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Effects of prostaglandin D2 on helper T cell functions
BBRC Biochemical and Biophysical Research Communications 316 (2004) 1009–1014 www.elsevier.com/locate/ybbrc
Effects of prostaglandin D2 on helper T cell functions Kazuya Tanaka,a,1 Hiroyuki Hirai,a,*,1 Shoichi Takano,a Masataka Nakamura,b and Kinya Nagataa a b
Department of Advanced Medicine and Development, BML, Inc., Saitama 350-1101, Japan Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo 113-8510, Japan Received 3 February 2004
Abstract Prostaglandin (PG) D2 is abundantly produced by mast cells in the sites of allergic inflammations and acts on various cell types through its receptors DP and CRTH2. Among human T cells, CRTH2 is preferentially expressed on Th2-type cells. However, distribution of DP among T cells and impacts of CRTH2- and DP-mediated signals on T cell functions are presently unclear. Here, we show that CD4þ and CD8þ T cells producing IFN-c and IL-2 were reduced by DP-mediated signals, while CRTH2-mediated signals enhanced IL-2, IL-4, IL-5, and IL-13 production by Th2 cells. CRTH2 signals also caused up-regulation of CD11b and CD40L in resting Th2 cells. RT-PCR analysis revealed distribution of DP among Th cell subsets. On CRTH2þ Th2 cells, the CRTH2-mediated PGD2 effects were dominantly observed. Thus, PGD2 favors Th2 functions through CRTH2 while restraining Th1 functions via DP, which may contribute to development of Th2-dominated status in allergic inflammations. Ó 2004 Elsevier Inc. All rights reserved.
Th1 and Th2 cells are subsets of helper T (Th) cells. In humans, Th1 cells secrete IFN-c, which elicit cellmediated immune responses, while Th2 cells secrete IL4, -5, and -13, which mount humoral immunity [1]. In type I allergy, accumulation of Th2 cells, in addition to accumulation of basophils and eosinophils, is observed in the sites of inflammations, which is often triggered by mediators from antigen-stimulated mast cells [2]. Th2 cell-derived cytokines induce IgE production by B cells, and growth and differentiation of mast cells and eosinophils [1]. Thus, Th2 cells play a central role in allergic inflammations. Activated mast cells secrete various mediators including prostaglandin (PG) D2 . PGD2 was also shown to be produced by antigen-presenting cells and a subset of Th2 cells [3,4]. Because of its high production in the sites of allergic inflammations, PGD2 has long been implicated in allergic diseases [4]. Several lines of evidence demonstrate contribution of PGD2 to the formation of allergic inflammations. For example, PGD synthase-transgenic mice showed enhanced allergic inflammations in an *
Corresponding author. Fax: +81-492-32-5480. E-mail address: [email protected] (H. Hirai). 1 These authors contributed equally to this manuscript. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.151
asthma model [5]. Conversely, mice deficient for the classical PGD2 receptor (called DP) showed reduced allergic inflammations in a similar asthma model system [6]. In these PGD synthase-transgenic or DP-deficient mice, IL-4 production in the sites of allergen challenge was increased or decreased, respectively, as compared to wild-type mice [5,6]. However, molecular mechanisms by which PGD2 favors Th2 development in the mouse allergy models remained unclear. In the human system, information as to the effects of PGD2 on T cell functions is little. It was reported that PGD2 induced cAMP generation in T cells [7]. Recently, we demonstrated that PGD2 elicits its biological actions not only via DP but also via the second receptor CRTH2 (also called DP2 ) and that the CRTH2 receptor is largely distinct from DP in its biological functions and cellular distribution [8,9]. In humans, CRTH2-expressing cells normally represent a very minor cell population in the immune system. CRTH2 is selectively expressed on Th2-type T cells (Th2 and Tc2), eosinophils, basophils, and a subset of monocytes [10,11]. Recent studies demonstrated that CRTH2mediated signals induce cell migration and activation [8,12,13]. On the other hand, DP expression is observed in eosinophils, basophils, and monocytes [8,13,14].
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It has been demonstrated that DP-mediated signals suppressed cell migration in DP-transfected Jurkat cells, eosinophils, dendritic cells, and fibroblasts, inhibited cell activation in eosinophils, and reduced IL-12 production in dendritic cells [8,12,14–17]. At present, however, cellular distribution of DP among T cells is unclear. Also, impacts of CRTH2- and DP-mediated signals on T cell functions remain to be elucidated. In this study, we examined the effects of PGD2 on the cytokine production and expression of functional cell-surface molecules in T cells using selective agonists and an antagonist for DP and CRTH2.
