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Originally published In Press as doi:10.1074/jbc.M310270200 on December 10, 2003

J. Biol. Chem., Vol. 279, Issue 9, 7663-7670, February 27, 2004
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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*

Eva Böhm{ddagger}, Gunter J. Sturm{ddagger}, Iris Weiglhofer{ddagger}, Hilary Sandig§, Michitaka Shichijo¶, Anne McNamee||, James E. Pease§, Manfred Kollroser**, Bernhard A. Peskar{ddagger}, and Akos Heinemann{ddagger}{ddagger}{ddagger}

From the Departments of {ddagger}Experimental and Clinical Pharmacology and **Forensic Medicine, Medical University Graz, A-8010 Graz, Austria, §Leukocyte Biology Section, Biomedical Sciences Division, Imperial College Faculty of Medicine, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, United Kingdom, Respiratory Research, Research Center Kyoto, Bayer Yakuhin Ltd., Kizu-cho Soraku-gun, Kyoto 619-0216, Japan, and ||Immunopharmacology Department, GlaxoSmithKline Place, Stevenage SG1 2NY, United Kingdom

Received for publication, September 16, 2003 , and in revised form, December 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thromboxane (TX) A2, a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB2, which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB2, but not the TXA2 analogue U46,619 or TXB2, activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB2 was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D2 has been shown to be its principal ligand. 11-Dehydro-TXB2 induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD2 but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB2 were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB2- and PGD2-induced shape change. 11-Dehydro-TXB2 also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB2 had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB2 is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB2/CRTH2axis may be of therapeutic relevance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulation of eosinophils at sites of allergic reactions, such as asthma and atopic rhinitis, is associated with tissue injury and lung dysfunction (1–4). Several chemoattractants can mediate eosinophil recruitment, in particular the CC-chemokines acting through the chemokine receptor CCR3 (5–7). The prostaglandin (PG)1D2 is a major mast cell mediator released during the allergic response (8) and is capable of inducing the chemotaxis of eosinophils, basophils, and TH2-type T cells by means of a novel receptor, the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) (9, 10). Moreover, CRTH2 is the most reliable marker for TH2-cells (11). We have shown recently that CRTH2 agonists also mediate the release of eosinophils from the bone marrow and prime eosinophils for chemotaxis toward other chemoattractants, such as the CCR3 ligand eotaxin/CCL11 (12). A significant contribution of PGD2 to the late-phase allergic reaction is suggested by enhanced eosinophilic lung inflammation and cytokine release in transgenic mice over-expressing PGD2 synthase (13).

Thromboxane (TX) A2, a further product of the arachidonic acid/cyclooxygenase pathway acting by means of the thromboxane receptor TP, has also been implicated in the pathogenesis of allergic diseases and asthma (14, 15), as it causes bronchoconstriction (16, 17), plasma extravasation (17, 18), and the growth of smooth muscle cells (16, 19). In addition, the wide range of TXA2 biological actions includes platelet aggregation (20), vasoconstriction (21), and the promotion of angiogenesis and tumor metastasis (22). TXA2 is short lived and very rapidly transformed non-enzymatically in aqueous solution to TXB2 (23). TXB2 is further metabolized enzymatically to a series of compounds, of which 11-dehydro-TXB2 is the major product found both in plasma and urine (24). The dehydrogenase-catalyzing 11-dehydro-TXB2 formation is tissue bound and widely expressed, with the highest occurrence being found in lung and kidney (24). Because of its stability, 11-dehydro-TXB2 has been used to monitor TXA2 production in vivo and in vitro (25, 26). In contrast to PGD2 metabolites, which retain potent TP (27) or CRTH2 (9, 10, 12, 28) agonistic activity, TXB2 and 11-dehydro-TXB2 have been considered as being devoid of TP agonistic activity (23). Considerable cross-reactivity of PGD2 in a radio-immunoassay for 11-dehydro-TXB2 has been reported (29), suggesting structural similarities between these prostanoids. In the current study, we observed that 11-dehydro-TXB2, but not TXB2 or the TXA2 mimetic U-46,619, caused calcium flux and chemotaxis in human eosinophils. Furthermore, we could demonstrate that these effects are mediated through activation of CRTH2 receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—All laboratory reagents were obtained from Sigma (Vienna, Austria), unless specified otherwise. Dulbecco's modified phosphate-buffered saline (PBS) (with or without Ca2+ and Mg2+) and RPMI 1640 medium was obtained from Invitrogen. Eotaxin/CCL11 was obtained from Peprotech EC (London, UK). CellFix and FACSFlow were obtained from Becton Dickinson Immunocytometry Systems (Vienna, Austria). Fixative solution was prepared by diluting CellFix 1/10 in distilled water and 1/4 in FACSFlow. Antibodies to HLA-DR (fluorescein isothiocyanate conjugate) were obtained from Sigma, and antibodies to CD123 (PE) and CD16 (PE) were obtained from Becton Dickinson. PGD2 and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) were obtained from Cayman Chemical (Ann Arbor, MI). 11-Dehydro-TXB2 and SQ29,548 were purchased from Biomol (Hamburg, Germany). Ramatroban (BAY u3405; (+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3,4-tetra-hydrocarbazole-9-propionic acid) was synthesized by Bayer Yakuhin Ltd. (Kyoto, Japan). ADP was obtained from Probe & Go (Endingen, Germany). The phosphatidylinositide-specific phospholipase C (PLC) inhibitor U73,122 and the control analogue U73,343 were supplied by Biomol.

