Induction of colitis.

DSS of the average molecular weight of 5,000 (Sigma Chemical Co., St. Louis, Missouri, USA) was administered to 8-week-old mice for 7 days at either 3% (low dose) or 7% (high dose) concentration in the drinking water. The addition of DSS or any drugs mentioned below to the drinking water did not affect water consumption of mice. Indomethacin also was added to the drinking water at a dose of 4 mg/kg/day and administered to the animals during the entire experimental period. This dose of indomethacin was reported to inhibit PGE₂ production in rats and mice in vivo (22, 23). An EP4 antagonist, ONO-AE3-208, 4-{4-Cyano-2-[2-(4-fluoronaphthalen-1-yl) propionylamino] phenyl} butyric acid (AE3-208), and an EP4 agonist, ONO-AE1-734, methyl-7-[(1R, 2R, 3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)-1-butenyl]-5-oxocyclopenthl]-5-thiaheptanoate (AE1-734), were kindly provided by Ono Pharmaceutical Co., Osaka, Japan. The K_i values of AE3-208 obtained by competition-binding isotherms to displace the radioligand binding to the respective prostanoid receptor are 1.3, 30, 790, 2,400 nM for EP4, EP3, FP, and TP, respectively, and more than 10,000 nM for the other prostanoid receptors. The K_i values of AE1-734 are 0.7, 56, and 620 nM for EP4, EP3, and EP2, respectively, and more than 10,000 nM for the rest of the prostanoid receptors. AE3-208 was administered (10 mg/kg/day) orally in the drinking water. When this compound was administered orally at 10 mg/kg as a bolus, a peak plasma concentration of 677 ng/ml was attained in 0.25 hours after the administration with 18% of bioavailability. The plasma half-life of this compound measured in an experiment of intravenous injection was 0.2 hours. AE1-734 was administered subcutaneously twice a day (0.1 mg/kg/each) from 1 day before the DSS treatment until the end of experiment. When AE1-734 was injected subcutaneously at this dose, the peak plasma concentration of 100 ng/ml was attained at 10 minutes after the injection with more than 70% for bioavailability. The plasma concentration declined with a half-life of 30 minutes.

To assess the extent of colitis, body weight, stool consistency, and occult blood in the stool were monitored daily (24). Diarrhea was scored as follows: 0, normal; 2, loose stools; 4, watery diarrhea. Hemoccult was scored as follows: 0, normal; 2, hemoccult positive; 4, gross bleeding. On the last day of the experiments, blood was collected from the animals by intracardiac puncture. The colon was removed for histological examination, and the spleen was weighed. Hematocrit (HCT) and the white blood cell (WBC) number were determined using a blood cell counter (Sysmex K4500; Sysmex Co., Long Grove, Illinois, USA).

To evaluate mucosal integrity, the FITC-dextran assay was used (25). Wild-type C57BL/6 mice were administered with either AE3-208 or vehicle in the drinking water. After 1 day, both groups of mice were fed with 3% DSS in the drinking water in the continued presence or absence of AE3-208, and 24 hours later, 200 μl of FITC-dextran (average molecular weight, 4,400; Sigma Chemical Co.) (2 mg/ml in saline) was administered orally. Serum concentration was determined 4 hours after the administration of FITC-dextran. EP4–/– mice and their wild-type control mice were similarly treated with 3% DSS and administered with FITC-dextran. The colon was snap-frozen and cryostat sections of 10-μm thickness were used for fluorescent microscopic analysis.

Histology.

The colon was divided into the proximal, middle, and distal portions and was fixed in Bouin’s fixative. Sections 5 μm thick were prepared and subjected to staining with hematoxylin and eosin (H&E). The histological examination was performed in a blinded fashion by two coauthors and scored as described (24). Two independent parameters were measured: the extent of inflammation (0, none; 1, slight; 2, moderate; 3, severe) and the extent of crypt damage (0, none; 1, the basal one-third portion damaged; 2, the basal two-thirds portion damaged; 3, the entire crypt damaged but the surface epithelium intact; 4, the entire crypt and epithelium lost). Each score was multiplied by a factor reflecting the percentage of involvement (1: 0–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%) and added. The histological injury score was the sum of the scores obtained in three sections, each taken from the proximal, middle, and distal portions of the colon. The maximal score is 84.

