachidonic acid and PGs. However, the major stimulatory effect in serum or plasma is not all due to IgG, since removal of IgG by a Protein A-agarose column did not remove the stimulatory effect of FBS and rabbit serum. The possibility of other factors, such as complement fragments, is discussed.     

An alternate mechanism for regulation of leukotriene production in leukocytes: studies with an anti-inflammatory drug, sodium diclofenac

Sodium diclofenac, a potent cyclooxygenase inhibitor, was recently shown to inhibit arachidonic acid conversion to leukotriene products in human leukocytes. This activity was confirmed by radioimmunoassay in calcium ionophore A 23187-stimulated leukocytes isolated from the rat peritoneal cavity and human peripheral blood. Studies with rat peritoneal leukocytes revealed that this effect was not mediated by inhibition of 5-lipoxygenase or phospholipase A2, but rather through modulation of arachidonic acid uptake and release. The potency of this effect was dependent upon cell type; macrophages being more sensitive to the drug than neutrophils. In leukocytes treated with sodium diclofenac, arachidonic acid released from phospholipids in response to A 23187 challenge was reincorporated into triacylglycerols. The drug enhanced the spontaneous uptake of arachidonic acid into the cellular triacylglycerol pool and, in this manner, decreased the availability of intracellular arachidonic acid. Therefore, sodium diclofenac, in addition to inhibition of cyclooxygenase, regulates leukotriene production of inflammatory cells by a mechanism mediated in part through the redistribution of arachidonic acid in lipid pools.     

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.     

Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hank's balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 micrograms/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandin E2 and thromboxane B2. Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.     

Serum and plasma stimulate prostaglandin production by alveolar macrophages

Fetal bovine serum (FBS) stimulated rabbit alveolar macrophages to synthesize prostaglandins (PG) and release lysosomal enzymes. This stimulatory actions was not entirely due to the effect of foreign protein in FBS, since rabbit serum and plasma, both homologous and autologous, also induced release of PGs and lysosomal enzymes. Rabbit serum and plasma are less effective than FBS as a stimulus for PG release, with rabbit serum being more potent than plasma at the same concentration. Bovine serum albumin elicited a dose-dependent increase of arachidonic acid release by macrophages, but not of PG production. Hence, the fatty acid “trapping” effect of albumin in serum and plasma is not responsible for the PG stimulation. The PG stimulating factors were stable at 56°C for 30 min., but lost half the activity after heating at 100°C for 10 min. Gel permeation chromatography of FBS showed several peaks of PG stimulating and arachidonic acid releasing activity. The molecular weight of the major one (150,000 daltons) is similar to that of immunoglobulin G. Rabbit IgG, when added to the macrophage culture, stimulated release of arachidonic acid and PGs. However, the major stimulatory effect in serum or plasma is not all due to IgG, since removal of IgG by a Protein A-agarose column did not remove the stimulatory effect of FBS and rabbit serum. The possibility of other factors, such as complement fragments, is discussed.     

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.     

Involvement of external calcium in the release of arachidonic acid by mouse peritoneal macrophages

The present investigation was undertaken to study the potential role of extracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages. Both in phorbol ester-treated and in Ca2(+)-depleted cells, a rapid release of arachidonic acid was seen in direct response to added Ca2+. The response was directly dependent on the extracellular Ca2+ concentration, with a Ca2+ threshold of 100 nM. These results support the notion that arachidonic acid release in macrophages is functionally coupled to influx of external calcium.     

Acyltransferase-catalyzed cleavage of arachidonic acid from phospholipids and transfer to lysophosphatides in lymphocytes and macrophages

The cleavage of fatty acyl moieties from phospholipids was compared in intact cells and homogenates of mouse lymphocytes (thymocytes, spleen cells) and macrophages. Liberation of free arachidonic acid during incubations of intact cells was only detectable in the presence of albumin. Homogenization of prelabeled thymocytes and further incubation of these homogenates at 37 degrees C resulted in a pronounced decrease of phospholipid degradation and cleavage of arachidonoyl residues, while further incubation of homogenates from prelabeled macrophages produced a greatly increased phospholipid degradation. Homogenates of macrophages but not those of thymocytes contain substantial activities of phospholipase A2 detectable using exogenous radiolabeled substrates. These findings indicate that in thymocytes cleavage of arachidonic acid from phosphatidylcholine is an active process that is not catalyzed by phospholipase A2. Addition of CoA and lysophosphatidylethanolamine to prelabeled thymocyte homogenates induced a fast breakdown of phosphatidylcholine and transfer of arachidonic acid to phosphatidylethanolamine, as in seen during incubations of intact thymocytes or macrophages. The transfer is restricted to arachidonic acid and does not require addition of ATP. Sodium cholate, a known inhibitor of the acyl-CoA:lysophosphatide acyltransferase, completely inhibited this transfer reaction. These results suggest that the CoA-mediated, ATP-independent breakdown of phosphatidylcholine and transfer of arachidonic acid is catalyzed by the acyl-CoA:lysophosphatide acyltransferase operating in reverse.     

Differential effect of diacylglycerols and phorbol ester on arachidonic acid metabolism in bone marrow-derived macrophages

Murine bone marrow-derived macrophages were induced to prostaglandin synthesis by activators of protein kinase C, the phorbolester TPA and the diacylglycerols dioctanoylglycerol (diC8) and diolein (diC18:1). As short term stimulation of prostaglandin synthesis is mainly dependent on the availability of free arachidonic acid, the modulation of arachidonic acid liberation and reacylation was investigated. DiC8 inhibited the reacylating enzyme lysophosphatide acyltransferase in the in vitro assay, but there was no evidence for an inhibitory effect of TPA or diacylglycerols on the activity of the lysophosphatide acyltransferase in whole cells. The release of arachidonic acid from prelabelled cells was stimulated by TPA and the diacylglycerols even in the presence of an inhibitor of reacylation, indicating an activation of phospholipase A2. An activation of phospholipase A2 was measured in membranes derived from TPA-stimulated macrophages. These data indicate that the enhanced pool of free arachidonic acid, which drives prostaglandin synthesis, is primarily due to a stimulation of the liberation of arachidonic acid from membrane phospholipids.