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. 2006 Jul;118(3):343–352. doi: 10.1111/j.1365-2567.2006.02376.x

E prostanoid 2 (EP2)/EP4-mediated suppression of antigen-specific human T-cell responses by prostaglandin E2

Mitsuhiro Okano 1, Yuji Sugata 1, Tazuko Fujiwara 1, Rie Matsumoto 1, Masahiro Nishibori 2, Kenji Shimizu 3, Megumi Maeda 4, Yoshinobu Kimura 4, Shin Kariya 1, Hisashi Hattori 1, Minehiko Yokoyama 5, Kosuke Kino 5, Kazunori Nishizaki 1
PMCID: PMC1782299  PMID: 16827895

Abstract

Prostaglandin E2 (PGE2) is a lipid mediator that displays important immunomodulatory properties, such as polarization of cytokine production by T cells. Recent investigations have revealed that the effect of PGE2 on cytokine production is greatly influenced by external stimuli; however, it is unclear whether PGE2 plays a significant role in major histocompatibility complex-mediated antigen-specific T-cell responses via binding to one of four subtypes of E prostanoid (EP) receptor alone or in combination. In the present study, we sought to determine the effect of PGE2 on antigen-specific CD4+ T-cell responses in humans, especially in terms of receptor specificity. We used purified protein derivative (PPD) and Cry j 1 as T helper type 1 (Th1) and Th2-inducing antigens, respectively. We generated several different Cry j 1- and PPD-specific T-cell lines (TCLs). PGE2 significantly and dose-dependently inhibited the proliferation and subsequent production of interleukin-4 by Cry j 1-specific TCLs and of interferon-γ by PPD-specific TCLs upon antigen stimulation. Administration of EP2 receptor agonist and EP4 receptor agonist suppressed these responses in an adenylate cyclase-dependent manner, while EP1 and EP3 receptor agonists did not. Messenger RNA for EP2, EP3 and EP4, but not EP1, receptors were detected in Cry j 1- and PPD-specific TCLs, and no differences in EP receptor expression were observed between them. Furthermore, PGE2 and EP2 receptor agonist significantly inhibited interleukin-5 and interferon-γ production by peripheral blood mononuclear cells in response to Cry j 1 and PPD stimulation, respectively. These results suggest that PGE2 suppresses both Th1- and Th2-polarized antigen-specific human T-cell responses via a cAMP-dependent EP2/EP4-mediated pathway.

Keywords: antigen, E prostanoid, human, prostaglandin E2, T cells

Introduction

The induction and exacerbation of allergic diseases, such as allergic rhinitis, are mediated by allergen-specific CD4+ T cells, particularly T helper type 2 (Th2) cells.1,2 Interleukin-4 (IL-4) derived from Th2 cells up-regulates the production of allergen-specific immunoglobulin E (IgE), and is suppressed by interferon-γ (IFN-γ) derived from Th1 cells.3,4 In addition, IL-5 derived from Th2 cells plays an important role in allergic inflammation because it selectively promotes the chemotaxis, activation and survival of eosinophils.5 Antigen-specific T cells require two distinct signals for functional activation. The first signal results from interaction of the antigen/major histocompatibility complex (MHC) complex with the T-cell receptor.6 The second requires costimulatory signals.7 In addition, the external and internal microenvironment, including various bacterial products and sex hormones, can affect T-cell function.8,9

PGE2 is a major prostanoid released from a variety of immune cells, including macrophages, via activation of cyclooxygenase enzymes and PGE2 synthase.10 PGE2 is known to affect the production of cytokines by CD4+ T cells. In general, PGE2 has either no effect or enhances the production of Th2 cytokines, such as IL-4 and IL-5, while dramatically inhibiting the production of Th1 cytokines, such as IFN-γ and IL-2.10–14 However, recent investigations suggest that this effect of PGE2 on cytokine production is highly subject to external influence.15–18 Surprisingly, most information was based on nominal T-cell receptor signals including mitogens, antibodies to membrane receptors, or other surface proteins, phorbol esters and ionophores. It is currently not known whether PGE2 plays a significant role in the MHC-mediated, antigen-specific proliferation and cytokine production of CD4+ T cells in humans.