To label S-phase cells, mice were injected intraperitoneally with 120 mg/kg of 5-bromo-2′-deoxyuridine (BrdU) and 12 mg/kg of fluorodeoxyuridine 120 minutes before sacrifice. The colon was excised and fixed as described above. Sections were incubated with a rat anti-BrdU mAb (BU1/75; Oxford Biotechnology, Oxford, United Kingdom), and staining was performed with a Ventana Basic DAB Detection Kit using the Ventana HX system (Ventana, Tucson, Arizona, USA) according to the manufacturer’s protocol. For immunohistochemistry, the colon was snap-frozen in liquid nitrogen and 10-μm thick cryostat sections were transferred on slides and air dried. After fixation for 10 minutes in pure acetone, samples were incubated with either a rat anti-mouse CD4 mAb (RM4-5; PharMingen, San Diego, California, USA), a rat anti-mouse CD8 mAb (53-6.7; PharMingen), a rat anti-mouse B220 mAb (RA3-6B2; PharMingen), or a rabbit anti-mouse IFN-γ polyclonal Ab (PBL Biomedical Laboratories, New Brunswick, New Jersey, USA), and staining was performed with a Ventana Basic DAB Detection Kit.

Isolation and culture of lamina propria mononuclear cells.

Lamina propria mononuclear cells (LPMNCs) were prepared from the colon as described (26). In brief, the colon was digested with collagenase type IV (0.5 mg/ml; Sigma Chemical Co.) in RPMI-1640 medium at 37°C, and the resulting suspension was filtered. The cells were pooled, washed, and layered on the top of discontinuous 40% and 70% Percoll gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden). After centrifugation for 20 minutes at 600 g, the cells at the interface were collected as LPMNCs.

The isolated LPMNCs were cultured in RPMI-1640 medium with 10% FBS at 37°C in a volume of 200 μl per well in 96-well round-bottom plates at a density of 106 cells/ml. In the proliferation assay, 0.5 μCi of [3H] thymidine was added at 24 hours, and the cells were collected at 72 hours on a cell harvester (Skatron Instruments AS, Lier, Norway). Incorporation of [3H] thymidine was determined using a liquid scintillation counter (Aloka Co., Tokyo, Japan). For measurement of IFN-γ and IL-2, the LPMNCs were incubated with either vehicle, LPS (10 μg/ml) alone, or LPS and concanavalin A (ConA) (2 μg/ml) in combination for 72 hours. The cytokine amounts in the culture supernatants were measured with an ELISA kit (Endogen Inc., Woburn, Massachusetts, USA) as described (26). All experiments were performed in triplicate.

DNA microarray analysis.

The colon was isolated from EP4+/+ and EP4–/– mice treated with or without 3% DSS for 7 days. Polyadenylated RNA was prepared as described (17) and used for the Affymetrix GeneChip Expression Analysis using a MG-U74Av2 chip (Affymetrix Inc., Santa Clara, California, USA) according to the manufacturer’s protocol. Data were analyzed with the Affymetrix GENECHIP Expression Analysis software (version 4.0).

Suppression by exogenous PGE₂ of the indomethacin-induced exacerbation of low-dose (3%) DSS-induced colitis.

Indomethacin inhibited the production of prostanoids by inhibition of both COX-1 and COX-2. Because it was reported that the level of PGE₂ was elevated locally in the intestine of IBD patients (10, 11), we wondered whether the exacerbation by indomethacin was due to the inhibition of PGE₂ production. To address this point, we administered intraperitoneally a stable PGE₂ analogue, dimethyl-PGE₂ (dm-PGE₂) to the mice treated with indomethacin and 3% DSS. Dm-PGE₂ significantly suppressed the weight loss in mice treated with indomethacin and 3% DSS in combination alone (Figure ​(Figure1a).1a). Dm-PGE₂ also prevented clinical symptoms, such as diarrhea and hemoccult, induced by indomethacin and 3% DSS. The diarrhea score was significantly improved on days 2, 3, 5, 6, and 7 (Figure ​(Figure1b).1b). The hemoccult score was also improved (Figure ​(Figure1c).1c). Moreover, dm-PGE₂ ameliorated the histological findings of the colon induced by the treatment with indomethacin and 3% DSS. There was no epithelial cell loss, crypt damage, or inflammatory cell aggregation in the colon from mice administered with dm-PGE₂ (Figure ​(Figure1e).1e). Consequently, dm-PGE₂ significantly suppressed the histological injury score, when compared with the mice treated with indomethacin and 3% DSS (Figure ​(Figure1d).1d). Hematological evaluation showed that the treatment with dm-PGE₂ corrected leukocytosis and anemia found with the treatment with indomethacin and 3% DSS in combination alone (Figure ​(Figure1,1, f and g) and resolved the splenomegaly found in the mice treated with indomethacin and 3% DSS in combination alone (Figure ​(Figure1h).1h). These results suggest that the exacerbation of DSS-induced colitis by indomethacin was mostly, if not solely, due to the inhibition of endogenous PGE₂ production.

