The expression and regulation of the PGE receptors, EP2 and EP4, both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP4 but not EP2 was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP4 was down-regulated by LPS but not by medium change. PGE2 increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP4 agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP2 agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-α and IL-12 production were equipotent with PGE2 at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE2 or dibutyryl cAMP alone, but not butaprost, reduced EP4 expression, and indomethacin reversed the LPS-induced down-regulation of EP4, indicating that the down-regulation of EP4 is mediated by LPS-induced PG synthesis and EP4 activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP4 expression was found in the cells treated with PGE2 alone, but not in LPS-treated cells. In contrast, up-regulation of EP2 expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP4 is involved mainly in the inhibition of cytokine release, and that the gene expression of EP2 and EP4 is differentially regulated during macrophage activation.

Prostaglandin E2 is a major arachidonate metabolite synthesized by cyclooxygenase (COX),3 and contributes to immune suppression. PGE2 inhibits B and T lymphocyte proliferation, as well as various macrophage functions (1, 2, 3, 4). Macrophages are known to produce a large amount of PGE2 in response to proinflammatory stimuli such as IL-1 and bacterial LPS (5). This production of PGE2 is believed to be driven by the COX-2 enzyme, which is also synthesized de novo upon stimulation with LPS (6, 7). Released PGE2 also acts on the macrophages themselves, and exhibits inhibitory effects on not only early but also late processes involved in macrophage activation, producing a negative feedback loop; PGE2 inhibits the production of various cytokines such as TNF-α, IL-1β, and IL-12 by macrophages (8, 9). A number of reports have stated that such inhibitory effects of PGE2 on cytokine production are mediated by an increase in intracellular cAMP. The effects of PGE2 are exerted by specific receptors on the plasma membrane of target cells (10, 11). Based on pharmacological and cDNA cloning studies, four subtypes of PGE receptor, EP1, EP2, EP3, and EP4, have been identified and have been shown to differ in their signal transduction pathways (12, 13, 14). Previous investigations have strongly suggested the coupling of PGE receptors to adenylate cyclase in activated macrophages (9). We and other groups have revealed that EP2 and EP4 receptors, both of which couple to the stimulation of adenylate cyclase, are expressed in cultured murine macrophage-like cell lines such as J774.1 and RAW264.7 (15, 16). However, it is not known whether the two receptors contribute equally to the inhibition of activated macrophage function. Furthermore, recent studies using receptor gene knockouts have focused on the regulation of PG receptor gene expression. In contrast, recent findings in the field of local mediators have led us to consider the possibility that gene expression of the receptors could also be induced by various kinds of stimuli (17). For example, Matsuoka et al. found that PGD receptor expression induced in alveolar epithelial cells upon Ag challenge plays a pivotal role in the appearance of allergic responses (18). Therefore, it is possible that PGE receptor gene expression changes during macrophage activation. However, there have been no reports stating such a point of view regarding PGE receptors in macrophages.

Here we designed experiments for two main purposes. First, to identify which receptor is mainly responsible for the inhibitory action of PGE2 on cytokine release, we investigated time-dependent mRNA expression of PGE receptors during LPS-induced macrophage activation, and examined the effects of EP-specific agonists on cytokine production. Second, to explore the possible mechanisms underlying regulation of receptor gene expression, we examined the effects of cAMP-elevating agents, an inhibitor of PG synthesis, and a spontaneous deficiency in LPS perception, which was recently identified to be a genetic mutation in one of the receptors, for a bacterial cell wall components. This study demonstrates that gene expression of EP4 is down-regulated via LPS-induced PG synthesis and EP4 activation, whereas that of EP2 is up-regulated by LPS stimulation via a Toll receptor-independent mechanism. Although the expression profiles suggested the possibility of a receptor switch from EP4 to EP2, the EP4 expressed initially appears mainly to contribute to the inhibition of cytokine production in macrophages.

LPS from Escherichia coli O55:B5, dibutyryl cAMP (dbcAMP) and indomethacin were obtained from Sigma (St. Louis, MO). Cycloheximide was purchased from Wako Chemicals (Osaka, Japan). The 125I-labeled cyclic AMP assay system was purchased from Amersham (Arlington Heights, IL), and ELISA kits for mouse TNF-α and for mouse IL-12 were purchased from BioSource International (Camarillo, CA) and R&D Systems (Minneapolis, MN), respectively. PGE2 in the medium was quantified using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). PGE2 was purchased from Funakoshi (Tokyo, Japan). Butaprost, an EP2-specific agonist, and ONO-604, an EP4-specific agonist, were generated, and their specificities were analyzed by measuring their binding affinities to the respective EPs expressed in Chinese hamster ovary (CHO) cells (Table I) (19). PGE2 bound to all EPs. Based on the results obtained from dose dependence analyses, we used 10 nM of ONO-604 and 1 μM of butaprost. These agonists at these concentrations selectively activate EP4 and EP2, respectively.

Table I.

Binding affinities of ONO-604, an EP4 agonist, and butaprost, an EP2 agonist, to the respective EPs expressed in CHO cellsa

Receptors: [3H]Ligands:EP13H-PGE2EP23H-PGE2EP33H-PGE2EP43H-PGE2
PGE2 0.018 0.038 0.005 0.0031 
ONO-604 0.61 0.28 1.5 0.0007 
Butaprost >10 0.092 >10 >10 
Receptors: [3H]Ligands:EP13H-PGE2EP23H-PGE2EP33H-PGE2EP43H-PGE2
PGE2 0.018 0.038 0.005 0.0031 
ONO-604 0.61 0.28 1.5 0.0007 
Butaprost >10 0.092 >10 >10 
a

Partially purified membranes of CHO cells expressing the respective EPs were used for the [3H]PGE2 binding assay, and the Ki values (micromolar concentrations) were calculated.

