Stimulation of prostaglandin E2 production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide

The effects of TPA (12-O-tetradecanoylphorbol 13-acetate)-type and non-TPA-type tumor promoters on prostaglandin E2 production by peritoneal macrophages of rats were examined. Among the TPA-type tumor promoters, aplysiatoxin was most potent in stimulating prostaglandin E2 production followed by dihydroteleocidin B, teleocidin, TPA and debromoaplysiatoxin. Prostaglandin E2 production by aplysiatoxin treatment was stimulated at doses up to 0.1 ng/ml. Palytoxin, a non-TPA-type tumor promoter, also stimulated both prostaglandin E2 production and the release of radioactivity from [3H]arachidonic acid-labeled macrophages. However, the dose required for the expression of these effects by palytoxin was up to 3 pg/ml. It was suggested that the tumor promoters are associated with the activity to stimulate arachidonic acid metabolism, irrespective of their type. Cycloheximide, a protein synthesis inhibitor, inhibited both prostaglandin E2 production and the release of radioactivity from prelabeled macrophages stimulated either by the TPA-type tumor promoters or by the non-TPA-type tumor promoter. It is possible that the tumor promoters may induce the synthesis of some proteins responsible for the stimulation of arachidonate metabolism.     

Inhibition by tectorigenin and tectoridin of prostaglandin E2 production and cyclooxygenase-2 induction in rat peritoneal macrophages.

Tectorigenin and tectoridin, isolated from the rhizomes of Korean Belamcanda chinensis (Iridaceae) which are used as Chinese traditional medicine for the treatment of inflammation, suppressed prostaglandin E2 production by rat peritoneal macrophages stimulated by the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the endomembrane Ca2+-ATPase inhibitor, thapsigargin. Tectorigenin inhibited prostaglandin E2 production more potently than tectoridin. Neither compound inhibited the release of radioactivity from [3H]arachidonic acid-labeled macrophages stimulated by TPA or thapsigargin. In addition, activities of isolated cyclooxygenase (COX)-1 and COX-2 were not inhibited by the two compounds. Western blot analysis revealed that the induction of COX-2 by TPA or thapsigargin was inhibited by the two compounds in parallel with the inhibition of prostaglandin E2 production. These findings suggest that one of the mechanisms of the anti-inflammatory activities of the rhizomes of Belamcanda chinensis is the inhibition of prostaglandin E2 production by tectorigenin and tectoridin due to the inhibition of the induction of COX-2 in the inflammatory cells.     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

Vitamin E inhibits PGE2 and O2- production in rat peritoneal macrophages.

In order to examine the possible role of vitamin E on the modulation of macrophages, we investigated the effect of vitamin E on O2- and PGE2 production in macrophages. The production of both PGE2 and O2- in rat peritoneal macrophages was dose-dependently stimulated by the addition of PMA and calcium ionophore A23187. However, the macrophages obtained after intraperitoneal injection of vitamin E for six successive days showed less PGE2 and O2- production when stimulated with PMA or A23187 as compared to those of control macrophages. O2- production in control macrophages stimulated with 139 nM PMA and 1 microM A23187 as 4.2 +/- 0.3 and 3.0 +/- 0.2 nmol/min per 10(6) cells, respectively. On the other hand, O2- production by the macrophages from vitamin E-treated rats was 1.5 +/- 0.4 nmol/min per 10(6) cells when stimulated with the PMA, and was not detectable when stimulated with A23187. As for the production of PGE2, control macrophages produced 2.59 +/- 0.70 ng/30 min per 10(6) cells when stimulated with PMA and 8.96 +/- 3.26 ng/30 min per 10(6) cells with the A23187, whereas PGE2 production by the macrophages from vitamin E-treated rats was reduced to 12-20% of the control. By analyzing alpha-tocopherol content and intracellular concentration of calcium ion [( Ca2+]i) in the macrophages isolated from control and vitamin E-treated rats, vitamin E treatment augmented alpha-tocopherol content (384.7 +/- 76.1 vs. 1.2 +/- 0.4 ng/10(6) cells) and decreased free [Ca2+]i when stimulated with A23187 (652 +/- 14 vs. 1201 +/- 223 nM).     

