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The Journal of Immunology, 2005, 175: 5744-5750.
Copyright © 2005 by The American Association of Immunologists

Physiological Levels of 15-Deoxy-{Delta}12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemotaxis on Human Eosinophils through Peroxisome Proliferator-Activated Receptor-{gamma} Ligation1

Yoshiki Kobayashi2, Shigeharu Ueki2, Gulixian Mahemuti, Takahito Chiba, Hajime Oyamada, Norihiro Saito, Akira Kanda, Hiroyuki Kayaba and Junichi Chihara3

Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), mainly produced by mast cells, is known as a potent lipid mediator derived from PGD2 in vivo. 15d-PGJ2 was thought to exert its effects on cells exclusively through peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), which are both expressed on human eosinophils. However, the physiological role of 15d-PGJ2 remains unclear, because the concentration generated in vivo is generally much lower than that required for its biological functions. In the present study we found that low concentrations (picomolar to low nanomolar) of 15d-PGJ2 and a synthetic PPAR{gamma} agonist markedly enhanced the eosinophil chemotaxis toward eotaxin, and the effect was decreased in a dose-dependent manner. Moreover, at a low concentration (10–10 M), 15d-PGJ2 and troglitazone primed eotaxin-induced shape change and actin polymerization. These priming effects were completely reversed by a specific PPAR{gamma} antagonist, but were not mimicked by CRTH2 agonist 13,14-dihydro-15-keto-PGD2, suggesting that the effects were mediated through PPAR{gamma} ligation. The effect exerted by 15d-PGJ2 parallels the enhancement of Ca2+ influx, but is not associated with the ERK, p38 MAPK, and NF-{kappa}B pathways. Furthermore, the time course and treatment of eosinophils with actinomycin D, an inhibitor of gene transcription, indicated that the transcription-independent pathway had a role in this process. PPAR{gamma} might interact with an eotaxin-induced cytosolic signaling pathway, because PPAR{gamma} is located in the eosinophil cytosol. Taken together with current findings, these results suggest that under physiological conditions, 15d-PGJ2 contributes to allergic inflammation through PPAR{gamma}, which plays a role as a biphasic regulator of immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Eosinophils are major effector cells in the pathogenesis of allergic inflammation such as bronchial asthma, and eosinophil migration to sites of inflammation is an important step. The chemotactic response of eosinophils is mostly mediated by CCR3 (1), a heteromeric G protein-coupled receptor that is known to transduce signals eliciting Ca2+ influx (2). This calcium-signaling pathway involves myosin L chain kinase activation by rearrangement of actin cytoskeleton, leading to chemotaxis (3). We and others have recently found that the CC chemokine eotaxin, the most potent, in keeping with its high affinity for CCR3 (4), activates ERK1/2 and p38 MAPK in eosinophils, and that these kinases are indispensable for eosinophil chemotaxis (5, 6).

PGD2 is a major arachidonic acid metabolite mainly released by Ag-activated mast cells (7), and a large amount of PGD2 is generated in the asthmatic lung (8, 9). PGD2 directly acts through the DP1, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2),4 which has been recently cloned as a Th2-selective surface molecule distinguished from Th1 cells (10). CRTH2 is also expressed on eosinophils and has been shown to induce eosinophil migration both in vitro (11) and in vivo (12). Furthermore, previous reports (11, 13) indicate that PGD2 metabolites are potent activators of eosinophils and other inflammatory cells, such as basophils and Th2 cells, suggesting their important role in allergic disease.

15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) is derived from PGD2 in vivo by dehydration in an albumin-independent manner (14), and it is a high-affinity ligand for peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) (15). PPAR{gamma} is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors that regulates lipid metabolism and glucose homeostasis. PPAR{gamma} is highly expressed not only in adipose tissue, but also in cells involved in the immune system, and it is implicated in the control of inflammation. We have previously shown that human eosinophils express PPAR{gamma}, and stimulation of eosinophils with a synthetic PPAR{gamma} agonist inhibited the factor-induced eosinophil activation, such as degranulation, survival, and chemotaxis (16, 17). Furthermore, administration of PPAR{gamma} agonist had beneficial effects on pathological conditions, including airway hyperresponsiveness and lung eosinophilia in a murine model of asthma (18). This evidence suggests that pharmacological PPAR{gamma} agonists may be a new therapeutic modality for the treatment of allergic diseases including asthma by negatively modulating eosinophil functions (19). However, micromolar levels of agonist are typically required for the anti-inflammatory effect (16), whereas the levels of 15d-PGJ2, a naturally occurring ligand for PPAR{gamma}, are in the low picomolar range in body fluid (20). Thus, its physiological role as an endogenous activator of PPAR{gamma} is a subject under discussion. In addition, the fact that 15d-PGJ2 binds two receptors, PPAR{gamma} and CRTH2 (21), which are both expressed on eosinophils, complicates understanding of the role of 15d-PGJ2 in allergic inflammation.