Preparation of Human Leukocytes—Blood was sampled from healthy volunteers according to a protocol approved by the Ethics Committee of the University of Graz and processed as described previously (12, 30, 31). Mixed peripheral blood leukocytes (eosinophils, neutrophils, basophils, monocytes, and lymphocytes) were obtained by dextran sedimentation of citrated whole blood. Preparations of polymorphonuclear leukocytes (containing eosinophils and neutrophils) and peripheral blood mononuclear cells (including basophils, monocytes, and lymphocytes) were prepared by Histopaque gradients as described. In some experiments, eosinophils were further purified from polymorphonuclear populations by negative magnetic selection using an antibody mixture from StemCell Technologies (Vancouver, Canada). 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 previously (12, 31). Stimulation of leukocytes by chemoattractants or chemokinetic agonists results in changes in the cell shape that are reflected by an increase of light scattering in flow cytometry. Mixed peripheral blood leukocytes were stained with anti-HLA-DR (fluorescein isothiocyanate) and anti-CD123 (PE) (1:100 dilution of each antibody) for 6 min and resuspended in assay buffer (composed of 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. 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. Samples were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences), and target cells were generally identified by their forward-scatter/side-scatter characteristics. In addition, eosinophils were identified according to their autofluorescence in FL-1 and FL-2, whereas neutrophils were identified by their lack of autofluorescence. Basophils were identified as CD123pos/HLA-DRneg cells (31, 32). Shape change responses in eosinophils, neutrophils, and basophils were quantified as the percentage of cells in a higher forward-scatter region, defined as a region that contained only 20% of cells with high forward-scatter values in a control sample. When subsequent samples from the same donor were exposed to a chemoattractant, e.g. eotaxin/CCL11, a concentration-dependent increase in the number of cells in the high forward-scatter region could be observed (31). At maximal stimulation, up to 80% of cells were present in this region. The various vehicles used (PBS, Me2SO, and ethanol) were without effect at the dilutions tested. In some experiments, the cells were pretreated with ramatroban, a mixed CRTH2/TP receptor antagonist (33), SQ29,548, a TP receptor antagonist (20), 11-dehydro-TXB2, TXB2, or the respective vehicles for 5 min at room temperature, with pertussis toxin (PTX) (1 µg/ml) or its vehicle for 60 min at 37 °C, or the PLC inhibitor U-73,122 (3.6 µM), or the control analogue U73,343 (30, 34) for 30 min at 37 °C, after which agonist-induced shape change was recorded.

Calcium Flux—Intracellular calcium levels were analyzed by flow cytometry as described (12, 28, 35). 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 washed in PBS without Ca2+/Mg2+. The leukocytes were then labeled with anti-CD16 (PE; 1:20 dilution of the antibody) 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. 950-µl aliquots 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). To investigate agonist-induced receptor desensitization, a first agonist was added to the cell sample followed by a second agonist 6 min later. Maximal calcium responses were determined by the addition of the calcium ionophore A23187 [GenBank] (10 µM) at the end of each experiment. To record calcium mobilization from intracellular stores, cells loaded with Fluo-3 were resuspended in assay buffer containing Ca2+ (0.9 mM) and Mg2+ (0.5 mM) to give a concentration of 30 x 106 leukocytes/ml. 950 µl of the same buffer, or assay buffer without Ca2+/Mg2+ but containing 2.22 mM EGTA, were added to 50-µl aliquots of the leukocyte suspension for 6 min, and responses to agonists were recorded thereafter.

