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Peritoneal Macrophagesby Lipopolysaccharide in C3H/HeN

and Their Different Regulation4 and EP2EPThe Expression of Prostaglandin E Receptors

IchikawaMaruyama, Hana Yamane, Soken Tsuchiya and AtsushiKatsuyama, Hisae Karahashi, Fumio Amano, Takayuki Reiko Ikegami, Yukihiko Sugimoto, Eri Segi, Masato

http://www.jimmunol.org/content/166/7/4689doi: 10.4049/jimmunol.166.7.4689

2001; 166:4689-4696; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/7/4689.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Expression of Prostaglandin E Receptors EP2 and EP4

and Their Different Regulation by Lipopolysaccharide inC3H/HeN Peritoneal Macrophages1

Reiko Ikegami,* Yukihiko Sugimoto,* Eri Segi,* Masato Katsuyama,* Hisae Karahashi,†

Fumio Amano,† Takayuki Maruyama, ‡ Hana Yamane,* Soken Tsuchiya,* andAtsushi Ichikawa2*

The expression and regulation of the PGE receptors, EP2 and EP4, both of which are coupled to the stimulation of adenylatecyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP4 but not EP2 was foundin nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNAexpression of EP4 was down-regulated by LPS but not by medium change. PGE2 increased the cAMP content of both LPS-treatedand nontreated cells. ONO-604, an EP4 agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPSfor 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 effectsof ONO-604 on TNF-a and IL-12 production were equipotent with PGE2 at any time point, but the inhibitory effects of butaprostwere only seen from 14 h after stimulation. PGE2 or dibutyryl cAMP alone, but not butaprost, reduced EP4 expression, andindomethacin reversed the LPS-induced down-regulation of EP4, indicating that the down-regulation of EP4 is mediated byLPS-induced PG synthesis and EP4 activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduc-tion in EP4 expression was found in the cells treated with PGE2 alone, but not in LPS-treated cells. In contrast, up-regulation ofEP2 expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP4 is involved mainly inthe inhibition of cytokine release, and that the gene expression of EP2 and EP4 is differentially regulated during macrophageactivation. The Journal of Immunology,2001, 166: 4689–4696.

Prostaglandin E2 is a major arachidonate metabolite synthe-sized by cyclooxygenase (COX),3 and contributes to im-mune suppression. PGE2 inhibits B and T lymphocyte pro-

liferation, as well as various macrophage functions (1–4).Macrophages are known to produce a large amount of PGE2 inresponse to proinflammatory stimuli such as IL-1 and bacterialLPS (5). This production of PGE2 is believed to be driven by theCOX-2 enzyme, which is also synthesized de novo upon stimula-tion with LPS (6, 7). Released PGE2 also acts on the macrophagesthemselves, and exhibits inhibitory effects on not only early butalso late processes involved in macrophage activation, producing anegative feedback loop; PGE2 inhibits the production of variouscytokines such as TNF-a, IL-1b, 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 in-tracellular cAMP. The effects of PGE2 are exerted by specific re-ceptors on the plasma membrane of target cells (10, 11). Based onpharmacological and cDNA cloning studies, four subtypes of PGEreceptor, EP1, EP2, EP3, and EP4, have been identified and havebeen shown to differ in their signal transduction pathways (12–14).Previous investigations have strongly suggested the coupling ofPGE 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, areexpressed in cultured murine macrophage-like cell lines such asJ774.1 and RAW264.7 (15, 16). However, it is not known whetherthe two receptors contribute equally to the inhibition of activatedmacrophage function. Furthermore, recent studies using receptorgene knockouts have focused on the regulation of PG receptorgene expression. In contrast, recent findings in the field of localmediators have led us to consider the possibility that gene expres-sion of the receptors could also be induced by various kinds ofstimuli (17). For example, Matsuoka et al. found that PGD receptorexpression induced in alveolar epithelial cells upon Ag challengeplays a pivotal role in the appearance of allergic responses (18).Therefore, it is possible that PGE receptor gene expressionchanges during macrophage activation. However, there have beenno reports stating such a point of view regarding PGE receptors inmacrophages.

Here we designed experiments for two main purposes. First, toidentify which receptor is mainly responsible for the inhibitoryaction of PGE2 on cytokine release, we investigated time-depen-dent mRNA expression of PGE receptors during LPS-inducedmacrophage activation, and examined the effects of EP-specificagonists on cytokine production. Second, to explore the possible

*Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, KyotoUniversity, Kyoto, Japan;†Department of Biochemistry and Cell Biology, NationalInstitute of Infectious Diseases, Tokyo, Japan; and‡Discovery Research LaboratoryI, Minase Research Institute, Ono Pharmaceutical, Osaka, Japan