Therefore, we investigated the functional role of 15d-PGJ2 on human eosinophils, especially its effect on eotaxin-induced migration at physiological concentrations, and we examined the involvement of PPAR{gamma} and CRTH2. Surprisingly, we found that at a low concentration (10–10 M), 15d-PGJ2 rapidly primed eotaxin-induced migration on human eosinophils. The effect exerted by 15d-PGJ2 parallels the intracellular calcium signal enhancement, but is not associated with NF-{kappa}B and MAPK. We also provide evidence that a nongenomic mechanism is involved in the priming effect of PPAR{gamma} ligation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Eosinophil purification

Peripheral blood was obtained from subjects with mild to moderate eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran, followed by centrifugation on 1.088 Percoll (Pharmacia Biotech) density gradients, as modified from the method reported by Hansel et al. (22, 23). The cells were additionally purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec). Eosinophils (>99% pure) were then suspended in HBSS (Invitrogen Life Technologies) with 1% FCS.

Chemotaxis assay

Chemotaxis of eosinophils was conducted in duplicate using 5-µm pore size, polycarbonate, polyvinylpyrrolidone-free membranes in Boyden chambers. Human eotaxin (R&D Systems) was diluted in RPMI 1640 (Invitrogen Life Technologies) with 1% FCS and placed in the lower wells (100 µl) at a 10-nM concentration. After incubation of the eosinophils with agonists or medium for 20 min at 37°C, the cells were washed twice. Aliquots of 100 µl of the cell suspension at 2.5 x 105 cells/ml were placed in the upper chambers. The loaded chambers were incubated at 37°C in humidified air with 5% CO2 for 1 h. Then the membrane was removed, followed by fixation and staining for 3 min in May-Grünwald solution. The cells that migrated and adhered to the lower surface of the membrane were counted from 10 fields by light microscopy. In this study we used the following agonists: 15d-PGJ2 (Calbiochem), troglitazone (Sankyo), and 13,14-dihydro-15-keto-PGD2 (DK-PGD2; Cayman Chemical). In some experiments the cells were mixed with some antagonists (GW9662 (Cayman Chemical) and ramatroban (Bayer Yakuhin)) or some pharmacological inhibitors (PD98059 (Cell Signaling Technology), SB203580 (BIOMOL), BAY11-7082 (Calbiochem), and actinomycin D (BIOMOL)) before being incubated with agonists.

Measurement of changes in eosinophil shape using cytometry

Eosinophil shape change was assessed using an autofluorescence/forward scatter (FSC) assay, modified from the method described by Sabroe et al. (24). Purified eosinophils were washed in buffer (PBS containing Ca2+ and Mg2+, 10 mM HEPES, 10 mM glucose, and 0.1% BSA (pH 7.4)) and preincubated for 30 min at 37°C. In all experiments after preincubation, the cells were washed again, resuspended in buffer containing Ca2+ and Mg2+, and left to stand for 5 min at room temperature to allow equilibration of intracellular and extracellular calcium. Aliquots of cells (5 x 105 cells) were incubated with agonists or medium in a 37°C shaking water bath for 20 min. The cells added to eotaxin (10 nM) or medium in a final volume of 100 µl and stimulated under the same conditions for 10 min, after which they were transferred to an ice-water bath, and 250 µl of ice-cold optimized fixative (2% formaldehyde solution in PBS) was added to terminate the reaction and maintain the cell shape change until analysis. In some experiments the cells were mixed with GW9662, a PPAR{gamma} antagonist, before being preincubated with agonists. The samples were then analyzed immediately on a FACScan flow cytometer (BD Biosciences). Data were acquired using the FL-2 fluorescence channel on a sensitive setting. The percent change in cell shape was calculated as follows: 100 x ([FSC (reagent) – FSC (medium)]/FSC (medium)). The medium used in this study had no effect in these shape change assays at the dilutions tested.

