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12-Prostaglandin J2, a Plasma Metabolite of Prostaglandin D2, Causes Eosinophil Mobilization from the Bone Marrow and Primes Eosinophils for Chemotaxis1
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* Department of Experimental and Clinical Pharmacology, Karl Franzens University, Graz, Austria;
Leukocyte Biology Section, Biomedical Sciences Division, Imperial College Faculty of Medicine, Imperial College of Science, Technology, and Medicine, South Kensington, London, United Kingdom; and
Section of Functional Genomics, Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom
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Abstract |
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12-PGJ2, the effect of which on eosinophil migration has not yet been characterized. In this study we found that 12-PGJ2 was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD2, PGJ2, or 15-deoxy-12,14-PGJ2. Moreover, pretreatment of eosinophils with 12-PGJ2 markedly enhanced the chemotactic response to eotaxin, and in this respect 12-PGJ2 was more effective than PGD2. 12-PGJ2-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that 12-PGJ2 signaled through the same receptor, CRTH2, as PGD2. Finally, 12-PGJ2 was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that 12-PGJ2 is present in the systemic circulation at relevant levels, a role for this PGD2 metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like other prostanoids PGD2 is short-lived and is rapidly metabolized in vivo through three pathways: enzymatically to 1) 11-epi-PGF2
or 2) dihydro-15-keto-PGD2, or nonenzymatically in aqueous solution to 3) PGJ2 (10). The apparent half-life of PGD2 in the blood has been reported to be 1.5 min (11), and PGD2 is rapidly metabolized in plasma to 12-PGJ2, which arises from catalytic conversion of PGJ2 by albumin (12, 13). The importance of 12-PGJ2 as a systemic PGD2 metabolite is also underlined by the fact that it can be detected in human urine in relevant amounts, which are increased after i.v. administration of PGD2 and decreased by cyclooxygenase inhibition (14). In addition to 12-PGJ2, incubation of PGD2 with albumin can yield smaller amounts of 15-deoxy-12,14-PGD2 and 15-deoxy-12,14-PGJ2 (13).
Several PGD2 metabolites, such as dihydro-15-keto-PGD2, 15-deoxy-12,14-PGJ2, and 15-deoxy-12,14-PGD2, have recently been characterized as potent eosinophil activators with respect to chemotaxis, actin polymerization, L-selectin shedding, and CD11b up-regulation (6, 7, 15). PGJ2 and 12-PGJ2 have also been shown to induce actin polymerization and CD11b up-regulation (15), and in the current study we show that PGJ2 and 12-PGJ2 are, in fact, potent eosinophil chemoattractants and inducers of respiratory burst. The rapid conversion of PGD2 to 12-PGJ2 in plasma (10), however, suggests that it is 12-PGJ2 rather than PGD2 that might have an impact on the intravascular pool of eosinophils. Therefore, we sought to investigate possible systemic effects of 12-PGJ2 on eosinophil function and found that this PGD2 metabolite can induce mobilization of eosinophils from the bone marrow and up-regulation of chemotactic responsiveness.
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Materials and Methods |
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All laboratory reagents were obtained from Sigma-Aldrich (Vienna, Austria), unless specified. Dulbecco’s modified PBS (with or without Ca2+ and Mg2+) was purchased from Invitrogen (Vienna, Austria). Chemokines were obtained from PeproTech EC (London, U.K.). CellFix and FACSFlow were purchased from BD Biosciences (Vienna, Austria). Fixative solution was prepared by diluting CellFix 1/10 in distilled water and 1/4 in FACS-Flow. Abs to CD63 (FITC conjugate) were obtained from Autogen Bioclear (Calne, U.K.), HLA-DR (FITC conjugate) were purchased from Sigma-Aldrich, and Abs to CD123 (PE) and CD16 (PE) were obtained from BD Biosciences. PGD2, PGJ2, 12-PGJ2, and 15-deoxy-12,14-PGJ2 were obtained from Cayman (Ann Arbor, MI). The p38 MAPK inhibitor SB-202190, the mitogen-activated protein kinase (MAPK)3/extracellular signal-regulated kinase kinase (MEK) inhibitor U-0126, and the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor LY-294002 were purchased from Biomol (Hamburg, Germany). Kimura’s stain for identification of eosinophils was prepared as previously described (16). The composition of the Tyrode solution used for perfusion experiments was 136.9 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 11.9 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM glucose.
