11,12-Epoxyeicosatrienoic Acid Attenuates Synthesis of Prostaglandin E 2 in Rat Monocytes Stimulated with Lipopolysaccharide

Abstract

Cytochrome P-450 monooxygenase (epoxygenase)-derived arachidonic acid (AA) metabolites, including 11,12-epoxyeicosatrienoic acid (11,12-EET), possess anti-inflammatory and antipyretic properties. Prostaglandin E₂ (PGE₂), a cyclooxygenase (COX)-derived metabolite of AA, is a well-defined mediator of fever and inflammation. We have tested the hypothesis that 11,12-EET attenuates synthesis of PGE₂ in monocytes, which are the cells that are indispensable for induction of fever and initiation of inflammation. Monocytes isolated from freshly collected rat blood were stimulated with lipopolysaccharide (LPS; 100 ng/2 × 10⁵ cells) to induce COX-2 and stimulate generation of PGE₂. SKF-525A, an inhibitor of epoxygenases, significantly augmented the lipopolysaccharide-provoked synthesis of PGE₂ in cell culture in a concentration-dependent manner. It did not affect, however, elevation of the expression of COX-2 protein in monocytes stimulated with LPS. 11,12-EET also did not affect the induction of COX-2 in monocytes incubated with lipopolysaccharide. However, 11,12-EET suppressed, in a concentration-dependent fashion, the generation of PGE₂ in incubates. Preincubation of a murine COX-2 preparation for 0–5 min with three concentrations of 11,12-EET (1, 5, and 10 μM) inhibited the oxygenation of [¹⁴C]-labeled AA by the enzyme. The inhibitory effect of 11,12-EET on COX-2 was time-and-concentration-dependent, suggesting a mechanism-based inhibition. Based on these data, we conclude that 11,12-EET suppresses generation of PGE₂ in monocytes via modulating the activity of COX-2. These data support the hypothesis that epoxygenasederived AA metabolites constitute a negative feedback on the enhanced synthesis of prostaglandins upon inflammation.

In addition to cyclooxygenases (COXs) and lipoxygenases (LOXs), the oxidative metabolism of arachidonic acid (AA) also includes multiple microsomal cytochrome P-450 enzymes collectively referred to as monooxygenases (1). They convert free AA into three types of eicosanoid products via reactions consisting of allylic oxidation, ω-terminal hydroxylation, and olefin epoxidation. The latter, also referred to as the epoxygenase reaction, results in the production of four cis-epoxyeicosatrienoic acids (EETs): 5,6-, 8,9-, 11,12-, and 14,15-EET (2). In mammals, expression of monooxygenases and synthesis of EETs has been detected, among other tissues, in the brain, lungs, liver and kidney, and in cells such as astrocytes, hepatocytes, and endothelium (1, 3). Monocytes, the cells playing a key role in inflammation (4), contain various P-450s capable of EET biosynthesis (5, 6) and, in addition, they express specific high affinity receptors for EETs (7).

The biology of COXs and LOXs has been extensively studied because their eicosanoid products play fundamental roles in health and disease and, increasingly, as therapeutic targeting agents (8). Inhibitors of COXs are the mainstays of current therapy aimed to modulate pain and inflammation and to control fever (9). It has been well documented that COX exists in two isoforms, COX-1 and COX-2 (10). In simplified terms, COX-1 is a constitutive housekeeping enzyme, whereas COX-2 is inducible and can be markedly upregulated during stress, pain, inflammation, and fever. COXs are bifunctional enzymes that catalyze the oxygenation of AA to unstable prostaglandin G₂ (PGG₂), followed by the reduction of the lipid hydroperoxide PGG₂ to alcohol PGH₂ (11). PGH₂ serves as substrate for the respective synthases generating thromboxane (in platelets), prostacyclin (in endothelial cells), and prostaglandins such as PGE₂ (in many cell types, including monocytes). Induction of COX-2, e.g., by an injection of fever-inducing inflammatory agents, such as lipopolysaccharide (LPS), and/or mediators, such as cytokines, is accompanied by a burst of large quantities of PGE₂, which can be detected in plasma and cerebrospinal fluid of the febrile animals (12, 13). PGE₂ is regarded as the proximal mediator of fever (14, 15). In contrast to COX-2, lipoxygenase products do not play a significant role in fever (see Ref. 16 and references therein).

The demonstration that AA is an endogenous substrate for cytochrome P-450 has stimulated investigations of the biologic roles of this pathway and its metabolites (1, 3, 17). Among other physiological activities, EETs have been suggested to be an endothelium-derived hyperpolarizing factor causing vasorelaxation (18, 19), a feature frequently possessed by anti-inflammatory and antipyretic agents (20). Node et al. (21) reported an anti-inflammatory activity of EETs, demonstrating that EETs inhibit the induction of cell adhesion molecules (CAMs) in endothelial cells stimulated with tumor necrosis factor-α and interleukin-1α (IL-1α), and with LPS. Maximal inhibition of CAM expression was achieved with 11,12-EET, followed by 8,9-EET, and 5,6-EET. Up-regulation of the expression of various CAMs in endothelium plays a central role in early stages of the inflammatory process. Similarly, a cytokine- and PGE₂-mediated LPS-induced fever is an early symptom of acute inflammation (22). In fact, it has been shown that efficacy of the induction of adhesion molecules depends on febrile temperature (23, 24), indicating that there is an intrinsic molecular coordination of these two critical components of acute inflammation. COX-2 and PGE₂ may be central to this mechanism since the induction of COX-2 accounts for both fever (25, 26) and stimulation of the expression of adhesion molecules (27). Findings demonstrating that EETs participate in the initial steps of inflammation implicate the involvement of these metabolites in fever.