PGE2 acts upon binding to one of four subtypes of receptor, E prostanoid 1 (EP1), EP2, EP3 or EP4, alone or in combination.10 The EP1 receptor is coupled to a Gq/p protein, resulting in phosphatidylinositol production and increased intracellular concentrations of Ca2+.10,19 The EP3 receptor is mostly coupled to a Gi protein, by which it inhibits adenylate cyclase.10,20 EP2 and EP4 receptors, on the other hand, are coupled to a Gs protein that stimulates adenylate cyclase.21 Although the production of T-cell-derived cytokines is known to be regulated by a cyclic-adenosine monophosphate (cAMP)-dependent pathway17, the specific role of each PGE2 receptor subtype in antigen-specific human T-cell responses is not fully understood.

The present study was performed to determine whether PGE2 affects antigen-specific CD4+ human T-cell responses using purified protein derivative (PPD) and Cry j 1 as Th1- and Th2-inducing antigens, respectively. In addition, we examined the expression of four subtypes of EP receptors by antigen-specific T cells, and sought to determine which receptor subtypes are involved in the action of PGE2. We believe that the findings presented in this study might provide new insight into the physiological role of PGE2 in human antigen-specific T-cell responses and inspire novel approaches to the treatment of allergic diseases.

Materials and methods

Subjects

Twelve Japanese patients (five men and seven women; age 18–49 years, mean 32·5 years) with Japanese cedar pollinosis were examined. Written informed consent was obtained from each subject. The patients showed elevations of serum IgE specific for Japanese cedar pollen using a radioallergosorbent test (capsulated hydrolic carrier polymer (CAP-RAST); Pharmacia, Uppsala, Sweden), with sensitivities ranging from 1·81 to 50·90 UA/ml (mean 19·40 UA/ml). None of the patients used immunosuppressive drugs or underwent immunotherapy during this study. As a control, six healthy Japanese volunteers not sensitized with Japanese cedar pollen as determined by skin scratch test were enrolled (three men and three women; age 18–46 years, mean 30·7 years).

Antigens and reagents

Cry j 1 was purified from crude extracts of Cryptomeria japonica pollen using a well-established procedure.22 Endotoxin contamination was considered to be negligible because an Endospec™ ES test was negative (Seikagaku Kogyo Corporation, Tokyo, Japan). PPD was purchased from Nihon BCG Seizo Co. (Tokyo, Japan). Ovalbumin was purchased from Sigma (St Louis, MO). Protein concentration was determined using a bicinchoninic acid assay, according to the manufacturer's instructions (Pierce, Rockford, IL). PGE2 was purchased from Cayman (Ann Arbor, MI). The receptor-selective agonists for EP1 (ONO-DI-004), EP2 (ONO-AE1-259-01), EP3 (ONO-AE-248) and EP4 (ONO-AE1-329) were provided by Ono Pharmaceuticals (Osaka, Japan). PGE2 and the agonists were dissolved to stock concentrations of 10−2 m in dimethylsulphoxide (DMSO; Sigma) and stored at −80° until use. SQ22536 and RP-8-Br-cAMPS were purchased from Sigma and BioLog Life Science (San Diego, CA), respectively.

Isolation and culture of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated and cultured as described previously.23 In brief, 1 × 106/ml cells were incubated in the presence or absence of either 10 μg/ml Cry j 1, 10 μg/ml ovalbumin or 2 μg/ml PPD, along with PGE2 and the receptor-selective agonists at 37° in a 5% CO2/air mixture. Culture supernatant was collected after 72 hr and stored at −80° until the time of cytokine production assay.