Actin polymerization assay

The content of F-actin was analyzed by flow cytometry with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin (Molecular Probes) staining, as modified from the method described by Howard et al. (25, 26). Purified eosinophils were resuspended in PBS containing Ca2+ and Mg2+ and were left to stand for 5 min at room temperature. Aliquots of cells (1 x 106 cells) were incubated with agonists or medium in a 37°C shaking water bath for 20 min. The cells were added to eotaxin (10 nM) or medium in a final volume of 100 µl. Twenty-five seconds later, they were fixed in 3.7% formaldehyde for 1 h. Cells were washed; resuspended in PBS; mixed with a staining mixture containing 3.7% formaldehyde, 3.3 x 107 M NBD-phallacidin, and 100 µg/ml lysophosphatidylcholine (Sigma-Aldrich; final concentrations); and incubated for 1 h in the dark. The samples were then analyzed fluorospectrometrically. Data were acquired using the FL-1 fluorescence channel on a sensitive setting. The relative F-actin content was expressed as the ratio of the mean channel fluorescence between agonist- and medium-stimulated cells.

Measurement of intracellular calcium

For intracellular calcium measurement, purified eosinophils (10 x 106 cells in HBSS/HEPES without Ca2+) in suspension were loaded with 2 µM fura 2-AM (Dojindo) for 40 min at room temperature in the dark. The cells were washed in HBSS/HEPES with 4% FCS and resuspended at a final concentration of 5 x 106 cells/ml in HBSS with Ca2+. Aliquots of cells (50 µl) were dispensed into cuvettes and equilibrated with 1 mM calcium at room temperature for 10 min before use. Changes in fluorescence were measured in a Fluorescence Drug Screening System 2000 (Hamamatsu Photoics) as previously described (27). Calculation of intracellular free calcium was derived from the fluorescence spectra (excitation wavelengths, 340 and 380 nm) in accordance with established methods (28). During the experiments the first agonist was added 90 s after commencing recording, and eotaxin (10 nM), as the second agonist, was added after incubation with the first agonist for 20 min at 37°C.

Preparation of cytosolic extracts

Purified eosinophils (106 cells) were incubated with or without 15d-PGJ2 (10–10 M) for 20 min at 37°C, followed by stimulation with and without eotaxin (10 nM) for 3 min. The reaction was terminated by the addition of 15 vol of ice-cold HBSS containing 1 mM Na3VO4. The cells were pelleted by centrifugation and lysed in a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3VO4, 1 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, 10% glycerol, and 1 µg/ml aprotinin, leupeptin, and pepstain). After 20 min on ice, detergent-insoluble materials were removed by centrifugation at 4°C at 15,000 x g. The supernatants were mixed with SDS sample buffer and boiled for 4 min.

Gel electrophoresis and Western blotting

SDS-polyacrylamide (10%) gels (Ready Gel J) were obtained from Bio-Rad. The electrophoresed gel was blotted onto Hybond ECL membranes (Amersham Biosciences). Blots were incubated in a blocking buffer containing 5% BSA in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, and 0.05% Tween 20 (pH 7.6)) for 30 min, followed by incubation in the primary Ab (0.1 µg/ml) to determine the phosphorylation state of signaling proteins with gentle agitation overnight at 4°C. After washing three times in TBS-T buffer, blots were incubated for 30 min with an HRP-conjugated secondary Ab (0.04 µg/ml) directed against the primary Ab. The blots were developed with the ECL substrate according to the manufacturer’s instructions. Afterward, blots were reprobed with another Ab to determine the nonphosphorylation state of signaling proteins after stripping in a buffer of 62.5 nM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS at 56°C for 30 min. The mouse mAb against phospho-ERK, rabbit polyclonal anti-ERK2, anti-p38, and HRP-conjugated goat anti-mouse and anti-rabbit Abs were obtained from Santa Cruz Biotechnology. The polyclonal anti-phospho-p38 Ab was obtained from Cell Signaling Technology.

Confocal microscopy

Purified eosinophils were incubated with and without 15d-PGJ2 (10–10 M) for 20 min at 37°C. The cells were then fixed with 4% formaldehyde in PBS for 10 min, permeabilized in PBS containing 0.1% Triton X-100 for 30 min, blocked with 3% BSA in PBS for 20 min, and rinsed with PBS. The fixed cells were incubated with anti-PPAR{gamma} rabbit polyclonal Ab (BIOMOL; 1/1000) overnight at 4°C and then with PE-conjugated anti-rabbit IgG Ab (Rockland Immunochemicals) for 30 min at 4°C. A confocal laser-scanning microscope system (LSM510; Carl Zeiss) was used to visualize the localization of PPAR{gamma}.