Received for publication December 9, 1999. Accepted for publication January22, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants-in-aid for scientific research from the Ministryof Education, Science, and Culture of Japan; scientific funds from the Japan HealthScience Foundation; and from the Mochida Memorial Foundation for Medical andPharmaceutical Research.2 Address correspondence and reprint requests to Dr. Atsushi Ichikawa, Departmentof Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University,Sakyo-ku, Kyoto 606-8501, Japan. E-mail address: aichikaw@pharm.kyoto-u.ac.jp3 Abbreviations used in the paper: COX, cyclooxygenase; dbcAMP, dibutyryl cAMP;TLR4, Toll-like receptor 4; CRE, cAMP response element; CHO, Chinese hamsterovary.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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mechanisms underlying regulation of receptor gene expression, weexamined the effects of cAMP-elevating agents, an inhibitor of PGsynthesis, and a spontaneous deficiency in LPS perception, whichwas recently identified to be a genetic mutation in one of the re-ceptors, for a bacterial cell wall components. This study demon-strates that gene expression of EP4 is down-regulated via LPS-induced PG synthesis and EP4 activation, whereas that of EP2 isup-regulated by LPS stimulation via a Toll receptor-independentmechanism. Although the expression profiles suggested the possi-bility of a receptor switch from EP4 to EP2, the EP4 expressedinitially appears mainly to contribute to the inhibition of cytokineproduction in macrophages.

Materials and MethodsReagents

LPS fromEscherichia coliO55:B5, dibutyryl cAMP (dbcAMP) and indo-methacin were obtained from Sigma (St. Louis, MO). Cycloheximide waspurchased from Wako Chemicals (Osaka, Japan). The125I-labeled cyclicAMP assay system was purchased from Amersham (Arlington Heights,IL), and ELISA kits for mouse TNF-a and for mouse IL-12 were purchasedfrom BioSource International (Camarillo, CA) and R&D Systems (Min-neapolis, MN), respectively. PGE2 in the medium was quantified using anenzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). PGE2 waspurchased from Funakoshi (Tokyo, Japan). Butaprost, an EP2-specific ag-onist, and ONO-604, an EP4-specific agonist, were generated, and theirspecificities were analyzed by measuring their binding affinities to the re-spective EPs expressed in Chinese hamster ovary (CHO) cells (Table I)(19). PGE2 bound to all EPs. Based on the results obtained from dosedependence analyses, we used 10 nM of ONO-604 and 1mM of butaprost.These agonists at these concentrations selectively activate EP4 and EP2,respectively.

Preparation and activation of mouse peritoneal macrophages

Six-week-old C3H/HeN and C3H/HeJ female mice were obtained fromJapan SLC (Hamamatsu, Japan) as specific pathogen-free animals. Themice were killed, and their peritoneal resident macrophages were collectedby washing the peritoneal cavity with 5 ml of ice-cold saline. After wash-ing with PBS, the macrophages (23 106 cells) were seeded onto plasticPetri 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°Cfor 1 h, nonadherent cells were removed by repeated washing. More than95% of the cells were macrophages as identified by their phagocytic ac-tivity toward zymosan, by Fc-rosette formation to the immune complexbetween sheep RBC and the specific Ab (SRBC-Ab), and by the nonspe-cific esterase reaction that catalyzesa-naphtyl butylate. The purity of thecell population was consistent throughout the experiments, and the viabilityof the macrophages remained greater than 98%.

The culture medium was replaced with fresh medium either with orwithout 100 ng/ml LPS, and the cells were then incubated at 37°C for theindicated number of hours. The cells were washed twice with PBS, scrapedwith 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 ofice-cold PBS and stored at280°C until use.

Immunoblotting of COX-2

Western blotting was performed as described (20). Fifty-microgram ali-quots of the cell extracts were loaded onto 7.5% SDS gels (21) and elec-trotransferred 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 with125I-labeled protein A at 37°C for 1 h. After repeated washing, the images werevisualized with the BAS-2000 bioimage analyzer (Fuji, Tokyo, Japan).

Northern blot analysis

Total RNA was isolated from 1–1.53 107 macrophages by the acid gua-nidinium thiocyanate-phenol-chloroform method (22). The RNA (10mg)was separated by electrophoresis on a 1.5% agarose gel and transferredonto a nylon membrane (BIODYNE; Pall, East Hills, NY). For the detec-tion of EP2 mRNA, theEcoRI insert DNA (1.7 kb) of ML202 (23) wasused as a hybridization probe. For EP4 mRNA detection, a 970-bp frag-ment of the mouse EP4 cDNA containing the putative first to seventh trans-membrane domains was used as a probe (15). The cDNA probes used formouse COX-1 and COX-2 were as reported previously (24). Hybridizationwas conducted under the conditions described previously (25), and RNAbands were visualized by autoradiography. The blots were then strippedand rehybridized with a32P-labeled DNA probe for GAPDH. Quantifica-tion of the hybridized signals was conducted with the BAS-2000 Bio-image analyzer. Northern blot experiments were independently repeatedthree times. Representative results are shown in the photographs, and EPmRNA levels were normalized to GAPDH mRNA levels as the mean6SEM of three independent experiments.

cAMP assay, PGE2 quantification, and ELISAs

cAMP levels in peritoneal macrophages were determined as reported pre-viously (26). Cells cultured in 24-well plates (13 106 cells/well) werewashed with 0.5 ml of Krebs-HEPES buffer (pH 7.4) with 10mM indo-methacin, and preincubated for 10 min. Reactions were started by the ad-dition of test reagents along with 100mM Ro-20-1724 and 10mM indo-methacin. After incubation for 10 min at 37°C, reactions were terminatedby the addition of 10% trichloroacetic acid. Then the cAMP content of thecells was measured by the cAMP radioimmunoassay kit.

