Abstract
We cloned, expressed, and characterized in vitro and in vivo the gene encoding the rat ortholog of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor for prostaglandin D2 (PGD2). Quantitative reverse transcription-polymerase chain reaction analysis demonstrated highest CRTH2 expression in the lung, brain, ovary, and spleen. Pharmacologically, rat CRTH2 stably transfected in mouse preB lymphoma L1.2 cells behaved very similar compared with the mouse and human orthologs, showing a binding affinity for PGD2 of 11 nM, a functional calcium mobilization when exposed to agonist, and similar sensitivity to agonists and antagonists. In vivo, selective activation of CRTH2 by 13,14-dihydro-15-keto (DK)-PGD2 injection into rats led to a dose- and time-dependent increase of the number of leukocytes in the peripheral blood. Specifically, eosinophils, lymphocytes, and neutrophils were recruited with maximum effects seen 60 min after the injection of 300 μg of DK-PGD2 per rat. Pretreatment of the animals with the CRTH2/thromboxane A2 receptor antagonist, ramatroban, completely abrogated DK-PGD2-induced eosinophilia, suggesting that CRTH2 might have a physiological and/or pathophysiological role in controlling leukocyte migration.
Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) has been identified by a comparative differential display approach in Th1 and Th2 lymphocytes (Nagata et al., 1999a,b). The initial attention this receptor gained due to its restricted expression pattern on Th2 cells, basophils, and eosinophils (Nagata et al., 1999a) was increased when it became clear that its ligand is the prostanoid prostaglandin D2 (Hirai et al., 2001; Monneret et al., 2001). This arachidonic acid product is predominantly released by activated mast cells, and its potential implication in allergic reactions has been known for many years (Lewis and Austen, 1981). Large amounts of PGD2 are released into the airways of asthmatics immediately after challenge (Murray et al., 1985, 1986) (Wenzel et al., 1991) and mice overexpressing the lipocalin-type PGD synthase show stronger inflammatory reactions in the lung upon antigen challenge (Fujitani et al., 2002). In contrast, mice lacking DP, the first PGD2 receptor identified, show a diminished response to ovalbumin challenge in animal models for asthma, highlighting the importance of PGD2 in allergic reactions (Matsuoka et al., 2000).
Despite binding the common ligand PGD2, CRTH2 and DP differ significantly from each other by showing low sequence homology and by being coupled to different classes of G proteins. Whereas DP couples to a stimulatory Gs protein leading to the activation of adenylyl cyclase and increasing levels of cAMP and Ca2+ (Hirata et al., 1994; Boie et al., 1995), CRTH2 activation inhibits cAMP formation but induces an increase in intracellular Ca2+ mobilization via Gi-dependent pathways (Hirai et al., 2001; Sawyer et al., 2002; Sugimoto et al., 2003). This difference in G protein coupling seems to allow CRTH2 to transmit promigratory signals in response to PGD2. For instance, CRTH2-transfected Jurkat cells can migrate along a gradient of PGD2, whereas DP-transfected Jurkat cells cannot (Hirai et al., 2001). In leukocytes of all species studied so far, PGD2 induces migration exclusively via CRTH2 (Hirai et al., 2001; Monneret et al., 2001; Gosset et al., 2003; Hata et al., 2003; Sugimoto et al., 2003).
Considering the ability of PGD2 to stimulate the migration of inflammatory cells, it is reasonable to speculate that CRTH2 may play a role in the selective recruitment of cellular components of the allergic response into sensitized or injured tissues. Expression studies in mice, however, have shown that a variety of tissues express mRNA for CRTH2 (Abe et al., 1999; Sawyer et al., 2002), suggesting that this receptor CRTH2 may have broader functions.
In this article, we describe the cloning and functional characterization of rat CRTH2 by comparing it with the mouse and human counterparts of the receptor. We further show the effect of the CRTH2/thromboxane A2 antagonist ramatroban (Sugimoto et al., 2003), a well established medicine for the treatment of allergic rhinitis, on PGD2-induced CRTH2 activation in vitro, and in animals exposed to the CRTH2-selective agonist 13,14-dihydro-15-keto-prostaglandin D2 (DKPGD2) (Giles and Leff, 1988; Hirai et al., 2001).