Materials and methods Cells. Isolation of PBMCs from consented healthy subjects, generation of Th1 and Th2 cells, and purification of CRTH2þ Th cells were performed as described previously [10]. Th1, Th2, and CRTH2þ Th2 cells were repeatedly stimulated in OKT3 (anti-CD3 mAb, Janssen-Kyowa, Tokyo, Japan)-coated culture plate every 10–14 days and maintained in the presence of IL-2 (100 U/ml). Only Th cells that had been cultured for more than 5 days after the last stimulation and showed good responses to monocyte chemotactic protein 1 in Ca2þ mobilization assay were used for experiments [8]. Analysis of cytokine-producing cells by FCM. Origin of reagents used in FCM was described previously [10,19]. Cells were pre-incubated with PGD2 , 13,14-dihydro-15-keto-PGD2 (DK-PGD2 ), BW245C (all from Cayman Chemicals, Ann Arbor, MI), or indomethacin (Sigma– Aldrich, St. Louis, MO) in the presence or absence of BWA868C (kindly provided by Dr. Richard J. Wilson of Glaxo Wellcome, Stevenage, UK) for 30 min to 1 h. Then, the cultures containing the cells and test compounds were transferred to OKT3-coated wells and cultured in the presence of 10 lg/ml of free KOLT-2 (mouse anti-CD28 mAb; Nichirei, Tokyo, Japan) for 6 h. Brefeldin A (Wako Pure Chemical, Osaka, Japan) was added to the cultures 1 h after the stimulation. After the 6 h stimulation, cells were stained as previously described [10,18]. Measurements of cell-surface expression of CD11b and CD40L. Th2 cells were suspended in Hanks’ balanced salt solution containing 10 mM Hepes (pH 7.4) and incubated with a test compound for 15 min at 37 °C. After the incubation, ice-cold FACSflow (Becton–Dickinson) was added to the mixture and cells were spun down. Afterwards, the cells were incubated for 30 min at 4 °C with PE-labeled anti-CD11b or FITClabeled CD40L (PharMingen, San Diego, CA, USA). After fixation with 1% of formaldehyde in PBS, stained cells were analyzed by FCM. RT-PCR. RT-PCR for CRTH2 and b-actin was performed as described previously [8]. RT-PCR for DP mRNA was done using PCR primers 50 -CCATGCGCAACCTCTATGCGAT (sense) and 50 -AAT TGCTGCACCGGCTCCTGTA (antisense), and the PCR condition was 45 cycles of 94 °C for 1 min, 68 °C for 1 min, and 72 °C for 1 min.
Results Effects of PGD2 on cytokine production of T cells To examine effects of PGD2 on cytokine production of T cells, PBMCs were stimulated with immobilized OKT3 and soluble KOLT-2 for 6 h in the presence or absence of PGD2 . The protein secretion inhibitor, brefeldin A, was added to the cultures during the last 5 h and the cells were analyzed for their intracellular cyto-
kine levels by FCM. Frequencies of IFN-c-producing cells among total CD4þ T cells were significantly reduced by PGD2 in a dose-dependent manner (Figs. 1A and B). Similar effects were observed by a DP-selective agonist BW245C [9] (Fig. 1C). A CRTH2-selective agonist DK-PGD2 [8] had no such effects (Fig. 1C). In addition, a DP-selective antagonist BWA868C [9] cancelled the effect of PGD2 (Fig. 1C). These results indicated that upon binding of PGD2 , the DP receptor, but not CRTH2, suppresses IFN-c production by CD4þ T cells. Similarly, PGD2 reduced IL-2-producing CD4þ T cells in a DP-dependent manner (Fig. 1D). The DPmediated effects of PGD2 on the IFN-c and IL-2 production were also observed in CD8þ T cells (Fig. 1E). In contrast to IFN-c- and IL-2-producing cells, IL-4producing cells were slightly increased among the CD3/ CD28-stimulated CD4þ PBMCs by the PGD2 treatment (Fig. 1A). These results were reminiscent of a CRTH2 function because CRTH2 is selectively expressed in Th2type cells. Therefore, we next used in vitro-generated and CRTH2-sorted Th2 cultures. Three CRTH2 agonists, PGD2 , DK-PGD2 , and indomethacin [19], consistently increased the numbers of IL-4-producing cells in a dose-dependent manner in the CRTH2þ cell population (Fig. 2A). The DP-selective antagonist BWA868C could not cancel the enhancing effects of PGD2 (Fig. 2B). In sharp contrast, DP-selective agonist BW245C decreased IL-4-producing cells in a dose-dependent mode (Fig. 2A), indicating that DP-mediated signals had