For the quantification of PGE2, peritoneal macrophages collected fromC3H/HeN mice were preincubated in 24-well plates at a density of 13 106

cells/well and then incubated with 0.5 ml of medium with or without 100ng/ml LPS for the indicated time periods. The concentration of PGE2 in themedium was then determined by an enzyme immunoassay kit, according tothe manufacturer’s protocol.

For the quantification of cytokine levels, cells were seeded at 0.53 106

cells/well and then incubated with 0.5 ml of medium containing 100 ng/mlLPS in the presence or absence of PGE2 (1 mM), butaprost (1mM), andONO-604 (10 nM). The culture supernatants were transferred to new tubesfor the indicated time periods, followed by the examination of TNF-a andIL-12 levels by ELISA kits, according to the manufacturer’s protocol.

Statistical analysis

For Northern analyses, data were expressed as the mean6 SEM of threeindependent experiments. For the determination of PGE2, cAMP, and cy-tokine levels, representative data were expressed as the mean6 SEM oftriplicate determinants. These experiments were independently repeatedthree times, and similar results were obtained. Statistical analyses wereperformed using Student’st test.

ResultsCOX expression and PGE2 synthesis in LPS-stimulatedmacrophages

In this study, we designed experiments to 1) clarify the mode ofPGE2 action in macrophages with respect to the PGE receptorsubtypes, and 2) investigate possible changes in receptor gene ex-pression during macrophage activation. Peritoneal resident macro-phages isolated from C3H/HeN mice were used for this purpose.

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-PGE2

EP23H-PGE2

EP33H-PGE2

EP43H-PGE2

PGE2 0.018 0.038 0.005 0.0031ONO-604 0.61 0.28 1.5 0.0007Butaprost .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 theKi values (micromolar concentrations) werecalculated.

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Macrophages themselves produce a large amount of PGE2 in re-sponse to stimuli such as LPS, and such endogenous PGE2 isthought to affect functions of macrophages in an autocrine manner.Therefore, we thought it is necessary to examine the time-depen-dent production of PGE2 from LPS-stimulated cells in our system(Fig. 1A). Basal PGE2 production in the absence of LPS treatmentfor 3 h was 0.441 ng/ml. In LPS-treated macrophages, a largeamount of PGE2 was detected; PGE2 production 3 and 7 h after

LPS treatment was 30.76 1.58 and 2866 13 ng/ml, respectively,which corresponds to 0.087 and 0.81mM of PGE2, respectively.When we examined the expression of COX isozyme mRNAs inthese cells, faint expression of COX-1 mRNA was detected innonstimulated macrophages, and expression levels did not changeupon LPS treatment (data not shown). In contrast, LPS treatmentrapidly induced the expression of COX-2 mRNA (Fig. 1B). West-ern blot analyses again showed that COX-2 proteins were inducedwhen cells were treated with LPS (Fig. 1C). LPS treatment stim-ulates PGE2 production in C3H/HeN macrophages, resulting inexposure of the cells to micromolar order concentrations of PGE2.Transcriptional induction of COX-2 may be a possible mechanismfor LPS-induced PGE2 production.

The effects of indomethacin and PGE2 on TNF-a and IL-12production in LPS-stimulated macrophages

PGE2 regulates a variety of functions in macrophages, includingcytokine production. This study demonstrated that LPS-stimulatedmacrophages are exposed to micromolar order concentrations ofPGE2. To explore the possible involvement of endogenous PGE2

in cytokine release, we examined the effects of indomethacin, aninhibitor of PG synthesis, on production of the most representativemacrophage cytokines, TNF-a and IL-12. As shown in Fig. 2,TNF-a was released immediately after LPS-treatment, reaching apeak after 3 h, and then diminished, possibly due to its rapid deg-radation (rapid response). In contrast, IL-12 production graduallyincreased until 14 h after treatment (slow response). When indo-methacin was added simultaneously with LPS, both TNF-a andIL-12 production was enhanced; a 5.7-fold increase in TNF-a at3 h and a 1.9-fold increase in IL-12 at 14 h was observed comparedwith treatment with LPS alone at the corresponding times. Theseresults indicated that endogenous PGs are involved in the suppres-sion of cytokine production. Even in this system, exogenous PGE2