Preparation of human leukocytes
Blood was sampled from healthy volunteers according to a protocol approved by the ethics committee of University of Graz. Preparations of polymorphonuclear leukocytes (containing eosinophils and neutrophils) and PBMC (including basophils, monocytes, and lymphocytes) were prepared by Histopaque gradients as previously described (17, 18). In some experiments eosinophils were further purified from granulocyte populations by negative magnetic selection using an Ab mixture from StemCell Technologies (Vancouver, Canada), (18). Resulting populations of eosinophils were typically >97%, with the majority of contaminating cells being neutrophils.
Leukocyte shape change assay
Eosinophil, basophil, and neutrophil shape change was assayed as described in previous work (17, 18). Stimulation of these leukocytes by chemoattractant and chemokinetic agonists results in changes in the cell shape that increase the scattering of light when illuminated in a flow cytometer (Fig. 1). Polymorphonuclear leukocytes or PBMC preparations were resuspended in assay buffer (comprising PBS with Ca2+/Mg2+ supplemented with 0.1% BSA, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 5 x 106 cells/ml, and 50-µl aliquots were mixed with 50 µl of agonists and stimulated for 4 min at 37°C. To stop the reaction samples were transferred to ice and fixed with 250 µl of fixative solution. Eosinophils were identified according to their autofluorescence in FL-1 and FL-2, while neutrophils were identified by their lack of autofluorescence (Fig. 1). To investigate basophil shape change, PBMC were stained with anti-HLA-DR (FITC) and anti-CD123 (PE; 1/100 dilution of each Ab) for 6 min, and basophils were identified as CD123pos/HLA-DRneg cells (17, 18, 19). Samples were immediately analyzed on a FACSort flow cytometer (BD Biosciences), and data were displayed as the percent increase in forward scatter compared with samples treated with buffer or vehicle alone. The various vehicles used (PBS and ethanol) were without effect in these shape change assays at the dilutions tested.
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Eosinophil degranulation was assayed by flow cytometric detection of the granule-associated marker CD63 (18, 20). Polymorphonuclear leukocyte preparations were labeled with anti-CD16 (PE) for 6 min at room temperature (1/100 dilution of the Ab), washed in PBS without Ca2+/Mg2+, and resuspended in assay buffer at 5 x 106 cells/ml in the presence of the anti-CD63 (FITC) at a 1/100 dilution of the Ab. Cells were then pretreated with cytochalasin B (5 µg/ml) for 5 min at 37°C, and 50-µl aliquots were mixed with 50 µl of agonists and stimulated for 30 min at 37°C. To stop the reaction samples were transferred to ice, washed in cold PBS without Ca2+/Mg2+, and fixed with 250 µl of fixative solution. Samples were immediately analyzed by flow cytometry. Eosinophils were identified by forward scatter/side scatter gating and were CD16 negative, while neutrophils were in a similar forward scatter region, showed lower side scatter, and were CD16 positive. Data were expressed as the percent change from a a control sample incubated with buffer alone.
Respiratory burst
Eosinophils purified by negative magnetic selection were resuspended in assay buffer at 5 x 105 cells/ml in the presence of 1 µM dihydrorhodamine 123, and 50-µl aliquots of cells were mixed with 50 µl of agonists and stimulated for 20 min at 37°C. To stop the reaction, samples were transferred to ice and fixed with 100 µl of fixative solution. Samples were immediately analyzed by flow cytometry. Respiratory burst was measured as an increase in eosinophil fluorescence in the FL-1 channel due to the oxidization by reactive oxygen species of the nonfluorescent dye dihydrorhodamine 123 into fluorescent rhodamine 123 (21), and responses were expressed as percent changes from a control sample incubated with buffer alone.
