Prostaglandin production by human blood monocytes and mouse peritoneal macrophages: synthesis dependent on in vitro culture conditions

The pattern of prostaglandin synthetase products from human peripheral blood monocytes was examined. Thromboxane and prostaglandin E were found to be the major products released by monocytes/macrophages on day one of culture following cell adherence. If these cells were studied 24h after cell adherence had occurred, then thromboxane synthesis was noted to be markedly reduced and PGE was the major secretory product. A day one type pattern, i.e. high thromboxane, high PGE could be elicited from day two cultured cells if cell adherence was delayed until day two of culture. Inflammatory stimuli caused a consistent rise in PGE release from day one and day two cultures, no consistent change in thromboxane was observed. It is suggested that activation of the thromboxane synthetase pathway in monocytes and macrophages is primarily a consequence of cell adherence. Prostaglandin E and prostacyclin (PGI) appear to be the major products released in response to inflammatory stimuli. These data demonstrate that the pattern and sequence of prostaglandins synthesized are in part a function of the in vitro culture conditions, time in culture and the species studied. Further, these findings offer a possible explanation to the discrepant reports in the literature.     

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-14C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D2, thromboxane (TX) B2, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15-hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE2 was also detected but neither 6-keto-PGF1 alpha nor PGF2 alpha were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB4) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD2, TXB2 and PGE2 was further confirmed by analyzing [3H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB4 and LTC4 by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD2, TXB2 and PGE2 from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10(-6) M).     

Prostaglandin-mediated suppression of macrophage phagocytosis of Listeria monocytogenes

Suppression of macrophage phagocytosis of Listeria monocytogenes has been shown to be due to a low-molecular-weight component of spleen cell culture supernatant. The possibility that the factor could be a prostaglandin was investigated. When murine peritoneal macrophages were treated with prostaglandin E2 (PGE2), L. monocytogenes was phagocytized at a rate comparable to that phagocytized when treated with a low-molecular-weight fraction of concanavalin A-generated spleen cell culture supernatant. Suppressive activity of the spleen cell culture supernatant was abrogated when supernatant was prepared in the presence of indomethacin, a prostaglandin synthetase inhibitor. Prostaglandins were identified in supernatants with thin-layer and high-pressure liquid chromatography. These results suggest a role for prostaglandins, particularly PGE2, as a modulator of macrophage phagocytosis of L. monocytogenes.     

Acrolein: a potent modulator of lung macrophage arachidonic acid metabolism

Resting rat pulmonary alveolar macrophages exposed to acrolein were stimulated to synthesize and release thromboxane B2 and prostaglandin E2 in a dose-dependent manner. Zymosan-activated pulmonary alveolar macrophages released approximately twice as much prostaglandin E2 as thromboxane B2, whereas acrolein-activated pulmonary alveolar macrophages released 4-5 times less prostaglandin E2 than thromboxane B2. In the zymosan-stimulated pulmonary alveolar macrophages, acrolein also induced a reversal in the relative amounts of prostaglandin E2 and thromboxane B2 synthesized and released into the culture medium. This reversal was achieved by a dose-dependent reduction in prostaglandin E2 synthesis. Although phagocytosis was also inhibited in a dose-dependent manner, the reduction in prostaglandin E2 appeared to be partially independent of particle ingestion since thromboxane B2 synthesis was not affected by low doses of acrolein. In fact, high doses induced a slight enhancement in thromboxane B2 synthesis. These results suggest that acrolein selectively inhibited the enzyme, prostaglandin endoperoxide E isomerase, necessary for the conversion of the endoperoxide to prostaglandin E2. Sulfhydryl reagents such as N-ethylmaleimide and 5,5'-dithiobis (2-nitrobenzoic acid) mimicked acrolein's effects, and reduced glutathione afforded protection against the effects of acrolein. These results indicated the possible involvement of acrolein's sulfhydryl reactivity in the inhibition of the isomerase enzyme. Propionaldehyde had no effect on macrophage arachidonic acid metabolism whereas crotonaldehyde mimicked the effects of acrolein. Pulmonary macrophages were unable to reverse the acrolein effects on arachidonate metabolite synthesis after 6 h in an acrolein-free environment. These data indicated the necessity of the unsaturated carbon bond for the acrolein effects on arachidonic acid metabolism and the relative irreversibility of acrolein's reaction with the macrophage.     