FIGURE 1. PGE2 synthesis and COX-2 expression in LPS-stimulatedC3H/HeN macrophages.A, PGE2 synthesis in LPS-stimulated macro-phages. Peritoneal macrophages collected from C3H/HeN mice were pre-incubated in 24-well plates at a density of 13 106 cells/well, followed byincubation in medium with (F) or without (E) 100 ng/ml LPS for theindicated time periods. The concentrations of PGE2 in the medium werethen determined as described inMaterials and Methods. The values ob-tained from the medium only were used as the value at 0 h. Values areexpressed as the mean6 SEM for triplicate determinants. The experimentswere independently repeated three times, and similar results were obtained.B, Expression of COX-2 mRNA in LPS-stimulated C3H/HeN macro-phages. Peritoneal macrophages were exposed to fresh medium with (LPS1) or without 100 ng/ml LPS (LPS2) for the indicated times, and totalRNA was isolated. Ten micrograms of each RNA sample was subjected toNorthern blot analysis. The blots were hybridized with a probe for COX-2,and then rehybridized with a probe for GAPDH.C, Detection of theCOX-2 protein in C3H/HeN macrophages. Peritoneal macrophages wereexposed to medium with (LPS1) or without 100 ng/ml LPS (LPS2) for4 h. The cells were collected, and the cell lysates were subjected to im-munoblot analysis for the COX-2 protein.

FIGURE 2. Effects of indomethacin, PGE2, and dbcAMP on LPS-in-duced TNF-a and IL-12 release in C3H/HeN macrophages. Peritonealmacrophages were exposed to fresh medium only (crosses) or mediumcontaining 100 ng/ml LPS supplemented with no reagents (F), 10mMindomethacin (M), 1mM PGE2 (E), or 1 mM dbcAMP (L) for the indi-cated times. The supernatants were recovered 3, 7, and 14 h after LPSstimulation, and the TNF-a and IL-12 contents were determined as de-scribed inMaterials and Methods. The values obtained in medium only areused for the time point 0 h. Values are expressed as the mean6 SEM fortriplicate determinants. The experiments were independently repeated threetimes, and similar results were obtained (p,p , 0.05 for LPS plus reagent-treated vs LPS-treated only cells).

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(1 mM) was effective for the inhibition of cytokine production,resulting in 17% of the value obtained with LPS alone for TNF-aproduction (3 h) and 48% for IL-12 production (14 h) (Fig. 2).Because PGE2 is known to be coupled to cAMP production in anumber of macrophage-like cells, we further examined the effectsof dbcAMP on cytokine production. In both cases, dbcAMP com-pletely mimicked the effects of PGE2, suggesting that suppressionof cytokine production by PGE2 is mediated by PGE receptorscoupling to the stimulation of adenylate cyclase, possibly by theEP2 and/or EP4 receptors.

The expression of EP2 and EP4 mRNA in LPS-stimulatedmacrophages

To investigate whether EP2 and EP4 receptors are expressed innonstimulated and LPS-stimulated macrophages, we examined theexpression of EP2 and EP4 mRNA (Fig. 3). In macrophages justbefore treatment (time 0), a significant amount of EP4 mRNA wasfound, but EP2 mRNA could not be detected in the Northern blot.When these macrophages were stimulated with LPS, the expres-sion of EP2 mRNA transiently increased to a maximum 3 h aftertreatment, then decreased by 6 h, but did not return to values be-fore the detection level. A slight but significant induction of EP2

mRNA was also observed in macrophages treated in a similarmanner in the absence of LPS. The expression level after LPStreatment is 3-fold that after medium change only. These resultsindicate that the expression levels of EP2 mRNA in peritonealmacrophages is below detection levels before any treatment, isslightly induced upon medium change, possibly due to other in-duction factors in the serum, and is strongly enhanced by LPS. Incontrast, the expression of EP4 mRNA was down-regulated 3 hafter LPS treatment, resulting in,10% of the control level. Thissuppression was still observed 6 h after LPS treatment, whereas nosuppression was observed upon medium change only. To test theexpression of EP1 and EP3 mRNA, we performed RT-PCR anal-yses on the RNA samples from freshly prepared macrophageswithout any treatment, macrophages treated with LPS for up to 6 h,and macrophages subjected to medium change only (data notshown). However, we failed to detect significant signals for EP1

and EP3 mRNA in all preparations. Peritoneal macrophages areunlikely to express the EP1 and EP3 subtypes of PGE receptors.