Generation and culture of antigen-specific T-cell lines

The CD4+ Cry j 1− and PPD-specific T-cell lines (TCLs) used were generated using a procedure described previously.24 In flat-bottomed microtitre plates (Corning Inc., Corning, NY), 2 × 104 cells from the TCLs were mixed with 1 × 105 irradiated autologous PBMCs (PBMCx) as antigen-presenting cells (APCs). Following this, the cells were cultured in the presence or absence of either 10 μg/ml Cry j 1, 10 μg/ml ovalbumin or 2 μg/ml PPD in 0·2 ml of culture medium containing serial concentrations of PGE2/receptor-selective agonists or control buffer (DMSO). Culture supernatant was collected to perform cytokine production assays and to harvest cells for proliferation assays, as previously described.25

To determine adenylate cyclase and protein kinase A type I activity, TCLs and/or APCs were incubated with SQ22536, an inhibitor of adenylate cyclase, or RP-8-Br-cAMPS, a protein kinase A (PKA) type I inhibitor, at 37° for 1 hr. Following this, the cells were washed with culture medium three times, after which they were mixed and cultured in the same manner described above.

Cytokine determination

Levels of IL-4, IL-5 and IFN-γ were measured in the culture supernatant by means of Opt EIA sets (BD Biosciences, San Jose, CA), according to the manufacturer's instructions.24 The detection limit of these assays was 3 pg/ml for IL-4, 20 pg/ml for IL-5 and 20 pg/ml for IFN-γ.

Reverse transcription—polymerase chain reaction (RT-PCR)

Cry j 1- and PPD-specific TCLs were immediately soaked in RNAlater™ RNA stabilization reagent (Qiagen, Tokyo, Japan) and stored at −30° until use. Total cellular RNA was extracted using the Rneasy™ mini kit (Qiagen), according to the manufacturer's instructions. The extracted material was then treated with amplification grade deoxyribonuclease I (Sigma) for 15 min at room temperature. Reverse transcription of the samples to generate cDNA was performed using a first-strand cDNA synthesis kit (Toyobo, Osaka, Japan), according to the manufacturer's instructions.

Real-time quantitative PCR assays were performed as described elsewhere.25 In brief, the assays were performed using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, CA, USA) with QuantiTect SYBR Green PCR (Qiagen). The PCR primer sequences and product sizes were as follows: EP1, forward 5′-CGCGCTGCCCATCTTCTCCAT-3′ and reverse 5′-CCCAGGCCGATGAAGCACCAC-3′ [(471 base pairs (bp)]; EP2, forward 5′-GCTGCTGCTTCTCATTGTCTCG-3′ and reverse 5′-TCCGACAACAGAGGACTGAACG-3′ (392 bp); EP3, forward 5′-GGACTAGCTCTTCGCATAACT-3′ and reverse 5′-GCAGTGCTCAACTGATGTCT-3′ (293 bp); EP4, forward 5′-ATCTTACTCATTGCCACC-3′ and reverse 5′-TCTATTGCTTTACTGAGCAC-3′ (212 bp); and glyceraldehyde 3-phosphate hydrogenase (GAPDH), forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′ (452 bp).26 The expression level of EP1, EP2, EP3 and EP4 was estimated by dividing each signal into the signal for GAPDH.

Statistical analysis

Statistical comparisons were performed using the Bartlett test, followed by Wilcoxon's signed-rank test and Mann–Whitney's U-test. A level of P < 0·05 was considered statistically significant. Values were given as means ± standard deviation (SD).

Results

Effect of PGE2 on Cry j 1- and PPD-specific cellular responses by TCLs

We generated a panel of both Cry j 1- and PPD-specific TCLs from three patients with Japanese cedar pollinosis. All six Cry j 1-specific TCLs proliferated in response to Cry j 1, and predominantly produced IL-4 (Fig. 1a–c). PGE2 significantly inhibited the Cry j 1-induced proliferative response, as well as IL-4 production, in a dose-dependent manner (Fig. 1a,b). As a whole, IL-4 production was significantly inhibited by 47·01 ± 18·92% (P = 0·028 by Wilcoxon's signed-rank test), 87·96 ± 11·61% (P = 0·028) and 96·72 ± 0·87% (P = 0·028), upon exposure to 0·01 μm, 0·1 μm and 1 μm of PGE2, respectively, compared to the buffer control (Fig. 2).

Figure 1.