Generation of BaF/3 Cells Expressing hCRTH2—The human open reading frame encoding CRTH2 was isolated after PCR amplification of genomic DNA. After ligation into the vector pCIN (Promega; Southampton, UK), DNA was linearized and introduced into the BaF/3 cell line by electroporation, as described previously (36). This cell line was maintained in RPMI 1640 medium containing 10% fetal calf serum and 5 ng/ml of mIL-3. Cells were selected 28 h later by the addition of 1 mg/ml G418 (Invitrogen) to the culture media, and individual resistant cells were isolated by limiting dilution. Clones were expanded after the selection of CRTH2-expressing cells by chemotaxis to PGD2 and fluorescence-activated cell sorting with anti-CRTH2 mAb BM2/16.

Chemotaxis Assays—Purified eosinophils were suspended in assay buffer at 2 x 106/ml, and 50 µl of the suspension were placed onto the top of a 48-well micro-Boyden chemotaxis chamber with a 5-µm pore-size polycarbonate filter (NeuroProbe Inc., Gaithersburg, MD), with 30 µl of agonists in the bottom well of the plate. Baseline migration was determined in wells containing only assay buffer. The plates were incubated at 37 °C in a humidified CO2 incubator for 1 h, and the membrane was carefully removed. Cells that had migrated to the lower chamber were enumerated by flow cytometry counting for 30 s, as described previously (30).

Chemotaxis of BaF/3 cells expressing hCRTH2 was carried out as described previously using ChemoTX plates (NeuroProbe Inc, Gaithersburg, MD) with 5-µm pore size (36). BaF/3 cells, naive or transfected, were washed and resuspended at 106/ml in RPMI 1640 medium and 0.1% bovine serum albumin. 20-µl aliquots of these cells were then placed onto the permeable membranes of the chamber and allowed to migrate for 5 h at 37 °C toward various concentrations of PGD2 or 11-dehydro-TXB2. The number of migrating cells was subsequently counted by microscopy.

Platelet Aggregation—Human platelet-rich plasma and platelet-poor plasma were prepared from citrated whole blood by centrifugation. Platelet aggregation was recorded at 37 °C with constant stirring (1000 rpm) in a four-channel Aggrecorder II aggregometer (KDK Corp., Kyoto, Japan) as described (37). Platelet aggregation was measured as the increase in light transmission for 5 min, starting with the addition of ADP (2.5–20 µM) as pro-aggregatory stimulus. CaCl2 at a final concentration of 1 mM was added 2 min before ADP. To record inhibition of ADP-induced aggregation, 11-dehydro-TXB2 (10 µM) or PGD2 (6.25–100 nM) were added 2 min before ADP. To probe for a possible antagonistic effect of 11-dehydro-TXB2 on PGD2-induced inhibition of platelet aggregation, 11-dehydro-TXB2 (10 µM) was added 5 min before PGD2. Data were expressed as the percent of maximum light transmission, with non-stimulated platelet-rich plasma being 0% and platelet-poor plasma 100%.

Statistics—Data are shown as mean ± S.E. for n observations. Comparisons of groups of data were performed using the Mann-Whitney U test or by analysis of variance, using Dunn's post test. Probability values of p < 0.05 were considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
11-Dehydro-TXB2 Causes Shape Change of Human Eosinophils and Basophils—Fig. 1A shows that 11-dehydro-TXB2 was a highly effective inducer of shape change in human eosinophils as measured by flow cytometry. 11-Dehydro-TXB2 also caused shape change in basophils, with a similar efficacy and potency as in eosinophils (Fig. 1B). In this respect, 11-dehydro-TXB2 was as effective as PGD2, whereas the potency of 11-dehydro-TXB2 on the shape change responses of both cell types was 300- to 500-times lower than that of PGD2. The effect of 11-dehydro-TXB2 in causing eosinophil and basophil shape change was concentration-dependent, with a threshold concentration of ~30 nM. The parent compound TXB2 and the stable TXA2 analogue U46,619 were devoid of any effect at concentrations examined up to 10 µM (Fig. 1, A and B). In contrast to eosinophils and basophils, neutrophils and monocytes did not respond with shape change to 11-dehydro-TXB2 or PGD2, although they responded to their respective positive controls of IL-8/CXCL8 and MCP-1/CCL2, respectively (data not shown, n = 5).