Data from our laboratory (28) and by others (29) revealed that EETs attenuate fever in rats injected with LPS. Similar to the in vitro results reported by Node et al. (21), 11,12-EET was also the most potent isomer inhibiting fever in rats (28, 29). In separate studies, we have shown that compounds known to induce various P-450s attenuated fever, while animals injected with inhibitors of monooxygenases, e.g., with SKF-525A or 1-aminobenzotriazole, displayed significantly higher fevers compared to that treated with control vehicle (16, and references therein). Therefore, in addition to the assumption that epoxygenase metabolites mediate anti-inflammation (21), we also concluded that the epoxygenase enzyme family is part of the endogenous antipyresis mechanism; the process that limits the activity of pro-febrile pathways and prevents fever from reaching lethal heights (16). The mechanism responsible for the antipyretic effects of EETs is unknown. We have found, however, that augmentation of fever in rats injected with LPS and treated with SKF-525A was associated with significantly higher levels of PGE₂ in plasma and cerebrospinal fluid, compared to rats treated with LPS and vehicle for the inhibitor (12). Based on these data, we hypothesize that EETs attenuate fever in rats in part via suppression of the synthesis of PGE₂. In the present report we demonstrate that 11,12-EET attenuates generation of PGE₂ in rat mononuclear cells stimulated with LPS. 11,12-EET did not affect the monocyte COX-2 protein levels analyzed by Western blotting. It did, however, inhibit the activity of purified COX-2 in a direct assay.

Materials and Methods

Animals.

Specific pathogen-free, young adult male Sprague–Dawley rats (Charles River, Portage, MI) weighing 250–300 g were used for the preparation of primary cultures of blood monocytes (see below). Animals were housed in plastic cages in a temperature-controlled room at 26 ± 1°C with a 12:12-hr light-dark cycle (lights on at 0600). Teklad Rodent Diet W8604 and drinking water were provided ad libitum.

Reagents.

SKF-525A (proadifen; 2-diethyl-aminoethyl-2,2-diphenyl-n-pentanoate) was purchased from BIOMOL (Plymouth Meeting, PA). Prostaglandin E₂, 11,12-EET, and COX-2 electrophoresis standard were supplied by Cayman Chemical Company (Ann Arbor, MI). Diethyl ether was purchased from Aldrich Chemical Co. (Milwaukee, WI). Methanol was from Burdick and Jackson (Muskegan, MI). Lipopolysaccharide (LPS; E. coli serotype 0111:B4), 1-aminobenzotriazole (ABT), butylated hydroxyanisole, hematin, phenol, and tris[hydroxymethyl]aminomethane (Tris) were purchased from Sigma (St. Louis, MO). [¹⁴C]AA was from NEN (Boston, MA). Silica gel 60A thin-layer chromatography plates were purchased from Whatman (Clifton, NJ). All other reagents and preparations were obtained as indicated.

Rat Mononuclear Cell Fraction Preparation.

Mononuclear cells were obtained as described elsewhere (30). Heparinized blood was collected from anesthetized rats (isoflurane inhaled anesthesia) by cardiac puncture. Whole blood was diluted 1:1 (vol/vol) with calcium- and magnesium-free Hanks’ balanced salt solution (HBSS; Gibco BRL Products, NY; Cat. No. 14170) at room temperature. A portion of 4 ml of diluted blood was carefully layered onto 3 ml of the separation medium in a 10 ml centrifuge tube and centrifuged for 25 min at 800g at room temperature. Separation medium was an aqueous solution of density 1.077 g/ml, consisting of 5.7 g Ficoll 400 and 9 g sodium diatriozate per 100 ml (Ficoll-Paque Plus, Amersham Pharmacia Biotech, code No. 17-0840-02). The mononuclear cell fractions were pooled and washed twice, each time in 2 vols of HBSS, and finally suspended at 1 × 10⁶ cells/ml in RPMI medium 1640 (Gibco BRL Products, NY; cat. no. 22400) containing L-glutamine and HEPES buffer, and supplemented with fatty acid-free rat albumin (Sigma, cat. no. A 2018) to a final concentration of 0.1%. The method results in a greater than 95% viable mononuclear cells in the suspension (by trypan blue dye exclusion assay; 26). Freshly isolated monocytes were immediately seeded at an exact density of 2 × 10⁵ cells/well (in a volume of 200 μl) into Costar 96-well tissue culture plates (Corning, NY). The cells were pre-incubated for 1 hr at 38.5°C, 5% CO₂, and 100% humidity prior to addition of LPS and other test agents to the culture (38.5°C was selected for the incubation since it reflects body temperature of the Sprague-Dawley rat in response to a pyrogenic dose of LPS) (12).

Stimulation of Monocytes.

After 1-hr preincubation (see above), the cells were stimulated with LPS added in a volume of 10 μl from stock (10 μg/ml; saline solution) to a final concentration of 100 ng/2 × 10⁵/well. Test reagents in a volume of 10 μl each (or appropriate control vehicle) were added simultaneously (see the Results section), and cells were further cultured at 38.5°C, 5% CO₂, and 100% humidity for the time as indicated in the figure legends. Supernatants were aspirated, centrifuged for 5 min at 1000g and stored frozen at −70°C before assays.