Figure 1

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Inhibition of antigen-specific human T-cell responses by PGE2. TCLs specific for Cry j 1 (a–c) and PPD (d–f) were cultured with APC and the respective antigen in the presence of serial concentrations of PGE2 (▪) or control buffer (DMSO: ○). Proliferation (a,d), IL-4 production (b,e), and IFN-γ production (c,f) were determined. The experiments were repeated at least six times using different TCLs. Typical results are shown in mean count per minutes (c.p.m.) ± SD from triplicate cultures for proliferation and mean concentration ± SD from triplicate cultures for cytokine production. Background proliferation and IL-4 production in the absence of Cry j 1 was 132 ± 25 c.p.m. and 0 pg/ml in YO-1, the Cry j 1-specific TCL (a–c), and the background proliferation and IFN-γ production in the absence of PPD was 155 ± 16 c.p.m. and 83 pg/ml in YO-P, the PPD-specific TCL (a–c).

Figure 2.

Figure 2

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PGE2-mediated inhibition of Cry j 1-induced IL-4 production by TCLs. Six Cry j 1-specific TCLs were mixed with APCs and cultured with 10 μg/ml of Cry j 1 for 65 hr in the presence of the following concentrations of PGE2 or control buffer: 0·01 μm (a), 0·1 μm (b), or 1 μm (c). Following incubation, supernatant was collected and the IL-4 concentration of each sample was determined by ELISA. P-values were determined using Wilcoxon's signed-rank test. Data on each TCLs are representative of two separate experiments.

All six PPD-specific TCLs proliferated in response to PPD and predominantly produced IFN-γ (Fig. 1d–f). As observed in the Cry j 1-specific TCLs, PGE2 significantly inhibited the PPD-induced proliferative response and IFN-γ production in a dose-dependent manner (Fig. 1d,f). As a whole, IFN-γ production was significantly inhibited by 25·93 ± 2·18% (P = 0·028), 53·10 ± 1·12% (P = 0·028) and 66·56 ± 1·48% (P = 0·028) upon exposure to 0·01 μm, 0·1 μm and 1 μm of PGE2, respectively, compared to the buffer control (Fig. 3). The baseline production of IL-4 by Cry j 1- specific TCLs and IFN-γ by PPD-specific TCLs in the absence of antigen was 0 ± 0 and 203 ± 314 pg/ml, respectively, and no additional proliferation or cytokine production over background was observed with ovalbumin, the irrelevant antigen (data not shown). PGE2 also significantly inhibited the PPD-induced proliferative response and IFN-γ production by PPD-specific TCLs from non-allergic healthy donors in a dose-dependent manner (data not shown).

Figure 3.

Figure 3

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PGE2-mediated inhibition of PPD-induced IFN-γ production by TCLs. Six PPD-specific TCLs were mixed with APCs and cultured with 2 μg/ml of PPD for 65 hr in the presence of the following concentrations of PGE2 or control buffer: 0·01 μm (a), 0·1 μm (b), or 1 μm (c). Following incubation, supernatant was collected and the IFN-γ concentration of each sample was determined by ELISA. P-values were determined using Wilcoxon's signed-rank test. Data on each TCLs are representative of two separate experiments.

Effect of EP receptor-selective agonists on antigen-specific cellular responses by TCLs

To determine which PGE2 receptor subtypes might mediate the inhibitory effect of PGE2 on antigen-specific cellular responses by TCLs, we used four EP receptor-selective agonists. Treatment with EP1 and EP3 receptor agonists did not affect the Cry j 1-specific proliferative response or IL-4 production. However, treatment with an EP2 receptor agonist strongly inhibited these responses especially the proliferation. Treatment with an EP4 receptor agonist also inhibited these responses, although to a much lesser degree. Combined treatment with EP2 and EP4 receptor agonists had an additive effect. Treatment with EP1 or EP3 receptor agonists did not alter the inhibitory effects of the EP2 or EP4 receptor agonists (Fig. 4a,b). EP2 and EP4 receptor agonists had similar inhibitory effects on the PPD-specific proliferative response and IFN-γ production by PPD-specific TCLs although the inhibitory effect seemed to be modest compared with the effect on Cry j 1- specific responses (Fig. 4c,d).

Figure 4.