Materials and Methods
Chemicals and Reagents. Ramatroban [(+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3,4-tetra-hydrocarbazole-9-propionic acid] was synthesized at Bayer Yakuhin Ltd. (Shiga, Japan), and ridogrel [(E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]-methylen]amino]oxy] pentanoic acid] was prepared by Bayer AG (Wuppertal, Germany). BWA868C was synthesized by SOGO Pharmaceutical Co. Ltd. (Tokyo, Japan) (http://www.sogo-pharma.co.jp/index.html). PGD2 was from Sigma-Aldrich (St. Louis. MO), and [3H]PGD2 was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Sodium butyrate was purchased from Wako Pure Chemicals (Osaka, Japan). Fluo-3/acetoxymethyl ester and pluronic F-127 were purchased from Molecular Probes (Eugene, OR). DK-PGD2 and BW245C and BW868C were purchased from Cayman Chemicals (Ann Arbor, MI). Pertussis toxin (PTX) was obtained from Calbiochem (La Jolla, CA). Ramatroban, BWA868C, BWC245C, and PGD2 were dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan). As confirmed in preliminary experiments, the concentrations of dimethyl sulfoxide in working dilutions used in this study (<0.1%) had no effect on receptor binding, Ca2+ mobilization and cell migration assays. All chemicals not further specified were obtained from Wako Pure Chemicals.
Cloning of Rat CRTH2. The mouse and human CRTH2 protein sequences were used to search for homologs in the rat EST subset of the GenBank database (http://www.ncbi.nlm.nih.gov) using the program tblastn. Three ESTs were identified, (accession numbers BE112439, BE100786, and BE113412, all derived from heart tissue) with high homology to the 5′ end of the mouse Crth2 transcript. The ESTs were then used to search the rat high-throughput genome sequences subset of GenBank to find sequence reads from the rat Crth2 genomic locus. The locus was identified on the sequence contig AC128991.3. After comparing the open reading frame of the locus with the coding sequence of mouse Crth2, PCR primers were designed to flank the rat coding sequence for amplification and sequencing. Primer sequences used were 5′-GGGTGCCAGGTTCAGCTCTCCTTT-3′ and 5′-ATGGGAGAGGCCTGGGATGTGTTG-3′.
For use as a template for amplification, cDNA was prepared from a pool of rat tissue (strain Sprague-Dawley) total RNAs (Ambion, Austin, TX). First-strand cDNA was synthesized with the Superscript first-strand synthesis system (Invitrogen, Carlsbad,, CA). PCR was then carried out with KOD-Plus-polymerase (Toyobo, Osaka, Japan; 2 min at 95°C, 15 s at 94°C, 15 s at 69°C, and 3 min at 72°C) for 35 cycles. The PCR products were cloned into a pGEM T easy vector (Promega, Madison, WI) for sequencing then subcloned into a pcDNA3.1 vector (Invitrogen) for expression. Clones were sequenced using the ABI Prism dye terminator cycle sequencing reaction kit and analyzed on an ABI Prism 377 sequencing system (Applied Biosystems, Foster City, CA). The sequence is available under GenBank accession number AY228550.
Expression Profiling. Total RNA samples prepared from rat tissues were purchased from Ambion. First-strand cDNA was synthesized with the Superscript first-strand synthesis system (Invitrogen). Semiquantitative PCR was carried out in 20-μl volumes containing 20 ng of each tissue cDNA as a template and 0.5 μM of each primer using Hot Star Taq master mix kit (QIAGEN, Valenica, CA). As the corresponding primers, cloning primers were used. PCR condition is as follows. All samples were preheated at 95°C for 15 min and then subjected to denaturing conditions at 95°C for 10 s. After annealing at 65° C for 15 s, genes were amplified at 72° C for 1.5 min (35 cycles).
Generation of CRTH2 Stable Transfectants. The rat and mouse Crth2 gene inserted into the pcDNA3.1(-) expression vector (Invitrogen) was transfected into L1.2 cell (a kind gift from Prof. Eugene Butcher, Standord, CA) by electroporation (250 V/1000 mF; Gene Pulser II; Bio-Rad, Hercules, CA). Stable transfectants were selected in the presence of G418 (0.5 mg/ml; Invitrogen). Human CRTH2 stable transfectants were generated as described previously (Sugimoto et al., 2003).