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  		FIG. 1.
11-Dehydro-TXB2 causes flow cytometric shape change and calcium flux in eosinophils and basophils. Eosinophils in peripheral blood leukocyte preparations were identified by their higher degree of autofluorescence (A), whereas basophils were identified as CD123pos/HL-DRAneg cells (B), and agonist-induced activation was quantified as the percentage of cells moving from a low to a high forward-scatter region which had previously been defined as containing 20% of cells in a non-stimulated sample. 11-Dehydro-TXB2 caused eosinophil and basophil shape change with the same efficacy but lower potency than PGD2, whereas TXB2 and U46,619 were ineffective. C, calcium flux was measured in polymorphonuclear cells loaded with Fluo-3 and labeled with anti-CD16 antibodies (PE). Eosinophils were gated as CD16-negative cells exhibiting higher side-scatter as compared with neutrophils (CD16-positive/low side-scatter) and changes in intracellular free calcium levels were detected as an increase in fluorescence intensity of the calcium-sensitive dye Fluo-3 in FL-1. Responses were quantified as percent fluorescence intensity with respect to baseline. 11-Dehydro-TXB2 caused effective calcium flux in eosinophils. Data are shown as mean ± S.E., n = 5–6.

 
11-Dehydro-TXB2-induced Calcium Flux and Cross-desensitization with PGD2—Calcium flux was recorded by flow cytometry in polymorphonuclear cell preparations loaded with Fluo-3 and labeled with anti-CD16 antibodies. In this assay, eosinophils were distinguished from neutrophils as CD16-negative cells that exhibited higher side-scatter. Fig. 1C illustrates that 11-dehydro-TXB2 caused effective calcium flux in eosinophils with a similar agonist concentration response-relationship as observed in the shape change assay (Fig. 1A).

The receptor usage of 11-dehydro-TXB2 was also investigated in calcium flux studies using homologous receptor desensitization (Fig. 2). Under control conditions, 11-dehydro-TXB2 (10 µM), PGD2 (10 nM), eotaxin/CCL11 (10 nM), and 5-oxo-ETE (150 nM) induced eosinophil calcium flux of comparable magnitude (Fig. 2A). However, no response to PGD2 could be elicited in cell samples previously stimulated with 11-dehydro-TXB2 (10 µM). This was consistent with PGD2 and 11-dehydro-TXB2 sharing the same receptor. Calcium responses to eotaxin/CCL11 and 5-oxo-ETE were not abolished by 11-dehydro-TXB2 (Fig. 2B), suggesting that these agonists utilize different receptors. Similarly, stimulation with PGD2 (10 nM) completely abrogated the consecutive calcium response to 11-dehydro-TXB2 (Fig. 2C), whereas a previous challenge with eotaxin/CCL11 (10 nM) or 5-oxo-ETE (150 nM) had little effect on the 11-dehydro-TXB2 response (Fig. 2, D and E). In contrast, neutrophils did not respond to 11-dehydro-TXB2 or PGD2, but effective calcium flux was elicited in neutrophils by 5-oxo-ETE (150 nM), C5a (10 nM), and A23187 [GenBank] (10 µm; data not shown, n = 4).



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  		FIG. 2.
Calcium flux cross-desensitization between 11-dehydro-TXB2 and PGD2 but not eotaxin/CCL11 or 5-oxo-ETE in eosinophils. Calcium flux was measured in polymorphonuclear cells loaded with Fluo-3 and labeled with anti-CD16 antibodies (PE). Eosinophils were gated as CD16-negative cells exhibiting higher side-scatter as compared with neutrophils (CD16-positive/low side-scatter), and changes in intracellular free calcium levels were detected as an increase in fluorescence intensity of the calcium-sensitive dye Fluo-3 in FL-1. To induce homologous receptor desensitization, cells were exposed to vehicle (control, A), 11-dehydro-TXB2 (10 µM, B), PGD2 (10 nM, C), eotaxin/CCL11 (10 nM, D) or 5-oxo-ETE (150 nM, E), and 6 min later, responses to agonists were recorded: 11-dehydro-TXB2 (10 µM), PGD2 (10 nM), eotaxin/CCL11 (10 nM), or 5-oxo-ETE (150 nM). Representative tracings are shown from three experiments with different blood donors.

 
11-Dehydro-TXB2-induced Shape Change Is Blocked by the CRTH2 Antagonist, Ramatroban—Eosinophil shape change in response to PGD2 was markedly inhibited by pretreatment of cells with the bi-specific CRTH2/TP receptor antagonist, ramatroban (1 µM) (33), as indicated by a 10-fold shift to the right of the PGD2 concentration response-curve (Fig. 3A). This effect was unrelated to nonspecific inhibition of eosinophil responsiveness, because ramatroban had no effect on eosinophil shape change induced by eotaxin/CCL11 (Fig. 3A). Ramatroban also inhibited 11-dehydro-TXB2 responses to a similar degree as it inhibited PGD2 responses (Fig. 3B). By contrast, the selective TP receptor antagonist SQ29,548 (1 µM) did not mimic the inhibitory effect of ramatroban (Fig. 3B), suggesting the inhibition of 11-dehydro-TXB2 responses by ramatroban was CRTH2- and not TP-specific. Identical results were also obtained in basophils (data not shown, n = 3–7).