inhibitory effects on cytokine production even in Th2 cells. Furthermore, CRTH2 agonists enhanced IL-2-, IL-5-, and IL-13-producing cells as well, in the CRTH2þ cell population (Fig. 2C). Expression levels of CRTH2 and DP mRNAs in Th1 and CRTH2þ Th2 cells Above pharmacological findings clearly indicated that DP is broadly expressed among T cell subsets. To confirm this at mRNA level we performed RT-PCR analysis using Th1 and Th2 cells. As shown in previous reports [8,10], CRTH2 mRNA was easily detected in Th2, but not Th1, cells by 30 cycles of PCR (Fig. 3). The CD3/CD28 stimulation caused complete down-regulation of CRTH2 mRNA in Th2 cells. On the other hand, although more PCR cycles (45 cycles) were needed, DP mRNA was detected evenly in Th1, CRTH2 Th2, and CRTH2þ Th2 cells (Fig. 3 and data not shown). Unlike CRTH2 mRNA, DP mRNA levels were not changed by the CD3/CD28 stimulation (Fig. 3). Changes in surface molecule expression levels in Th2 cells by PGD2 We examined effects of PGD2 on the expression levels of various cell-surface molecules in Th2 cells. Expression
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Fig. 1. Effects of PGD2 and related compounds on cytokine production CD4þ and CD8þ T cells of PBMCs from healthy donors. (A) IL-4- and IFNc-producing profiles of CD4þ lymphocytes stimulated in the presence (right panel) or absence (center panel) of 100 nM PGD2 are shown. Quadrants were set according to the control staining (left panel). Percentages of cells are given in each quadrant. (B) Effects of PGD2 on IFN-c production in CD4þ lymphocytes. The vertical axis shows the rate in comparison with control condition (three donors, mean SEM). (C) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), BW245C (300 nM), and BWA868C (1 lM) on IFN-c production in CD4þ lymphocytes (n ¼ 3, mean SEM). (D) Effects of PGD2 (300 nM) and BW245C (300 nM) on IL-2 production in CD4þ lymphocytes (n ¼ 3, mean SEM). (E) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), and BW245C (300 nM) on IFN-c and IL-2-production in CD8þ lymphocytes (n ¼ 3, mean SEM). These results are representative two experiments similar in design.
levels of CD40L and CD11b were rapidly and transiently up-regulated by PGD2 (Fig. 4). DK-PGD2 and indomethacin, but not BW245C, also enhanced expression levels of these surface molecules, indicating that the enhancing effects of PGD2 were mediated by CRTH2. Expression levels of CD4, CD25, CD45RA, CD45RO, CD49d, and CD69 were not significantly changed by agonists for CRTH2 and DP (Fig. 4 and data not shown).
Discussion PGD2 has dual receptor systems, DP and CRTH2 with different signaling pathways, Gas-type G protein and Gai- and, in some cases, Gaq-type G proteins, respectively [8,9,11,20]. Consequently, DP and CRTH2 frequently exhibit opposite functions in the immune system. For example, DP and CRTH2, respectively, have inhibitory and stimulatory/enhancing effects on
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Fig. 4. Up-regulation of cell-surface CD11b and CD40L expression by PGD2 in Th2 cells. CRTH2þ Th2 cells were stimulated by PGD2 (100 nM), DK-PGD2 (100 nM), indomethacin (1 lM), or BW245C (1 lM) for 15 min, and expression levels of CD11b, CD40L, and CD4 were determined by FCM (n ¼ 3, mean SEM). MFI, Mean fluorescence intensity. These results are representative two experiments similar in design.
Fig. 2. Effects of PGD2 and related compounds on cytokine production in CRTH2þ Th2 cells. CRTH2þ cells (purity more than 70%) were prepared from Th2 cultures. Proportions of cytokine-producing cells in the CD3/CD28-stimulated CRTH2þ cell cultures were determined by FCM. (A) Effects of PGD2 (circles), DK-PGD2 (triangles), indomethacin (squares), and BW245C (crosses) on IL-4 production in CRTH2þ Th2 cells. (B) Effects of PGD2 (300 nM) and BWA868C (1 lM) on IL-4 production in CRTH2þ Th2 cells (n ¼ 3, mean SEM). (C) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), indomethacin (3 lM), and BW245C (300 nM) on IL-2, IL-5, and IL-13 production in CRTH2þ Th2 cells. These results are representative two experiments similar in design. NT, non-tested.