Statistical analysis

Comparisons of two groups of data were performed using Student’s t test. Other data were analyzed by ANOVA, using Dunnett’s post-test. The difference was considered significant at p < 0.05. The results were expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
15d-PGJ2 has a rapid, but transient, priming effect on eotaxin-induced eosinophil chemotaxis

First, to test whether 15d-PGJ2 is involved in allergic inflammation with respect to eosinophil migration, we examined the effect of 15d-PGJ2 on eotaxin-induced chemotaxis using Boyden chambers. Eosinophils were exposed to 15d-PGJ2 or a selective PPAR{gamma} agonist, troglitazone (29), in a wide range of concentrations. It has been shown that PGD2 and its metabolites are potent chemoattractants for eosinophils (13). We also observed that nanomolar to micromolar concentrations of 15d-PGJ2 itself effectively induced eosinophil chemotaxis (data not shown; n = 3). For these reasons, to avoid affecting the chemotactic responses to eotaxin, stimulated cells were washed twice just before being applied to the upper chambers. The lower chambers contained 10 nM eotaxin, approximately half the maximally effective concentration. Fig. 1A shows that short pretreatment of eosinophils with 15d-PGJ2 amplified the migration at physiological concentrations (picomolar to low nanomolar). A maximal effect was found at a concentration of 10–10 M, and the priming effect decreased in a dose-dependent manner. Eosinophils treated with troglitazone (Fig. 1A) or ciglitazone, which is another synthetic PPAR{gamma} agonist, had a dose-response curve similar to that observed for 15d-PGJ2 (ciglitazone; data not shown). Consistent with our previous report (16), at the highest concentration (10–4 M) tested, troglitazone significantly reduced eosinophil migration. Interestingly, we observed that the ability of 15d-PGJ2 to enhance chemotaxis toward eotaxin was diminished time-dependently and dissolved after 180-min incubation (Fig. 1B). Pretreatment with GW9662, a selective PPAR{gamma} antagonist (30), abrogated the priming effect (Fig. 1C). In contrast, pretreatment with ramatroban, a selective thromboxane A2 receptor/CRTH2 antagonist (31), failed to reverse the effect of 15d-PGJ2, and DK-PGD2, a selective CRTH2 agonist (11), had no effect on eotaxin-induced chemotaxis (Fig. 1D). Taken together, this rapid priming effect of 15d-PGJ2 (at a low concentration) appears to depend on PPAR{gamma} ligation, but not CRTH2. In all cases, we observed no cytotoxic effect, as evidenced by the absence of a significant number of cells stained by trypan blue.



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  		FIGURE 1. Effect of 15d-PGJ2 on eotaxin-induced eosinophil chemotaxis. Migration assays were performed using Boyden chambers. In some experiments, purified eosinophils were mixed with antagonists for 10 min before being preincubated with agonists. The chemotactic response to 10 nM eotaxin (494 ± 76% vs medium alone) was considered as 100%. A, Dose-response curve of 15d-PGJ2 or the PPAR{gamma} agonist, troglitazone, on eosinophil chemotaxis. Purified eosinophils were preincubated with increasing concentrations of 15d-PGJ2 or troglitazone for 20 min. B, The effect of 15d-PGJ2 is rapid, but transient. The cells were preincubated with 15d-PGJ2 (10–10 M) for varying time periods as indicated. C, Effect of PPAR{gamma} antagonist (GW9662; 10 µM). GW9662 reversed the priming effect of 15d-PGJ2 and troglitazone (10–10 M). D, Effect of CRTH2 antagonist (ramatroban; 10 µM) and CRTH2 agonist (DK-PGD2; 10–10 M). Ramatroban failed to reverse the effect of 15d-PGJ2 (10–10 M). DK-PGD2 had no effect on eotaxin-induced chemotaxis. Data are expressed as the mean of three to seven (A) or three (B–D) experiments ± SEM. *, p < 0.01 vs control (medium; eotaxin alone).

 
15d-PGJ2 primes eotaxin-induced shape change via PPAR{gamma}

Eosinophil shape change is consistent with a relationship between chemokine-induced responses and the earliest phases of leukocyte recruitment from the microcirculation (24). Because shape change must occur before or during its movement, we next examined eosinophil shape change using the modified autofluorescence/FSC assay, which allows quantitative measurement of chemoattractant-induced cell shape change by flow cytometry. In this assay, to minimize the interaction by other contaminated cells, we used purified eosinophils, unlike the originally devised method (24). A low concentration (10–10 M) of agonists (15d-PGJ2, troglitazone, or DK-PGD2) did not, by itself, affect eosinophil shape change (Fig. 2i). In contrast, pretreatment with 15d-PGJ2 and troglitazone, but not the CRTH2 agonist, DK-PGD2, increased the effect on eotaxin-induced shape change (Fig. 2ii). Furthermore, GW9662 abrogated the priming effect, suggesting 15d-PGJ2 primed eotaxin-induced eosinophil shape change through PPAR{gamma} at a low concentration.