Prostaglandin E2, monocyte adherence and interleukin-1 in the regulation of human natural killer cell activity by monocytes

Monocytes have previously been shown both to augment and suppress human natural killer (NK) cell activity depending upon the conditions. An interleukin-1/interleukin-2 (IL-1/IL-2)-dependent mechanism has been shown to be involved in the augmentative effect. In the current study, the role of the method of monocyte isolation was evaluated. Monocytes isolated by Percoll gradient centrifugation were ineffective for modulating NK activity, but monocytes isolated by adherence from most donors exhibited increased augmentation with increased interval of adherence (up to 1 h). However, monocytes isolated by adherence from certain donors reproducibly exhibited increased suppression with increased interval of adherence. The observation of augmentation was correlated with an increase in the balance between IL-1 production and prostaglandin E (PGE) production by the monocytes. The roles of PGE2 and IL-1 were therefore examined by mixing these cytokines with enriched null lymphocyte preparations in the absence or presence of monocytes in the NK assay system. The participation of PGE2 was further examined using monocytes treated with indomethacin (10(-6) M), and the participation of monocyte-membrane-bound IL-1 was evaluated using monocytes fixed with 1% paraformaldehyde. The results revealed that PGE2 production is involved in the suppression of human NK activity by human monocytes, and the functional balance between IL-1 and PGE2 determines whether suppression or augmentation is observed. The data of this and previous studies are consistent with the suggestion that membrane-associated IL-1 is the important IL-1 moiety for the augmentation of human NK activity by monocytes.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids

The production of prostaglandins by phagocytic cells of the thymic reticulum in culture (P-TR) was studied by using high pressure liquid chromatography and radioimmunoassay. Radioimmunologic determinations showed that thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (6 keto-PGF1 alpha) were the major compounds released into the culture medium, whereas prostaglandin F2 alpha (PGF2 alpha) was only a minor component. Indomethacin and dexamethasone exerted a similar pattern of differential inhibition of the secretion of prostanoids. PGE2 and 6-keto PGF1 alpha productions were markedly decreased by these anti-inflammatory drugs, whereas those of TXB2 and PGF2 alpha were not or were only slightly affected. Experiments performed with an antiglucocorticoid compound (RU 38486) showed that the steroid-induced inhibition of prostanoid secretion is a classical receptor-mediated action. These results demonstrated that phagocytic cells of the thymic reticulum, which resemble the thymic interdigitating cells, produce several types of prostaglandins. Because it has been described that P-TR regulate thymocyte proliferation in vitro via the secretion of both interleukin 1 and PGE2, these results suggest that anti-inflammatory agents may be able to modulate the thymic microenvironment and, consequently, thymocyte proliferation.     

Cyclooxygenase products formed by primary cultures of cells from human chorion laeve: influence of steroids

Cells were isolated from human chorion laeve obtained at term (38-40 weeks gestation) by elective caesarean section and were maintained in primary culture for 1 week in defined media supplemented with 10% fetal calf serum. The production of various cyclooxygenase products by the cultures was examined. Little or no prostaglandin (PG) F2 alpha, 6-keto-PGF1 alpha, thromboxane B2, or 13,14-dihydro-15-keto-PGF2 alpha was found. In contrast, the cells produced PGE2 which was low on day 0, increased during culture to a maximum on day 1 or 2, then declined to low levels. When cells were grown in the presence of media containing cortisol, dexamethasone, progesterone, and estradiol (at 10(-7) or 10(-9) M), the glucocorticoids (at 10(-7) and 10(-9) M), but not estrogen or progesterone, markedly inhibited the increase in PGE2 output. There was no difference in the protein content and thymidine incorporation of cells grown in the presence of glucocorticoids when compared with controls. This inhibitory effect was not sensitive to cycloheximide (1 microgram/mL) indicating protein synthesis may not be involved in the process. These studies indicate that PGE2 is the major prostaglandin formed by primary cultures of chorion laeve and that prostaglandin metabolism in the chorion is sensitive to glucocorticoid inhibition.     