Chemotaxis
Fifty microliters of purified eosinophils suspended in assay buffer at 2 x 106/ml were placed onto the top of a 96-well chemotaxis chamber with 5-µm pore size polycarbonate filter (NeuroProbe, Gaithersburg, MD) with 30 µl of agonists in the bottom well of the plate. The plates were incubated at 37°C in a humidified CO2 incubator for 1 h, and the membrane was carefully removed. Cells in the lower chamber were counted by flow cytometry as previously described (18). In each chemotaxis plate, migration in response to agonists was expressed as a ratio of the migration in response to the buffer control (chemotactic index). In some experiments the eosinophils were mixed with varying concentrations of 12-PGJ2 (4–250 nM) or PGD2 (1–64 nM) before being applied to the chemotaxis chamber, and migration toward eotaxin (10 nM) or 12-PGJ2 (1000 nM) was determined. The role of intracellular signaling pathways was investigated by pretreating eosinophils with the p38 MAPK inhibitor SB-202190 (10 µM) (22), the MEK inhibitor U-0126 (2 µM) (23), or the PI-3 kinase inhibitor LY-294002 (20 µM) (24) for 30 min at 37°C before the chemotaxis assay.
Calcium flux
Intracellular calcium levels were analyzed by flow cytometry as previously described (15). Polymorphonuclear leukocytes (107 cells/ml) were treated with 2 µM of the acetoxymethyl ester of Fluo-3 in the presence of 0.02% Pluronic F-127 for 60 min at room temperature and were washed in PBS without Ca2+/Mg2+. The leukocytes were then labeled with anti-CD16 (PE; 1/20 dilution of the Ab) for 6 min at room temperature, washed, and resuspended in assay buffer without Ca2+/Mg2+ to give a concentration of 3 x 106 leukocytes/ml. Aliquots (950 µl) of the leukocyte suspension were removed and treated with 50 µl of PBS containing Ca2+ (36 mM) and Mg2+ (20 mM) for 5 min. Changes in intracellular free calcium levels were detected by flow cytometry as an increase in fluorescence intensity of the calcium-sensitive dye Fluo-3 in the FL-1 channel for eosinophils (CD16 negative/high side scatter) and neutrophils (CD16 positive/low side scatter; Fig. 4A). Maximal calcium responses were determined by addition of the calcium ionophore A23187 (10 µM) at the end of each experiment. Time kinetics of calcium flux were analyzed using FlowJo software (Tree Star, San Carlos, CA).
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Adult guinea pigs (TRIK strain, either sex, 350–450 g body weight) were used. The guinea pig hind limb was perfused as previously described (25). The external iliac artery and vein were exposed, and the caudal abdominal artery, superficial iliac circumflex artery, and pudendoepigastric trunk along with their satellite veins were ligated. Polyethylene cannulas (0.8 mm outside diameter) were inserted into the external iliac artery and vein, and Tyrode bicarbonate buffer (37°C, gassed with 95% O2/5% CO2; composition detailed above) was infused (4 ml/min) via the arterial cannula and removed from the venous cannula using a peristaltic pump. Perfusate fractions were collected every 10 min and centrifuged at 300 x g for 10 min, and the cell pellet was resuspended in Kimura’s stain. Nucleated leukocytes and Kimura-positive eosinophils were counted in a Neubauer hemocytometer.
Statistics
Data are shown as the mean ± SEM for n observations. Comparisons of groups of data were performed by Mann-Whitney U test or ANOVA, using Dunn’s post-test. Probability values of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12-PGJ2 and PGJ2 cause shape change of human eosinophils and basophils
Fig. 1A illustrates the changes in the shape of human eosinophils in mixed leukocyte populations as measured by flow cytometry, showing a marked increase in forward scatter of eosinophils in response to 12-PGJ2. In causing shape change, 12-PGJ2 was as potent as PGJ2 and 15-deoxy-12,14-PGJ2, but was 10- to 30-fold less potent than PGD2 or eotaxin in both eosinophils and basophils (Fig. 1, B and C). Maximal shape change effects of 12-PGJ2 were similar to those observed with PGD2, indicating that this PGD2 metabolite was a full receptor agonist. In contrast, neutrophils selectively responded to IL-8, but to none of the PGD2 analogs (data not shown; n = 5).