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  		FIGURE 2. Effect of 15d-PGJ2 on eotaxin-induced shape change at a low concentration. Purified eosinophils were preincubated with 10–10 M PPAR{gamma} agonists (15d-PGJ2 and troglitazone) or CRTH2 agonist (DK-PGD2) for 20 min, followed by stimulation with medium (i) or eotaxin (10 nM; ii) for 10 min and fixation with formaldehyde. In some experiments, cells were mixed with GW9662 (10 µM) for 10 min before being preincubated with agonists. The autofluorescence/FSC assays were performed using flow cytometry. 15d-PGJ2 and troglitazone had no direct effect on eosinophil shape (i). When cells were stimulated with eotaxin (10 nM), 15d-PGJ2 and troglitazone significantly enhanced the shape change, and GW9662 reversed the effect of 15d-PGJ2 (ii). Data are expressed as the mean of three experiments ± SEM. *, p < 0.01 vs control (without agonists).

 
15d-PGJ2 primes eotaxin-induced cytoskeletal rearrangement via PPAR{gamma}

Although we can visually detect the priming effect by autofluorescence/FSC assay, it may also involve contributions from changes in cell size (volume). To examine cytoskeletal rearrangement at shape change, we measured F-actin content using an actin polymerization assay. As shown in Fig. 3i, a low concentration (10–10 M) of agonist (15d-PGJ2, troglitazone, or DK-PGD2) alone did not alter F-actin content (Fig. 3i). In contrast, when eosinophils were stimulated with 10 nM eotaxin, the minimum effective concentration in our system, pretreatment with 15d-PGJ2 or troglitazone, but not DK-PGD2, enhanced eotaxin-induced actin polymerization. Furthermore, the effect was completely abrogated by GW9662 (Fig. 3ii). These data indicate that the priming effect of 15d-PGJ2 on eosinophil cytoskeletal rearrangement is PPAR{gamma} dependent as well as the shape change assay.



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  		FIGURE 3. Effect of 15d-PGJ2 on eotaxin-induced actin polymerization at a low concentration. Purified eosinophils were preincubated with 10–10 M PPAR{gamma} agonists (15d-PGJ2 and troglitazone) or CRTH2 agonist (DK-PGD2) for 20 min, followed by stimulation with medium (i) or eotaxin (10 nM; ii) for 25 s, fixation with formaldehyde, and staining of F-actin with NBD-phallacidin. The F-actin content was measured by flow cytometry. In some experiments cells were mixed with GW9662 (10 µM) before being preincubated with agonists. 15d-PGJ2 and troglitazone had no effect on actin polymerization (i). When cells were stimulated with eotaxin (10 nM), 15d-PGJ2 and troglitazone significantly enhanced the actin polymerization, and GW9662 reversed the effect of 15d-PGJ2 (ii). Data are expressed as the mean of three experiments ± SEM. *, p < 0.01 vs control (without agonists).

 
15d-PGJ2 enhances eotaxin-induced calcium flux via PPAR{gamma}

We examined whether 15d-PGJ2 modulates the surface expression CCR3 on eosinophils. However, a flow cytometric study revealed a low concentration (10–10 M) of PPAR{gamma} agonists (15d-PGJ2 and troglitazone), and the CRTH2 agonist, DK-PGD2, had no effect on CCR3 surface expression (data not shown; n = 3). Consequently, in an attempt to investigate chemotactic signaling pathways that might be affected by 15d-PGJ2, we examined calcium flux, which is crucial for eosinophils to allow CCR3-mediated transmigration (3). A low concentration (10–10 M) of agonists (15d-PGJ2, troglitazone, or DK-PGD2) alone did not induce calcium mobilization on eosinophils (Fig. 4, left). In contrast, the cells incubated with 15d-PGJ2 or troglitazone, but not the CRTH2 agonist, DK-PGD2, had a much greater stimulatory effect than the control (medium) on eotaxin-induced calcium mobilization (Fig. 4, right). Eosinophils treated with ciglitazone (10–10 M), another synthetic PPAR{gamma} agonist, had the same enhanced effect (data not shown).