PGE₂ Assay in Supernatant of Stimulated Monocytes.

Prostaglandin levels were determined in duplicate using a highly sensitive colorimetric assay kit from R&D Systems (Minneapolis, MN; cat. no. DE2100). Briefly, 50 μl of standard/sample was pipetted into a precoated goat anti-mouse 96-well plate. Aliquots (50 μl) of a mouse polyclonal PGE₂ antibody and IgG conjugated alkaline phosphatase were added to each well and allowed to incubate at room temperature for 1 h. After incubation, wells were washed six times with 400 μl of 1× PBS + 0.5% Tween 20, followed by the addition of 150 μl of 3,3′,5,5′-tetramethylbenzidine substrate. Wells were read at 670 nm with an ELISA reader (model EL 312; Biotek Instruments) 30 min after addition of substrate.

Detection of COX-2 Protein in Monocytes by Western Blot.

In separate experiments, cells (2 × 10⁵/well) were harvested for the times indicated in the figure legends, briefly centrifuged and resuspended in 100 μl of HBSS. An equal volume of 125 mM Tris-HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.005% bromophenol blue, and 4% β-mercaptoethanol was added and the sample boiled for 7 min. Total protein (100 μg; determined by the method of Bradford) was separated by SDS-PAGE (10% acrylamide) along with low molecular weight markers (Bio-Rad) and 72 kDa COX-2 denaturated standard for electrophoresis (Cayman Chemical, Ann Arbor, MI; cat. no. 360120) and transferred to hybond-ECL membranes (Amersham Pharmacia Biotech). Membranes were blocked for 1 h with 5% nonfat milk in 1× Tris-buffered saline, 0.1% Tween 20, and were then probed for 1 hr with primary antibody (anti-COX-2 polyclonal goat antibody specific for human, rat, and mouse COX-2; Santa Cruz Biotechnology, Santa Cruz, CA) followed by 1 hr with β-actin (Sigma) goat antibodies, diluted in 5% nonfat milk in 1× Tris-buffered saline, 0.1% Tween 20. The membranes were washed in PBS, 0.1% Tween and incubated for 20 min with horseradish peroxidase-linked anti-goat immunoglobulin (Amersham). COX-2 and β-actin protein were visualized using the Renaissance Western blot chemiluminescence reagent (NEN Life Science Products). Relative densitometry values were assessed using Quantity One scanning densitometry software.

Incubation of COX-2 with [¹⁴C]AA and 11,12-EET.

Wild-type murine COX-2 was a generous gift of Dr. Lawrence J. Marnett and was expressed in SF-9 cells (Novagen, Madison, WI), and purified as previously described (31). The COX-2 preparation (specific activity 109 mol AA/min/mol enzyme) was preincubated on ice for 20 min with 2 M equivalents of hematin in Tris-HCl buffer pH 8.0, 500 μM phenol. To examine the time-dependency for COX-2 inhibition, the solution was preincubated from 0–5 min at 37°C in the presence or absence of 11,12-EET (final concentration 10 μM). For subsequent inhibition assays, the COX-2-hematin solution was preincubated for 5 min at 37°C in the presence or absence of 11,12-EET (final concentration 1, 5, and 10 μM). 20 μl of [¹⁴C]AA (4.8 nCi, 0.5 μM final concentration) in Tris HCl buffer pH 8.0 was warmed at 37°C for 2 min. The reaction was initiated by adding COX-2 (10 nM final concentration) to a total volume of 200 μl. The reaction was terminated after 8 sec by the addition of 200 μl of ice-cold diethyl ether: methanol: 4 M citric acid (30:4:1) containing 8 μg of butylated hydroxyanisole as antioxidant and 8 μg of unlabeled AA as a carrier.

The organic layer was loaded on a silica plate and eluted with the organic phase of ethyl acetate: isooctane: water: glacial acetic acid (45:25:50:1). TLC plates were analyzed for radioactivity by a Bioscan AR-2000 imaging scanner (Bioscan, Washington, DC). Graphical analysis was performed using Win-Scan software (Bioscan, Washington, DC). COX-2 activity was expressed as activity relative to control (no inhibitor). Activity was determined as pmol AA converted to product per minute per pmol enzyme. Experiments were performed in duplicate on two separate occasions. Time-dependence experiments were performed on a single occasion.

Statistical Analysis.

Results, expressed as mean ± SEM, were analyzed by analysis of variance followed by the Newman-Keuls Student’s t test with the level of significance set at P < 0.05.

Results

SKF-525A and ABT, Inhibitors of Monooxygenases, Augment the Secretion of PGE₂ from Monocytes Stimulated with LPS.

LPS is a potent activator of prostaglandin synthesis and, as expected, triggered a rapid release of PGE₂ from purified rat monocytes (Fig. 1). As can be seen, supernatants from the cells incubated with LPS (100 ng/2 × 10⁵ cells) show that the release of PGE₂ reached a peak level after two hours of incubation. Therefore, for the following experiments we have chosen a 2-hr incubation schedule to examine the effects of cytochrome P-450 modulators and epoxygenase metabolites on LPS-induced synthesis and release of PGE₂ from rat mononuclear cells.