Fig. 3. mRNA levels of CRTH2 and DP in Th1 and CRTH2þ Th2 cells. Cells indicated were cultured for 8 h with (+) or without ()) CD3/ CD28 stimulation, and their mRNA levels for CRTH2 (30 cycles), DP (45 cycles), and b-actin (internal control, 25 cycles) were analyzed by RT-PCR.
human eosinophils with respect to their migratory behavior, cell adhesion molecule expression, and activation [8,12]. In this study, we for the first time show that
DP and CRTH2 have opposing effects even on T cell functions as well. We showed that DP signal reduces while CRTH2 signal enhances cytokine-producing T cells. Recently, Trottein’s group reported that DPmediated PGD2 signal inhibits the production of IL-12 in dendritic cells and thereby impacts on the orientation of the immune response by favoring a Th2 response [13,17]. Our data suggest that PGD2 also contributes at the T cell level to the formation of Th2-skewed situations in the cases where CRTH2 expression is induced. In this study, we revealed through pharmacological and RT-PCR analyses that DP is broadly expressed among T cell subsets including Th1, Th2, and CD8þ T cells. Although expression levels of DP mRNA in these T cell subsets appeared to be at relatively low levels, our pharmacological analysis showed potent effect for DP in these T cells. Since DP has been shown to be up-regulated by some inflammatory stimuli [5,6] the DP-mediated signals might be elicited more strongly in T cells in the sites of PGD2 -involving inflammations. Thus, it is currently conceivable that, in an inflamed site where CRTH2 expression occurs only at negligible levels, PGD2 exclusively activates the DP receptor, which leads to general attenuation in T cell cytokine production. Such a mechanism may, at least partly, explain reported anti-inflammatory functions of PGD2 [21]. In contrast to broad distribution of DP in T cell subsets, CRTH2 expression is strictly limited to a minor T cell population with Th2 phenotype [10,22–24]. In fact, CRTH2-positive Th cells have been shown to significantly increase in the circulation of patients with atopic dermatitis, a typical Th2-related allergic disease [18]. Since CRTH2 signals induce cell migration toward
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its agonists [8], CRTH2þ T cells are thought to accumulate to the sites of allergic inflammation where antigen-activated mast cells secrete a lot of PGD2 . CD11b is consists of Mac-1, a member of integrin family, associating with CD18, and binds to ICAM-1 or selectin [25]. Mac-1 is thought to play important roles in leukocyte trafficking into inflamed tissues and cell-to-cell interaction. CD40L is mainly expressed on activated helper T cells, and binding of CD40L to CD40 molecules on B cells and dendritic cells promotes their IgE production and antigen-presenting ability, respectively [26]. Therefore, as well as enhancement of Th2 cytokine production, up-regulation of CD11b and CD40L by CRTH2-mediated signals is thought to accelerate Th2 trafficking and functions. Thus, PGD2 /CRTH2 signals may serve as an amplifier of Th2 cells. PGD synthase-transgenic mice demonstrated increased levels in Th2 cytokine production and in accumulation of eosinophils and lymphocytes in the lung of OVA-induced asthma models [5]. Recently, Honda and co-workers [27] showed that, even in OVA-sensitized wild-type mice, prior exposure to aerosolized PGD2 enhanced production of IL-4 and IL-5 and accumulation of eosinophils, lymphocytes, and macrophages in the lung after low-dose OVA challenge. To what degrees CRTH2 or DP contributes to the formation of allergic inflammation in vivo is presently unclear. However, Sugimoto et al. [29] recently found that a clinically used thromboxane A2 antagonist, ramatroban, which has been shown to improve allergen-induced asthma and skin reaction models in mice [28], has a potent and selective antagonistic activity against CRTH2. Some of the pharmaceutical effects of ramatroban may be explained by the blockade of the PGD2 /CRTH2 system. Furthermore, it was shown that PGD2 and its derivatives induce eosinophil mobilization from the bone marrow, through CRTH2 function [30]. Taken together with findings currently available, CRTH2 signals likely promote allergic inflammations through its stimulatory nature in immune cell types. In contrast, roles of DP in inflammations appear to vary with the types of inflammations and target cells. DP has been shown to be involved in the formation of allergic asthma, to induce vasodilation, and to promote eosinophil survival and mucin secretion from a colonic adenocarcinoma cell line [6,14,31,32]. Also, our findings show that DP signals restrain Th1 functions. Therefore, both DP and CRTH2 should be favorable targets for the treatments and prevention of allergic inflammations.