Induction of severe colitis by 3% DSS in EP4-deficient mice but not in mice deficient in other prostanoid receptors.

PGE₂ elicits its physiological effects by acting on four different subtypes of EP receptors (9). Previously, we generated mice deficient in each EP subtype individually (14–16). To clarify which subtype of the EP receptor mediates the protective action of PGE₂ in DSS-induced colitis, we subjected EP1–/–, EP2–/–, EP3–/–, and EP4–/– mice to 3% DSS treatment. C57BL/6 mice were used as controls for EP1–/–, EP2–/–, and EP3–/– mice, and the progeny of wild-type littermates (EP4+/+) in the mixed background of C57BL/6 and 129/Ola were used as controls for EP4–/– mice. These mice neither developed gastrointestinal inflammation spontaneously nor exhibited any clinical manifestations, such as diarrhea, fecal occult blood, or anemia, when they were raised under SPF conditions. When these animals were treated with 3% DSS, only EP4–/– mice developed severe bloody diarrhea and body weight loss, while the body weight loss, diarrhea, and hemoccult of EP1–/–, EP2–/–, and EP3–/– mice were similar to those of control C57BL/6 mice. EP4–/– mice treated with 3% DSS lost their weight progressively during the entire experimental period and significantly as compared with control EP4+/+ mice (Figure ​(Figure2a).2a). The diarrhea score of EP4–/– mice was significantly high compared with EP4+/+ mice (Figure ​(Figure2b).2b). These mice showed loose stool already on day 1, and some of them exhibited watery diarrhea after day 6. EP4–/– mice also showed hemoccult already on day 1, and most of them exhibited gross bleeding in the stool after day 6, yielding significantly higher hemoccult scores than the EP4+/+ mice throughout the experimental period (Figure ​(Figure2c).2c). The macroscopic finding of the intestine of EP4–/– mice treated with 3% DSS and sacrificed on day 7 showed edema and hemorrhagic redness all through the colon and cecum (Figure ​(Figure2d).2d). These mice also showed splenomegaly (data not shown). While the histological examination of the colon obtained from EP4+/+ mice treated with 3% DSS appeared normal, the histological results from EP4–/– mice treated with 3% DSS showed epithelial loss, crypt damage, partial ulceration, and infiltration of inflammatory cells (Figure ​(Figure2e,2e, upper and middle). The inflammatory cells included neutrophils, lymphocytes, and eosinophils as found at high magnification (Figure ​(Figure2e,2e, lower). Consistently, the histological injury score obtained from six EP4–/– mice was significantly higher than that from six control mice (Figure ​(Figure2f).2f). The time course of weight loss, the character of bloody diarrhea, and histological findings obtained from EP4–/– mice treated with 3% DSS were similar to those obtained from C57BL/6 mice treated with 3% DSS and indomethacin in combination. These findings suggest that the indomethacin-induced exacerbation of colitis was mostly, if not solely, due to its inhibition of the PGE₂-EP4 signaling. To confirm this point further and to assess the contribution of prostanoids other than PGE₂, we subjected mice deficient in either DP, FP, IP, or TP to the 3% DSS-induced colitis model. As shown in Figure ​Figure2,2, g and h, none of the DP–/–, IP–/–, FP–/–, and TP–/– mice developed body weight loss, diarrhea, or bloody stool to an extent similar to that found in EP4–/– mice. These results suggest that contribution of other prostanoids is, if present, much limited.

