Direct evidence for the presence of selective binding sites for (3H) prostaglandin E2 on rat peritoneal macrophages

A method is presented which provides for a simple and rapid determination of PGE2 receptors on viable peritoneal macrophages. Incubation of the harvested cells with (3H)PGE2 revealed specific binding of (3H)PGE2 by use of the Millipore filter assay system. Maximum binding was attained in the presence of 1 mM EDTA. Specific binding was saturable at 65 fmol/mg protein with an equilibrium dissociation constant (Kd) of 3.2 X 10(-8)M. Inhibition of (3H)PGE2 binding with unlabelled prostaglandins revealed a potency series of PGE2 greater than PGE2 greater than PGI2. The PGE2 concentration which displaced 50% of the labelled ligand was 10(-7)M. Comparable kinetic data were obtained for adenylate cyclase stimulation, since the concentration which showed a halfmaximal stimulation of cAMP production was 2 X 10(-7)M of PGE2. Since PGE1 and PGI2 compete with (3H)PGE2 binding in a non-parallel manner compared to PGE2 itself, it is proposed that macrophages possess different types of PG receptors.     

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.     

Regulation of prostaglandin E2 receptors in vivo by dietary fatty acids in peritoneal macrophages from rats

Groups of rats were pretreated with 4-week diets containing 12.5% corn oil or linseed oil. At the end of this period peritoneal macrophages were elicited and isolated. These cells were used for binding experiments with 3H-PGE2 and for estimation of prostaglandin-stimulated cAMP production. Specific binding of 3H-PGE2 was saturable, reversible, protein-dependent, and correlated with stimulation of cAMP production, indicating that specific binding referred to receptor binding. PGE1 and PGI2 were far less effective than PGE2 in competition of binding with 3H-PGE2, indicating receptor selectivity for PGE2. Scatchard analysis of the specific binding data revealed a high affinity component (Kd 17 nM) and low affinity component. The total number of high- and low-affinity binding sites, respective Kd values, and PG stimulation of cAMP production of cells from rats fed the linseed oil diet were comparable to controls. The corn oil diet, however, resulted in a twofold increase in total number of high- and low-affinity binding sites, while respective Kd values were unchanged. This enhancement of binding capacity could be explained by an increased density of binding sites on the cells, and may itself be responsible for the increased sensitivity of the macrophages in this diet group for PG-stimulated cAMP production. The data suggest a regulatory mechanism at the receptor level and are discussed in terms of possible altered bioavailability of arachidonic acid-derived PGE2.     

Regulation of macrophage-mediated tumor cell killing by prostaglandins: comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), the latter detected as its stable metabolite, 6-keto PGF1 alpha. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10(-7)M for PGI2 and 3 x 10(-8)M for PGE2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE2 and PGI2; however, only PGE2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

Potentiation of PGE2-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.     

Differential mRNA expression of prostaglandin receptor subtypes in macrophage activation

Assessing the regulation of macrophage receptors for prostaglandin (PGE2) is essential to understanding the control which that potent lipid mediator has in modulating macrophage activities. The purpose of this study was to assess the differential mRNA expression of PGE2 receptor subtypes (EP) during macrophage exposure to activating and transducing agents. RAW 264.7 macrophages constitutively expressed mRNA for EP2,EP3 and EP4 receptor subtypes. Messenger RNA for EP4 was expressed at a much higher level when compared to EP2 in unstimulated macrophages as assessed by kinetic quantitative RT-PCR. When macrophages were stimulated with LPS, EP2 m RNA levels were 12-fold higher when compared to unstimulated macrophages, while EP4 m RNA remained unchanged. Conversely, mRNA levels of both EP2 and EP4 receptors were lower after macrophages were treated with IFN-gamma. Messenger RNA levels of both receptors were lower in macrophages after treatment with PGE2 or dibutyryl (db) cAMP Addition of the PKA inhibitor H89 reversed the effects of PGE2 and dbcAMP to varying degrees. Proteosome and p38 MAP kinase inhibitors blocked the LPS-stimulated increase in EP2 mRNA levels. Those inhibitors had no effect on EP4 mRNA.Thus, activating agents such as LPS and IFN-gamma may differentially regulate mRNAfor PGE2 receptor types in macrophages but the ligand and its associated signal transducing factors probably have similar regulatory effects.     

