Preliminary studies on the induction of a 120 000 molecular weight protein in resident peritoneal macrophages by arachidonic acid

Exogenous arachidonic acid induced the synthesis of a 120 000 molecular weight protein in resident peritoneal macrophages. The induction of this protein is specific to the presence of arachidonic acid in the culture medium and is not induced by the presence of other fatty acids, irrespective of their chain length or degree of unsaturation. The protein induced is not a secretory protein and is not formed as a result of the processing of preexisting proteins in macrophages. In addition to arachidonic acid, prostaglandin E₂ also induced the synthesis of 120 000 molecular weight protein in macrophages.     

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.     

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.     

Polycations as prostaglandin synthesis inducers. Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(L-lysine) and other synthetic polycations

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.     

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages.

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E₂ but did not affect the zymosan-induced release of arachidonic acid, PGD₂, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A₂, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.     

The diacylglycerols dioctanoylglycerol and oleoylacetylglycerol enhance prostaglandin synthesis by inhibition of the lysophosphatide acyltransferase.

Prostanoids are synthesized by resident macrophages upon stimulation with diacylglycerols. Oleoylacetylglycerol and dioctanoylglycerol induced prostaglandin E and thromboxane synthesis in a time- and concentration-dependent manner. Both diacylglycerols inhibited the lysophosphatide acyltransferase, which is the key enzyme in the reacylation of arachidonic acid. By this mechanism the pool of free arachidonic acid available for prostanoid synthesis is increased. Both diacylglycerols were able to inhibit the membrane-bound lysophosphatide acyltransferase by a direct interaction independent of protein kinase C. Thus lysophosphatide acyltransferase could be shown to be a new target of these diacylglycerols, known as activators of protein kinase C.     

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E₂ but did not affect the zymosan-induced release of arachidonic acid, PGD₂, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A₂, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

Expression of macrophage p120 depends on early protein synthesis

We have previously identified a group of early proteins preceding the expression of a 120-kDa protein (p120) which coincides with tumoricidal activation in peritoneal macrophages. In the present report, we have asked whether the in vitro induction of new or enhanced expression of p120 depends on early protein synthesis and RNA synthesis during the treatment period. Expression of p120 was sensitive to pretreatment of the macrophages with either actinomycin D or cycloheximide, indicating that both active protein synthesis and RNA synthesis were required. When poly-adenylated RNA isolated from various macrophage populations was translated in a rabbit reticulocyte in vitro translation system, only mRNA isolated from cells which express p120 was able to direct synthesis of a 120-kDa polypeptide. This product showed identical mobility to p120 induced in intact activated macrophages radiolabeled with [35S]methionine. The presence of translatable p120 mRNA was dependent upon treatment of thioglycollate-elicited macrophages with both IFN-gamma plus LPS at low doses, as is expression of p120 in intact cells. Accumulation of translatable p120 mRNA was blocked by treatment with cycloheximide, indicating that active protein synthesis was required during the induction period. These results suggest that the presence of specific translatable mRNA encoding the p120 polypeptide is dependent upon the expression of early macrophage gene products.     

Control of prostanoid synthesis: Role of reincorporation of released precursor fatty acids

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A₂ activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A₂ activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.     

Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A2 activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A2 activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.     

Oxysterol activation of arachidonic acid release and prostaglandin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase C activity [corrected]

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

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.     

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.     

A possible role of protein kinase C in regulating prostaglandin synthesis of mouse peritoneal macrophages

The addition of the analogue of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), to resident macrophages isolated from the peritoneal cavity of mice led to a dose and time dependent increase in the synthesis of prostaglandin E. This was likely due to an enhanced amount of arachidonic acid available for eicosanoid synthesis as OAG suppressed the incorporation of arachidonic acid into cellular phospholipids by inhibiting acyl-CoA:lysophosphatide acyltransferase. Since OAG has been shown to activate protein kinase C in various cells, these data lead us to suggest that synthesis of eicosanoids in peritoneal macrophages is mediated by the activation of protein kinase C.     

Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase

The regulation of arachidonic acid conversion by the 5-lipoxygenase and the cyclooxygenase pathways in mouse peritoneal macrophages has been studied using particulate and soluble agonists. Particulate agonists, zymosan and latex, stimulated the production of cyclooxygenase metabolites as well as the 5-lipoxygenase product, leukotriene C4. In contrast, incubation with the soluble agonist phorbol myristate acetate or exogenous arachidonic acid led to the production of cyclooxygenase metabolites but not leukotriene C4. We tested the hypothesis that the 5-lipoxygenase, unlike the cyclooxygenase, requires activation by calcium before arachidonic acid can be utilized as a substrate. Addition of phorbol myristate acetate to macrophages in the presence of calcium ionophore (A23187) at a concentration which alone did not stimulate arachidonate metabolism resulted in a synergistic increase (50-fold) in leukotriene C4 synthesis compared to phorbol ester or A23187 alone. No such effect on the cyclooxygenase pathway metabolism was observed. Exogenous arachidonic acid in the presence of A23187 produced similar results yielding a 10-fold greater synthesis of leukotriene C4 over either substance alone without any effects on the cyclooxygenase metabolites. Presumably, calcium ionophore unmasked the synthesis of leukotriene C4 from phorbol myristate acetate-released and exogenous arachidonate by elevating intracellular calcium levels enough for 5-lipoxygenase activation. These data indicate that once arachidonic acid is released from phospholipid by an agonist, it is available for conversion by both enzymatic pathways. However, leukotriene synthesis may not occur unless intracellular calcium levels are elevated either by phagocytosis of particulate agonists or with calcium ionophore.     

Phospholipase C-diglyceride lipase is a major pathway for arachidonic acid release in macrophages

Macrophages are a rich source of arachidonic acid oxygenated metabolites and play a remarkable role in a number of physiopathological situations. The synthesis and secretion of arachidonic acid metabolites are triggered at the cytoplasmic membrane level. The present study was outlined to further investigate the cellular mechanisms controlling arachidonic acid release in macrophages. The results presented here strongly suggest that the amount of arachidonic acid released in macrophages in response to phagocytic challenge could be accounted for by a phospholipase C-diglyceride lipase system being unnecessary the presence of phospholipase A2 whose activity, on the other hand, was found vanishingly small in macrophage homogenates.     

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.     

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.