Six-week-old C3H/HeN and C3H/HeJ female mice were obtained from Japan SLC (Hamamatsu, Japan) as specific pathogen-free animals. The mice were killed, and their peritoneal resident macrophages were collected by washing the peritoneal cavity with 5 ml of ice-cold saline. After washing with PBS, the macrophages (2 × 106 cells) were seeded onto plastic Petri dishes in Ham’s F-12 medium (LPS < 10 pg/ml; Flow Laboratories, McLean, VA), supplemented with 10% (v/v) heat-inactivated FBS (LPS < 30 pg/ml; Life Technologies, Gaithersburg, MD). After incubation at 37°C for 1 h, nonadherent cells were removed by repeated washing. More than 95% of the cells were macrophages as identified by their phagocytic activity toward zymosan, by Fc-rosette formation to the immune complex between sheep RBC and the specific Ab (SRBC-Ab), and by the nonspecific esterase reaction that catalyzes α-naphtyl butylate. The purity of the cell population was consistent throughout the experiments, and the viability of the macrophages remained greater than 98%.

The culture medium was replaced with fresh medium either with or without 100 ng/ml LPS, and the cells were then incubated at 37°C for the indicated number of hours. The cells were washed twice with PBS, scraped with a cell scraper (Costar) in ice-cold PBS, and pelleted by centrifugation (1200 rpm) at 4°C. The resultant cell pellet was washed with 0.5 ml of ice-cold PBS and stored at −80°C until use.

Western blotting was performed as described (20). Fifty-microgram aliquots of the cell extracts were loaded onto 7.5% SDS gels (21) and electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The proteins on the membrane were treated with a polyclonal anti-COX-2 Ab (Oxford Biomedical Research, Oxford, MI). The immune complexes on the membrane were then treated with 125I-labeled protein A at 37°C for 1 h. After repeated washing, the images were visualized with the BAS-2000 bioimage analyzer (Fuji, Tokyo, Japan).

Total RNA was isolated from 1–1.5 × 107 macrophages by the acid guanidinium thiocyanate-phenol-chloroform method (22). The RNA (10 μg) was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane (BIODYNE; Pall, East Hills, NY). For the detection of EP2 mRNA, the EcoRI insert DNA (1.7 kb) of ML202 (23) was used as a hybridization probe. For EP4 mRNA detection, a 970-bp fragment of the mouse EP4 cDNA containing the putative first to seventh transmembrane domains was used as a probe (15). The cDNA probes used for mouse COX-1 and COX-2 were as reported previously (24). Hybridization was conducted under the conditions described previously (25), and RNA bands were visualized by autoradiography. The blots were then stripped and rehybridized with a 32P-labeled DNA probe for GAPDH. Quantification of the hybridized signals was conducted with the BAS-2000 Bio-image analyzer. Northern blot experiments were independently repeated three times. Representative results are shown in the photographs, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments.

cAMP levels in peritoneal macrophages were determined as reported previously (26). Cells cultured in 24-well plates (1 × 106 cells/well) were washed with 0.5 ml of Krebs-HEPES buffer (pH 7.4) with 10 μM indomethacin, and preincubated for 10 min. Reactions were started by the addition of test reagents along with 100 μM Ro-20-1724 and 10 μM indomethacin. After incubation for 10 min at 37°C, reactions were terminated by the addition of 10% trichloroacetic acid. Then the cAMP content of the cells was measured by the cAMP radioimmunoassay kit.

For the quantification of PGE2, peritoneal macrophages collected from C3H/HeN mice were preincubated in 24-well plates at a density of 1 × 106 cells/well and then incubated with 0.5 ml of medium with or without 100 ng/ml LPS for the indicated time periods. The concentration of PGE2 in the medium was then determined by an enzyme immunoassay kit, according to the manufacturer’s protocol.

For the quantification of cytokine levels, cells were seeded at 0.5 × 106 cells/well and then incubated with 0.5 ml of medium containing 100 ng/ml LPS in the presence or absence of PGE2 (1 μM), butaprost (1 μM), and ONO-604 (10 nM). The culture supernatants were transferred to new tubes for the indicated time periods, followed by the examination of TNF-α and IL-12 levels by ELISA kits, according to the manufacturer’s protocol.

For Northern analyses, data were expressed as the mean ± SEM of three independent experiments. For the determination of PGE2, cAMP, and cytokine levels, representative data were expressed as the mean ± SEM of triplicate determinants. These experiments were independently repeated three times, and similar results were obtained. Statistical analyses were performed using Student’s t test.