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Effects of prostaglandin D2 on helper T cell functions Kazuya Tanaka,a,1 Hiroyuki Hirai,a,*,1 Shoichi Takano,a Masataka Nakamura,b and Kinya Nagataa a b
Department of Advanced Medicine and Development, BML, Inc., Saitama 350-1101, Japan Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo 113-8510, Japan Received 3 February 2004
Abstract Prostaglandin (PG) D2 is abundantly produced by mast cells in the sites of allergic inflammations and acts on various cell types through its receptors DP and CRTH2. Among human T cells, CRTH2 is preferentially expressed on Th2-type cells. However, distribution of DP among T cells and impacts of CRTH2- and DP-mediated signals on T cell functions are presently unclear. Here, we show that CD4þ and CD8þ T cells producing IFN-c and IL-2 were reduced by DP-mediated signals, while CRTH2-mediated signals enhanced IL-2, IL-4, IL-5, and IL-13 production by Th2 cells. CRTH2 signals also caused up-regulation of CD11b and CD40L in resting Th2 cells. RT-PCR analysis revealed distribution of DP among Th cell subsets. On CRTH2þ Th2 cells, the CRTH2-mediated PGD2 effects were dominantly observed. Thus, PGD2 favors Th2 functions through CRTH2 while restraining Th1 functions via DP, which may contribute to development of Th2-dominated status in allergic inflammations. Ó 2004 Elsevier Inc. All rights reserved.
Th1 and Th2 cells are subsets of helper T (Th) cells. In humans, Th1 cells secrete IFN-c, which elicit cellmediated immune responses, while Th2 cells secrete IL4, -5, and -13, which mount humoral immunity [1]. In type I allergy, accumulation of Th2 cells, in addition to accumulation of basophils and eosinophils, is observed in the sites of inflammations, which is often triggered by mediators from antigen-stimulated mast cells [2]. Th2 cell-derived cytokines induce IgE production by B cells, and growth and differentiation of mast cells and eosinophils [1]. Thus, Th2 cells play a central role in allergic inflammations. Activated mast cells secrete various mediators including prostaglandin (PG) D2 . PGD2 was also shown to be produced by antigen-presenting cells and a subset of Th2 cells [3,4]. Because of its high production in the sites of allergic inflammations, PGD2 has long been implicated in allergic diseases [4]. Several lines of evidence demonstrate contribution of PGD2 to the formation of allergic inflammations. For example, PGD synthase-transgenic mice showed enhanced allergic inflammations in an *
Corresponding author. Fax: +81-492-32-5480. E-mail address: [email protected] (H. Hirai). 1 These authors contributed equally to this manuscript. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.151
asthma model [5]. Conversely, mice deficient for the classical PGD2 receptor (called DP) showed reduced allergic inflammations in a similar asthma model system [6]. In these PGD synthase-transgenic or DP-deficient mice, IL-4 production in the sites of allergen challenge was increased or decreased, respectively, as compared to wild-type mice [5,6]. However, molecular mechanisms by which PGD2 favors Th2 development in the mouse allergy models remained unclear. In the human system, information as to the effects of PGD2 on T cell functions is little. It was reported that PGD2 induced cAMP generation in T cells [7]. Recently, we demonstrated that PGD2 elicits its biological actions not only via DP but also via the second receptor CRTH2 (also called DP2 ) and that the CRTH2 receptor is largely distinct from DP in its biological functions and cellular distribution [8,9]. In humans, CRTH2-expressing cells normally represent a very minor cell population in the immune system. CRTH2 is selectively expressed on Th2-type T cells (Th2 and Tc2), eosinophils, basophils, and a subset of monocytes [10,11]. Recent studies demonstrated that CRTH2mediated signals induce cell migration and activation [8,12,13]. On the other hand, DP expression is observed in eosinophils, basophils, and monocytes [8,13,14].
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It has been demonstrated that DP-mediated signals suppressed cell migration in DP-transfected Jurkat cells, eosinophils, dendritic cells, and fibroblasts, inhibited cell activation in eosinophils, and reduced IL-12 production in dendritic cells [8,12,14–17]. At present, however, cellular distribution of DP among T cells is unclear. Also, impacts of CRTH2- and DP-mediated signals on T cell functions remain to be elucidated. In this study, we examined the effects of PGD2 on the cytokine production and expression of functional cell-surface molecules in T cells using selective agonists and an antagonist for DP and CRTH2.