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.     

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.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Stimulation of histamine release and arachidonic acid metabolism in rat peritoneal mast cells by thapsigargin, a non-TPA-type tumor promoter

Thapsigargin, a non-TPA-type tumor promoter, releases histamine and stimulates arachidonic acid metabolism in rat peritoneal mast cells. In order to clarify the relationship between the histamine-releasing activity and the arachidonic acid metabolism-stimulating activity of thapsigargin in mast cells, the effects of cyclooxygenase inhibitors, indomethacin and ibuprofen, a lipoxygenase inhibitor, AA861, and dual inhibitors for cyclooxygenase and lipoxygenase, nordihydroguaiaretic acid and BW755C, on histamine release and arachidonic acid metabolism were examined. High-performance liquid chromatography analysis revealed that the peritoneal mast cells preferentially produce prostaglandin D2 by thapsigargin treatment. These inhibitors suppressed thapsigargin-induced prostaglandin D2 production in a dose-dependent manner, but failed to inhibit histamine release, suggesting that the mechanisms for stimulation of histamine release by thapsigargin is not dependent on increased arachidonic acid metabolism. Time-course experiments of histamine release and the release of radioactivity from [3H]arachidonic acid-labeled mast cells also provide evidence for a difference in mechanism.     

Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages

We have investigated the effect of tumor necrosis factor on the release of interleukin-1 and PGE2 from murine resident peritoneal macrophages. Tumor necrosis factor causes an increase in the production of interleukin-1 and PGE2 with a maximum induction for both noted at 5.9 X 10(-8) M. While indomethacin decreased tumor necrosis factor induced PGE2 production, this cyclooxygenase inhibitor augmented tumor necrosis factor induced interleukin-1 production. Our data suggests that tumor necrosis factor may be an important immunopotentiating agent in addition to its previously described cytolytic and metabolic activities.     

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.     

Endothelin-1 and endothelin-3 stimulate prostaglandin E2 secretion from the rat median eminence.

The in vitro effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the release of prostaglandin (PG)E2 from the rat median eminence were investigated. The addition of ET-1 from 10(-9) M to 10(-6) M stimulated PGE2 release in a dose-dependent manner (from 10.5 +/- 2.1 to 54.4 +/- 5.6 pg/ME fragment/30 min; mean +/- SEM, p less than 0.001). ET-3 also stimulated the release of PGE2 from 10(-7) M to 10(-5) M dose dependently (from 18.1 +/- 0.7 to 60.9 +/- 17.4 pg/ME fragment/30 min p less than 0.05). The time course effect of ET-3 (10(-6) M) showed that PGE2 release was stimulated within five minutes (control, 1.5 +/- 0.5; ET-3, 15.8 +/- 3.0 pg/ME fragment/5 min, p less than 0.01). These results suggest that ET-1 and ET-3 have some physiological effects on the rat median eminence.     

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.     

Structure-activity relations of inhibitory effects of various flavonoids on lipopolysaccharide-induced prostaglandin E2 production in rat peritoneal macrophages: comparison between subclasses of flavonoids.

A study of the structure-activity relations of the inhibitory effect of flavonoids on lipopolysaccharide (LPS)-induced prostaglandin production was carried out via a comparative examination of 39 flavonoids and related compounds. A comparison of the subclasses showed that flavones were most effective, followed by flavanones. Flavonols were less effective than those two groups. These results suggest that the C2-C3 double-bond and 4-oxo functional group of the C-ring are important factors for the high inhibition activities. Flavonoids showed the strongest inhibitory effect on the expression of Cox-2 protein. These results help to explain part of the reason for the pharmacological efficacy of flavonoids as anti-inflammatory compounds.     

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.     