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  		FIGURE 4. Effect of 15d-PGJ2 on eotaxin-induced intracellular calcium flux at a low concentration. Purified eosinophils loaded with 2 µM fura 2 were pre-equilibrated with Ca2+. Eosinophil preparations were then stimulated with medium, 10–10 M PPAR{gamma} agonists (15d-PGJ2 and troglitazone), or CRTH2 agonist (DK-PGD2; closed arrows, left). After 20 min of incubation, eotaxin (10 nM) was added (closed arrows, right). Changes in intracellular free calcium levels were detected as the increase in fluorescence intensity of the calcium-sensitive dye, fura 2. The calcium flux response to eotaxin was enhanced by incubation with 15d-PGJ2 or troglitazone (open arrows). The results are representative of experiments with similar results using eosinophils from three different donors.

 
Neither the MAPK, NF-{kappa}B, nor genomic pathway is involved in the 15d-PGJ2-induced priming effect at low concentration

To explore the mechanisms of the 15d-PGJ2-induced priming effect on eotaxin-directed migration, we examined the MAPK signaling pathway. A low concentration (10–10 M) of 15d-PGJ2 did not affect the eotaxin-induced phosphorylation of ERK1/2 and p38 (Fig. 5A). In line with our Western blot data, pretreatment of eosinophils with an effective concentration of the MEK inhibitor PD98059 or the p38 MAPK inhibitor SB203580 did not alter the ability of 15d-PGJ2 to enhance chemotaxis to eotaxin (Fig. 5B), suggesting that neither the ERK1/2 nor the p38 MAPK pathway was involved in the 15d-PGJ2-induced priming effect. Subsequently, we examined the NF-{kappa}B pathway, which is regulated by 15d-PGJ2 in a PPAR{gamma}-independent manner (32). Blockade of the NF-{kappa}B pathway with the pharmacological inhibitor BAY11-7082 could not reverse the ability of 15d-PGJ2 to enhance chemotaxis (Fig. 5B). We also examined the effect of actinomycin D, an inhibitor of gene transcription, on the priming effect. 15d-PGJ2 enhanced eotaxin-induced migration in the presence of actinomycin D (Fig. 5B), suggesting that the priming effect might be independent of gene transcription. In this assay we used the optimal concentration of inhibitors without any cell toxicity.



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  		FIGURE 5. Examination of the effect of the intracellular signaling pathway on the 15d-PGJ2-induced priming effect. A, Effect of a low concentration of 15d-PGJ2 on eotaxin-induced phosphorylation of ERK1/2 and p38 MAPK. Purified eosinophils were incubated with or without 15d-PGJ2 (10–10 M) for 20 min. After incubation, eosinophils were stimulated with eotaxin (10 nM) for 1 min (for ERK1/2) or 3 min (for p38). Phosphorylated ERK1/2 (p-ERK), total ERK2 (ERK2), phosphorylated p38 MAPK (p-p38), and total p38 MAPK (p38) were determined by Western blot analysis. 15d-PGJ2 did not affect the eotaxin-induced phosphorylation of ERK1/2 and p38. Experiments were repeated at least three times with different cell preparations. B, Purified eosinophils were mixed with or without the indicated inhibitors for 30 min before being preincubated with a low concentration of 15d-PGJ2 (10–10 M). The MAPK kinase inhibitor, PD98059 (50 µM); the p38 MAPK inhibitor, SB203580 (20 µM); the NF-{kappa}B inhibitor, BAY11-7082 (2.5 µM); or the gene transcription inhibitor, actinomycin D (5 µg/ml), did not alter the priming effect of 15d-PGJ2 on eotaxin (10 nM)-induced chemotaxis. The chemotactic response to eotaxin alone was considered as 100%. Data are expressed as the mean of three experiments ± SEM. *, p < 0.05, significant differences between the indicated treatment groups.

 
PPAR{gamma} is predominantly located in cytosol in eosinophils

Although we have previously shown that human eosinophils express PPAR{gamma} (16), the localization in eosinophils has been unknown. To investigate the possibility that PPAR{gamma} interacts with eotaxin-induced signaling in cytosol, we tried to detect the localization of PPAR{gamma} using confocal microscopy. PPAR{gamma} was mainly located in the cytosol, rather than the nucleus, in human fresh eosinophils (Fig. 6). Previous reports indicate ligand-activated PPAR{gamma} translocates from the cytosol into the nucleus (33, 34). In this study, however, short pretreatment (20 min) of eosinophils with 15d-PGJ2 (10–10 M) did not induce translocation of PPAR{gamma} into the nucleus (data not shown).