In this study, we designed experiments to 1) clarify the mode of PGE2 action in macrophages with respect to the PGE receptor subtypes, and 2) investigate possible changes in receptor gene expression during macrophage activation. Peritoneal resident macrophages isolated from C3H/HeN mice were used for this purpose. Macrophages themselves produce a large amount of PGE2 in response to stimuli such as LPS, and such endogenous PGE2 is thought to affect functions of macrophages in an autocrine manner. Therefore, we thought it is necessary to examine the time-dependent production of PGE2 from LPS-stimulated cells in our system (Fig. 1,A). Basal PGE2 production in the absence of LPS treatment for 3 h was 0.441 ng/ml. In LPS-treated macrophages, a large amount of PGE2 was detected; PGE2 production 3 and 7 h after LPS treatment was 30.7 ± 1.58 and 286 ± 13 ng/ml, respectively, which corresponds to 0.087 and 0.81 μM of PGE2, respectively. When we examined the expression of COX isozyme mRNAs in these cells, faint expression of COX-1 mRNA was detected in nonstimulated macrophages, and expression levels did not change upon LPS treatment (data not shown). In contrast, LPS treatment rapidly induced the expression of COX-2 mRNA (Fig. 1,B). Western blot analyses again showed that COX-2 proteins were induced when cells were treated with LPS (Fig. 1 C). LPS treatment stimulates PGE2 production in C3H/HeN macrophages, resulting in exposure of the cells to micromolar order concentrations of PGE2. Transcriptional induction of COX-2 may be a possible mechanism for LPS-induced PGE2 production.

FIGURE 1.

PGE2 synthesis and COX-2 expression in LPS-stimulated C3H/HeN macrophages. A, PGE2 synthesis in LPS-stimulated macrophages. Peritoneal macrophages collected from C3H/HeN mice were preincubated in 24-well plates at a density of 1 × 106 cells/well, followed by incubation in medium with (•) or without (○) 100 ng/ml LPS for the indicated time periods. The concentrations of PGE2 in the medium were then determined as described in Materials and Methods. The values obtained from the medium only were used as the value at 0 h. Values are expressed as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained. B, Expression of COX-2 mRNA in LPS-stimulated C3H/HeN macrophages. Peritoneal macrophages were exposed to fresh medium with (LPS +) or without 100 ng/ml LPS (LPS −) for the indicated times, and total RNA was isolated. Ten micrograms of each RNA sample was subjected to Northern blot analysis. The blots were hybridized with a probe for COX-2, and then rehybridized with a probe for GAPDH. C, Detection of the COX-2 protein in C3H/HeN macrophages. Peritoneal macrophages were exposed to medium with (LPS +) or without 100 ng/ml LPS (LPS −) for 4 h. The cells were collected, and the cell lysates were subjected to immunoblot analysis for the COX-2 protein.

FIGURE 1.

PGE2 synthesis and COX-2 expression in LPS-stimulated C3H/HeN macrophages. A, PGE2 synthesis in LPS-stimulated macrophages. Peritoneal macrophages collected from C3H/HeN mice were preincubated in 24-well plates at a density of 1 × 106 cells/well, followed by incubation in medium with (•) or without (○) 100 ng/ml LPS for the indicated time periods. The concentrations of PGE2 in the medium were then determined as described in Materials and Methods. The values obtained from the medium only were used as the value at 0 h. Values are expressed as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained. B, Expression of COX-2 mRNA in LPS-stimulated C3H/HeN macrophages. Peritoneal macrophages were exposed to fresh medium with (LPS +) or without 100 ng/ml LPS (LPS −) for the indicated times, and total RNA was isolated. Ten micrograms of each RNA sample was subjected to Northern blot analysis. The blots were hybridized with a probe for COX-2, and then rehybridized with a probe for GAPDH. C, Detection of the COX-2 protein in C3H/HeN macrophages. Peritoneal macrophages were exposed to medium with (LPS +) or without 100 ng/ml LPS (LPS −) for 4 h. The cells were collected, and the cell lysates were subjected to immunoblot analysis for the COX-2 protein.

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PGE2 regulates a variety of functions in macrophages, including cytokine production. This study demonstrated that LPS-stimulated macrophages are exposed to micromolar order concentrations of PGE2. To explore the possible involvement of endogenous PGE2 in cytokine release, we examined the effects of indomethacin, an inhibitor of PG synthesis, on production of the most representative macrophage cytokines, TNF-α and IL-12. As shown in Fig. 2, TNF-α was released immediately after LPS-treatment, reaching a peak after 3 h, and then diminished, possibly due to its rapid degradation (rapid response). In contrast, IL-12 production gradually increased until 14 h after treatment (slow response). When indomethacin was added simultaneously with LPS, both TNF- α and IL-12 production was enhanced; a 5.7-fold increase in TNF-α at 3 h and a 1.9-fold increase in IL-12 at 14 h was observed compared with treatment with LPS alone at the corresponding times. These results indicated that endogenous PGs are involved in the suppression of cytokine production. Even in this system, exogenous PGE2 (1 μM) was effective for the inhibition of cytokine production, resulting in 17% of the value obtained with LPS alone for TNF-α production (3 h) and 48% for IL-12 production (14 h) (Fig. 2). Because PGE2 is known to be coupled to cAMP production in a number of macrophage-like cells, we further examined the effects of dbcAMP on cytokine production. In both cases, dbcAMP completely mimicked the effects of PGE2, suggesting that suppression of cytokine production by PGE2 is mediated by PGE receptors coupling to the stimulation of adenylate cyclase, possibly by the EP2 and/or EP4 receptors.

FIGURE 2.

Effects of indomethacin, PGE2, and dbcAMP on LPS-induced TNF-α and IL-12 release in C3H/HeN macrophages. Peritoneal macrophages were exposed to fresh medium only (crosses) or medium containing 100 ng/ml LPS supplemented with no reagents (•), 10 μM indomethacin (□), 1 μM PGE2 (○), or 1 mM dbcAMP (⋄) for the indicated times. The supernatants were recovered 3, 7, and 14 h after LPS stimulation, and the TNF-α and IL-12 contents were determined as described in Materials and Methods. The values obtained in medium only are used for the time point 0 h. Values are expressed as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.05 for LPS plus reagent-treated vs LPS-treated only cells).