Open in a separate window

Figure 2

Induction of severe colitis by 3% DSS in EP4-deficient mice and not in mice deficient in other prostanoid receptors. (a–c) Wild-type C57BL/6 (filled circles), EP1^–/– (filled triangles), EP2^–/– (filled squares), EP3^–/– (open circles), EP4^+/+ (open triangles), and EP4^–/– (open squares) mice were treated with 3% DSS for 7 days and monitored for body weight loss (a), diarrhea (b), and hemoccult (c). The numbers of mice used in this experiment were 25 (C57BL/6), 7 (EP1^–/–), 10 (EP2^–/–), 14 (EP3^–/–), 16 (EP4^–/–), and 12 (EP4^+/+). (d–f) On day 7, EP4^+/+ and EP4^–/– mice treated with 3% DSS were sacrificed. The macroscopic examination of the large intestine is shown (d). The colon was dissected for histological analysis with H&E staining (e), and the histological injury scores were obtained (f). (g and h) Body weight loss (g) and clinical scores (h) of mice deficient in DP, FP, IP, and TP in 3% DSS colitis. DP^–/–, FP^+/+, FP^–/–, IP^–/–, and TP^–/– mice were treated with 3% DSS for 7 days and monitored for body weight, diarrhea, and hemoccult. The body weight loss and clinical scores (diarrhea score plus hemoccult score) of these mice are compared with those of EP4^+/+, EP4^–/–, and C57BL/6 wild-type mice. The numbers of mice used in this experiment were 12 (EP4^+/+), 16 (EP4^–/–), 18 (C57BL/6), 11 (DP^–/–), 5 (FP^+/+), 4 (FP^–/–), 8 (IP^–/–), and 5 (TP^–/–). Data in a–c and f–h are means ± SEM. *P < 0.05 versus EP4^+/+ (t test) in a–c. Scale bars, 1 cm in d; 200 μm in upper and middle panel of e; 40 μm in lower panel of e.

Reproduction of the EP4–/– phenotype in wild-type mice by administration of the EP4 antagonist.

To confirm that the above phenotype of EP4–/– mice is due to the lack of EP4 receptor, we used an EP4-selective antagonist, AE3-208 (Figure ​(Figure3a,3a, left). AE3-208 was added to 3% DSS in the drinking water and administered to C57BL/6 mice for 7 days. The mice treated with the EP4 antagonist and 3% DSS together showed severe weight loss and bloody diarrhea. Significant differences in the weight loss and the diarrhea and hemoccult scores were found between these mice and mice treated with 3% DSS alone (Figure ​(Figure3b).3b). Consistently, the histological injury score of mice treated with the EP4 antagonist and 3% DSS in combination was also significantly high compared with those treated with 3% DSS alone (Figure ​(Figure3c).3c). To verify the specificity of AE3-208 in vivo, we applied this compound to EP4–/– mice together with 3% DSS. AE3-208 did not exacerbate the colitis further in EP4–/– mice; the body weight loss of 10.2% ± 4.2% and 8.7% ± 3.2%, the diarrhea score of 1.3 ± 0.3 and 1.8 ± 0.3, and the hemoccult score of 3.3 ± 0.5 and 3.3 ± 0.5 were observed on day 7 in EP4–/– mice treated with AE3-208 and untreated EP4–/– mice, respectively (n = 5, each).

Open in a separate window

Figure 3

Effect of EP4-selective drugs on DSS-induced colitis. (a) Structures of ONO-AE3-208 (left) and ONO-AE1-734 (right). (b and c) Reproduction of the EP4^–/– phenotype in wild-type mice by administration of an EP4 antagonist. AE3-208, an EP4 antagonist, was added to 3% DSS in the drinking water and administered to C57BL/6 mice for 7 days. Body weight loss, diarrhea, and hemoccult scores on day 7 of 3% DSS-treated mice administered with vehicle (open bars) or AE3-208 (closed bars) is shown in b, and histological injury scores of the colon are shown in c. (d–f) Amelioration of high-dose (7%) DSS-induced colitis by an EP4 agonist. High-dose (7%) DSS was administered to C57BL/6 mice together with vehicle or AE1-734, an EP4 agonist, for 7 days. Body weight loss, diarrhea, and hemoccult scores on day 7 of 7% DSS-treated mice with vehicle (open bars) or the EP4 agonist (closed bars) are shown in d, and H&E staining of the colon (f) and the histological injury scores (e) are shown. Data are means ± SEM from five to ten mice. *P < 0.05 versus mice treated with 3% or 7% DSS alone (t test). Scale bars, 200 μm in f.