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.     

Essential fatty acids and immune response

The implication that essential fatty acids (EFA) can affect immune response was based on the observation that EFA deficiency can accentuate or improve symptoms of certain autoimmune diseases in animals, and that supplementation of linoleic acid to animals reversed such effects. Furthermore, treatment of animals with cyclooxygenase inhibitors abrogated the effect of linoleic acid. Administration of cyclooxygenase inhibitors to animals enhanced both cell-mediated and humoral immune responses. In vitro studies have shown that prostaglandin E (PGE) group inhibits both T and B lymphocyte functions; it is suggested that effects of EFA on immune response are, in part, mediated through eicosanoids. Growing evidence now suggests that the PGE group of prostaglandins can serve as a negative feedback modulator of immune response. However, in vitro effects of other cyclooxygenase-derived products, such as PGI2 and thromboxane A2 (TXA2) have not been well established, perhaps because of their instability in aqueous media. Unlike the PGE group, some of lipoxygenase-derived products of arachidonic acid have shown immunostimulatory effects, as assessed by lymphokine production in vitro. Whether such effects can be seen in vivo remains to be determined. Some lipoxygenase-derived products with strong chemotactic action may indirectly influence immune response by modulating the population of antigen-presenting macrophages in tissues. Thus, the net effect of eicosanoids synthesized in macrophages on modulating immune response may depend on relative amounts of cyclooxygenase-derived products as compared with lipoxygenase-derived products. Macrophages are the major source of eicosanoids among immunocompetent cells. The profile of eicosanoids, produced in vitro by macrophages, varies with type of stimuli and anatomical sites. It can also be affected by the fatty acid composition of tissue lipids, which in turn can be modified by the composition of dietary EFA. Whether manipulating dietary EFA can modulate immune response in normal humans and animals needs to be determined.     

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.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release.

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence-dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell-derived extracellular matrix or a denatured collagen/fibronectin-coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin-coated wells demonstrated significantly less cytolytic activity (15 +/- 2% lysis) when compared with either tissue culture plastic adherent PM (43 +/- 7% lysis) or PM adherent to extracellular matrix (59 +/- 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 +/- .9 nmoles/10(6) PM) than PM adherent to either tissue culture plastic (43 +/- 1 nmoles/10(6) PM) or the extracellular matrix (60 +/- 0.5 nmoles/10(6) PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dose-dependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.     

Sex differences in arachidonate cyclo-oxygenase products in elicited rat peritoneal macrophages

Peritoneal macrophages were elicited by Freund's incomplete adjuvant from adult male and female Fisher 344 rats. The release of prostaglandin E2 and thromboxane B2 from these macrophages was determined by radioimmunoassay. The basal release of these products was the same for males and females. The macrophages of the female rats released, in a dose-dependent manner, significantly more prostaglandin E2 and thromboxane B2 than macrophages from the male, following challenge with either a particulate stimulus, zymosan (25-150 micrograms/ml) or a soluble stimulus, calcium ionophore A23187 (1 X 10(-7) -1 X 10(-6) M). These results may relate to gender differences in immune responses.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE2. Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE2 upon LPS stimulation. Resident mPGES-1(-/-) peritoneal macrophages exhibited severely impaired PGE2-releasing activity but retained some LPS-inducible PGE2 production capacity. Both macrophage types showed a 50% decrease in PGE2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80-90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B2, PGD2, PGF(2alpha), and 6-keto-PGF(1alpha)) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1.     

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins

The effect of exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH . The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.