Receptor Binding Assay. CRTH2/L1.2 cells (2 × 105 cells) were mixed with 3H-labeled PGD2 and various concentrations of test compounds in 100 μl of binding buffer (50 mM Tris-HCl, pH 7.4, 40 mM MgCl2, 0.1% bovine serum albumin, 0.1% NaN3) in 96-well U-bottom polypropylene plates. After incubation for 60 min at room temperature, the cell suspension was transferred to a filtration plate (#MAFB; Millipore Corporation, Bedford, MA), and radioactivity was measured by a scintillation counter (TopCount; PerkinElmer Life Sciences, Boston, MA). Nonspecific binding was determined by incubations in the presence of 1 μM unlabeled PGD2.
Ca2+Mobilization Assay. Ca2+ loading buffer was prepared by mixing 1 mM Fluo-3/acetoxymethyl ester and pluronic F-127 (Molecular Probes) in Ca2+ assay buffer (20 mM HEPES, pH 7.6, 0.1% bovine serum albumin, 1 mM probenecid, Hanks' solution). The CRTH2 transfectants established were resuspended in Ca2+ loading buffer at 1 × 107 cells/ml and incubated for 60 min at room temperature. After the incubation, cells were washed and resuspended in Ca2+ assay buffer, and then dispensed into transparent-bottomed 96-well plates (3631; Coaster, Corning, NY) at 2 × 105 cells/well. Cells were incubated with various concentrations of ramatroban for 5 min at room temperature. The emitted 480-nm fluorescence was measured on a FDSS6000 fluorometer (Hamamatsu Photonics, Hamamatsu, Japan).
Animal Experiments. Male Brown Norway or Wistar rats (7 weeks) were purchased from Charles River (Yokohama, Japan). Animals were kept under standard conditions in a 12-h day/night rhythm with free access to food and water ad libitum. All animals received humane care in accordance with international guidelines and national law.
DK-PGD2, supplied as a solution in methyl acetate, was evaporated under a gentle stream of nitrogen, and reconstituted in ethanol to prepare a stock solution of 30 mg/ml. Before injection, the adequate volume of PBS was added to obtain diluted ethanol solutions as specified in figure legends.
Rats were injected intravenously with DK-PGD2, or the corresponding volume of the respective solvent with or without pretreatment by ramatroban (dissolved in NaOH, pH-neutralized by HCl addition, and given in a 10% cremophor solution) under slight anesthetization with ether. At the time points indicated after injection, peripheral blood was collected from the abdominal vein under anesthetization by i.p. injection of urethane (2 g/kg). After blood collection, animals were sacrificed by complete bleeding. Immediately after the bleeding, the left femur was isolated and the femoral head and condoles were removed. The displaceable cells were recovered by flushing the lumen of the femur shaft with 4 ml of PBS. Total white blood cells in the samples were counted under the microscope using a hemocytometer. Differential cell counts were performed on the blood smears stained with May-Gruenwald's and Giemsa's solution based on standard morphologic and histological criteria (200 cells counted in total).
Statistics. Unless otherwise stated, data are expressed as means ± S.D. of at least three independent experiments. Statistical significance was determined using the unpaired Student's t test if applicable or with the Dunnett's or Welch test if variances were nonhomogeneous using commercially available statistical software (GraphPad Software Inc., San Diego, CA). Values of p < 0.05 were considered as statistically significant (*p < 0.05, **p < 0.01).
Results
Molecular Cloning, Sequence Alignment, and Tissue Expression of Rat CRTH2. The gene encoding the rat ortholog of CRTH2 was cloned by search for homologs in the rat EST subset of the GenBank database using the program tblastn, and the full cDNA sequence is shown in Fig. 1. The sequence of the rat CRTH2 protein bears 89% identity with mouse CRTH2 and 75% identity with human CRTH2, the three differing primarily in their cytoplasmic tail regions (Fig. 2).