Figure 4

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Effect of EP receptor-selective agonists on antigen-specific human T-cell responses. TCLs specific for Cry j 1 (a,b) and PPD (c,d) were cultured with APC and the respective antigen in the presence of PGE2, an EP receptor-selective agonist or control buffer, each at a concentration of 0·2 μm. Experiments were also performed to examine the effects of 0·1 μm of each EP receptor agonist in combination. Proliferation (a,c), IL-4 production (b) and IFN-γ production (d) were determined. Typical results are shown in mean c.p.m. ± SD from triplicate cultures for proliferation and mean concentration ± SD from triplicate cultures for cytokine production. The baseline proliferation and IL-4 production by Cry j 1-specific TCLs in the absence of Cry j 1 were 83 ± 14 c.p.m. and 0 pg/ml, respectively and the baseline proliferation and IFN-γ production by PPD-specific TCLs in the absence of antigen were 1250 ± 96 c.p.m. and 17 pg/ml, respectively. Data are representative of at least three separate experiments. Similar results were seen when EP receptor agonists were added into the culture at 1 μm.

Reversal of EP2/EP4-induced inhibition of antigen-specific T-cell responses by an inhibitor of adenylate cyclase that acts on APCs and T cells

EP2 and EP4 are coupled to a Gs protein that stimulates adenylate cyclase.20 Thus, we sought to determine whether the inhibitory effect of PGE2 mediated by EP2/EP4, is dependent on the activity of adenylate cyclase. Pretreatment of Cry j 1-specific TCLs alone with SQ22536, an inhibitor of adenylate cyclase, followed by the coculture with intact APC partially suppressed inhibition of the Cry j 1-specific proliferative response by EP2 and EP4 receptor agonists. Marked inhibition was observed when APCs alone were pretreated with SQ22536 followed by the coculture with intact TCL. In addition, pretreatment of both TCLs and APCs with SQ22536 completely reversed the inhibitory effects of EP2 and EP4 receptor agonists on the Cry j 1-specific response (Fig. 5a,b). Pretreatment with SQ22536 was also observed to inhibit EP2- and EP4-induced PPD-specific T-cell responses (Fig. 5c,d). In addition, pretreatment of TCLs and/or APCs with RP-8-Br-cAMPS, a PKA type I inhibitor, partially reversed the EP2 and EP4 receptor agonist-induced inhibition of both Cry j 1- and PPD-specific responses (data not shown).

Figure 5.

Figure 5

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Reversal of EP2/EP4-induced inhibition of amtigen-specific T-cell responses with an adenylate cyclase inhibitor. Cry j 1-specific (a,b) and PPD-specific (c,d) TCL alone, APC alone or both TCL and APC were pretreated with SQ22536 at 37° for 1 hr. Following incubation, the cells were washed with culture medium three times, after which they were mixed and cultured with the respective antigen in the presence of an EP2 receptor agonist (a,c) or an EP4 receptor agonist (b,d) at a concentration of 0·2 μm for 72 hr. Typical proliferative responses are shown in mean c.p.m. ± SD from triplicate cultures. The baseline proliferations in the absence of Cry j 1 were 69 ± 5, 56 ± 14 and 66 ± 38 for OG-J1 (a), YJ-15 (b) and YP-11 (c,d), respectively. Data are representative of at least three separate experiments.

Effect of PGE2 and EP receptor-selective agonists on Cry j 1- and PPD-specific PBMC responses

Next, we investigated the effect of PGE2 on Cry j 1- and PPD-specific T-cell responses in PBMCs. In patients with Japanese cedar pollinosis 10−6 m PGE2 significantly inhibited IL-5 and IFN-γ production by PBMCs in response to stimulation with Cry j 1 and PPD, respectively (Fig. 6). Among four EP receptor-selective agonists, only the EP2 receptor agonist significantly inhibited Cry j 1-specific IL-5 production (Fig. 6a). On the other hand, both EP2 and EP4 receptor agonists significantly inhibited PPD-specific IFN-γ production (Fig. 6b). The EP1 and EP3 receptor agonists had no effects on antigen-specific cytokine production by PBMCs. PBMCs from control subjects not sensitized with Japanese cedar pollen did not produce IL-5 in response to Cry j 1 (data not shown). However, those produced a significant amount of IFN-γ in response to PPD (mean 5953 pg/ml). Only the EP2 receptor agonist significantly inhibited PPD-specific IFN-γ production (mean 2558 pg/ml: P = 0·028).