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  		FIGURE 6. Visualization of the localization of PPAR{gamma} by confocal laser microscopy. For immunolocalization of PPAR{gamma}, purified eosinophils were fixed with formaldehyde and treated with anti-PPAR{gamma} polyclonal Ab, followed by PE-conjugated anti-rabbit IgG Ab. PPAR{gamma} was predominantly located in the cytosol in eosinophils.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recently, it has been reported that the amount of 15d-PGJ2 (at low picomolar concentrations) generated in vivo might be insufficient for it to play a physiological role in modulating most of the response, including activation of PPAR{gamma} (35). In this study we investigated the functional role of 15d-PGJ2 on human eosinophils with regard to chemotaxis. Our present data demonstrated that the eosinophils preincubated with low concentrations of 15d-PGJ2 (picomolar to low nanomolar) enhanced the chemotaxis toward eotaxin, reaching a maximum priming effect at a concentration of 10–10 M. To our knowledge, this is the first report indicating the possibility that physiological levels of 15d-PGJ2 play a role in modulating inflammatory responses.

15d-PGJ2 primed eosinophil activation, such as chemotaxis, shape change, and actin polymerization, at much lower concentrations than those required for the proposed proinflammatory effects of PGD2 metabolites via CRTH2. 15d-PGJ2 as well as PGD2 have stimulatory effects (EC50, ~10 nM) on eosinophils, including calcium mobilization, actin polymerization, and CD11b expression through CRTH2 (36). Heinemann et al. (13) reported that at concentrations as low as 16 nM, the PGD2 metabolite, {Delta}12-PGJ2, markedly enhanced the eosinophil chemotactic response to eotaxin, which is likely to be mediated by CRTH2. Thus, the involvement of CRTH2 may explain why the magnitude of the priming effect of 15d-PGJ2 appeared to be more than that produced by troglitazone at nanomolar concentrations (Fig. 1A). However, the maximum priming effect of 15d-PGJ2 was observed at a concentration of 10–10 M, which was mediated through PPAR{gamma}. PGD2 and {Delta}12-PGJ2 are low affinity ligands for PPAR{gamma} (15); hence, it is possible that they exert a proinflammatory effect partly through PPAR{gamma}. Furthermore, several prostanoids, including the PGD2 and PGJ2 series, can act on eosinophils as DP1 receptor agonists as well as CRTH2 receptor agonists (11). 15d-PGJ2, however, hardly binds to DP1 receptor (Ki, >30,000 nM) (21), and BW245C, a selective DP1 agonist, did not affect eotaxin-induced eosinophil chemotaxis (data not shown). Taken together, we conclude that the priming effect of 15d-PGJ2 is mediated through PPAR{gamma}, but not CRTH2 or DP1, at a low concentration (10–10 M).

It is interesting that the proinflammatory effects we observed were mediated through PPAR{gamma} ligation, because a number of studies have proposed anti-inflammatory effects of PPAR{gamma} agonists both in vivo and vitro (18, 19). We also previously demonstrated that at high concentrations, the synthetic PPAR{gamma} agonist inhibited IL-5-induced survival, degranulation, and eotaxin-induced chemotaxis (16, 17). One explanation for this discrepancy is that PPAR{gamma} could have an opposing effect mediated by its ligand concentration. In support of this idea, Fukunaga et al. (37) reported that synthetic PPAR{gamma} agonists, such as troglitazone, significantly stimulated DNA synthesis of endothelial cells at low nanomolar concentrations, but they significantly suppressed it at micromolar concentrations. In other words, exogenous PPAR{gamma} agonists in therapeutic concentrations might act as a negative immunomodulator, whereas endogenous or natural ligands in physiological concentrations might have some proinflammatory effects. It is also worth noting that naturally occurring fatty acid-derived molecules, such as 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, which are also known as ligands of PPAR{gamma}, contribute to the pathophysiology of allergic airway inflammation (38, 39). Thus, additional studies are necessary to clarify the physiological role of PPAR{gamma}.