cAMP production by PGE receptor-selective agonists in LPS-stimulated macrophages

To confirm the dynamic changes in the expression of these recep-tors, it was considered best to investigate the EP2 and EP4 recep-tors at the protein level using Western blot analyses. However,unfortunately, there are currently no EP2 or EP4 receptor Absavailable for Western blot detection, and we chose to study theexpression of the EP2 and EP4 receptors using functional analysesby examining the effects of PGE2 and EP-selective agonists oncAMP production in C3H/HeN macrophages at the indicated timepoints after LPS treatment (Fig. 4). As shown in Fig. 1, becausethese macrophages produce PGE2 at micromolar order concentra-tions, we used 1mM of PGE2 to induce cAMP production. Weused 1mM of butaprost, an EP2-specific agonist, and 10 nM ofONO-604, an EP4-selective agonist, both of which have equipotentefficacies for inducing cAMP production as that of 1mM of PGE2,without affecting other receptor functions as evaluated in the CHOexpression system (Table I). In macrophages without LPS treat-ment, both PGE2 and ONO-604, but not butaprost increased cAMPproduction, suggesting that the PGE2-induced cAMP production ismediated via EP4, but not by EP2 in this preparation. Similar re-sults were obtained 1 and 3 h after LPS treatment. In contrast, inmacrophages stimulated with LPS for 6 h, butaprost increased

cAMP to levels generated by PGE2, but ONO-604 did not. Theseresults indicate that the PGE receptor responsible for cAMPswitches from EP4 to EP2 by 6 h after LPS treatment. In this assay,basal cAMP production increased and the response generated byagonists decreased with time after LPS treatment despite the pres-ence of indomethacin in the assay solution. One of the reasonscould be because a variety of responses occur in LPS-stimulatedmacrophages, and other cAMP-producing factors may be releasedduring the assay or the sensitivity of PGE2 in PGE receptors maybe altered. Thus, this assay may not reflect the exact amount ofeach receptor expressed at the protein level.

The effects of PGE agonists on the production of TNF-a andIL-12 in LPS-stimulated macrophages

To investigate the contribution of the EP2 and EP4 receptors inmacrophage functions, we examined the effects of EP-selective

FIGURE 3. Time course of EP2 and EP4 mRNA expression in LPS-stimulated C3H/HeN macrophages. Peritoneal macrophages were pre-pared, exposed to fresh medium with (LPS1 or F) or without (LPS2 orE) 100 ng/ml LPS for the indicated times, and then collected. The mac-rophages just before exposure to fresh medium were collected as the timepoint 0 h. Total RNA (10mg) isolated from each sample was subjected toNorthern blot analysis of EP2 and EP4. The blots were rehybridized with aprobe for GAPDH. The results shown in theupper panelsare representa-tive of three separate experiments. The blots were subjected to radioactiveimage analysis, and EP mRNA levels were normalized to GAPDH mRNAlevels as the mean6 SEM of three independent experiments shown in thelower panels. The EP2/GAPDH values are represented as fold of the valuefor LPS (2) 3 h, and the EP4/GAPDH values are represented as fold of thevalue at 0 h. (p,p , 0.01 for LPS-stimulated vs nonstimulated cells).

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agonists on the LPS-induced production of TNF-a and IL-12. Re-garding TNF-a production measured 3 h after LPS treatment, PGE2

inhibited TNF- a production by 83% and ONO-604 by 61%, butinhibition by butaprost was only by 9.6% (Fig. 5). As suggested in thecAMP formation assays, EP4 works as a predominant PGE receptoragainst TNF-a production around 3 h after stimulation. RegardingIL-12 production, both PGE2 and ONO-604 showed inhibitory effectat every time point examined, and the inhibitory potency of ONO-604was close to that of PGE2. This is an interesting observation, as theEP4 receptor is likely not to work around 6 h after stimulation, asdeduced from the cAMP formation experiments. Activation of EP4

for just the first several hours after LPS treatment may be enough tocause an inhibition of this cytokine to near maximum levels. On thecontrary, the inhibitory effect of butaprost was not significant at 3 and7 h, but apparent at 14 h (29.3% inhibition), indicating that the EP2

receptor works only during late time points. These results indicatedthat the effects of PGE2 are mediated by both the EP2 and EP4 re-ceptors, but that each receptor contributes to the inhibition of activatedmacrophage function in a different manner via changes in their ex-pression patterns.

PGs mediate the down-regulation of EP4 expression but not theup-regulation of EP2 expression

We next focused on the regulatory mechanisms underlying thechanges in expression of EP2 and EP4 mRNAs upon LPS treat-ment. LPS stimulation was able to influence the transcriptionalexpression of various proteins in a direct or indirect manner inmacrophages. To explore whether the up-regulation of the EP2

gene and the down-regulation of the EP4 gene require general pro-tein synthesis, we examined the effect of cycloheximide on the EP2

and EP4 mRNA expression (Fig. 6). Cycloheximide (0.1mg/ml)failed to affect LPS-induced EP2 gene expression, but completelyreversed the LPS-induced down-regulation of EP4 gene expres-sion. These results suggest that the up-regulation of EP2 gene ex-pression does not require the protein synthesis, and is possiblydirectly activated by LPS signals, but that the down-regulation ofEP4 gene expression requires protein synthesis, possibly indirectlycontrolled by LPS via the synthesis of other proteins. COX-2 isundoubtedly one of the proteins synthesized rapidly in response toLPS treatment in macrophages. PGE2 release is believed to be a