In the experiments shown in Figure 2 , LPS (100 ng/2 × 10⁵ cells) and SKF-525A (upper panel) or ABT (lower panel) were administered simultaneously to the cell cultures at concentrations as shown. The levels of PGE₂ in supernatants of monocytes treated with LPS and, separately, with two monooxygenase inhibitors were significantly higher (P < 0.05; average of three experiments, assay in duplicates) from that of LPS and vehicle (saline) for all concentrations studied. The amount of PGE₂ in supernatants from incubates treated with SKF-525A and ABT at concentrations of 0.1 mM were significantly greater than that at 0.01 mM (P = 0.001, n = 3 and 4 experiments for each inhibitor, respectively; PGE₂ assay in duplicates). In the experiment with SKF-525A, the difference between 0.1 mM and 1 mM was not significant (P > 0.05), indicating that the SKF-525A-provoked exaggeration of PGE₂ release reached saturation at a concentration of 0.1 mM in our incubation assay. In the experiment using ABT, however, supernatants from incubates containing 1 mM of the inhibitor showed depleted amount of PGE₂ compared to that of harvested from monocytes treated with ABT at 0.1 mM. It may indicate that ABT at a concentration of 1 mM suppressed the activity of cyclooxygenases or affected functional integrity of the cells. This effect of ABT at 1 mM concentration has not been further investigated in our studies.

Overall, these in vitro data demonstrate that two structurally dissimilar inhibitors of monooxygenases exacerbate the LPS-induced generation of PGE₂ in cultured monocytes. These data complement our previous in vivo observation (12) that injection of the inhibitor of monooxygenase into febrile rats resulted in exacerbation of the plasma and cerebrospinal fluid levels of PGE₂. We reasoned, based on these data, that one of the possibilities that monooxygenase inhibitors exacerbated the levels of PGE₂ was the result of a suppression of negative feedback on the prostaglandin synthesis machinery, which, under normal conditions, is presumably imposed by monooxygenase-dependent metabolites. To test this hypothesis, we determined the effect of 11,12-EET on the release of PGE₂ by rat monocytes stimulated with LPS.

11,12-EET Inhibits PGE₂ Secretion by Monocytes Stimulated with LPS and Attenuates the Enhancing Effect of SKF-525A on the Release of PGE₂.

As can be seen in Figure 3, addition of 11,12-EET to the culture inhibited the release of PGE₂ from the cells stimulated with LPS (mean ± SE for three experiments). The effect was concentration-dependent. Levels of PGE₂ in supernatants collected from incubates supplemented with 10 μM and 100 μM 11,12-EET, except of 1 μM 11,12-EET, were significantly lower from that of LPS/vehicle group (P = 0.001). The contents of PGE₂ of all three 11,12-EET groups were significantly different from each other (P < 0.05).

To further test the hypothesis that the effect of SKF-525A on the generation of PGE₂ was due to the inhibition of monooxygenases, we added 11,12-EET back to rat monocytes stimulated with LPS and incubated with SKF-525A. Figure 4 demonstrates the average data representative of four separate experiments (four repetitions). SKF-525A (100 μM) potentiated the effect of LPS (100 ng/2 × 10⁵ cells) on the generation of PGE₂ by monocytes. Addition of 11,12-EET (10 μM) blunted the effect of SKF-525A.

11,12-EET Does Not Affect the LPS-Induced Elevation of COX-2 Protein in Monocytes.

Up- and/or downregulation of COX-2 induction may account for the enhancement and reduction of PGE₂ synthesis in monocytes stimulated with LPS in the presence of SKF-525A and/or 11,12-EET, respectively. To test this hypothesis we used Western blot analysis to assess the expression of COX-2 protein in monocytes stimulated with LPS, with and without the respective agent. As expected, the amount of COX-2 protein in control monocytes increased following LPS addition; COX-2 expression was evident at 2 hr (Fig. 5 ).

We have not been able to determine any significant effect of SKF-525A and/or 11,12-EET on the LPS-induced elevation of the expression of COX-2 in monocytes analyzed at 2 h of incubation (Fig. 6 ). Collectively, these data indicate that changes in the LPS-stimulated levels of PGE₂ discussed above were not due to respective alterations in the induction of COX-2 in monocytes. We hypothesized, therefore, that 11,12-EET affected PGE₂ synthesis via inhibition of the enzymatic activity of COX-2.

11,12-EET Inhibits the Activity of COX-2 in a Concentration- and Time-Dependent Fashion.

Preincubation of the murine COX-2 preparation for 5 min with three concentrations of 11,12-EET (1, 5, and 10 μM) led to the inhibition of the activity of the enzyme in a concentration-dependent manner (Fig. 7 ) with an IC₅₀ of 6.43 ± 1.58 μM. The inhibitory effect of 11,12-EET on COX-2 was also time-dependent, i.e., the longer pre-incubation time of COX-2 with 11,12-EET the greater inhibition of the activity of COX-2 (Fig. 8 ). Interestingly, when the reaction was initiated without a prior co-incubation between COX-2 and 11,12-EET, the oxygenation of AA was enhanced compared to the control experiment in which no 11,12-EET was added (Fig. 8 ).

Discussion

Previous reports from our laboratory (28) and others (29) revealed that epoxygenase-derived eicosanoids, EETs, induce antipyresis, with the maximal inhibition of LPS-induced fever achieved with 11,12-EET, followed by 14,15-EET, and 8,9-EET (28). The mechanisms responsible for this inhibition have not been investigated. As noted, PGE₂, a COX-derived eicosanoid, is regarded as the proximal mediator of fever (14, 15). Therefore, we have investigated the influence of 11,12-EET on PGE₂ generation induced by bacterial pyrogen. The present results demonstrate that 11,12-EET, at low μM concentrations (Fig. 3), decreases in vitro PGE₂ production in rat monocytes stimulated with LPS. To our knowledge, the observation that 11,12-EET can affect the generation of PGE₂ in inflammatory cells stimulated with a bacterial product has not been previously reported.