PGJ2 and 12-PGJ2 cause respiratory burst, but not CD63 expression, in human eosinophils
Eosinophil respiratory burst (Fig. 2A) and CD63 up-regulation, a degranulation-associated marker (Fig. 2B), were strongly induced by C5a. In contrast, PGD2 and its analogs PGJ2 and 12-PGJ2 were unable to elicit any changes in eosinophil CD63 expression at the concentrations tested (Fig. 2B), but were effective inducers of respiratory burst in eosinophils (Fig. 2A). Again, PGJ2 and 12-PGJ2 were 10-fold less potent than PGD2, but retained full agonistic activity in stimulating the production of reactive oxygen species.
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12-PGJ2 facilitates chemotaxis of human eosinophils
Purified eosinophils were placed in the top wells of a chemotaxis chamber separated by a filter membrane from the chemoattractants in the bottom wells. When present in the bottom well, 12-PGJ2 induced migration of eosinophils into the bottom chamber of the chemotaxis plate (Fig. 2C). The potency of 12-PGJ2 to induce eosinophil migration appeared to be 10-fold lower than that of PGD2, but similar to that of PGJ2. The observed migration of eosinophils was due to chemotaxis rather than chemokinesis, since no significant increase in eosinophil numbers in the bottom well was observed when 12-PGJ2 was present, together with the cells, in the top well only (Fig. 2D). In keeping with this, addition of a submaximal concentration of 12-PGJ2 (250 nM) to the top of the chemotaxis chamber impaired the chemotactic responses to a higher (1000 nM) agonist concentration in the bottom well (Fig. 2D).
In addition to being chemotactic by itself, 12-PGJ2 revealed the potential to prime signaling to other eosinophil-active mediators. Fig. 3A shows that a short pretreatment of eosinophils with 12-PGJ2 effectively amplified the migration of eosinophils toward eotaxin in the bottom well of the chemotaxis plate. On the average, the chemotactic response to eotaxin (10 nM), the half-maximally effective concentration in our system (18), was enhanced by 100% when 12-PGJ2 (250 nM) was added to the cells immediately before they were applied to the top wells of the chemotaxis plate. The magnitude of this effect depended on the concentration of 12-PGJ2 in the top well, and a significant enhancement of the eotaxin response was observed at concentrations as low as 16 nM 12-PGJ2 (Fig. 3B). Similarly, pretreatment of eosinophils with PGD2 (1–64 nM) augmented the eotaxin-induced migration, although PGD2 was less effective in this respect than 12-PGJ2 (Fig. 3B). In an attempt to analyze the molecular mechanisms underlying the ability of 12-PGJ2 to amplify the responsiveness of eosinophils to eotaxin, we pretreated the cells with effective concentrations of inhibitors of intracellular signaling pathways, the p38 MAPK inhibitor SB-202190, the MEK inhibitor U-0126, and the PI-3 kinase inhibitor LY-294002, but we found that none of the inhibitors was able to reverse the ability of 12-PGJ2 to facilitate the responsiveness of eosinophils to eotaxin (Fig. 3C). Although a combination of these inhibitors substantially reduced the eotaxin-induced migration, thus confirming the involvement of these pathways in the chemotactic response, 12-PGJ2-induced enhancement of eosinophil responsiveness was still evident (data not shown; n = 3). Therefore, these observations suggest that in a setting when it is present in the systemic circulation, 12-PGJ2 might augment the effect of other chemoattractants to cause eosinophil accumulation at sites of inflammation independently from p38 MAP, MEK, and PI-3 kinases.