result of COX-2 protein induction, as shown in Fig. 1. Thus wehypothesized that the LPS-induced PGE2 may affect EP4 gene ex-pression itself in a negative-feedback manner. Based on this hy-pothesis, we examined the effect of PGE2 alone or indomethacinwith LPS on EP4 mRNA expression (Fig. 7). Incubation of PGE2(1 mM) without LPS for 3 h inhibited the basal expression of EP4

mRNA, and indomethacin added simultaneously with LPS stimu-lation reversed the LPS-diminished expression of EP4 mRNA, sug-gesting that the decrease in EP4 mRNA expression was a result offeedback regulation by PGE2. dbcAMP (1 mM) and ONO-604 (10nM, data not shown) mimicked the effects of PGE2, but butaprost(1 mM) failed to inhibit EP4 mRNA expression. These results sug-gested that EP4-induced cAMP formation negatively regulates theexpression of the EP4 gene itself, and the effect of cycloheximidemay 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 expressionupon LPS treatment is independent of PG synthesis.

LPS treatment elicited the up-regulation of EP2 mRNA but notthe down-regulation of EP4 mRNA in C3H/HeJ macrophages

One of the reasons we chose macrophages from the C3H/HeNstrain is that this strain has a genetically comparable mutant strain,

FIGURE 4. cAMP-elevating potencies of PGE analogs in C3H/HeN mac-rophages at different time points after LPS treatment. Peritoneal macrophageswere prepared, and untreated cells or cells treated with 100 ng/ml LPS for theindicated time periods were exposed to medium supplemented with no PGEanalogs (h), 1 mM PGE2 (■), 1 mM butaprost (p), or 10 nM ONO-604 (1)for 10 min. Test reagents contain 10mM indomethacin and 100mM Ro-20-1724. The cAMP contents were then determined as described inMaterials andMethods. Values are expressed as the mean6 SEM of triplicate determinants.The experiments were independently repeated three times, and similar resultswere obtained (p, p , 0.005 for PGE analog-treated vs PG-untreated cells).

FIGURE 5. Effects of PGE analogs on LPS-induced TNF-a and IL-12release in C3H/HeN macrophages. Peritoneal macrophages were preparedand exposed to fresh medium only (crosses) or medium containing 100ng/ml LPS supplemented with no PG analogs (F), 1mM PGE2 (E), 1 mMbutaprost (‚), or 10 nM ONO-604 (Œ) for the indicated time periods. Thesupernatants were recovered, and the TNF-a and IL-12 contents were de-termined as described inMaterials and Methods. The contents obtainedfrom medium only were used as time 0. The results are shown as themean6 SEM for triplicate determinants. The experiments were indepen-dently repeated three times, and similar results were obtained (p,p , 0.05for PGE analog-treated vs nontreated cells).

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C3H/HeJ, in which the macrophages hardly respond to LPS. Re-cently, C3H/HeJ was found to have a point mutation within thecoding region of the Toll-like receptor 4 (TLR4), a number of aprotein family containing proteins that have been implicated inLPS-induced cell signaling (27). Indeed, C3H/HeJ macrophagesdid produce a significant amount of PGE2, but much less than thatin C3H/HeN; the PGE2 contents in the medium was,1 nM evenafter 7 h of LPS stimulation (Fig. 8A). COX-2 expression levelswere analyzed by Northern and Western analyses, showing thatCOX-2 induction was faint in LPS-treated C3H/HeJ macrophages(data not shown). If the down-regulation of EP4 gene expressionrequires more than a nanomolar order concentration of PGE2, itshould not occur in this strain. As expected, no suppression of EP4

mRNA expression was observed after LPS treatment (Fig. 8B). Inaddition, when C3H/HeJ macrophages were stimulated with PGE2

or dbcAMP, there was a decrease in EP4 mRNA, as was seen in theC3H/HeN macrophages (Fig. 8C). These results again indicate thatthe expression of EP4 mRNA is down-regulated via an EP4-me-diated cAMP-dependent pathway. In contrast, surprisingly and un-expectedly, a slight induction of EP2 mRNA was again seen uponjust medium change, and this induction was greatly enhanced byLPS treatment, as was seen in C3H/HeN macrophages. These re-sults 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.