Our results complement reports showing that EETs at micromolar concentrations reduce in vitro prostanoid production in non-immune cells such as smooth muscle cells (32) and platelets (33). It is worth noting however, that EETs are thought to exert their in vivo effects in sub-micromolar concentrations (1–3), and the higher concentrations employed in our studies and by others (32, 33) may not represent (patho)physiological concentrations. It is speculative that at sites of inflammation EET concentrations could reach micromolar levels, although this has not yet been investigated. In line with this speculation, one can hypothesize that intracellular colocalization of COX-2 and monooxygenases could lead to a locally high (e.g., micromolar) concentration of EET in the proximity of COX-2. In addition, the concentrations used in vitro may be better understood in light of a limited stability of EETs and their ability to penetrate cell membranes. For example, EETs may undergo hydroxylation by respective epoxy-hydrolases or may be incorporated into the membrane glycerophospholipids (3), thereby masking the action of these compounds when applied in vitro at physiological concentrations. Studies in our laboratory (28) and by others (29) have shown that EETs are antipyretic when administered into the rat brain at low micromolar concentrations. Nevertheless, the inhibitory effect of EETs on PGE₂ generation reported in in vitro studies needs to be confirmed in the in vivo settings.

Although the exact nature of the suppressive effect of EETs on the in vitro PGE₂ generation is unclear, other investigations suggest that EETs may directly inhibit COX activity. In studies by Fitzpatrick et al. (33), 11,12-EET at micromolar concentration inhibited the activity of COX-1 purified from a ram seminal vesicle by approximately 50%. Fang et al. (32) studied the effect of a 14,15-EET isomer on PGE₂ generation in smooth muscle cells. They reported that low micromolar concentrations of the EET inhibited PGE₂ production via a mechanism that did not reduce COX protein expression, concluding that 14,15-EET directly blocked AA metabolism by COX. Results of our studies using 11,12-EET in monocytes stimulated with LPS are consistent with this conclusion.

We demonstrate that 11,12-EET does not affect induction of COX-2 in monocytes stimulated with LPS. A direct inhibitory effect of 11,12-EET on the activity of COX-2 may therefore account for the suppression of PGE₂ generation by the monocytes. In support of this hypothesis, we show that 11,12-EET inhibits the activity of purified murine COX-2 (Figs. 7 and 8 ). The suppression of COX-2 activity by 11,12-EET was time-dependent, which is not characteristic of a competitive inhibitor. The potency of inhibition of COX-2 by 11,12-EET (IC₅₀ = 6.43 ± 1.58 μM) was approximately 58-fold greater than that of acetaminophen (IC₅₀ = 372 μM; ref. 34), and ca. 120-fold greater than that of sodium salicylate (IC₅₀ = 763 μM; ref. 35). It was, however, ca. 11-fold lower than that of ibuprofen (IC₅₀ = 0.60 μM; unpublished data).

The induction of COX-2 in immune and non-immune cells during inflammation and the resultant generation of PGE₂ represent important steps in the regulation of the inflammatory cascade. For this reason, there has been a great deal of research focused on the endogenous and exogenous factors activating prostaglandin synthesis, as well as on the pharmacological inhibition of COXs (8, 10). Although relatively less attention has been paid to the physiological (i.e., nonpharmacological) factors suppressing prostanoid synthesis, it appears that prostaglandin generation can be negatively controlled by endogenous systems. Glucocorticoids/lipocortins have been among the earliest discovered and most thoroughly studied endogenous systems that downregulate the synthesis of prostanoids (36). Inhibitory effects of steroids on the arachidonate cascade may occur by destabilizing the COX-2 transcript (37), and also via inhibiting the activity of phospholipase A₂, an enzyme responsible for the liberation of AA from cellular membrane phospholipids (38, 39). However, cytokines, such as IL-4, IL-10, transforming growth factor-β, and epidermal growth factor, are capable of inhibiting the generation of PGE₂ via a downregulation of the COX-2 mRNA in immune cells (40, 41). Also several members of the natriuretic peptide family have recently been reported to attenuate expression of COX-2 protein and mRNA, and, consequently, inhibit generation of PGE₂ in LPS-activated murine bone marrow-derived macrophages (42). Nitric oxide (NO) represents yet another endogenous factor that can modulate PGE₂ synthesis. It has been reported that NO inhibits expression of COX-2 mRNA in LPS-stimulated macrophages (43). However, the effect of NO on COX-2 transcription may be tissue- and/or stimulus-specific, since amplification of IL-1β-induced COX-2 expression in rat mesangial cells by NO and other cGMP-elevating factors have been demonstrated (44). In macrophages, however, analogs of both cAMP and cGMP, as well as stimulants of cAMP including PGE₂ itself, were reported to inhibit the expression of COX-2 (45).

Results of our studies using monocytes stimulated with LPS, as well as data presented by others using different cell types (32, 33), implicate EETs as endogenous suppressors of the synthesis of PGE₂. They, however, appear to directly inhibit COX-2 activity rather than the LPS-induced stimulation of COX-2 transcription. The generation of prostanoids during inflammation, therefore, is modulated by various endogenous factors acting at every level of the arachidonate cascade; modulating the cleavage of AA from membrane phospholipids, regulating the synthesis of COX-2 protein and, finally, modulating the catalytic activity of the enzyme.