Semiquantitative RT-PCR analysis revealed that the tissue distribution of rat (Fig. 3) resembles the pattern reported for the mouse CRTH2 mRNA (Abe et al., 1999), showing highest expression in the lung, brain, ovary, and spleen but differs from that in humans, where it is most highly expressed in heart, stomach, small intestine, and thymus (Sawyer et al., 2002).
Pharmacological Characterization of Rat CRTH2. To pharmacologically characterize the rat CRTH2 receptor, we generated stable rat CRTH2 expressing L1.2 cells and compared them with clones expressing the mouse or human ortholog. Saturation analysis experiments of 3H-labeled PGD2-specific binding to recombinant rat, mouse, and human CRTH2 transfectants were performed. The three orthologs showed similar binding affinities for PGD2, similar total binding, and similar numbers of binding sites per cell, respectively (Fig. 4; Table 1). Nonlabeled PGD2 inhibited the binding of 3H-labeled PGD2 to rat, mouse, and human CRTH2 transfectants in a concentration-dependent manner with IC50 values of 6.1, 2.6, and 2.3 nM, respectively. The CRTH2-specific agonist DK-PGD2 was comparable with PGD2 in binding affinities to all three orthologs. Interestingly, BWA868C, known as a DP-specific antagonist, showed weak but significant inhibition of 3H-labeled PGD2 binding at micromolar concentrations. Ramatroban, a CRTH2/thromboxane A2 antagonist, showed inhibitory effects on the binding of 3H-labeled PGD2 to rat, mouse, and human CRTH2 transfectants with IC50 values around 50 nM. At concentrations equal to or exceeding 1 μM, weak but significant inhibition of PGD2 binding to CRTH2 was noted for BW245C and ridogrel, questioning their absolute specificity for the DP and thromboxane A2 receptor, respectively (Fig. 5; Table 2).
To determine the functional expression of rat CRTH2, PGD2-, DK-PGD2-, and BW245C-stimulated calcium mobilization was monitored. PGD2 and DK-PGD2 induced Ca2+ mobilization in a concentration-dependent manner with EC50 values of 6.9 and 4.9 nM in human CRTH2 transfectants, 16 and 40 nM in mouse CRTH2 transfectants, and 39 and 90 nM in rat CRTH2 transfectants, respectively. These effects were completely suppressed by pretreatment with a Gαi inhibitor, PTX. The DP-specific agonist BW245C failed to induce Ca2+ mobilization in all three transfectants (Fig. 6A; data not shown). Ramatroban concentration dependently inhibited PGD2-induced Ca2+ mobilization in rat, mouse, and human CRTH2 transfectants with similar IC50 values in the range of 120 to 130 nM. In line with its weak inhibitory effect on PGD2 binding BW868C diminished the calcium response at 10 μM. Again, ridogrel was without any effect (Fig. 6B; Table 3). Unlike indomethacin, ramatroban did not show any agonistic effects on Ca2+ signaling in CRTH2 transfectants (data not shown).
Induction of Leukocyte Recruitment from the Bone Marrow by the CRTH2 Agonist DK-PGD2. PGD2 stimulation evoked a weak but significant migratory response of CRTH2-transfected L1.2 cells, which was fully abrogated by ramatroban (data not shown). Splenocytes and bone marrow cells isolated from rats showed a distinct mRNA signal for CRTH2. Exposure of the cells to PGD2 or DK-PGD2, however, did not trigger significant cell migration, whereas a response to other chemoattractants such as platelet activation factor, leukotriene B4, or IL-5 was observed. In addition, pretreatment of cells with IL-5 did not prime leukocytes for CRTH2 agonists nor did DK-PGD2 preincubation modify the responses to any chemoattractant investigated (data not shown).
Due to these weak responses in vitro, we sought to investigate CRTH2-triggered migration in vivo. A bolus injection of PGD2 (data not shown) or the CRTH2-specific agonist DK-PGD2 led to a time- and dose-dependent leukocyte recruitment from the bone marrow in rats (Figs. 7 and 8), mice, and guinea pigs (data not shown). In all species investigated, the maximum increases in leukocyte numbers in peripheral blood were reached 60 min after injection (Fig. 7). Increases in peripheral blood cell counts were paralleled, but not fully compensated, by a drop of cell counts in the bone marrow (Fig. 8). Significant effects were seen at DK-PGD2 doses equal or higher to 100 μg/animal (data not shown). As expected, eosinophils and lymphocytes were affected in particular as exemplified in Fig. 7, however, neutrophils also showed a similar tendency.