DiscussionA functional switch from EP4 to EP2 during macrophage activation

Although there have been many reports on the mechanism ofCOX-2 expression after LPS stimulation and the effect of PGE2 onLPS-activated macrophages, there have been very few reports thatfocus on the expression and function of the PGE receptors inmonocytes/macrophages (28–30). Here we examined the time-de-pendent expression of EP2 and EP4 mRNA during activation inresident peritoneal macrophages, and found that the EP2 gene istransiently up-regulated, whereas the EP4 gene is down-regulatedupon LPS stimulation. Based on the results obtained from thecAMP assays using subtype-selective agonists, we conclude that

EP4 is the dominant PGE receptor in nonstimulated macrophagesor macrophages up to 3 h after LPS treatment. This is supported byour results showing that TNF-a production 3 h after LPS treatmentis completely inhibited by an EP4 agonist, but not an EP2 agonist.However, the cAMP assay also indicated that the EP4 receptor isno longer active by 6 h after LPS treatment, and, in turn, EP2

seems to become the dominant receptor by 6 h. This is also sup-ported by the result that the inhibitory effect of an EP2 agonist onIL-12 production was apparent at this time point. In the presenceof an EP2 agonist, EP2 is likely to work by 6 h after LPS treatment,resulting in no increase observed in production of IL-12 between6 and 14 h. In contrast, the inhibitory effect of an EP4 agonist onIL-12 production was observed for a longer time than we expected,even beyond 6 h after LPS treatment. Because IL-12 productioninitiated by LPS includes de novo synthesis, the EP4-inducedcAMP increase during the initial few hours after LPS addition mayexert continuous inhibition on IL-12 production. It is possible thatthe EP4-induced cAMP increase and the resultant activation ofcAMP-dependent kinase during initiation of IL-12 gene activationmay have a crucial effect on the following gene expression of thiscytokine. A number of reports have established that the cAMP-dependent pathway inhibits IL-12 production at the transcriptionallevel, possibly by affecting cAMP response element (CRE) bind-ing complexes (31–33). Thus, EP4 gene expression is down-reg-ulated upon stimulation with LPS, but this receptor appears to havea 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 co-operate elaborately with each other with regard to inhibition of netresponses of macrophages. Such transcriptional switching fromEP4 to EP2 may have more profound roles other than inhibition ofcytokine production. The precise physiological significance of EP2

FIGURE 6. Effects of cycloheximide on EP2 and EP4 mRNA expressionin LPS-treated and nontreated C3H/HeN macrophages. Peritoneal macro-phages were prepared, exposed to medium supplemented with a combinationof 100 ng/ml LPS and 0.1mg/ml cycloheximide (CHX) for 3 h, and collected.Total RNA (10mg) isolated from each sample was subjected to Northern blotanalysis. The results shown in theupper panelsare representative of threeseparate experiments. The blots were subjected to radioactive image analysis,and EP mRNA levels were normalized to GAPDH mRNA levels as themean6 SEM of three independent experiments as shown in thelower panels.The EP2/GAPDH and the EP4/GAPDH values are shown as the fold of valuesobtained from the sample exposed to medium only (p, p , 0.01 for CHX (1)and LPS (1) vs CHX (2) and LPS (1) cells).

FIGURE 7. Effects of cAMP-generating agents on basal mRNA expres-sion of EP receptors and the effects of indomethacin on up-regulated EP2

mRNA expression or down-regulated EP4 mRNA expression in C3H/HeNmacrophages. Peritoneal macrophages were exposed to medium supple-mented with 1mM PGE2, 1 mM butaprost, or 1 mM dbcAMP for 3 h, orcells were alternatively exposed to medium containing 100 ng/ml LPS inthe presence (LPS1 Indo.) or absence of 10mM indomethacin (LPS) for3 h. Total RNA (10mg) isolated from each collected sample was subjectedto Northern blot analyses. Theupper panelsare representative results ofthree separate experiments. The blots were subjected to radioactive imageanalysis, and EP mRNA levels were normalized to GAPDH mRNA levelsas the mean6 SEM of three independent experiments shown in thelowerpanels. The EP2/GAPDH and the EP4/GAPDH values are shown as thefold of values obtained from the cells exposed to the medium only for 3 h(p, p , 0.01 for PGE2- or dbcAMP-treated vs nontreated cells; †,p , 0.01for LPS-treated vs LPS and indomethacin-treated cells).

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and EP4 in macrophages currently remains unknown, but should beaddressed by receptor-knockout studies in the near future.

Mechanisms underlying the down-regulation of EP4 geneexpression and the up-regulation of EP2 gene expression inLPS-treated macrophages

One of the remarkable findings in this study is that both EP2 andEP4 gene expression are affected by LPS stimulation, but that themechanisms of their regulation appear quite different. This studydemonstrated that the down-regulation of EP4 gene expression ismediated by PGE2 itself, produced upon LPS-stimulation. Becausean EP4 agonist, as well as dbcAMP, but not an EP2 agonist affectedEP4 expression, EP4-mediated cAMP accumulation is thought tobe involved in its own down-regulation. Moreover, the reversalaction of cycloheximide may be due to its inhibitory effect on theendogenous synthesis of COX-2 protein. Such effects of LPS onEP4 expression were different from our previous observations inthe J774.1 macrophage-like cell line, in which EP4 mRNA wasslightly induced upon LPS-stimulation. One possibility for thisdifference is that J774.1 cells produce a smaller amount of PGE2 inresponse to LPS treatment. Indeed, the amount of PGE2 productionin the current experiment 14 h after LPS treatment (Fig. 1) is 10times greater than the value obtained from J774.1 cells in the samecondition (34). One of the reasons we chose peritoneal residentmacrophages in this study is that these cells maintain characteris-tics close to that of native macrophages. It is interesting in thisrespect that peritoneal macrophages are able to produce a muchlarger amount of PGE2 compared with the J774.1 cell line. In