Synthetic prostacyclin analogs differentially regulate macrophage function via distinct analog-receptor binding specificities

PGI(2) (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI(2), in the regulation of both innate and acquired immunity. As PGI(2) is unstable, we sought to define the effects of various PGI(2) analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G(alphas) protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI(2) analogs and more closely resembled the effects of PGE(2). Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI(2) analogs. These studies are the first to explore the capacity of PGI(2) to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.     

3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.     

Prostaglandins as endogenous mediators of interleukin 1 production

We examined the role of cyclooxygenase (CO)-derived metabolites of arachidonic acid (AA) in the regulation of interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated murine resident peritoneal macrophages. The use of LPS proved to be an efficacious probe, because it stimulated both IL 1 production and AA metabolism via only the CO pathway. The production of the CO metabolites prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2; measured as its stable metabolite 6-Keto prostaglandin F1 alpha) by LPS-stimulated macrophages was demonstrated by high pressure liquid chromatography and radioimmunoassay. The addition of exogenous PGE2 or PGI2 resulted in a dose-dependent suppression of macrophage IL 1 production. Inhibitors of the CO pathway (indomethacin, piroxicam, and ibuprofen) caused a dose-dependent augmentation in the LPS-induced IL 1 response. This augmentation directly correlated with the efficacy of the compounds as CO inhibitors. Similar results were found when macrophage-derived fibroblast growth factor was assessed. The addition of exogenous IL 1 to macrophage cultures caused an increase in the levels of PGE2, over a narrow dose range (0.05 to 0.6 IL 1 units). These studies provide detailed evidence that AA metabolites synthesized via the CO pathway can modulate the production of growth factors by LPS-stimulated macrophages. In addition, our data support the concept that IL 1, as with classical hormones, can regulate its own production through a self-induced inhibitor, PGE2.     

Factors affecting the termination of cardiovascular actions of 10,10-difluoro,13-dehydroprostacyclin

Experiments were conducted to determine why 10,10-difluoro,13-dehydroprostacyclin (DF2-PGI2) has a long vascular relaxant activity in vitro but like PGI2 has a short duration of effect in vivo. DF2-PGI2 produced depressor responses in anesthetized dogs which were not affected by nephrectomy suggesting that the kidney was not responsible for the termination of action. DF2-PGI2 given intravenously or into the ascending aorta produced depressor responses of a similar magnitude but injection of the same doses into the hepatic portal circulation resulted in a large attenuation of responses. The data suggest hepatic, but not pulmonary, metabolism of DF2-PGI2. Injection or infusion of PGI2 and DF2-PGI2 into the hindlimb circulation caused vasodilatation of a similar duration suggesting diffusion from tissue sites as another mechanism of termination of action.     

Characterization of prostaglandin (PG)-binding sites expressed on human basophils. Evidence for a prostaglandin E1, I2, and a D2 receptor.

Recent data suggest that prostaglandins (PGs) are involved in the regulation of basophil activation. The aim of this study was to characterize the basophil PG-binding sites by means of radioreceptor assays using 3H-labeled PGs. Scatchard analysis for pure (greater than 95%) chronic myeloid leukemia (CML) basophils revealed two classes of PGE1-binding sites differing in their affinity for the natural ligand (Bmax1 = 217 +/- 65 fmol/10(8) cells; Kd1 = 0.5 +/- 0.2 nM; Bmax2 = 2462 +/- 381 fmol/10(8) cells; Kd2 = 47 +/- 20 nM; IC50 = PGE1 less than PGI2 less than PGD2 less than PGE2 less than PGF2 alpha) as well as two classes of PGI2 (iloprost)-binding sites (Bmax1 = 324 +/- 145 fmol/10(8) cells; Kd1 = 0.5 +/- 0.3 nM; Bmax2 = 2541 +/- 381; Kd2 = 27 +/- 6 nM; IC50 = PGI2 less than PGE1 less than PGD2 less than PGE2 less than PGF2 alpha. In addition, CML basophils exhibited a single class of PGD2-binding sites (Bmax = 378 +/- 98 fmol/10(8) cells; Kd = 13 +/- 4 nM; IC50: PGD2 less than PGI2 less than PGE1 less than PGE2 less than PGF2 alpha). In contrast, we were unable to detect specific saturable PGE2-binding sites. Primary and immortalized (KU812) CML basophils revealed an identical pattern of PG receptor expression. Basophils (KU812) expressed significantly (p less than 0.001) lower number of PGE1 (PGI2)-binding sites (Bmax1: 9% (20%) of control; Bmax2: 36% (50%) of control) when cultured with recombinant interleukin 3 (rhIL-3), a basophil-activating cytokine, whereas rhIL-2 had no effect on PG receptor expression. Functional significance of binding of PGs to basophils was provided by the demonstration of a dose-dependent increase in cellular cAMP upon agonist activation, with PGE1 (ED50 = 1.7 +/- 1.1 nM) and PGI2 (ED50 = 2.8 +/- 2.3 nM) being the most potent compounds. These findings suggest that human basophils express specific receptors for PGE1, PGI2 as well as for PGD2.     

Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides

PGE2 has previously been shown to suppress various leukocyte functions. In this study, we examined whether PGE2 would affect release of TNF-alpha from rat resident peritoneal macrophages. Two different, dose-dependent effects were observed: low PGE2 concentrations (0.1 to 10 ng/ml) stimulated, whereas higher concentrations (greater than 10 ng/ml) suppressed TNF-alpha release. PGE2-stimulated TNF-alpha production was dependent on de novo protein synthesis and was associated with an intracellular rise of cGMP. The importance of cGMP as an intracellular messenger for PGE2 was confirmed by the following evidence: (1) low PGE2 concentrations preferentially increased cGMP and not cAMP and (2) cGMP, either exogenously added or endogenously generated by sodium nitroprusside, were efficient stimulators of TNF-alpha production. In contrast, agents increasing intracellular cAMP concentrations such as PGE1, higher PGE2 doses, isoproterenol, and theophylline, all suppressed TNF-alpha synthesis. Only resident, but not casein-elicited or Corynebacterium parvum-activated macrophages, were stimulated by low PGE2 concentrations to increase TNF-alpha production. In tumor cytotoxicity assays, PGE2-activated macrophages were active only against TNF-alpha-sensitive target cells. These findings demonstrate that TNF-alpha synthesis in macrophages is up-regulated by cGMP and down-regulated by cAMP, which indicates that cyclic nucleotides act as intracellular messengers for extracellular signals of macrophage activation.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

Binding to prostaglandin (PG) receptors and activation of adenyl cyclase by 20-isopropylidene-PGS in rabbit platelet membranes

20-Isopropylidene-PGE1 (Isop-PGE1) was about 10 times more potent than PGE1 in inhibition of thrombin-induced aggregation of rabbit washed platelets. Likewise, 20-isopropylidene-17(R)-methyl-carbacyclin (CS-570), a stable PGI2 analogue, was more potent than carbacyclin in the anti-aggregatory activity. In order to define the platelet-prostaglandin interactions, a binding assay was done using platelet membranes with [3H]-PGE1 as a radioligand. Isop-PGE1 (IC50 = 0.18 microM) bound to the PG receptors more potently than PGE1 (IC50 = 2.1 microM). CS-570 (IC50 = 0.39 microM) was more potent than carbacyclin (IC50 = 1.9 microM). These indicate that introduction of an isopropylidene group to the carbon 20 of PGs increases the binding ability to the receptors. These PGE1 and PGI2 analogues activated platelet membrane adenyl cyclase and increased intracellular cAMP levels with the same potency series obtained in the binding experiments. All these results suggest that the binding to the receptors by these PGs is coupled to the activation of adenyl cyclase, followed by the increase in cAMP levels in platelets and the inhibition of platelet aggregation. Thus, the increased anti-aggregatory activity of 20-isop-PGs may be explained by their increased affinity for the PG receptors and stimulation of adenyl cyclase. 15-Epimeric-20-isopropylidene-PGE1 (15-Epi-isop-PGE1), which has an unnatural configuration of the 15-hydroxyl group, was much less potent than isop-PGE1 in the binding experiment and the other three investigations. This indicates that the configuration of the 15-hydroxyl group is important for the binding to the PG receptors and the consequent activities in platelets.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.