FIGURE 2.

Effects of indomethacin, PGE2, and dbcAMP on LPS-induced TNF-α and IL-12 release in C3H/HeN macrophages. Peritoneal macrophages were exposed to fresh medium only (crosses) or medium containing 100 ng/ml LPS supplemented with no reagents (•), 10 μM indomethacin (□), 1 μM PGE2 (○), or 1 mM dbcAMP (⋄) for the indicated times. The supernatants were recovered 3, 7, and 14 h after LPS stimulation, and the TNF-α and IL-12 contents were determined as described in Materials and Methods. The values obtained in medium only are used for the time point 0 h. Values are expressed as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.05 for LPS plus reagent-treated vs LPS-treated only cells).

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To investigate whether EP2 and EP4 receptors are expressed in nonstimulated and LPS-stimulated macrophages, we examined the expression of EP2 and EP4 mRNA (Fig. 3). In macrophages just before treatment (time 0), a significant amount of EP4 mRNA was found, but EP2 mRNA could not be detected in the Northern blot. When these macrophages were stimulated with LPS, the expression of EP2 mRNA transiently increased to a maximum 3 h after treatment, then decreased by 6 h, but did not return to values before the detection level. A slight but significant induction of EP2 mRNA was also observed in macrophages treated in a similar manner in the absence of LPS. The expression level after LPS treatment is 3-fold that after medium change only. These results indicate that the expression levels of EP2 mRNA in peritoneal macrophages is below detection levels before any treatment, is slightly induced upon medium change, possibly due to other induction factors in the serum, and is strongly enhanced by LPS. In contrast, the expression of EP4 mRNA was down-regulated 3 h after LPS treatment, resulting in <10% of the control level. This suppression was still observed 6 h after LPS treatment, whereas no suppression was observed upon medium change only. To test the expression of EP1 and EP3 mRNA, we performed RT-PCR analyses on the RNA samples from freshly prepared macrophages without any treatment, macrophages treated with LPS for up to 6 h, and macrophages subjected to medium change only (data not shown). However, we failed to detect significant signals for EP1 and EP3 mRNA in all preparations. Peritoneal macrophages are unlikely to express the EP1 and EP3 subtypes of PGE receptors.

FIGURE 3.

Time course of EP2 and EP4 mRNA expression in LPS-stimulated C3H/HeN macrophages. Peritoneal macrophages were prepared, exposed to fresh medium with (LPS + or •) or without (LPS − or ○) 100 ng/ml LPS for the indicated times, and then collected. The macrophages just before exposure to fresh medium were collected as the time point 0 h. Total RNA (10 μg) isolated from each sample was subjected to Northern blot analysis of EP2 and EP4. The blots were rehybridized with a probe for GAPDH. The results shown in the upper panels are representative of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments shown in the lower panels. The EP2/GAPDH values are represented as fold of the value for LPS (−) 3 h, and the EP4/GAPDH values are represented as fold of the value at 0 h. (∗, p < 0.01 for LPS-stimulated vs nonstimulated cells).

FIGURE 3.

Time course of EP2 and EP4 mRNA expression in LPS-stimulated C3H/HeN macrophages. Peritoneal macrophages were prepared, exposed to fresh medium with (LPS + or •) or without (LPS − or ○) 100 ng/ml LPS for the indicated times, and then collected. The macrophages just before exposure to fresh medium were collected as the time point 0 h. Total RNA (10 μg) isolated from each sample was subjected to Northern blot analysis of EP2 and EP4. The blots were rehybridized with a probe for GAPDH. The results shown in the upper panels are representative of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments shown in the lower panels. The EP2/GAPDH values are represented as fold of the value for LPS (−) 3 h, and the EP4/GAPDH values are represented as fold of the value at 0 h. (∗, p < 0.01 for LPS-stimulated vs nonstimulated cells).

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To confirm the dynamic changes in the expression of these receptors, it was considered best to investigate the EP2 and EP4 receptors at the protein level using Western blot analyses. However, unfortunately, there are currently no EP2 or EP4 receptor Abs available for Western blot detection, and we chose to study the expression of the EP2 and EP4 receptors using functional analyses by examining the effects of PGE2 and EP-selective agonists on cAMP production in C3H/HeN macrophages at the indicated time points after LPS treatment (Fig. 4). As shown in Fig. 1, because these macrophages produce PGE2 at micromolar order concentrations, we used 1 μM of PGE2 to induce cAMP production. We used 1 μM of butaprost, an EP2-specific agonist, and 10 nM of ONO-604, an EP4-selective agonist, both of which have equipotent efficacies for inducing cAMP production as that of 1 μM of PGE2, without affecting other receptor functions as evaluated in the CHO expression system (Table I). In macrophages without LPS treatment, both PGE2 and ONO-604, but not butaprost increased cAMP production, suggesting that the PGE2-induced cAMP production is mediated via EP4, but not by EP2 in this preparation. Similar results were obtained 1 and 3 h after LPS treatment. In contrast, in macrophages stimulated with LPS for 6 h, butaprost increased cAMP to levels generated by PGE2, but ONO-604 did not. These results indicate that the PGE receptor responsible for cAMP switches from EP4 to EP2 by 6 h after LPS treatment. In this assay, basal cAMP production increased and the response generated by agonists decreased with time after LPS treatment despite the presence of indomethacin in the assay solution. One of the reasons could be because a variety of responses occur in LPS-stimulated macrophages, and other cAMP-producing factors may be released during the assay or the sensitivity of PGE2 in PGE receptors may be altered. Thus, this assay may not reflect the exact amount of each receptor expressed at the protein level.