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12-PGJ2 and PGD2 responses
The receptor usage of 12-PGJ2 was investigated in calcium flux studies using the calcium-sensitive dye Fluo-3, CD16-labeled polymorphonuclear leukocytes, and flow cytometry (Fig. 4A). Like PGD2 (100 nM), 12-PGJ2 (250 nM) induced eosinophil calcium flux, which amounted to 70–90% of the response to the calcium ionophore A23187 (10 µM; Fig. 4B). However, no response to 12-PGJ2 could be observed in eosinophils after calcium flux had previously been induced by PGD2 in the same cell sample. This effect was consistent with receptor desensitization, since the consecutive eotaxin-induced calcium response was not abolished by PGD2. Similarly, stimulation with 12-PGJ2 completely abrogated the calcium response to PGD2, while a previous challenge with eotaxin had little effect on the PGD2 response (Fig. 4B). Receptor desensitization was also observed with PGJ2, 12-PGJ2, and PGD2, respectively (data not shown; n = 3), thus suggesting that these PGD2 metabolites activate eosinophils through CRTH2 receptors. In contrast, neutrophils did not respond to PGD2 or 12-PGJ2, but effective calcium flux was elicited in neutrophils by C5a (10 nM) and A23187 (data not shown; n = 4).
12-PGJ2 induces rapid mobilization of eosinophils from the bone marrow of guinea pigs
When infused into the guinea pig isolated perfused hind limb 12-PGJ2 (10 and 100 nM) increased the number of eosinophils in the effluent perfusate in a concentration-dependent manner, indicating mobilization of eosinophils from the femoral bone marrow (Fig. 5). This effect occurred within 10 min and declined 20 min after the infusion had been stopped. IL-5 (0.5 nM), a cytokine known to induce eosinophil mobilization from the bone marrow (25), also increased the number of eosinophils in the perfusate, but with more delayed kinetics than 12-PGJ2, reaching a maximum 40 min after the start and 20 min after the end of the infusion (Fig. 5). The number of other nucleated cells released into the perfusate remained unchanged during the entire test period without respect to the infused compounds (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12-PGJ2, which is present in human plasma (12, 13), not only induces chemotaxis and respiratory burst of eosinophils, but also mobilizes eosinophils from the bone marrow and effectively facilitates their migration toward other chemoattractants, thus suggesting two novel roles of PGD2 metabolites in the regulation of eosinophil function in allergic disease.
We found that PGJ2, 15-deoxy-12,14-PGJ2, and 12-PGJ2 induced a direct shape change response in eosinophils in a manner similar to the chemoattractant agonists eotaxin and PGD2, reaching a maximum shape change response at a 12-PGJ2 concentration of 100 nM. In addition, we observed that PGJ2, 15-deoxy-12,14-PGJ2, and 12-PGJ2 induced shape change responses in basophils, but not neutrophils or monocytes. The potency of 12-PGJ2 was similar to those of PGJ2 and 15-deoxy-12,14-PGJ2, but 30 times lower than that of eotaxin or PGD2. Similar concentration-response relationships had previously been reported for eosinophil actin polymerization induced by these PGD2 analogs (15). From these data it appeared likely that PGJ2 and 12-PGJ2 might also act as chemoattractants, and our data show that these PGD2 metabolites did induce concentration-dependent chemotaxis of purified eosinophils with the same efficacy as the other eosinophil-stimulating agonists, eotaxin and PGD2. This effect was indeed due to chemotaxis rather than chemokinesis, since 12-PGJ2, if applied directly to the cells in the top of the chemotaxis chamber, did not induce migration of eosinophils. In addition, the chemotactic response to 12-PGJ2 was inhibited if the chemoattractant was present in both the bottom and top wells. 12-PGJ2 was equally potent as PGJ2 and 10–20 times less potent than PGD2 in causing eosinophil chemotaxis. These figures are at variance with receptor binding studies by Hirai et al. (6), who reported that in CRTH2 transfectants the receptor affinity of 12-PGJ2 to CRTH2 was >100 times lower than that of PGD2. Subtle differences between cells transfected with or naturally expressing CRTH2 receptors might possibly account for the altered receptor pharmacology. Similarly, we have recently observed that signaling through CRTH2 in eosinophils is insensitive to pertussis toxin (18), while functional responses in CRTH2 transfectants are abolished by pertussis toxin (6).