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  		FIG. 3.
11-Dehydro-TXB2-induced shape change responses are inhibited by the TP/CRTH2 receptor antagonist ramatroban but not by the selective TP antagonist SQ29,548. Eosinophils in peripheral blood leukocyte preparations were identified by their higher degree of autofluorescence, and agonist-induced activation was quantified as the percent of cells moving from a low to a high forward-scatter region which had previously been defined as containing 20% of eosinophils in a non-stimulated sample. A, ramatroban (1 µM) effectively inhibited PGD2 in causing eosinophil shape change but was without effect on eotaxin/CCL11-induced responses. B, ramatroban (1 µM) also inhibited shape change in response to 11-dehydro-TXB2, whereas SQ29,548 (1 µM) was ineffective. Data are shown as mean ± S.E., n = 3–7. *, p < 0.05 versus control.

 
11-Dehydro-TXB2 Induces Chemotaxis of Human Eosinophils and Cells Transfected with hCRTH2—In addition to its effects on cell shape change and calcium flux, 11-dehydro-TXB2 also induced the migration of eosinophils into the bottom wells of the chemotaxis chamber (Fig. 4A). The potency of 11-dehydro-TXB2 to induce eosinophil migration seemed to be 300- to 600-fold lower than that of PGD2, whereas its efficacy was comparable with that of PGD2. The observed migration of eosinophils was due to chemotaxis rather than chemokinesis, because no significant increase in eosinophil numbers in the bottom well was observed when 11-dehydro-TXB2 was present, together with the cells, in the top well only (Fig. 4A). Similarly, BAF/3 cells expressing hCRTH2 effectively migrated in response to 11-dehydro-TXB2 and PGD2, whereas naive non-transfected cells did not (Fig. 4B), suggesting that the chemotactic responses to 11-dehydro-TXB2 observed in eosinophils are mediated by means of CRTH2.



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  		FIG. 4.
11-Dehydro-TXB2 induces chemotaxis of eosinophils and hCRTH2-transfected BAF/3 cells. A, purified eosinophils were placed into the top wells of a micro-Boyden chamber with 11-dehydro-TXB2 present in the top or the bottom wells. Eosinophils were allowed to migrate for 1 h, and cells that migrated into the bottom wells were enumerated by flow cytometry. 11-Dehydro-TXB2 was found to be chemotactic rather than chemokinetic, because it did not induce significant migration if present only in the top wells. B, BaF/3 cells, naive or transfected with hCRTH2, were placed on top of 96-well chemotaxis plates and incubated for 5 h. Cells that had migrated into the bottom wells were counted by microscopy. Data are shown as mean ± S.E., n = 3–7.

 
11-Dehydro-TXB2 Lacks CRTH2 Antagonistic Activity and Is not a Ligand for DP Receptors—To date, all of our assays have highlighted the hypothesis that 11-dehydro-TXB2 is an agonist of CRTH2 that gives responses of similar magnitude to those obtained with PGD2, albeit with reduced potency. To rule out the possibility that 11-dehydro-TXB2 might be a partial CRTH2 agonist, and therefore display antagonistic activity against PGD2 at lower concentrations, cells were pretreated with 30 nM 11-dehydro-TXB2. Fig. 5A shows that pre-treatment of eosinophils with this concentration of 11-dehydro-TXB2 (the threshold concentration for 11-dehydro-TXB2-induced eosinophil activation, Fig. 1A) did not inhibit PGD2-induced shape change in eosinophils. Thus, 11-dehydro-TXB2 can be regarded as a full agonist of CRTH2. In addition, pretreatment of eosinophils with TXB2 (1 µM) or the TP receptor antagonist SQ29,548 (1 µM) did not antagonize shape change responses induced by PGD2 (Fig. 5A), suggesting that TXB2 and SQ29,548 do not interact with CRTH2.