Materials and methods Cells. Isolation of PBMCs from consented healthy subjects, generation of Th1 and Th2 cells, and purification of CRTH2þ Th cells were performed as described previously [10]. Th1, Th2, and CRTH2þ Th2 cells were repeatedly stimulated in OKT3 (anti-CD3 mAb, Janssen-Kyowa, Tokyo, Japan)-coated culture plate every 10–14 days and maintained in the presence of IL-2 (100 U/ml). Only Th cells that had been cultured for more than 5 days after the last stimulation and showed good responses to monocyte chemotactic protein 1 in Ca2þ mobilization assay were used for experiments [8]. Analysis of cytokine-producing cells by FCM. Origin of reagents used in FCM was described previously [10,19]. Cells were pre-incubated with PGD2 , 13,14-dihydro-15-keto-PGD2 (DK-PGD2 ), BW245C (all from Cayman Chemicals, Ann Arbor, MI), or indomethacin (Sigma– Aldrich, St. Louis, MO) in the presence or absence of BWA868C (kindly provided by Dr. Richard J. Wilson of Glaxo Wellcome, Stevenage, UK) for 30 min to 1 h. Then, the cultures containing the cells and test compounds were transferred to OKT3-coated wells and cultured in the presence of 10 lg/ml of free KOLT-2 (mouse anti-CD28 mAb; Nichirei, Tokyo, Japan) for 6 h. Brefeldin A (Wako Pure Chemical, Osaka, Japan) was added to the cultures 1 h after the stimulation. After the 6 h stimulation, cells were stained as previously described [10,18]. Measurements of cell-surface expression of CD11b and CD40L. Th2 cells were suspended in Hanks’ balanced salt solution containing 10 mM Hepes (pH 7.4) and incubated with a test compound for 15 min at 37 °C. After the incubation, ice-cold FACSflow (Becton–Dickinson) was added to the mixture and cells were spun down. Afterwards, the cells were incubated for 30 min at 4 °C with PE-labeled anti-CD11b or FITClabeled CD40L (PharMingen, San Diego, CA, USA). After fixation with 1% of formaldehyde in PBS, stained cells were analyzed by FCM. RT-PCR. RT-PCR for CRTH2 and b-actin was performed as described previously [8]. RT-PCR for DP mRNA was done using PCR primers 50 -CCATGCGCAACCTCTATGCGAT (sense) and 50 -AAT TGCTGCACCGGCTCCTGTA (antisense), and the PCR condition was 45 cycles of 94 °C for 1 min, 68 °C for 1 min, and 72 °C for 1 min.
Results Effects of PGD2 on cytokine production of T cells To examine effects of PGD2 on cytokine production of T cells, PBMCs were stimulated with immobilized OKT3 and soluble KOLT-2 for 6 h in the presence or absence of PGD2 . The protein secretion inhibitor, brefeldin A, was added to the cultures during the last 5 h and the cells were analyzed for their intracellular cyto-
kine levels by FCM. Frequencies of IFN-c-producing cells among total CD4þ T cells were significantly reduced by PGD2 in a dose-dependent manner (Figs. 1A and B). Similar effects were observed by a DP-selective agonist BW245C [9] (Fig. 1C). A CRTH2-selective agonist DK-PGD2 [8] had no such effects (Fig. 1C). In addition, a DP-selective antagonist BWA868C [9] cancelled the effect of PGD2 (Fig. 1C). These results indicated that upon binding of PGD2 , the DP receptor, but not CRTH2, suppresses IFN-c production by CD4þ T cells. Similarly, PGD2 reduced IL-2-producing CD4þ T cells in a DP-dependent manner (Fig. 1D). The DPmediated effects of PGD2 on the IFN-c and IL-2 production were also observed in CD8þ T cells (Fig. 1E). In contrast to IFN-c- and IL-2-producing cells, IL-4producing cells were slightly increased among the CD3/ CD28-stimulated CD4þ PBMCs by the PGD2 treatment (Fig. 1A). These results were reminiscent of a CRTH2 function because CRTH2 is selectively expressed in Th2type cells. Therefore, we next used in vitro-generated and CRTH2-sorted Th2 cultures. Three CRTH2 agonists, PGD2 , DK-PGD2 , and indomethacin [19], consistently increased the numbers of IL-4-producing cells in a dose-dependent manner in the CRTH2þ cell population (Fig. 2A). The DP-selective antagonist BWA868C could not cancel the enhancing effects of PGD2 (Fig. 2B). In sharp contrast, DP-selective agonist BW245C decreased IL-4-producing cells in a dose-dependent mode (Fig. 2A), indicating that DP-mediated signals had