Early events of the exogenously provided L-Carnitine in murine macrophages,T- and B-lymphocytes: modulation of prostaglandin E1 and E2 production in response to arachidonic acid

L-carnitine is an essential energy-providing compound to the cell since it transports long chain fatty acids through the mitochondrial membrane and delivers them to the beta-oxidation pathway for catabolism and/or entrance to biosynthetic pathways. Some of the early events taking place in immune cells after L-carnitine inoculation in vitro are defined in this report. Using arachidonic acid as a fatty acid source, we determined the utilization rate of L-carnitine by murine T-, B-lymphocytes and macrophages within two hours of cell culture, its effect on prostaglandin E1 and E2 production and the levels of beta-hydroxy-butyrate. The results show that although all immune cells consume a small portion of L-carnitine, beta-hydroxy-butyrate decreases upon addition of arachidonic acid and/or L-carnitine indicating that active biosynthetic pathways are induced. L-carnitine is shown to increase the arachidonic acid-induced production of prostaglandins E1 and E2 in macrophages, while their secretion from T- and B-lymphocytes is decreased. These findings indicate the L-carnitine may very rapidly alter the activation state of immune cells and lead to the development of various reactions, beneficial or not to the organism.     

Competition for adenyl cyclase coupled (3H)-prostacyclin binding sites with prostaglandin E2 in rat peritoneal macrophages

Prostaglandins regulate macrophage function by their action on membrane-associated adenyl cyclase. In order to define more directly macrophage-prostaglandin interactions, a binding assay has been developed for macrophage receptors using (3H)-PGI2 as ligand. (3H)-PGI2 binding was specific, saturable and reversible. Moreover, specific binding showed to be enriched in a membrane-enriched fraction of the cells. The assay conditions ensured stability of (3H)-PGI2 during incubations and should exclude intracellular accumulation of the ligand in macrophages. Unlabelled PGE2 and PGI2 competed for (3H)-PGI2 specific binding in both macrophages and membrane preparations. PGE2 showed to be more potent in this respect than PGI2, a phenomena which was also observed for prostaglandin activation of cAMP production in macrophages. The data suggest an interaction at receptor level of endogenously released PGE2 and PGI2 by peritoneal macrophages in vivo and provide support for a previously proposed mechanism of action of low concentrations of PGE2, counteracting stimulation of cAMP production by PGI2 in macrophages.     

Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E2 and cAMP

Human rTNF-alpha stimulates the metabolism of murine peritoneal macrophages as demonstrated by an increased consumption of arginine and an increased release of L-ornithine. This TNF-mediated effect is augmented by several substances that raise the intracellular concentration of cAMP, including PGE2, cholera toxin, and dibutyryl-cAMP. TNF also stimulates the endogenous production of PGE2 in cultures of peritoneal macrophages. The addition of the cyclo-oxygenase inhibitor, indomethacin, suppresses the TNF-mediated metabolic activation of macrophages, and this suppressive effect of indomethacin is overcome if exogenous PGE2 or cholera toxin is added to the culture. Taken together, the experiments indicate that the TNF-induced production of PGE2 and the PGE2-induced increase of the intracellular cAMP concentration are essential elements of an auto-regulatory loop that controls the magnitude of the TNF-mediated effect in the macrophage.     

Role of prostaglandin E2 in peptidoglycan mediated iNOS expression in mouse peritoneal macrophages in vitro

Many extracellular stimuli, e.g. microbial products, cytokines etc., result in the expression of inducible nitric oxide synthase (iNOS) in macrophages. However, it is not known whether expression of the iNOS gene in response to microbial products is a primary response of macrophages, or is the result of paracrine/autocrine signalling induced by endogenous biomolecules that are synthesised as a result of host cell-microbe interaction. In this paper we demonstrate that iNOS expression in mouse peritoneal macrophages in response to bacterial peptidoglycan (PGN) is a secondary effect requiring autocrine signalling of endogenously produced prostaglandin E2, and that PGN stimulation is mandatory, but not sufficient in itself, for induction of iNOS expression.