addition, it should be noted that the concentration of PGE2 that wasadded exogenously is in the same range as that derived from LPS-stimulated macrophages. This suggests that down-regulation ofEP4 gene expression in macrophages may take place in the peri-toneal cavity when they are subjected to inflammatory conditions.It has indeed been reported that PGE2 is present at greater thannanomolar order concentrations in exudates from mice with peri-tonitis (35). However, at present, it remains unknown as to whatkinds of transcription factors are involved in the regulation of EP4

receptor gene expression, regarding both its constitutive expres-sion as well as its expression upon induction by stimuli. The EP4

gene contains a NF-kB site in its 59 flanking region (16), andinteraction between cAMP-induced CRE and NF-kB is a likelyregulatory mechanism because cAMP-elevating agents reducedNF-kB binding through stabilization of IkBa in several cell types(36). However, EP4 gene expression is already present in macro-phages and other macrophage-like cells in the absence of LPStreatment (37). Therefore, it is unlikely that NF-kB is involved inthe basal expression of the EP4 gene.

We previously reported that the EP2 gene has potential NF-kB,NF-IL6, progesterone response element, and CRE binding sites inits promoter region, and that this gene has two transcriptional startsites specific to macrophages and uterine epithelial cells (38).These results suggested that EP2 gene expression may be regulatedby many kinds of stimuli in a cell type-specific manner. This studydemonstrated that EP2 gene expression is induced upon mediumchange, and that this induction is up-regulated by LPS stimulation.A slight induction of EP2 gene expression was also observed in

FIGURE 8. PGE2 production and mRNA expression of EP2 and EP4 in C3H/HeJ macrophages.A, PGE2 synthesis in LPS-stimulated C3H/HeN andC3H/HeJ macrophages. Peritoneal macrophages were collected and exposed to medium with (LPS1) or without (LPS2) 100 ng/ml LPS for 7 h. Thesupernatants were recovered, and their PGE2 content was determined as described inMaterials and Methods. Values are expressed as the mean6 SEMof three independent experiments (p,p , 0.01 for LPS-treated cells vs cells treated with medium change only in C3H/HeN mice; †,p , 0.05 for LPS-treatedcells 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 (LPS1 or F) or without (LPS2 or E) 100 ng/ml LPS. The macrophages before mediumchange were used for values at 0 h (p, p , 0.01 for LPS-treated cells vs cells treated with medium change only).C, Effects of cAMP-elevating agents onbasal mRNA expression of EP4 in C3H/HeJ macrophages. Peritoneal macrophages were treated with medium containing 1mM PGE2, 1 mM butaprost, or1 mM dbcAMP for 3 h (p, p , 0.01 for PGE2- or dbcAMP-treated cells vs cells treated with medium change only). InB andC, total RNA (10mg) isolatedfrom each sample was subjected to Northern blot analyses. The blots were subjected to radioactive image analysis, and EP mRNA levels were normalizedto GAPDH mRNA levels as the mean6 SEM of three independent experiments. The EP2/GAPDH values are represented as the fold of the value for LPS(2) 3 h, and the EP4/GAPDH values are represented as the fold of the values at 0 h.

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C3H/HeJ macrophages. This induction upon medium change couldbe due to growth factors present in the fetal serum. Indeed, we andother groups have found that EP2 gene expression is affected bystimulation with not only LPS but also by hormones and cytokines(15, 39). In addition, the most remarkable finding in this study isthat an up-regulation of EP2 by LPS stimulation was observed inC3H/HeJ macrophages. A mutation in the gene for TLR4 has beenfound to be the main cause of LPS hyporesponsiveness in C3H/HeJ mice (27), and TLR4 is now thought to be one of the receptorsrecognizing LPS in vivo (40). However, this study indicates thatTLR4 is not necessary for LPS-induced up-regulation of EP2 geneexpression. As shown by PGE2 synthesis, C3H/HeJ macrophageswere still sensitive to LPS even though the response was much lessthan that of C3H/HeN macrophages, indicating that some mecha-nism that responds to LPS treatment exists other than that viaTLR4 in C3H/HeJ macrophages. Such mechanisms of LPS-in-duced signaling may play a role in the up-regulation of the EP2

gene. Indeed, the activation of NF-kB upon LPS treatment in C3H/HeJ macrophages has been reported (41), and this event may beinvolved in the up-regulation of the EP2 gene.

AcknowledgmentsWe thank H. A. Popiel for careful reading of the manuscript, andS. Terai for secretarial assistance.

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