Figure 6.

Figure 6

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PGE2/EP receptor-selective agonists-mediated inhibition of antigen-specific cytokine production by PBMCs. PBMCs from 12 patients with Japanese cedar pollinosis were cultured with 10 μg/ml of Cry j 1 (a), or 2 μg/ml of PPD (b), in the presence of PGE2 and an EP receptor agonist or control buffer, each at a concentration of 1 μm for 72 hr. Following incubation, supernatant was collected and concentrations of IL-5 (a) and IFN-γ (b) were determined in each sample using ELISA. P-values were obtained using Wilcoxon's signed-rank test. The baseline productions of IL-5 and IFN-γ in the absence of antigen were 0 ± 0 and 0 ± 0 pg/ml, respectively.

Expression of the four EP receptors on Cry j 1- and PPD-specific TCLs

Finally, messenger RNA expression of the four EP receptors was examined in five Cry j 1- and five PPD-specific TCLs by RT-PCR. EP1 expression was almost undetectable in all TCLs. However, EP2 and EP4 mRNA were clearly detected in all TCLs. EP3 expression varied among the cells (Fig. 7). Relative expression levels of the four EP receptors were not observed to differ among Cry j 1- and PPD-specific TCLs (EP2: P = 0·148, EP3: P = 0·917, EP4: P = 0·117, using Mann—Whitney's U-test). However, significantly increased expression of the EP2 receptor, compared to the other subtypes, was observed in both types of TCLs (Fig. 8).

Figure 7.

Figure 7

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Expression of EP receptors by human TCLs. Messenger RNA was extracted from five Cry j 1-specific TCLs (lanes 1–5) and five PPD-specific TCLs (lanes 6–10), after which levels of EP1, EP2, EP3, EP4 and GAPDH were detected by RT-PCR as described in the Materials and methods section. M, molecular marker; P, positive control (genomic DNA).

Figure 8.

Figure 8

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Comparison of EP receptor expression among TCLs. The expression levels of four EP receptors were determined in five Cry j 1-specific TCLs (○) and five PPD-specific TCLs (▴) using real-time RT-PCR. Each bar represents the median expression level of each messenger. P-values were obtained using Wilcoxon's signed-rank test.

Discussion

In the present study, we examined the effect of PGE2 on antigen-specific human T-cell responses. PGE2 dose-dependently inhibited Cry j 1- and PPD-induced T-cell responses in cultured TCLs and freshly isolated PBMCs. In addition, EP2 and EP4 receptor agonists also inhibited these antigen-specific responses, and the inhibition was restored by the addition of an adenylate cyclase inhibitor. These results suggest that PGE2 suppresses both Th1- and Th2-polarized antigen-specific human T-cell responses via a cAMP-dependent EP2 and/or EP4-mediated pathway.

Early reports showed inhibition of IFN-γ and IL-2 production from CD4+ T cells by PGE2, however, no changes in IL-4 production were observed.11,12 More recent reports, however, have revealed that the effects of PGE2 on cytokine production are highly influenced by external stimuli.15–18 For example, PGE2 has been observed to inhibit IL-4 gene expression in anti-CD3- plus anti-CD28-activated T cells, however, not when the cells are stimulated with phorbol 12-myristate 13-acetate plus a calcium ionophore.16 In addition, Dooper et al. recently reported that PGE2 inhibited concanavalin A-stimulated IFN-γ but not IL-2 production by PBMC.18 These results suggest that the effect of PGE2 on cytokine production may differ depending on whether T cells are stimulated with MHC-mediated antigen-specific signals or nominal T-cell receptor signals. Since most of what is known about PGE2 has been demonstrated in the presence of nominal T-cell receptor signals, we sought to determine the role of PGE2 alone in antigen-specific human T-cell responses. To the best of our knowledge, this is the first report to demonstrate the effect of PGE2 and PGE2 receptor agonists on antigen-specific human T-cell responses.