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  		FIG. 5.
11-Dehydro-TXB2 does not display antagonistic activity on CRTH2 or DP receptor-mediated responses. A, eosinophils in peripheral blood leukocyte preparations were identified by their higher degree of autofluorescence, and agonist-induced activation was quantified as the percentage of cells moving from a low to a high forward-scatter region which had previously been defined as containing 20% of eosinophils in a non-stimulated sample. Pretreatment of cells with 11-dehydro-TXB2 (30 nM), TXB2 (1 µM), or the TP receptor antagonist SQ29,548 (1 µM) had no effect on eosinophil responses to PGD2. B, 11-dehydro-TXB2 (10 µM) did not by itself promote platelet aggregation, did not inhibit ADP-induced platelet aggregation, and did not attenuate the inhibitory effect of PGD2 on ADP-induced platelet aggregation. The concentrations of ADP (2.5–20 µM) and PGD2 (6.25–100 nM) were adjusted in each individual experiment to give sub-maximal aggregation and sub-maximal inhibition of aggregation, respectively. Results were expressed as the percent of maximum light transmission, with non-stimulated platelet-rich plasma = 0% and platelet-poor plasma = 100%. Data are shown as mean ± S.E., n = 4–6.

 
Finally, we investigated whether 11-dehydro-TXB2 was also a ligand for the alternative PGD2 receptor, DP, which mediates the PGD2-induced inhibition of platelet aggregation. Fig. 5B demonstrates that 11-dehydro-TXB2 (10 µM) did not promote platelet aggregation by itself. Moreover, 11-dehydro-TXB2 did not inhibit ADP-induced platelet aggregation and did not attenuate the inhibitory effect of PGD2 on ADP-induced platelet aggregation (Fig. 5B). These data show that 11-dehydro-TXB2 was not an activator of pro-aggregatory TP receptors, nor did it interact with anti-aggregatory DP receptors in an agonistic, or antagonistic, fashion.

Similar Signaling Pathways of 11-Dehydro-TXB2 and PGD2—We have shown previously that PGD2 is an effective inducer of calcium flux (12) and shape change in eosinophils by means of PTX-insensitive activation of PLC (30). In the current study, we observed that the calcium flux as elicited by PGD2 (10 nM) and also 11-dehydro-TXB2 (10 µM) was not reduced in the presence of EGTA (2 mM) in the assay buffer, as compared with control responses (Fig. 6). Similarly, responses to eotaxin/CCL11 (10 nM) were not altered by EGTA, suggesting that CRTH2- and CCR3-mediated calcium flux was mainly attributed to mobilization from intracellular calcium stores. In contrast, important differences in CRTH2 and CCR3 signaling were noted in the shape change assay: pretreatment with PTX (1 µg/ml) abolished the responses to eotaxin/CCL11, whereas shape change elicited either by PGD2 or 11-dehydro-TXB2 was left largely unaltered (Fig. 7A). Common signaling pathways for PGD2 and 11-dehydro-TXB2 were also suggested by the ability of the phosphatidylinositide-specific PLC inhibitor U73,122 (3.6 µM) to attenuate the responses to both prostanoids to a comparable degree (Fig. 7B). This inhibitory effect of U73,122 was specific for PLC, because it was not mimicked by the structurally related, but inactive analogue, U73,343.



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  		FIG. 6.
Intracellular calcium mobilization by 11-dehydro-TXB2, PGD2, and eotaxin/CCL11 in eosinophils. Calcium flux was measured in polymorphonuclear cells loaded with Fluo-3 and labeled with anti-CD16 antibodies (PE). Eosinophils were gated as CD16-negative cells exhibiting higher side-scatter as compared with neutrophils (CD16-positive/low side-scatter), and changes in intracellular free calcium levels were detected as an increase in fluorescence intensity of the calcium-sensitive dye Fluo-3 in FL-1. To determine the portion of calcium flux originating from intracellular stores, cells were incubated in the absence (control, A) or the presence (B) of 2 mM EGTA for 6 min before being stimulated with 11-dehydro-TXB2 (10 µM), PGD2 (10 nM), or eotaxin/CCL11 (10 nM). The results suggest that a major part of the calcium flux in response to these agonists originates from intracellular calcium stores. Representative tracings are shown from three experiments with different blood donors.

 