Under normal conditions, inflammation is usually self-limited. Systemic/behavioral responses to proinflammatory stimuli, such as fever, hyperalgesia, suppression of the physical, and alimentary activities, as well as local responses such as edema, vasodilation, and increased blood flow, involve the AA cascade and generation of prostaglandins, which act synergistically with other inflammatory mediators. It is still not entirely clear how these adaptive systemic and behavioral responses are regulated and contained within safe limits (22). It is also not clear what processes account for the resolution of the inflammatory reaction (46). Results of our studies contribute to the hypothesis that AA metabolism yields both pro- and anti-inflammatory eicosanoids through a cascade inherently requiring tight regulation. This regulation of the AA pathway helps to control both the magnitude and duration of local and systemic/behavioral inflammatory reactions. Our previous studies on the role of EETs in the pathogenesis of fever support this assumption.

Fever is currently regarded as an adaptive response, and under normal circumstances its magnitude does not reach unsafe high levels (22). The current hypothesis of endogenous antipyresis predicts involvement of endogenous antipyretics (or cryogens) to prevent fever from reaching lethal heights (22). We have reported data supporting a role of cytochrome P-450, and EETs in particular, in the mechanism of limiting the height of fever (16). Induction of COX-2 and generation of PGE₂ is thought to be a key mechanism of pyrogenesis (14). Therefore, we speculate that the inhibition of COX-2 activity by epoxyeicosanoids may account for the antipyretic effect of these molecules. Because eicosanoids in general are clinically important metabolites of AA, to understand the regulation between PGs and EETs production is important in therapeutic practice.

Figure 1.

Concentration of PGE₂ (pg/ml) over time in culture supernatants from rat monocytes incubated with LPS (100 ng/ 2 × 10⁵ cells; closed circles) and saline as control (open circles). Concentration of PGE₂ was determined by as described in the Methods section. P = 0.001 at 2 hr for supernatants from LPS-treated versus control cells. Results are an average (± SE) of three experiments. Asterisks indicate significant elevation of PGE₂ in supernatants collected already after 1 and 2 hr of incubation of rat monocytes with LPS to that of control (saline). Supernatants collected 4, 8, and 12 h after the start of the incubation of monocytes with LPS also revealed significantly higher PGE₂ levels compared to control (saline), but not to that collected after 2 hr of incubation with LPS.

Figure 2.

Effect of SKF-525A (upper panel) and ABT (lower panel) on PGE₂ levels in supernatants of rat monocytes stimulated with LPS. Cells were prepared as described in the Methods section. Ten microliters of LPS dissolved in saline was added to a final concentration of 100 ng/well/2 × 10⁵ cells (saline, vehicle for LPS, was added to the control cell incubates). Immediately after the addition of LPS, 10 μl of SKF-525A and/or ABT dissolved in RPMI were added to final concentrations of 0.01, 0.1, and 1 mM/well. RPMI vehicle was added as control. Incubations were terminated after 2 hr. Data are representative of three experiments. *Indicates significant difference from control incubate. ^#Depicts significant difference of LPS/SKF-525A and LPS/ABT groups from LPS/vehicle groups in both studies. ^@Depicts significant difference in PGE₂ levels between supernatants from the LPS incubates supplemented with 0.01 and 0.1 mM of either inhibitor. Effect of inhibitor at a concentration of 1 mM did not differ significantly from that of 0.1 mM.

Figure 3.

Effect of 11,12-EET on PGE₂ levels in supernatants of rat monocytes stimulated with LPS. After separation, washing, and plating (see the Methods section), the cells were equilibrated for 1 hr in the CO₂ incubator. Then, 10 μl of LPS solution was added to a final concentration of 100 ng/well/2 × 10⁵ cells. Saline (vehicle for LPS) was added to the control cell incubates. Immediately after the addition of LPS, 10 μl of 11,12-EET reconstituted with RPMI medium 1640 was added to a final concentration of 1, 10, and 100 μM/well. RPMI vehicle was used in the respective control incubates. Incubations were carried on for 2 hr. Data are representative of three experiments. *Depicts significant difference of the respective group from the control vehicle/11,12-EET (10 μM) incubates. ^#Indicates significant difference of the respective group from the LPS/vehicle group. ^@Depicts significant difference between groups indicated. Data are average (± SE) of three experiments.

Figure 4.

Effect of 11,12-EET on the SKF-525A-provoked enhancement of PGE₂ levels in supernatants of rat monocytes stimulated with LPS. Ten microliters of LPS solution was added to a final concentration of 100 ng/well/2 × 10⁵ cells (saline, vehicle for LPS, was added to the control cell incubates). Immediately after the addition of LPS, 11,12-EET and SKF-525A (10 μl each) were added as shown, to a final concentration of 10 and 100 μM/well, respectively. Incubations were terminated after 2 hr. Data are representative of four experiments. * and ^|P% Depict significant difference between groups as indicated.

Figure 5.

Expression of 72-kDa COX-2 protein as determined by Western blot analysis of lysates from monocytes cultured with LPS (100 ng/2 × 10⁵ cells) for 0, 1, 2, 4, 8, and 12 hr. Also shown are 45-kDa β-actin and 72-kDa COX-2 standard (Std).

Figure 6.

Effect of SKF-525A and 11,12-EET on expression of 72 kDa COX-2 induced by LPS in rat monocytes. Cells were cultured for 2 hr with agents as shown, and protein expression was determined by a Western blot analysis. Densitometry analysis revealed no difference in LPS-induced COX-2 protein expression regardless of the presence of 11,12-EET and/or SKF-525A.