Amelioration of high-dose (7%) DSS-induced colitis by an EP4 agonist.

Because the above study showed that the EP4 antagonist exacerbated low-dose DSS-induced colitis, we wondered whether an EP4 agonist ameliorates severe colitis. To examine this issue, we used the colitis induced by high-dose DSS as a model. Administration of high-dose (7%) DSS in the drinking water to wild-type mice induced fulminant colitis characterized by the bloody diarrhea and loss of body weight; the body weight loss, diarrhea score, and hemoccult score on day 7 were 9.8% ± 2.7%, 2.2 ± 0.1, and 3.8 ± 0.2, respectively (Figure ​(Figure3d).3d). Upon histological examination, epithelial loss, goblet cell loss, and aggregation of inflammatory cells were observed in the mice treated with 7% DSS (Figure ​(Figure3f).3f). When we administered an EP4 agonist, AE1-734 (Figure ​(Figure3a,3a, right), subcutaneously twice a day to 7% DSS-treated mice, we found that the EP4 agonist significantly suppressed the weight loss and bloody diarrhea induced by 7% DSS treatment in C57BL/6 mice. The body weight loss, diarrhea score, and hemoccult score on day 7 were 3.3% ± 1.0% versus 9.8% ± 2.7%, 1.1 ± 0.3 versus 2.2 ± 0.1, and 1.4 ± 0.4 versus 3.8 ± 0.2, respectively (Figure ​(Figure3d).3d). Upon histological examination, there were neither loss of epithelial and goblet cells nor aggregation of inflammatory cells in the colon of the mice treated with the EP4 agonist in addition to 7% DSS (Figure ​(Figure3f).3f). Consistently, the EP4 agonist lowered the histological injury score to 7.5 ± 1.1 from 22.3 ± 3.5 found in mice treated with 7% DSS alone (Figure ​(Figure3e).3e). This preventive action of AE1-734 was most likely due to stimulation of the EP4 receptor, because administration of this compound to EP4–/– mice treated with 7% DSS did not ameliorate any symptoms of the colitis throughout the experi-mental period. The body weight loss, the diarrhea score, and the hemoccult score on day 7 were 23.4% ± 1.4% and 22.0% ± 2.2%, 4.0 ± 0.0 and 4.0 ± 0.0, and 4.0 ± 0.0 and 4.0 ± 0.0 for EP4–/– mice treated with AE1-734 and untreated EP4–/– mice, respectively (n = 5, each).

Impaired mucosal integrity in mice with the EP4 deficiency.

The above studies taken together have suggested that the PGE₂-EP4 signaling plays a critical role in protection of DSS-induced colitis. To obtain a mechanistic insight into the protective action of the PGE₂-EP4 signaling, we first used FITC-dextran assay and evaluated the mucosal barrier function in the induction phase of colitis. The EP4 antagonist (AE3-208) was administered in the drinking water (10 mg/kg/day) to C57BL/6 mice 1 day before and throughout the treatment of 3% DSS. Twenty-four hours after DSS treatment, FITC-dextran was given orally, and 4 hours later blood was drawn. The serum level of FITC-dextran in the mice treated with the EP4 antagonist and DSS was significantly higher than that in nontreated mice, while that in mice treated with 3% DSS alone or AE1-734 alone was not significantly higher than that in nontreated mice (Figure ​(Figure4a).4a). We also administered FITC-dextran orally to EP4+/+ and EP4–/– mice treated with 3% DSS for 1 day. Four hours after FITC-dextran administration we excised the colon for fluorescent microscopic examination. The colon from EP4–/– mice treated with 3% DSS showed significant infiltration of FITC-dextran into submucosa, while no infiltration was found in that from EP4+/+ mice (Figure ​(Figure44b).