inhibitory effects on cytokine production even in Th2 cells. Furthermore, CRTH2 agonists enhanced IL-2-, IL-5-, and IL-13-producing cells as well, in the CRTH2þ cell population (Fig. 2C). Expression levels of CRTH2 and DP mRNAs in Th1 and CRTH2þ Th2 cells Above pharmacological findings clearly indicated that DP is broadly expressed among T cell subsets. To confirm this at mRNA level we performed RT-PCR analysis using Th1 and Th2 cells. As shown in previous reports [8,10], CRTH2 mRNA was easily detected in Th2, but not Th1, cells by 30 cycles of PCR (Fig. 3). The CD3/CD28 stimulation caused complete down-regulation of CRTH2 mRNA in Th2 cells. On the other hand, although more PCR cycles (45 cycles) were needed, DP mRNA was detected evenly in Th1, CRTH2 Th2, and CRTH2þ Th2 cells (Fig. 3 and data not shown). Unlike CRTH2 mRNA, DP mRNA levels were not changed by the CD3/CD28 stimulation (Fig. 3). Changes in surface molecule expression levels in Th2 cells by PGD2 We examined effects of PGD2 on the expression levels of various cell-surface molecules in Th2 cells. Expression
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Fig. 1. Effects of PGD2 and related compounds on cytokine production CD4þ and CD8þ T cells of PBMCs from healthy donors. (A) IL-4- and IFNc-producing profiles of CD4þ lymphocytes stimulated in the presence (right panel) or absence (center panel) of 100 nM PGD2 are shown. Quadrants were set according to the control staining (left panel). Percentages of cells are given in each quadrant. (B) Effects of PGD2 on IFN-c production in CD4þ lymphocytes. The vertical axis shows the rate in comparison with control condition (three donors, mean SEM). (C) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), BW245C (300 nM), and BWA868C (1 lM) on IFN-c production in CD4þ lymphocytes (n ¼ 3, mean SEM). (D) Effects of PGD2 (300 nM) and BW245C (300 nM) on IL-2 production in CD4þ lymphocytes (n ¼ 3, mean SEM). (E) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), and BW245C (300 nM) on IFN-c and IL-2-production in CD8þ lymphocytes (n ¼ 3, mean SEM). These results are representative two experiments similar in design.
levels of CD40L and CD11b were rapidly and transiently up-regulated by PGD2 (Fig. 4). DK-PGD2 and indomethacin, but not BW245C, also enhanced expression levels of these surface molecules, indicating that the enhancing effects of PGD2 were mediated by CRTH2. Expression levels of CD4, CD25, CD45RA, CD45RO, CD49d, and CD69 were not significantly changed by agonists for CRTH2 and DP (Fig. 4 and data not shown).
Discussion PGD2 has dual receptor systems, DP and CRTH2 with different signaling pathways, Gas-type G protein and Gai- and, in some cases, Gaq-type G proteins, respectively [8,9,11,20]. Consequently, DP and CRTH2 frequently exhibit opposite functions in the immune system. For example, DP and CRTH2, respectively, have inhibitory and stimulatory/enhancing effects on
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Fig. 4. Up-regulation of cell-surface CD11b and CD40L expression by PGD2 in Th2 cells. CRTH2þ Th2 cells were stimulated by PGD2 (100 nM), DK-PGD2 (100 nM), indomethacin (1 lM), or BW245C (1 lM) for 15 min, and expression levels of CD11b, CD40L, and CD4 were determined by FCM (n ¼ 3, mean SEM). MFI, Mean fluorescence intensity. These results are representative two experiments similar in design.
Fig. 2. Effects of PGD2 and related compounds on cytokine production in CRTH2þ Th2 cells. CRTH2þ cells (purity more than 70%) were prepared from Th2 cultures. Proportions of cytokine-producing cells in the CD3/CD28-stimulated CRTH2þ cell cultures were determined by FCM. (A) Effects of PGD2 (circles), DK-PGD2 (triangles), indomethacin (squares), and BW245C (crosses) on IL-4 production in CRTH2þ Th2 cells. (B) Effects of PGD2 (300 nM) and BWA868C (1 lM) on IL-4 production in CRTH2þ Th2 cells (n ¼ 3, mean SEM). (C) Effects of PGD2 (300 nM), DK-PGD2 (1 lM), indomethacin (3 lM), and BW245C (300 nM) on IL-2, IL-5, and IL-13 production in CRTH2þ Th2 cells. These results are representative two experiments similar in design. NT, non-tested.