FIGURE 4.

cAMP-elevating potencies of PGE analogs in C3H/HeN macrophages at different time points after LPS treatment. Peritoneal macrophages were prepared, and untreated cells or cells treated with 100 ng/ml LPS for the indicated time periods were exposed to medium supplemented with no PGE analogs (□), 1 μM PGE2 (▪), 1 μM butaprost (▧), or 10 nM ONO-604 (▦) for 10 min. Test reagents contain 10 μM indomethacin and 100 μM Ro-20-1724. The cAMP contents were then determined as described in Materials and Methods. Values are expressed as the mean ± SEM of triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.005 for PGE analog-treated vs PG-untreated cells).

FIGURE 4.

cAMP-elevating potencies of PGE analogs in C3H/HeN macrophages at different time points after LPS treatment. Peritoneal macrophages were prepared, and untreated cells or cells treated with 100 ng/ml LPS for the indicated time periods were exposed to medium supplemented with no PGE analogs (□), 1 μM PGE2 (▪), 1 μM butaprost (▧), or 10 nM ONO-604 (▦) for 10 min. Test reagents contain 10 μM indomethacin and 100 μM Ro-20-1724. The cAMP contents were then determined as described in Materials and Methods. Values are expressed as the mean ± SEM of triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.005 for PGE analog-treated vs PG-untreated cells).

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To investigate the contribution of the EP2 and EP4 receptors in macrophage functions, we examined the effects of EP-selective agonists on the LPS-induced production of TNF-α and IL-12. Regarding TNF-α production measured 3 h after LPS treatment, PGE2 inhibited TNF- α production by 83% and ONO-604 by 61%, but inhibition by butaprost was only by 9.6% (Fig. 5). As suggested in the cAMP formation assays, EP4 works as a predominant PGE receptor against TNF-α production around 3 h after stimulation. Regarding IL-12 production, both PGE2 and ONO-604 showed inhibitory effect at every time point examined, and the inhibitory potency of ONO-604 was close to that of PGE2. This is an interesting observation, as the EP4 receptor is likely not to work around 6 h after stimulation, as deduced from the cAMP formation experiments. Activation of EP4 for just the first several hours after LPS treatment may be enough to cause an inhibition of this cytokine to near maximum levels. On the contrary, the inhibitory effect of butaprost was not significant at 3 and 7 h, but apparent at 14 h (29.3% inhibition), indicating that the EP2 receptor works only during late time points. These results indicated that the effects of PGE2 are mediated by both the EP2 and EP4 receptors, but that each receptor contributes to the inhibition of activated macrophage function in a different manner via changes in their expression patterns.

FIGURE 5.

Effects of PGE analogs on LPS-induced TNF-α and IL-12 release in C3H/HeN macrophages. Peritoneal macrophages were prepared and exposed to fresh medium only (crosses) or medium containing 100 ng/ml LPS supplemented with no PG analogs (•), 1 μM PGE2 (○), 1 μM butaprost (▵), or 10 nM ONO-604 (▴) for the indicated time periods. The supernatants were recovered, and the TNF-α and IL-12 contents were determined as described in Materials and Methods. The contents obtained from medium only were used as time 0. The results are shown as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.05 for PGE analog-treated vs nontreated cells).

FIGURE 5.

Effects of PGE analogs on LPS-induced TNF-α and IL-12 release in C3H/HeN macrophages. Peritoneal macrophages were prepared and exposed to fresh medium only (crosses) or medium containing 100 ng/ml LPS supplemented with no PG analogs (•), 1 μM PGE2 (○), 1 μM butaprost (▵), or 10 nM ONO-604 (▴) for the indicated time periods. The supernatants were recovered, and the TNF-α and IL-12 contents were determined as described in Materials and Methods. The contents obtained from medium only were used as time 0. The results are shown as the mean ± SEM for triplicate determinants. The experiments were independently repeated three times, and similar results were obtained (∗, p < 0.05 for PGE analog-treated vs nontreated cells).