Figure 7.

Effect of 11,12-EET on COX-2 activity measured as pmol AA converted to product per minute per pmol enzyme. The enzyme was preincubated for 5 min with 11,12-EET (at a concentration of 1, 5, and 10 μM) before initiation of the reaction. The reaction with [¹⁴C]AA (final concentration 0.5 μM) was initiated by the addition of the enzyme/EET mixture and terminated after 8 sec. Each data point represents the mean (± SE) of four reactions. The COX activity is expressed as a percentage of the control to which no 11,12-EET was added.

Figure 8.

Inhibition of COX-2 by 11,12-EET is time-dependent. The reaction with [¹⁴C]AA (final concentration 0.5 μM) was initiated by the addition of the enzyme/EET mixture and terminated after 8 sec. 11,12-EET (final concentration 10 μM) was either added to the [¹⁴C]AA (time 0) or preincubated with COX-2 (for the time indicated) prior to initiation of the reaction. Each data point represents the mean of two reactions. The COX activity is expressed as a percentage of the control to which no 11,12-EET was added.

Footnotes

This work was supported in part by the National Institutes of Health Grant GM 07569.

1

To whom requests for reprints should be addressed at Department of Physiology, Medical College of Georgia, 1120 Fifteenth Street, Augusta, GA 30912–3000. E-mail:

2

Present address and affiliation: Department of Medicine, University of Michigan Health System, Ann Arbor, MI 48109.

References

Capdevila JH, Harris RC, Falck JR. Microsomal cytochrome P450 and eicosanoid metabolism. Cell Mol Life Sci 59 : 780–789, 2002.

Oliw EH. Oxygenation of polyunsaturated fatty acids by cytochrome P450 monooxygenases. Prog Lipid Res 33 : 329–354, 1994.

Imig JD. Epoxygenase metabolites: Epithelial and vascular actions. Mol Biotech 16 : 233–251, 2000.

Burger D, Dayer JM. The role of human T-lymphocyte-monocyte contact in inflammation and tissue destruction. Arthritis Res 4 : S169–S176, 2002.

Baron JM, Zwadlo-Klarwasser G, Jugert F, Hamann W, Rubben A, Mukhtar H, Merk HF. Cytochrome P450 1B1: A major P450 isoenzyme in human blood monocytes and macrophage subsets. Biochem Pharmacol 56 : 1105–1110, 1998.

Carcillo JA, Parise RA, Adedoyin A, Frye R, Branch RA, Romkes M. CYP2D6 mRNA expression in circulating peripheral blood mononuclear cells correlates with in vivo debrisoquine hydroxylase activity in extensive metabolizers. Res Commun Mol Pathol Pharmacol 91 : 149–159, 1996.

Wong PY, Lai PS, Falck JR. Mechanism of signal transduction of 14 R, 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes. Prostagl Lipid Mediat 62 : 321–333, 2000.

Fletcher JR. Eicosanoids: Critical agents in the physiological process and cellular injury. Arch Surg 128 : 1192–1196, 1993.

Vane JR. Towards a better aspirin. Nature 367 : 215–216, 1994.

10.

Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 38 : 97–120, 1998.

11.

Marnett LJ. Cyclooxygenase mechanism. Curr Opinion Chem Biol 4 : 545–552, 2000.

12.

Kozak W, Mayfield KP, Kozak A, Kluger MJ. Proadifen (SKF-525A), an inhibitor of cytochrome P-450, augments LPS-induced fever and exacerbates prostaglandin-E₂ levels in the rat. J Thermal Biol 25 : 45–50, 2000.

13.

Rotondo D, Abul HT, Milton AS, Davidson J. Pyrogenic immunomodulators increase the level of prostaglandin E2 in the blood simultaneously with the onset of fever. Eur J Pharmacol 154 : 145–152, 1988.

14.

Blatteis CM, Sehic E. Prostaglandin E₂: A putative fever mediator. In: Mackowiak PA, Ed. Fever: Basic Mechanism and Management. New York: Raven-Lippincott, pp117–148, 1997.

15.

Milton AS, Wendlandt S. A possible role for prostaglandin E1 as a modulator for temperature regulation in the central nervous system of the cat. J Physiol 207 : 76P–77P, 1970.

16.

Kozak W, Kluger MJ, Tesfaigzi J, Wachulec M, Kozak A, Dokladny K. Molecular mechanism of fever and antipyresis. Ann NY Acad Sci 917 : 121–134, 2000.

17.

Carroll MA, McGiff JC. A new class of lipid mediators: cytochrome P450 arachidonate metabolites. Thorax 55 : S13–S16, 2000.

18.

Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78 : 415–423, 1996.

19.

Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, Busse R. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 401 : 493–497, 1999.

20.

Keeble J, Al-Swayeh OA, Moore PK. Vasorelaxant effect of nitric oxide releasing steroidal and nonsteroidal anti-inflammatory drugs. Br J Pharmacol 133 : 1023–1028, 2001.

21.

Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, Liao JK. Anti-inflammatory properties of cytochrome P-450 epoxygenase-derived eicosanoids. Science 285 : 1276–1279, 1999.

22.

Kluger MJ. Fever: Role of pyrogens and cryogens. Physiol Rev 71 : 93–127, 1991.

23.

Evans SS, Bain MD, Wang WC. Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion. Int J Hypertherm 16 : 45–59, 2000.

24.