Since classical chemoattractants such as C5a or fMLP also potently induce degranulation of granulocytes, we investigated the effects of PGJ2 and 12-PGJ2 on eosinophil degranulation. While C5a effectively enhanced the expression of the granule-associated marker CD63, PGD2 and its metabolites had no effect, suggesting that they are not involved in triggering eosinophil degranulation. In contrast, PGD2, PGJ2, and 12-PGJ2 induced respiratory burst in eosinophils, although with less potency than C5a. Eosinophils express two receptors for PGD2, DP and CRTH2. Of these two receptors, CRTH2 is selectively responsible for eosinophil chemotaxis, actin polymerization, CD11b up-regulation, and calcium mobilization in response to PGD2, while DP receptors have been shown to modulate eosinophil apoptosis only (6, 7, 30). In keeping with this, we found that the DP-selective agonist BW-245C was unable to cause eosinophil shape change (18) or respiratory burst (unpublished observations), and 12-PGJ2 has been reported to have very low affinity to the DP receptor (6). We therefore hypothesized that PGJ2 and 12-PGJ2 were acting via the CRTH2 receptor, in keeping with their PGD2-like activities on eosinophils. In fact, we observed cross-desensitization of calcium responses among PGD2, PGJ2, and 12-PGJ2, but not with eotaxin. As reported previously the DP-selective agonist BW-245C did not induce calcium mobilization in eosinophils (6), thus suggesting that PGJ2 and 12-PGJ2 activate eosinophils via CRTH2.
The above data suggest that PGJ2 and 12-PGJ2 might be involved in eosinophil recruitment to sites of inflammation if formed locally in the tissue. These degradation products of PGD2 are, however, also present in plasma (12, 13) and might therefore have important systemic effects on the allergic response. We found that 12-PGJ2 caused eosinophil mobilization from the bone marrow with similar magnitude as reported for eotaxin and IL-5 (26). The selectivity for eosinophils and the rapid onset of eosinophil release within 10 min and return to baseline within 30 min after removal of the stimulant were in keeping with those of the direct eosinophil chemoattractant, eotaxin (26), and might hence indicate the involvement of similar cellular mechanisms. In contrast, we observed that eosinophil release by IL-5 followed a more delayed time course compared with 12-PGJ2, suggesting different mechanisms. In addition to increasing the intravascular pool of eosinophils ready to enter the tissue, the current data indicate that 12-PGJ2 might also up-regulate eosinophil responsiveness to chemoattractants and thus facilitate eosinophil extravasation. The chemokine receptor CCR3 is emerging as a key molecule in mediating eosinophil accumulation in the tissue (3, 4) and is the receptor for multiple chemokines, including eotaxin; eotaxin-2 and -3; monocyte chemotactic peptide-2, -3, and -4; and RANTES (4), which are all expressed during the allergic response. We observed that eosinophil chemotaxis toward the CCR3 agonist eotaxin was up-regulated 
2-fold in the presence of 12-PGJ2 or PGD2. In these experiments the prostanoids were added directly to the eosinophils, which were separated from the chemoattractant eotaxin by a membrane, thus mimicking the effect of plasma 12-PGJ2 on eosinophil migration in vivo. A similar magnitude of priming eosinophil migration toward eotaxin has been reported for IL-5 (26). It is also worth noting that 12-PGJ2 did not induce chemokinesis, a mechanism proposed to underlie the enhancement of eosinophil chemotaxis by IL-3, IL-4, or IL-5 (25, 27, 28). Interestingly, the threshold concentration of 12-PGJ2 to induce chemotaxis or respiratory burst (64 nM; Fig. 2) was considerably higher than the minimal concentrations of 12-PGJ2 required for eosinophil mobilization from the bone marrow (10 nM; Fig. 5) or up-regulation of the chemotactic response (16 nM; Fig. 3), suggesting that these latter effects might reflect primary actions of 12-PGJ2. PGD2 likewise enhanced the chemotactic responsiveness to eotaxin, but was less effective than 12-PGJ2, which might reflect rapid degradation of PGD2 due to the presence of albumin in the assay buffer (13).