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  		FIG. 7.
11-Dehydro-TXB2-induced shape change responses are PTX-insensitive and involve PLC. Eosinophils in peripheral blood leukocyte preparations were identified by their higher degree of autofluorescence, and agonist-induced activation was quantified as the percentage of cells moving from a low to a high forward-scatter region which had previously been defined as containing 20% of eosinophils in a non-stimulated sample. A, a 60-min incubation of cells with PTX (1 µg/ml) abolished the responses to eotaxin/CCL11 but had no effect on PGD2- or 11-dehydro-TXB2-induced responses. B, pretreatment of cells with the PLC inhibitor U73,122 (3.6 µM) for 30 min attenuated the responsiveness of eosinophils to PGD2 and 11-dehydro-TXB2, whereas the inactive analogue U73,343 was ineffective. Data are shown as mean ± S.E., n = 5. *, p < 0.05 PTX versus vehicle, or U73,122 versus U73,343, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has been proposed that TXA2 modulates leukocyte function, based on observations of TXA2 analogues enhancing the endothelial expression of adhesion molecules and chemokines (38, 39), enhancing neutrophil migration (40), inhibiting neutrophil aggregation, {beta}-glucuronidase release and superoxide production (41), and stimulating cytokine release from monocytes (42). In this report, we show that 11-dehydro-TXB2, a stable product of thromboxane metabolism, is an effective CRTH2 activator in human eosinophils and basophils, whereas the parent metabolite TXB2 is inactive. In addition to PGD2 itself, several of its metabolites have been shown to retain CRTH2 agonistic activity (9, 10, 12, 43). However, all of these agonists arise from PGD-synthase, whereas our present observations imply, for the first time, that CRTH2 can be activated through alternative endogenous pathways. Hence, CRTH2 might respond to a wider range of mediators than it has been assumed to date and might play an important role in eosinophil activation also in cellular contexts, where PGD-synthase is not expressed.

The notion that 11-dehydro-TXB2 is a CRTH2 agonist is supported by several lines of evidence. (i) 11-Dehydro-TXB2 induced flow cytometric shape change and calcium flux selectively in eosinophils and basophils, but not in neutrophils and monocytes, thus mimicking the cell type-selective effects of PGD2. Like PGD2, 11-dehydro-TXB2 also induced the chemotaxis of eosinophils. Although less potent than PGD2, 11-dehydro-TXB2 was as efficacious as PGD2 in inducing both eosinophil and basophil activation. (ii) 11-Dehydro-TXB2 cross-desensitized PGD2 responses in calcium flux studies, i.e. a first challenge of eosinophils with 11-dehydro-TXB2 rendered cells unresponsive to PGD2. Similarly, pretreatment with PGD2 abolished consecutive eosinophil responses to 11-dehydro-TXB2. In contrast, pretreatment of eosinophils with 11-dehydro-TXB2 or PGD2 did not abolish responsiveness to other eosinophil activators such as eotaxin/CCL11 or 5-oxo-ETE, which act through CCR3 (7) and TG1019 (44), respectively. These data are consistent with agonist-induced cross-desensitization by 11-dehydro-TXB2 and PGD2 of a common receptor, most likely CRTH2. By using the same approach, several PGD2 analogues and the non-steroidal anti-inflammatory drug, indomethacin, have previously been described as CRTH2 agonists (10, 12, 28, 45). Thirdly, shape change responses to 11-dehydro-TXB2 were effectively inhibited by ramatroban, a TP receptor antagonist, which has been described recently as having activity against CRTH2 (33). Because a second TP antagonist, SQ29,548, used at a supramaximal-effective concentration (20), did not inhibit 11-dehydro-TXB2 or PGD2 responses, it is most likely that 11-dehydro-TXB2-induced eosinophil responses are mediated by CRTH2. In addition, the TXA2 analogue U46,619 did not cause eosinophil activation, arguing against the involvement of TP receptors in eosinophil responses to 11-dehydro-TXB2. In support of this, CRTH2 transfectants, but not naive cells, migrated in response to 11-dehydro-TXB2 with the same efficacy as toward PGD2. Interestingly, CRTH2 transfectants were at least 10-fold more sensitive to PGD2 and 11-dehydro-TXB2 than eosinophils, which probably relates to the fact that CRTH2 expression on eosinophils is quite low,2 whereas our transfectants over-expressed it.

Intracellular signaling pathways of 11-dehydro-TXB2 were likewise similar to PGD2 but differed from CCR3-mediated responses to eotaxin/CCL11: although PTX pretreatment completely abrogated the shape change after eosinophil stimulation with eotaxin/CCL11, responses to both prostanoids were maintained. Responses to 11-dehydro-TXB2 and PGD2 were sensitive to inhibition of PLC, but did not depend on the presence of extracellular calcium. These data might suggest, therefore, that 11-dehydro-TXB2 activates eosinophils through CRTH2, which is coupled to PLC via PTX-insensitive G proteins (probably Gq), causing the mobilization of intracellular calcium stores.