Open in a separate window

Figure 4

Impaired mucosal integrity caused by the EP4-deficiency. (a) Effect of the EP4 antagonist. C57BL/6 mice were treated with either vehicle (None), 3% DSS alone, AE3-208 (EP4 antagonist) alone, or 3% DSS and AE3-208 in combination for 1 day. FITC-dextran was administered by mouth, and the serum level was measured 4 hours later. Data are means ± SEM from six mice per group. *P < 0.05 versus the vehicle-treated mice (t test). (b) DSS-induced submucosal infiltration of FITC-dextran in EP4-deficient mice. FITC-dextran was administered by mouth to EP4^+/+ and EP4^–/– mice pretreated with 3% DSS for 1 day. Four hours later the colon was excised and subjected to fluorescent microscopic examination. Scale bars, 1 mm.

Impaired epithelial regeneration and enhanced CD4+ T cell activation in mice treated with an EP4 antagonist during the recovery period after high-dose DSS-induced colitis.

The results above revealed that the PGE₂-EP4 signaling is important in the maintenance of the mucosal barrier in induction of colitis. This impairment of the mucosal barrier would lead to the exposure of submucosa to luminal antigens. We next examined whether EP4 plays a role in prevention of the disease progression and/or in repair of epithelial damage. Since EP4 was reported to be expressed on epithelial cells in the large intestine (27), we hypothesized that the intestinal epithelial growth after injury was modulated by the PGE₂-EP4 signaling. To clarify this point, we used the BrdU incorporation assay. After high-dose (7%) DSS treatment for 7 days, mice were treated with or without the EP4 antagonist (AE3-208) in the absence of DSS treatment for 3 days and sacrificed. BrdU was added intraperitoneally 2 hours before sacrifice. While the vehicle-treated mice quickly recovered from the weight loss and bloody diarrhea, the EP4 antagonist–treated mice continued to exhibit severe colitis (data not shown). The histological results from the H&E-stained colon from the vehicle-treated mice appeared normal, while those from the EP4 antagonist–treated mice showed that epithelial and crypt loss remained as that seen in the colon from mice treated with 7% DSS for 7 days (Figure ​(Figure5,5, a and b). Moreover, in the mice treated with the EP4 antagonist, infiltration of inflammatory cells, including lymphocytes and neutrophils in submucosa, became more pronounced (Figure ​(Figure5c).5c). As for BrdU staining, a number of positive cells were found in the crypt epithelium of the colon from the vehicle-treated mice, suggesting that repair from epithelial damage was occurring (Figure ​(Figure5,5, d and e). On the other hand, in mice treated with the EP4 antagonist after DSS treatment, BrdU-positive epithelial cells were barely detected, indicating that the PGE₂-EP4 signal blockade suppressed epithelial regeneration (Figure ​(Figure5,5, f and g). On the other hand, there existed a large number of BrdU-positive mononuclear cells in the submucosal area of the EP4 antagonist–treated mice (Figure ​(Figure5,5, f and h). Immunohistochemistry showed that CD4⁺ T cells comprised the majority of these inflammatory cells (Figure ​(Figure5i).5i). This pronounced infiltration of CD4⁺ T cells was not seen in the colons from mice treated with 7% DSS for 7 days (data not shown). CD8- or B220-positive cells were also seen, but they did not make clusters as did CD4⁺ cells but were scattered all over the lamina propria (Figure ​(Figure5,5, j and k). Neutrophils and macrophages were also detected (data not shown). Most of the infiltrated cells were also stained positive with anti–IFN-γ Ab, while IFN-γ–positive cells were found only occasionally in the submucosa from the vehicle-treated mice (Figure ​(Figure5,5, l and m). These results suggest that the activation of T cells occurred upon prolonged PGE₂-EP4 signal blockade.

Open in a separate window

Figure 5

Impaired epithelial proliferation and enhanced CD4⁺ T cell proliferation in mice treated with an EP4 antagonist. Mice pretreated with 7% DSS for 7 days were then administered with either vehicle (a, d, e, and l) or the EP4 antagonist (AE3-208) (b, c, f–k, and m) for 3 days without DSS and sacrificed. BrdU was added intraperitoneally 2 hours before sacrifice. The colon was dissected for histological examination with H&E staining (a–c) and immunohistochemical analysis for BrdU (d–h), CD4 (i), CD8 (j), B220 (k), and IFN-γ (l and m). (e and g) Enlarged figures of d and f, respectively, showing the epithelial area; (h) an enlarged figure of f showing the submucosal area. (i–m) The submucosal area. Scale bars, 200 μm in a, b, d, f; 100 μm in c, e, g, h; 40 μm in i–m.