Eotaxin-induced eosinophil responses have been shown to involve signaling pathways dependent upon p38 MAPK and PI-3 kinase (18, 22, 31), which can couple to pathways regulating actin polymerization (32), integrin-dependent eosinophil adhesion (33), and chemokinesis (25, 28). In addition, eosinophil adhesion in response to IL-5 and platelet-activating factor has been shown to involve MEK (33). Recently, we have shown that these pathways also regulate CRTH2-induced signaling in eosinophils (18). Therefore, we hypothesized that 12-PGJ2-induced up-regulation of the chemotactic response to eotaxin might result from preactivation of these pathways. Preincubation of eosinophils with the p38 MAPK inhibitor SB-202190, the MEK inhibitor U-0126, or the PI-3 kinase inhibitor LY-294002, however, did not attenuate the effect of 12-PGJ2 in facilitating chemotaxis toward eotaxin. Enhancement of calcium mobilization in response to eotaxin was not involved in the up-regulation of chemotaxis by 12-PGJ2 as determined by calcium flux studies following a 30-min preincubation with the CRTH2 agonist (our unpublished observation). Therefore, the mechanisms contributing to 12-PGJ2-induced modulation of eosinophil chemotaxis remain to be determined.
In summary, the current data extend the range of known PGD2 actions in the allergic response. Along with previous reports, our findings suggest a key role for PGD2 and related products by acting 1) systemically to induce eosinophil release from the bone marrow and up-regulation of the chemotactic response to other chemoattractants (Fig. 6), and 2) locally to cause eosinophil accumulation, respiratory burst, and, via the DP receptor, enhancement of eosinophil survival at sites of inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Akos Heinemann, Department of Experimental and Clinical Pharmacology, Universitaetsplatz 4, A-8010 Graz, Austria. E-mail address: akos.heinemann{at}uni-graz.at
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; PI-3 kinase, phosphatidylinositol 3-kinase.
Received for publication August 13, 2002. Accepted for publication February 27, 2003.
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I. Spik, C. Brenuchon, V. Angeli, D. Staumont, S. Fleury, M. Capron, F. Trottein, and D. Dombrowicz Activation of the Prostaglandin D2 Receptor DP2/CRTH2 Increases Allergic Inflammation in Mouse J. Immunol., March 15, 2005; 174(6): 3703 - 3708. [Abstract] [Full Text] [PDF]
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Y. Shiraishi, K. Asano, T. Nakajima, T. Oguma, Y. Suzuki, T. Shiomi, K. Sayama, K. Niimi, M. Wakaki, J. Kagyo, et al. Prostaglandin D2-Induced Eosinophilic Airway Inflammation Is Mediated by CRTH2 Receptor J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 954 - 960. [Abstract] [Full Text] [PDF]
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A. Hartnell, A. Heinemann, D. M. Conroy, R. Wait, G. J. Sturm, M. Caversaccio, P. J. Jose, and T. J. Williams Identification of Selective Basophil Chemoattractants in Human Nasal Polyps as Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2 J. Immunol., November 15, 2004; 173(10): 6448 - 6457. [Abstract] [Full Text] [PDF]
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 Z.-Z. Shan, K. Masuko-Hongo, S.-M. Dai, H. Nakamura, T. Kato, and K. Nishioka A Potential Role of 15-Deoxy-{Delta}12,14-prostaglandin J2 for Induction of Human Articular Chondrocyte Apoptosis in Arthritis J. Biol. Chem., September 3, 2004; 279(36): 37939 - 37950. [Abstract] [Full Text] [PDF]
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E. Bohm, G. J. Sturm, I. Weiglhofer, H. Sandig, M. Shichijo, A. McNamee, J. E. Pease, M. Kollroser, B. A. Peskar, and A. Heinemann 11-Dehydro-thromboxane B2, a Stable Thromboxane Metabolite, Is a Full Agonist of Chemoattractant Receptor-homologous Molecule Expressed on TH2 Cells (CRTH2) in Human Eosinophils and Basophils J. Biol. Chem., February 27, 2004; 279(9): 7663 - 7670. [Abstract] [Full Text] [PDF]
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M. Shichijo, H. Sugimoto, K. Nagao, H. Inbe, J. A. Encinas, K. Takeshita, K. B. Bacon, and F. Gantner Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells Activation in Vivo Increases Blood Leukocyte Counts and Its Blockade Abrogates 13,14-Dihydro-15-keto-prostaglandin D2-Induced Eosinophilia in Rats J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 518 - 525. [Abstract] [Full Text] [PDF]
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