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We next focused on the regulatory mechanisms underlying the changes in expression of EP2 and EP4 mRNAs upon LPS treatment. LPS stimulation was able to influence the transcriptional expression of various proteins in a direct or indirect manner in macrophages. To explore whether the up-regulation of the EP2 gene and the down-regulation of the EP4 gene require general protein synthesis, we examined the effect of cycloheximide on the EP2 and EP4 mRNA expression (Fig. 6). Cycloheximide (0.1 μg/ml) failed to affect LPS-induced EP2 gene expression, but completely reversed the LPS-induced down-regulation of EP4 gene expression. These results suggest that the up-regulation of EP2 gene expression does not require the protein synthesis, and is possibly directly activated by LPS signals, but that the down-regulation of EP4 gene expression requires protein synthesis, possibly indirectly controlled by LPS via the synthesis of other proteins. COX-2 is undoubtedly one of the proteins synthesized rapidly in response to LPS treatment in macrophages. PGE2 release is believed to be a result of COX-2 protein induction, as shown in Fig. 1. Thus we hypothesized that the LPS-induced PGE2 may affect EP4 gene expression itself in a negative-feedback manner. Based on this hypothesis, we examined the effect of PGE2 alone or indomethacin with LPS on EP4 mRNA expression (Fig. 7). Incubation of PGE2 (1 μM) without LPS for 3 h inhibited the basal expression of EP4 mRNA, and indomethacin added simultaneously with LPS stimulation reversed the LPS-diminished expression of EP4 mRNA, suggesting that the decrease in EP4 mRNA expression was a result of feedback regulation by PGE2. dbcAMP (1 mM) and ONO-604 (10 nM, data not shown) mimicked the effects of PGE2, but butaprost (1 μM) failed to inhibit EP4 mRNA expression. These results suggested that EP4-induced cAMP formation negatively regulates the expression of the EP4 gene itself, and the effect of cycloheximide may be due to inhibition of COX-2 protein synthesis. In contrast, none of the reagents tested affected the expression levels of EP2 mRNA, confirming that the increase in EP2 mRNA expression upon LPS treatment is independent of PG synthesis.

FIGURE 6.

Effects of cycloheximide on EP2 and EP4 mRNA expression in LPS-treated and nontreated C3H/HeN macrophages. Peritoneal macrophages were prepared, exposed to medium supplemented with a combination of 100 ng/ml LPS and 0.1 μg/ml cycloheximide (CHX) for 3 h, and collected. Total RNA (10 μg) isolated from each sample was subjected to Northern blot analysis. The results shown in the upper panels are representative of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments as shown in the lower panels. The EP2/GAPDH and the EP4/GAPDH values are shown as the fold of values obtained from the sample exposed to medium only (∗, p < 0.01 for CHX (+) and LPS (+) vs CHX (−) and LPS (+) cells).

FIGURE 6.

Effects of cycloheximide on EP2 and EP4 mRNA expression in LPS-treated and nontreated C3H/HeN macrophages. Peritoneal macrophages were prepared, exposed to medium supplemented with a combination of 100 ng/ml LPS and 0.1 μg/ml cycloheximide (CHX) for 3 h, and collected. Total RNA (10 μg) isolated from each sample was subjected to Northern blot analysis. The results shown in the upper panels are representative of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments as shown in the lower panels. The EP2/GAPDH and the EP4/GAPDH values are shown as the fold of values obtained from the sample exposed to medium only (∗, p < 0.01 for CHX (+) and LPS (+) vs CHX (−) and LPS (+) cells).

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FIGURE 7.

Effects of cAMP-generating agents on basal mRNA expression of EP receptors and the effects of indomethacin on up-regulated EP2 mRNA expression or down-regulated EP4 mRNA expression in C3H/HeN macrophages. Peritoneal macrophages were exposed to medium supplemented with 1 μM PGE2, 1 μM butaprost, or 1 mM dbcAMP for 3 h, or cells were alternatively exposed to medium containing 100 ng/ml LPS in the presence (LPS + Indo.) or absence of 10 μM indomethacin (LPS) for 3 h. Total RNA (10 μg) isolated from each collected sample was subjected to Northern blot analyses. The upper panels are representative results of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments shown in the lower panels. The EP2/GAPDH and the EP4/GAPDH values are shown as the fold of values obtained from the cells exposed to the medium only for 3 h (∗, p < 0.01 for PGE2- or dbcAMP-treated vs nontreated cells; †, p < 0.01 for LPS-treated vs LPS and indomethacin-treated cells).

FIGURE 7.

Effects of cAMP-generating agents on basal mRNA expression of EP receptors and the effects of indomethacin on up-regulated EP2 mRNA expression or down-regulated EP4 mRNA expression in C3H/HeN macrophages. Peritoneal macrophages were exposed to medium supplemented with 1 μM PGE2, 1 μM butaprost, or 1 mM dbcAMP for 3 h, or cells were alternatively exposed to medium containing 100 ng/ml LPS in the presence (LPS + Indo.) or absence of 10 μM indomethacin (LPS) for 3 h. Total RNA (10 μg) isolated from each collected sample was subjected to Northern blot analyses. The upper panels are representative results of three separate experiments. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments shown in the lower panels. The EP2/GAPDH and the EP4/GAPDH values are shown as the fold of values obtained from the cells exposed to the medium only for 3 h (∗, p < 0.01 for PGE2- or dbcAMP-treated vs nontreated cells; †, p < 0.01 for LPS-treated vs LPS and indomethacin-treated cells).

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One of the reasons we chose macrophages from the C3H/HeN strain is that this strain has a genetically comparable mutant strain, C3H/HeJ, in which the macrophages hardly respond to LPS. Recently, C3H/HeJ was found to have a point mutation within the coding region of the Toll-like receptor 4 (TLR4), a number of a protein family containing proteins that have been implicated in LPS-induced cell signaling (27). Indeed, C3H/HeJ macrophages did produce a significant amount of PGE2, but much less than that in C3H/HeN; the PGE2 contents in the medium was <1 nM even after 7 h of LPS stimulation (Fig. 8,A). COX-2 expression levels were analyzed by Northern and Western analyses, showing that COX-2 induction was faint in LPS-treated C3H/HeJ macrophages (data not shown). If the down-regulation of EP4 gene expression requires more than a nanomolar order concentration of PGE2, it should not occur in this strain. As expected, no suppression of EP4 mRNA expression was observed after LPS treatment (Fig. 8,B). In addition, when C3H/HeJ macrophages were stimulated with PGE2 or dbcAMP, there was a decrease in EP4 mRNA, as was seen in the C3H/HeN macrophages (Fig. 8 C). These results again indicate that the expression of EP4 mRNA is down-regulated via an EP4-mediated cAMP-dependent pathway. In contrast, surprisingly and unexpectedly, a slight induction of EP2 mRNA was again seen upon just medium change, and this induction was greatly enhanced by LPS treatment, as was seen in C3H/HeN macrophages. These results suggest that the mechanism underlying the induction and up-regulation of EP2 gene expression may be independent of TLR4-mediated signals induced by LPS treatment.