Wang WC, Goldman LM, Schleider DM, Appenheimer MM, Subjeck JR, Repasky EA, Evans SS. Fever-range hyperthermia enhances L-selectin-dependent adhesion of lymphocytes to vascular endothelium. J Immunol 160 : 961–969, 1998.

25.

Cao C, Matsumura K, Shirakawa N, Maeda M, Jikihara I, Kobayashi S, Watanabe Y. Pyrogenic cytokines injected into the rat cerebral ventricle induce cyclooxygenase-2 in brain endothelial cells and also upregulate their receptors. Eur J Neurosci 13 : 1781–1790, 2001.

26.

Li S, Wang Y, Matsumura K, Ballou LR, Morham SG, Blatteis CM. The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2 −/−, but not in cyclooxygenase-1 −/− mice. Brain Res 825 : 86–94, 1999.

27.

Koll S, Goppelt-Struebe M, Hauser I, Goerig M. Monocytic-endothelial cell interaction: regulation of prostanoids synthesis in human coculture. J Leuk Biol 61 : 679–688, 1997.

28.

Kozak W, Kluger MJ, Kozak A, Wachulec M, Dokladny K. Role of cytochrome P-450 in endogenous antipyresis. Am J Physiol 279 : R455–R460, 2000.

29.

Nakashima T, Yoshida Y, Miyata S, Kiyohara T. Hypothalamic 11,12-epoxyeicosatrienoic acid attenuates fever induced by central interleukin-1beta in the rat. Neurosci Lett 310 : 141–144, 2001.

30.

Kozak W, Milton AS, Abul H, Davidson J, Rotondo D. Lipopolysaccharide, muramyl dipeptide and polyinosinic: policytidylic acid induce the accumulation of inositol phosphates in blood monocytes and lymphocytes. Cell Signal 4 : 345–365, 1989.

31.

Rowlinson SW, Crews BC, Lanzo CA, Marnett LJ. The binding of arachidonic acid in the cyclooxygenase active site of mouse prostaglandin endoperoxide synthase-2 (COX-2). A putative L-shaped binding conformation utilizing the top channel region. J Biol Chem 274 : 23305–23310, 1999.

32.

Fang X, Moore SA, Stoll LL, Rich G, Kaduce TL, Weintraub NL, Spector AA. 14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E₂ production in vascular smooth muscle cells. Am J Physiol. 275 : H2113–H2121, 1998.

33.

Fitzpatrick FA, Ennis MD, Baze ME, Wynalda MA, McGee JE, Liggett WF. Inhibition of cyclooxygenase activity and platelet aggregation by epoxyeicosatrienoic acids. J Biol Chem 261 : 15334–15338, 1986.

34.

Boutaud O, Aronoff DM, Richardson JH, Marnett LJ, Oates JA. Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases. Proc Nat Acad Sci 99 : 7130–7135, 2002.

35.

Aronoff D, Boutaud O, Marnet L, Oates J. Inhibition of prostaglandin H₂ synthases by salicylate is dependent on the oxidative state of the enzymes. J Pharmacol Exp Ther 304 : 589–595, 2003.

36.

Saeed SA, McDonald-Gibson WJ, Cuthbert J, Copas JL, Schneider C, Gardiner PJ, Butt NM, Collier HOJ. Endogenous inhibitor of prostaglandin synthase. Nature 270 : 32–36, 1977.

37.

Ristimaki A, Narko K, Hla T. Down-regulation of cytokine-induced cyclo-oxygenase-2 transcript isoforms by dexamethasone: Evidence for post-transcriptional regulation. Biochem J 318 : 325–331, 1996.

38.

Flower RJ. Background and discovery of lipocortins. Agents Actions 17 : 255–262, 1986.

39.

Sebaldt RJ, Sheller JR, Oates JA, Roberts LJ, FitzGerald GA. Inhibition of eicosanoid biosynthesis by glucocorticoids in humans. Proc Nat Acad Sci USA 87 : 6974–6978, 1990.

40.

Brown NL, Alvi SA, Elder MG, Bennett PR, Sullivan MH. The regulation of prostaglandin output from term intact fetal membranes by anti-inflammatory cytokines. Immunology 99 : 124–133, 2000.

41.

Minghetti L, Polazzi E, Nicolini A, Levi G. Opposite regulation of prostaglandin E2 synthesis by transforming growth factor-beta1 and interleukin 10 in activated microglial cultures. J Neuroimmunol 82 : 31–39, 1998.

42.

Kiemer AK, Lehner MD, Hartung T, Vollmar AM. Inhibition of cyclooxygenase-2 by natriuretic peptides. Endocrinology 143 : 846–852, 2002.

43.

Tanaka Y, Igimi S, Amano F. Inhibition of prostaglandin synthesis by nitric oxide in RAW 264.7 macrophages. Arch Biochem Biophys 392 : 207–217, 2001.

44.

Tetsuka T, Daphna-Iken D, Miller BW, Guan Z, Baier LD, Morrison AR. Nitric oxide amplifies interleukin-1-induced cyclooxygenase-2 expression in rat mesangial cells. J Clin Invest 97 : 2051–2056, 1996.

45.

Pang L, Hoult JR. Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E₂ and other cyclic AMP stimulants in J774 macrophages. Biochem Pharmacol 53 : 493–500, 1997.

46.

Willoughby DA, Moore AR, Colville-Nash PR, Gilroy D. Resolution of inflammation. Int J Immunopharmacol 22 : 1131–1135, 2000.