Up-regulation of CCR3 expression might be associated with the augmentative effect of eotaxin (40). However, in this study, because PPAR{gamma} agonists did not alter CCR3 surface expression at a low concentration (data not shown), it may be assumed that PPAR{gamma} agonists modulate the downstream signaling of CCR3 to enhance the response to eotaxin. Eosinophil chemotaxis is regulated by multiple signaling pathways that involve calcium signaling, the ERK1/2 and p38 MAPK pathways (3). In this study we demonstrated that eosinophils incubated with 15d-PGJ2 or troglitazone enhanced the Ca2+ response to eotaxin (Fig. 4). In contrast, neither the ERK1/2 nor the p38 MAPK pathway was involved in the priming effect (Fig. 5). Consistent with our results, the synthetic PPAR{gamma} agonists regulate monocyte migration without affecting MCP-1-directed ERK MAPK phosphorylation, indicating that PPAR{gamma} agonists target downstream of MAPK or are independent from this pathway (41).

We also examined the effect of the NF-{kappa}B inhibitor, because many studies have shown that 15d-PGJ2 affects the NF-{kappa}B system in a PPAR{gamma}-independent manner (32). NF-{kappa}B as a transcription factor regulates the immune response and inflammation (42), although its role in eosinophil chemotaxis has not been determined. The fact that blockade of the NF-{kappa}B pathway significantly reduced chemotaxis (Fig. 5B) suggests the importance of NF-{kappa}B in eosinophil activation. However, this pathway is not involved in the priming effect, because the NF-{kappa}B inhibitor did not alter the ability of 15d-PGJ2. Taken together, our data suggest that the PPAR{gamma} agonist-induced functional up-regulation is, at least in part, involved in Ca2+ influx in eosinophils.

Ca2+ influx is a rapid cytosolic event mediated by ligand-activated, membrane-bound, G protein-coupled receptors. In contrast, PPAR{gamma} is a nuclear receptor that forms a heterodimer with retinoid X receptors, binds to a specific DNA sequence, PPAR response element, and activates target gene transcription, which requires a relatively long period (43). However, we found that the priming effect of 15d-PGJ2 occurred within 20 min and disappeared after incubation with 15d-PGJ2 over a long period of time (>180 min; Fig. 1B). This time course is too rapid to encompass PPAR response element-dependent gene transcription. Additional study using the inhibitor of gene transcription (Fig. 5B) led us to believe that the 15d-PGJ2-induced priming effect is independent of transcription.

One possibility is that ligand-activated PPAR{gamma} exerts direct effects on cytosolic molecules, inducing depolarization of membrane potential more intensely. This hypothesis is supported by the cytosolic and perinuclear distribution of PPARs that underwent nuclear translocation upon ligand activation (33). Indeed, our study using immunofluorescence coupled with confocal microscopy revealed that PPAR{gamma} is predominantly located in cytosol in fresh eosinophils (Fig. 6). It has been shown that in endothelial cells, exposure to troglitazone inhibits vascular endothelial growth factor-induced phosphorylation of the cytosolic protein, Akt (44). Moreover, it should be noted that epidermal growth factor-stimulated MAPK phosphorylates PPAR{gamma} and modulates transcriptional activity (45). Thus, PPAR{gamma} could play a role in cross-talk with cytosolic signal molecules. In contrast, steroid hormones act not only via the classical genomic pathway, but also via rapid nongenomic effects, which are mediated by membrane-bound receptors (46). Therefore, as another possibility, a rapid nongenomic pathway by other unknown receptors for PPAR{gamma} agonists is considered (47).

In conclusion, the results of the present study demonstrate that at low concentrations (near physiological levels), 15d-PGJ2 has a rapid priming effect on eotaxin-induced eosinophil migration through PPAR{gamma}, rather than CRTH2. This is a novel finding, different from the current recognition that 15d-PGJ2 has anti-inflammatory effects via PPAR{gamma} at micromolar levels and proinflammatory effects via CRTH2 at low nanomolar levels. 15d-PGJ2 might augment the ability of chemoattractants, such as eotaxin, to facilitate eosinophil accumulation at sites of inflammation in allergic diseases. Finally, we also provide evidence that PPAR{gamma} might play a role as a biphasic regulator of the immune response.


   Acknowledgments
 
We are grateful to Yoko Nakamura and Hikari Kato for outstanding technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a grant from the 21st Century Center of Excellence Program supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Y.K. and S.U. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Junichi Chihara, Department of Clinical and Laboratory Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543 Japan. E-mail address: chihara{at}hos.akita-u.ac.jp Back

4 Abbreviations used in this paper: CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; DK-PGD2, 13,14-dihydro-15-keto-PGD2; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; FSC, forward scatter; NBD, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}. Back

Received for publication March 18, 2005. Accepted for publication August 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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