FIGURE 8.

PGE2 production and mRNA expression of EP2 and EP4 in C3H/HeJ macrophages. A, PGE2 synthesis in LPS-stimulated C3H/HeN and C3H/HeJ macrophages. Peritoneal macrophages were collected and exposed to medium with (LPS +) or without (LPS −) 100 ng/ml LPS for 7 h. The supernatants were recovered, and their PGE2 content was determined as described in Materials and Methods. Values are expressed as the mean ± SEM of three independent experiments (∗, p < 0.01 for LPS-treated cells vs cells treated with medium change only in C3H/HeN mice; †, p < 0.05 for LPS-treated cells vs cells treated with medium change only in C3H/HeJ mice). B, Time course of EP2 and EP4 mRNA expression in LPS-treated C3H/HeJ macrophages. C3H/HeJ macrophages were exposed to fresh medium with (LPS + or •) or without (LPS − or ○) 100 ng/ml LPS. The macrophages before medium change were used for values at 0 h (∗, p < 0.01 for LPS-treated cells vs cells treated with medium change only). C, Effects of cAMP-elevating agents on basal mRNA expression of EP4 in C3H/HeJ macrophages. Peritoneal macrophages were treated with medium containing 1 μM PGE2, 1 μM butaprost, or 1 mM dbcAMP for 3 h (∗, p < 0.01 for PGE2- or dbcAMP-treated cells vs cells treated with medium change only). In B and C, total RNA (10 μg) isolated from each sample was subjected to Northern blot analyses. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments. The EP2/GAPDH values are represented as the fold of the value for LPS (−) 3 h, and the EP4/GAPDH values are represented as the fold of the values at 0 h.

FIGURE 8.

PGE2 production and mRNA expression of EP2 and EP4 in C3H/HeJ macrophages. A, PGE2 synthesis in LPS-stimulated C3H/HeN and C3H/HeJ macrophages. Peritoneal macrophages were collected and exposed to medium with (LPS +) or without (LPS −) 100 ng/ml LPS for 7 h. The supernatants were recovered, and their PGE2 content was determined as described in Materials and Methods. Values are expressed as the mean ± SEM of three independent experiments (∗, p < 0.01 for LPS-treated cells vs cells treated with medium change only in C3H/HeN mice; †, p < 0.05 for LPS-treated cells vs cells treated with medium change only in C3H/HeJ mice). B, Time course of EP2 and EP4 mRNA expression in LPS-treated C3H/HeJ macrophages. C3H/HeJ macrophages were exposed to fresh medium with (LPS + or •) or without (LPS − or ○) 100 ng/ml LPS. The macrophages before medium change were used for values at 0 h (∗, p < 0.01 for LPS-treated cells vs cells treated with medium change only). C, Effects of cAMP-elevating agents on basal mRNA expression of EP4 in C3H/HeJ macrophages. Peritoneal macrophages were treated with medium containing 1 μM PGE2, 1 μM butaprost, or 1 mM dbcAMP for 3 h (∗, p < 0.01 for PGE2- or dbcAMP-treated cells vs cells treated with medium change only). In B and C, total RNA (10 μg) isolated from each sample was subjected to Northern blot analyses. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalized to GAPDH mRNA levels as the mean ± SEM of three independent experiments. The EP2/GAPDH values are represented as the fold of the value for LPS (−) 3 h, and the EP4/GAPDH values are represented as the fold of the values at 0 h.

Close modal

Although there have been many reports on the mechanism of COX-2 expression after LPS stimulation and the effect of PGE2 on LPS-activated macrophages, there have been very few reports that focus on the expression and function of the PGE receptors in monocytes/macrophages (28, 29, 30). Here we examined the time-dependent expression of EP2 and EP4 mRNA during activation in resident peritoneal macrophages, and found that the EP2 gene is transiently up-regulated, whereas the EP4 gene is down-regulated upon LPS stimulation. Based on the results obtained from the cAMP assays using subtype-selective agonists, we conclude that EP4 is the dominant PGE receptor in nonstimulated macrophages or macrophages up to 3 h after LPS treatment. This is supported by our results showing that TNF-α production 3 h after LPS treatment is completely inhibited by an EP4 agonist, but not an EP2 agonist. However, the cAMP assay also indicated that the EP4 receptor is no longer active by 6 h after LPS treatment, and, in turn, EP2 seems to become the dominant receptor by 6 h. This is also supported by the result that the inhibitory effect of an EP2 agonist on IL-12 production was apparent at this time point. In the presence of an EP2 agonist, EP2 is likely to work by 6 h after LPS treatment, resulting in no increase observed in production of IL-12 between 6 and 14 h. In contrast, the inhibitory effect of an EP4 agonist on IL-12 production was observed for a longer time than we expected, even beyond 6 h after LPS treatment. Because IL-12 production initiated by LPS includes de novo synthesis, the EP4-induced cAMP increase during the initial few hours after LPS addition may exert continuous inhibition on IL-12 production. It is possible that the EP4-induced cAMP increase and the resultant activation of cAMP-dependent kinase during initiation of IL-12 gene activation may have a crucial effect on the following gene expression of this cytokine. A number of reports have established that the cAMP-dependent pathway inhibits IL-12 production at the transcriptional level, possibly by affecting cAMP response element (CRE) binding complexes (31, 32, 33). Thus, EP4 gene expression is down-regulated upon stimulation with LPS, but this receptor appears to have a pivotal role in autocrine regulation of cytokine release, and EP2 might contribute to extend the inhibitory effect of PGE2 on a day-scale duration. In any case, these two receptors are likely to cooperate elaborately with each other with regard to inhibition of net responses of macrophages. Such transcriptional switching from EP4 to EP2 may have more profound roles other than inhibition of cytokine production. The precise physiological significance of EP2 and EP4 in macrophages currently remains unknown, but should be addressed by receptor-knockout studies in the near future.