Fig. 3. mRNA levels of CRTH2 and DP in Th1 and CRTH2þ Th2 cells. Cells indicated were cultured for 8 h with (+) or without ()) CD3/ CD28 stimulation, and their mRNA levels for CRTH2 (30 cycles), DP (45 cycles), and b-actin (internal control, 25 cycles) were analyzed by RT-PCR.
human eosinophils with respect to their migratory behavior, cell adhesion molecule expression, and activation [8,12]. In this study, we for the first time show that
DP and CRTH2 have opposing effects even on T cell functions as well. We showed that DP signal reduces while CRTH2 signal enhances cytokine-producing T cells. Recently, Trottein’s group reported that DPmediated PGD2 signal inhibits the production of IL-12 in dendritic cells and thereby impacts on the orientation of the immune response by favoring a Th2 response [13,17]. Our data suggest that PGD2 also contributes at the T cell level to the formation of Th2-skewed situations in the cases where CRTH2 expression is induced. In this study, we revealed through pharmacological and RT-PCR analyses that DP is broadly expressed among T cell subsets including Th1, Th2, and CD8þ T cells. Although expression levels of DP mRNA in these T cell subsets appeared to be at relatively low levels, our pharmacological analysis showed potent effect for DP in these T cells. Since DP has been shown to be up-regulated by some inflammatory stimuli [5,6] the DP-mediated signals might be elicited more strongly in T cells in the sites of PGD2 -involving inflammations. Thus, it is currently conceivable that, in an inflamed site where CRTH2 expression occurs only at negligible levels, PGD2 exclusively activates the DP receptor, which leads to general attenuation in T cell cytokine production. Such a mechanism may, at least partly, explain reported anti-inflammatory functions of PGD2 [21]. In contrast to broad distribution of DP in T cell subsets, CRTH2 expression is strictly limited to a minor T cell population with Th2 phenotype [10,22–24]. In fact, CRTH2-positive Th cells have been shown to significantly increase in the circulation of patients with atopic dermatitis, a typical Th2-related allergic disease [18]. Since CRTH2 signals induce cell migration toward
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its agonists [8], CRTH2þ T cells are thought to accumulate to the sites of allergic inflammation where antigen-activated mast cells secrete a lot of PGD2 . CD11b is consists of Mac-1, a member of integrin family, associating with CD18, and binds to ICAM-1 or selectin [25]. Mac-1 is thought to play important roles in leukocyte trafficking into inflamed tissues and cell-to-cell interaction. CD40L is mainly expressed on activated helper T cells, and binding of CD40L to CD40 molecules on B cells and dendritic cells promotes their IgE production and antigen-presenting ability, respectively [26]. Therefore, as well as enhancement of Th2 cytokine production, up-regulation of CD11b and CD40L by CRTH2-mediated signals is thought to accelerate Th2 trafficking and functions. Thus, PGD2 /CRTH2 signals may serve as an amplifier of Th2 cells. PGD synthase-transgenic mice demonstrated increased levels in Th2 cytokine production and in accumulation of eosinophils and lymphocytes in the lung of OVA-induced asthma models [5]. Recently, Honda and co-workers [27] showed that, even in OVA-sensitized wild-type mice, prior exposure to aerosolized PGD2 enhanced production of IL-4 and IL-5 and accumulation of eosinophils, lymphocytes, and macrophages in the lung after low-dose OVA challenge. To what degrees CRTH2 or DP contributes to the formation of allergic inflammation in vivo is presently unclear. However, Sugimoto et al. [29] recently found that a clinically used thromboxane A2 antagonist, ramatroban, which has been shown to improve allergen-induced asthma and skin reaction models in mice [28], has a potent and selective antagonistic activity against CRTH2. Some of the pharmaceutical effects of ramatroban may be explained by the blockade of the PGD2 /CRTH2 system. Furthermore, it was shown that PGD2 and its derivatives induce eosinophil mobilization from the bone marrow, through CRTH2 function [30]. Taken together with findings currently available, CRTH2 signals likely promote allergic inflammations through its stimulatory nature in immune cell types. In contrast, roles of DP in inflammations appear to vary with the types of inflammations and target cells. DP has been shown to be involved in the formation of allergic asthma, to induce vasodilation, and to promote eosinophil survival and mucin secretion from a colonic adenocarcinoma cell line [6,14,31,32]. Also, our findings show that DP signals restrain Th1 functions. Therefore, both DP and CRTH2 should be favorable targets for the treatments and prevention of allergic inflammations.
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