In this study, antigen-induced production of IL-4 from Cry j 1-specific TCLs, and IFN-γ from PPD-specific TCLs, was suppressed by treatment with PGE2. This result differs from the results of two previous studies.11,12 However, our finding is consistent with a recent report by He and Stuart indicating that PGE2 inhibits the production of IL-2 and IFN-γ, as well as IL-4, IL-5, and IL-10, by human CD4+ T-cell clones stimulated with anti-CD3 monoclonal antibody.27 The baseline production of IL-4 by Cry j 1-specific TCLs and of IFN-γ by PPD-specific TCLs in the absence of antigen were 0 ± 0 and 203 ± 314 pg/ml, respectively, and no additional proliferation or cytokine production over background was observed with ovalbumin, the irrelevant antigen. In addition, endotoxin contamination was considered to be negligible because the result of an Endospec™ ES test was negative. These results suggest that the cellular responses presented here represent bona fide antigen-specific restimulation of rested T cells.

It is unclear why PGE2 might inhibit the production of both IL-4 and IFN-γ by TCLs. It is possible that T cells stimulated by specific antigens are more sensitive to inhibition by PGE2 than when stimulated by other stimulants, including mitogens and antibodies against surface proteins. PGE2 has been reported to selectively inhibit human CD4+ T cells secreting small amounts of IL-2 and IL-4. Although we have not yet determined the amount of IL-2 produced by the TCLs examined in this experiment, it does not appear that low levels of cytokine production resulted in an increased response to PGE2 in the present study. This was supported by the fact that Cry j 1-specific TCLs almost failed to produce IL-4 in the presence of 1 μm PGE2 (96·72 ± 0·87% inhibition), regardless of the initial level of IL-4 production noted following antigen stimulation (from 53·0 to 685·0 pg/ml, mean 322·2 ± 228·3 pg/ml. Fig. 2). Again, this inconsistency might be because of differences in exposure of the TCLs to external stimuli in the two studies.

PGE2 also inhibited the production of Cry j 1-induced IL-5 production, as well as PPD-induced IFN-γ production, from PBMCs, respectively. It is well known that PBMCs from patients with Japanese cedar pollinosis produce IL-5 in response to Cry j 1 but that PBMCs from asymptomatice subjects do not.28 Thus we used IL-5 as a marker of the Th2 response in PBMCs because, unlike Cry j 1-specific TCLs, IL-4 production by PBMCs in response to Cry j 1 is marginal.23 Together with the result that the contamination of endotoxin in Cry j 1 is negligible by endospec assay (SeiKagaku Kogyo Corporation, Tokyo, Japan), it is suggested that IL-5 is indeed produced by PBMCs from the patients in an antigen-specific manner. Since PGE2-induced cellular responses are known to differ between cultured T cells and freshly isolated PBMCs,11 the present results suggest that the inhibitory effect of PGE2 on antigen-specific cytokine production was also seen in a more physiological situation. In addition, the effect of PGE2 on IL-5 production by human T cells remains controversial.11–14 Our results match a report by Snijdewint et al. demonstrating that IL-5 production by PBMCs stimulated with anti-CD2 plus anti-CD28 monoclonal antibodies is significantly inhibited by the addition of 1 nm PGE2. 11

A few reports have demonstrated the role of EP receptor isoforms in immune responses influenced by PGE2.29–31 Nataraj et al. observed that the EP2 receptor plays a dominant role in PGE2-mediated inhibition of mixed lymphocyte reactions in mice.29 Walker and Rotondo recently reported that suppressive effects of PGE2 on IL-12 and IL-18-induced IFN-γ synthesis by natural killer cells are mediated via EP2 receptors.31 Among four EP receptor-selective agonists, we observed the EP2 receptor agonist to have the greatest inhibitory effect on both Cry j 1-specific and PPD-specific cellular responses in TCLs and PBMCs. The EP4 receptor agonist also had an inhibitory effect; however, this effect was weak compared with that of the EP2 receptor agonist and did not result in inhibition of Cry j 1-specific IL-5 production by PBMCs. EP1 and EP3 receptor agonists, on the other hand, had no effect. Since EP2 and EP4, but not EP1 or EP3, are Gs-coupled receptors, our results suggest that the inhibitory effect of PGE2 on antigen-specific cellular responses might be mediated by activation of the Gs protein through binding of EP2 and/or EP4.21 It seems to be a bimodal distribution: high and low responders in the 12 patitents with allergic rhinitis especially for IL-5 production. However, the EP2 receptor agonist significantly inhibited Cry j 1-specific IL-5 production by PBMC from both high (n = 5: P = 0·043) and low (n = 7: P = 0·028) responders.