One of the remarkable findings in this study is that both EP2 and EP4 gene expression are affected by LPS stimulation, but that the mechanisms of their regulation appear quite different. This study demonstrated that the down-regulation of EP4 gene expression is mediated by PGE2 itself, produced upon LPS-stimulation. Because an EP4 agonist, as well as dbcAMP, but not an EP2 agonist affected EP4 expression, EP4-mediated cAMP accumulation is thought to be involved in its own down-regulation. Moreover, the reversal action of cycloheximide may be due to its inhibitory effect on the endogenous synthesis of COX-2 protein. Such effects of LPS on EP4 expression were different from our previous observations in the J774.1 macrophage-like cell line, in which EP4 mRNA was slightly induced upon LPS-stimulation. One possibility for this difference is that J774.1 cells produce a smaller amount of PGE2 in response to LPS treatment. Indeed, the amount of PGE2 production in the current experiment 14 h after LPS treatment (Fig. 1) is 10 times greater than the value obtained from J774.1 cells in the same condition (34). One of the reasons we chose peritoneal resident macrophages in this study is that these cells maintain characteristics close to that of native macrophages. It is interesting in this respect that peritoneal macrophages are able to produce a much larger amount of PGE2 compared with the J774.1 cell line. In addition, it should be noted that the concentration of PGE2 that was added exogenously is in the same range as that derived from LPS-stimulated macrophages. This suggests that down-regulation of EP4 gene expression in macrophages may take place in the peritoneal cavity when they are subjected to inflammatory conditions. It has indeed been reported that PGE2 is present at greater than nanomolar order concentrations in exudates from mice with peritonitis (35). However, at present, it remains unknown as to what kinds of transcription factors are involved in the regulation of EP4 receptor gene expression, regarding both its constitutive expression as well as its expression upon induction by stimuli. The EP4 gene contains a NF-κB site in its 5′ flanking region (16), and interaction between cAMP-induced CRE and NF-κB is a likely regulatory mechanism because cAMP-elevating agents reduced NF-κB binding through stabilization of IκBα in several cell types (36). However, EP4 gene expression is already present in macrophages and other macrophage-like cells in the absence of LPS treatment (37). Therefore, it is unlikely that NF-κB is involved in the basal expression of the EP4 gene.

We previously reported that the EP2 gene has potential NF-κB, NF-IL6, progesterone response element, and CRE binding sites in its promoter region, and that this gene has two transcriptional start sites specific to macrophages and uterine epithelial cells (38). These results suggested that EP2 gene expression may be regulated by many kinds of stimuli in a cell type-specific manner. This study demonstrated that EP2 gene expression is induced upon medium change, and that this induction is up-regulated by LPS stimulation. A slight induction of EP2 gene expression was also observed in C3H/HeJ macrophages. This induction upon medium change could be due to growth factors present in the fetal serum. Indeed, we and other groups have found that EP2 gene expression is affected by stimulation with not only LPS but also by hormones and cytokines (15, 39). In addition, the most remarkable finding in this study is that an up-regulation of EP2 by LPS stimulation was observed in C3H/HeJ macrophages. A mutation in the gene for TLR4 has been found to be the main cause of LPS hyporesponsiveness in C3H/HeJ mice (27), and TLR4 is now thought to be one of the receptors recognizing LPS in vivo (40). However, this study indicates that TLR4 is not necessary for LPS-induced up-regulation of EP2 gene expression. As shown by PGE2 synthesis, C3H/HeJ macrophages were still sensitive to LPS even though the response was much less than that of C3H/HeN macrophages, indicating that some mechanism that responds to LPS treatment exists other than that via TLR4 in C3H/HeJ macrophages. Such mechanisms of LPS-induced signaling may play a role in the up-regulation of the EP2 gene. Indeed, the activation of NF-κB upon LPS treatment in C3H/HeJ macrophages has been reported (41), and this event may be involved in the up-regulation of the EP2 gene.

We thank H. A. Popiel for careful reading of the manuscript, and S. Terai for secretarial assistance.

1

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan; scientific funds from the Japan Health Science Foundation; and from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

3

Abbreviations used in the paper: COX, cyclooxygenase; dbcAMP, dibutyryl cAMP; TLR4, Toll-like receptor 4; CRE, cAMP response element; CHO, Chinese hamster ovary.

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