Downregulation of LPMNC proliferation and Th1 cytokine production by the PGE₂-EP4 signaling.

The results above showed that the enhanced proliferation of CD4⁺ T cells in the lamina propria occurred with prolonged inhibition of the EP4 signaling in colitis. EP4 was reported to be present on CD4⁺ T cells in the submucosa of the large intestine (27). These findings taken together suggest that EP4 activation may downregulate the proliferation of CD4⁺ T cells to modulate immunological response in the lamina propria. To examine this issue, we isolated LPMNCs from C57BL/6 mice and examined the effect of the EP4 agonist and antagonist on their proliferative response by measuring [³H] thymidine incorporation. LPMNCs treated with the EP4 antagonist (AE3-208) or indomethacin exerted a significant increase in the proliferative response compared with those treated with vehicle (Figure ​(Figure6a).6a). There was no additive effect of the EP4 antagonist and indomethacin in combination compared with the EP4 antagonist alone or indomethacin alone. The addition of the EP4 agonist suppressed the increase in the proliferative response by indomethacin (Figure ​(Figure6a),6a), while it did not suppress the proliferation of LPMNCs without indomethacin treatment (data not shown). Control experiments using LPMNCs from EP4–/– mice showed that the EP4 agonist and antagonist, as well as indomethacin, did not affect the proliferative response (Figure ​(Figure6b).6b). These results suggest that stimulation of the proliferative response of wild-type LPMNCs by the EP4 antagonist or indomethacin was due to the inhibition of the action of endogenous PGE₂ on the EP4 receptor of these cells. This experiment thus clearly corroborates the immunosuppressive action of EP4 on LPMNCs and suggests that the enhanced proliferation of CD4⁺ T cells seen in the colitis of the animals treated with the EP4 antagonist AE3-208 was caused by prolonged inhibition of the EP4 signaling in T cells. We next examined the effect of the EP4 signaling on cytokine production. We either incubated LPMNCs for 72 hours without stimulation or stimulated them for 72 hours with LPS or LPS and ConA in combination and examined the effects of the EP4 agonist or antagonist on the levels of Th1 cytokines produced under these conditions. In all cases, the levels of IFN-γ significantly increased with the treatment of the EP4 antagonist and decreased with the EP4 agonist (AE1-734) treatment (Figure ​(Figure6c).6c). Similarly, the levels of IL-2 in the culture medium significantly increased with the EP4 antagonist treatment and decreased with the EP4 agonist treatment (Figure ​(Figure6d).6d). These results suggest that the PGE₂-EP4 signaling regulates the proliferation and Th1 cytokine production of LPMNCs.

Open in a separate window

Figure 6

Modulation of the LPMNC proliferation and Th1 cytokine production by EP4 signaling. (a and b) LPMNCs were prepared from C57BL/6 mice (a) or from EP4^–/– mice (b), cultured with either vehicle (None), the EP4 antagonist (AE3-208), the EP4 agonist (AE1-734), or indomethacin (IND) either alone or in indicated combination, and examined for the proliferation by measuring [³H] thymidine uptake. Data are means ± SEM (n = 3). *P < 0.05 versus None (Tukey multiple comparison test). ^#P < 0.05 versus IND (Tukey multiple comparison test). (c and d) LPMNCs were stimulated for 72 hours with vehicle, LPS, or LPS and ConA in combination in the presence of vehicle (None), the EP4 antagonist (AE3-208), or the EP4 agonist (AE1-734), and the levels of IFN-γ (c) and IL-2 (d) in the culture medium were measured with an ELISA kit. Data are means ± SEM (n = 3). *P < 0.05 versus None (Dunnett multiple comparison test).

We are grateful to T. Dohi of the International Medical Center of Japan; S. Fukuyama, M-N. Kweon, and H. Kiyono of Osaka University for helpful discussions; S. Kitajima of Saga Medical School for advice on the FITC-dextran assay; C Hamada of Kyoto University for advice on statistical analysis; K. Deguchi for animal care and breeding; and T. Arai for secretarial assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Organization for Pharmaceutical Safety and Research.