Little is known about the expression of EP receptors on human T cells. We detected messenger RNA expression of EP2, EP3 and EP4, but not EP1, receptors in both Cry j 1- and PPD-specific TCLs. This result differs from the observations of Nataraj et al. who observed messenger RNA expression of EP1, EP2 and EP4, but not EP3, receptors on splenic T cells in mice.29 It is possible that T-cell expression of the four subtypes of EP receptors varies among different species. In addition, more research is needed to determine the expression of these receptors on various T-cell subsets, such as naive and memory T cells. However, the fact that we observed a predominance of EP2 receptors on TCLs is probably related to the marked inhibitory effect of the EP2 receptor agonist on antigen-specific cellular responses. This predominance may explain why signals through EP3, which can inhibit adenylate cyclase, had little effect on EP2-mediated suppression (Fig. 2).

Pretreatment of both TCLs and APCs with SQ22536 completely reversed the inhibitory effects of EP2 and EP4 receptor agonists on antigen-specific cellular responses. This result probably relates to the fact that EP2 and EP4 both stimulate adenylate cyclase.21 These results are consistent with a previous report indicating that the cAMP-dependent signalling pathway inhibits the production of Th1- and Th2-related cytokines.17 In addition, a recent report has demonstrated that cAMP inhibits T-cell activation by triggering PKA type I.32 This is supported by our finding that pretreatment of TCLs and/or APCs with Rp-8-bromo-cAMP-phosphorothiate, a PKA type I antagonist, partially reversed the inhibitory effects of EP2 and EP4 receptor agonists on Cry j 1-specific and PPD-specific T-cell responses.

PGE2 seems to influence the antigen-specific cellular responses of both T cells and APCs because reversal of inhibition was seen by pretreatment of both with SQ22536. Pretreatment of APCs with SQ22536 caused a more marked reversal of the PGE2-mediated response than pretreatment of TCLs, suggesting that PGE2 may have a greater effect on the antigen-specific cellular responses of APCs. PGE2 is known to affect various other APC functions, such as expression of MHC class II molecules and cytokine production of tumour necrosis factor-α and IL-12.33,34 In addition, we have recently reported that PGE2 inhibits the expression of several costimulatory molecules through EP2/EP4 in human monocytes.30

In conclusion, we have provided in vitro evidence that PGE2 inhibits both Th2- and Th1-polarized antigen-specific human T-cell responses. Stimulation of EP2 and EP4 and subsequent activation of adenylate cyclase and PKA type I might mediate these effects. These observations might provide a basis for future therapeutic approaches in the management of diseases, such as type I allergy and autoimmune diseases, in which antigen-specific T-cell responses are involved.

Acknowledgments

The authors would like to thank Ono Pharmaceutical for providing ONO-DI-004, ONO-AE1-259-01, ONO-AE-248, and ONO-AE1-329. They are also grateful to Dr Teruhiro Ogawa for his helpful advice and Yuko Okano for her editorial assistance. This work was supported in part by grants from Research on Allergic Disease and Immunology of the Ministry of Health, Labour and Welfare (no. 14210301 to M.O.).

Abbreviations

APC

antigen-presenting cell

bp

base pair

cAMP

cyclic adenosine-3′5′-

c.p.m.

counts per minute

EP

E prostanoid

IFN

interferon-γ

IgE

immunoglobulin E

IL-4

interleukin-4

MHC

major histocompatibility complex

PBMC

peripheral blood mononuclear cell

PGE2

prostaglandin E2

PKA

protein kinase A

PPD

purified protein derivative

RT-PCR

reverse transcription-polymerase chain reaction

TCL

T-cell line

Th1

T helper type 1

References

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