Agonistic activity of 11-dehydro-TXB2 was restricted to CRTH2, as the second PGD2 receptor, DP, did not seem to bind 11-dehydro-TXB2. This was inferred from the finding that 11-dehydro-TXB2 did not mimic the inhibitory effect of PGD2 on ADP-induced platelet aggregation, which is known to involve DP-mediated cAMP biosynthesis (46, 47). Moreover, 11-dehydro-TXB2 did not inhibit the anti-aggregatory effect of PGD2, suggesting that this thromboxane metabolite is not acting as a DP antagonist. We also tested the hypothesis that 11-dehydro-TXB2 might be a partial CRTH2 agonist and therefore might, at lower concentrations, display antagonistic activity against PGD2. However, a concentration of 11-dehydro-TXB2, which was the threshold concentration for 11-dehydro-TXB2-induced eosinophil activation, did not inhibit PGD2-induced shape change in eosinophils. Like 11-dehydro-TXB2, the parent compound TXB2 did not inhibit PGD2 responses in eosinophils, showing that these thromboxane metabolites are not CRTH2 antagonists.

The above data suggest that 11-dehydro-TXB2 may be involved in eosinophil recruitment to sites of inflammation if formed locally in the tissue. The EC50 of 300 nM of 11-dehydro-TXB2 in eliciting eosinophil shape change was similar to the potency of the TXA2 analogue U46,619 in inducing platelet aggregation (37) and 10-fold higher than the EC50 of U46,619 in causing human bronchial (48) and arterial constriction (49). In our hands, the potency of 11-dehydro-TXB2 in causing eosinophil activation was ~500 times lower than that of PGD2. However, this does not necessarily preclude a role for 11-dehydro-TXB2 under in vivo conditions, because the dehydrogenase catalyzing the formation of 11-dehydro-TXB2 is predominantly expressed in the lung (24), and by being a stable compound, high concentrations of 11-dehydro-TXB2 may accumulate in lung tissue. Several studies have shown that serum and/or urinary levels of 11-dehydro-TXB2 are elevated in asthma and chronic obstructive lung disease (50–52) and are further increased after allergen challenge, thus reflecting increased TXA2 biosynthesis (26, 29, 50, 53). It has been suggested that TXA2 is a mediator of allergic diseases and asthma through its ability to induce bronchoconstriction (16, 17), plasma extravasation (17, 18), and growth of smooth muscle cells (16, 19). However, inhibition of thromboxane biosynthesis has been reported to be more efficacious than TP receptor blockade in controlling asthma (54), which indicates a role for thromboxane metabolites acting through receptors other than TP. CRTH2 has been shown to mediate the activation of eosinophils, basophils, and TH2 lymphocytes (9, 10, 43), cells which are known to be crucial to allergic diseases. Thus, effective CRTH2 activation by 11-dehydro-TXB2, as observed in the present study, might account for TP-independent roles of the thromboxane pathway in the allergic response.

In summary, the current data extend the range of known thromboxane actions in the inflammatory response. In addition to TXA2, which is capable of causing bronchoconstriction, plasma extravasation, and chemokine expression, its stable metabolite 11-dehydro-TXB2 might also be directly involved in the recruitment of eosinophils, basophils, and presumably TH2 lymphocytes to sites of inflammation. Our observations indicate that this effect is mediated through the activation of a PGD2 receptor, CRTH2, and that antagonism of this interaction may be of therapeutic use in the treatment of allergic inflammation.


   FOOTNOTES
 
* This work was supported by the Royal Society, the Austrian Academy of Sciences, Jubiläumsfonds of the Austrian National Bank Grant 10005 (to A. H.), Austrian Science Fund Grant P15453 [GenBank] (to A. H.), GlaxoSmithKline/Biotechnology and Biological Sciences Research Council Case Studentship (to H. S.), and Wellcome Trust Programme Grant 038775/Z/96/A (to J. E. P.). 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. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Experimental and Clinical Pharmacology, Medical University Graz, Universitaetsplatz 4, A-8010 Graz, Austria. Tel.: 43-316-380-4508; Fax: 43-316-380-9645; E-mail: akos.heinemann{at}uni-graz.at.

1 The abbreviations used are: PG, prostaglandin; CRTH2, chemoattractant receptor-homologous molecule expressed on TH2 cells; TX, thromboxan; TP, thromboxane receptor; PBS, phosphate-buffered saline; PE, phycoerythrin; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; DP, alternative PGD2 receptor; FACS, fluorescence-activated cell sorting; PLC, phospholipase C; PTX, pertussis toxin;. Back

2 J. E. Pease, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. David P. Andrew and Dr. Ashley Barnes (GlaxoSmithKline, Stevenage, UK) for generating the CRTH2 transfectants, and Dr. Rufina Schuligoi (Graz, Austria), Dr. Ian Sabroe (Sheffield, UK) and Dr. Kevin B. Bacon (Bayer Yakuhin, Kyoto, Japan) for helpful comments. We also thank Martina Ofner and Carmen Prenn for excellent technical assistance.



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 DISCUSSION
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