We then set out to examine whether this DK-PGD2-induced peripheral blood eosinophilia was mediated by CRTH2 and pretreated rats with ramatroban before DK-PGD2 injection. Indeed, a dose-dependent reduction of total leukocytes and eosinophils in the peripheral blood was noted in rats pretreated with the CRTH2 antagonist. At the highest ramatroban dose tested, i.e., at 30 mg/kg, cell counts were comparable with levels of solvent controls, proving that blockade of the CRTH2 receptor in vivo abrogates DK-PGD2-induced cell recruitment (Fig. 9).
Discussion
In the present study, we identified the rat CRTH2 gene, examined the organ expression pattern and the homology of this gene to its human and mouse counterparts, characterized its pharmacology using gene-transfected cells, and, finally, established an in vivo animal model suitable for CRTH2 antagonist evaluation.
Overall identity between the human and rat CRTH2 sequences was 75.6%, and 89.1% between the mouse and rat sequences. Phylogenetic homology comparison studies evidenced high overall similarity to other rat chemoattractant receptors such as LTB4, N-formyl peptide, C3a, and C5a (data not shown).
RT-PCR analysis revealed that rat CRTH2 mRNA was expressed in various tissues including kidney, embryo, liver, brain, thymus, heart, lung, spleen, testis, and ovary. Like the mouse (Abe et al., 1999), the rat CRTH2 mRNA signal was strongest in lung, brain, and spleen, suggesting together with the high percentage of sequence identity, high functional similarity between rat and mouse CRTH2. The tissue expression pattern of rodents, however, differs from the human tissue expression profile (Sawyer et al., 2002), pointing toward potential physiological and/or pathophysiological disparities. Indeed, some biological responses reported for its selective agonist DK-PGD2 in secretory organs and tissues (Larsen et al., 2002) suggest that CRTH2 has more functions than inducing the migration of leukocytes, inducing shape changes, and regulating the expression of adhesion molecules in Th2 cells, eosinophils, and/or basophils (Hirai et al., 2001; Heinemann et al., 2003). Nevertheless, these well established functions of CRTH2 fit very nicely into the concept of PGD2 being a crucial player in the pathogenesis of asthma (Murray et al., 1985; Murray et al., 1986; Wenzel et al., 1991; Matsuoka et al., 2000; Fujitani et al., 2002). Because various animal models for asthma exist to study the underlying disease processes, the investigation of rodent CRTH2 receptor biology and its comparison with human CRTH2 is very helpful and might lead to a better understanding of PGD2's function under physiological and pathophysiological conditions.
With regard to ligand binding, ligand specificity, G protein coupling, second messenger generation, and induction of migration, CRTH2 receptors from all species studied so far seem to behave alike (c.f. Figs. 4, 5, 6; Tables 1, 2, 3). Rat CRTH2 binds PGD2 with high affinity (Kd = 11 nM), comparable with human (Kd = 6.3 nM) and mouse (Kd = 9.1 nM). Equilibrium competition binding assays confirmed that DK-PGD2 is a high-affinity ligand for CRTH2. Interestingly, BWA868C, reported to be a selective DP antagonist, showed competitive binding to CRTH2 at high concentrations (c.f. Table 2). This partial antagonism might explain why BWA868C showed a weak inhibitory effect on PGD2-induced Ca2+ flux (Fig. 6B) and human eosinophil migration (Sugimoto et al., 2003).
Ligand-triggered calcium mobilization was observed in rat, mouse, and human CRTH2 transfectants after PGD2 or DKPGD2 stimulation, respectively. Very recently, Hata et al. (2003) failed to detect changes in intracellular calcium in their mouse transfectants using ER293 host cells. The host cells we used, L1.2 mouse B cell leukemia cells, clearly are capable of generating a calcium flux in response to chemokine ligand (Gallatin et al., 1983; Yoshida et al., 1998) and thus CRTH2-triggered calcium mobilization was comparatively easy to measure. All other data presented in the report by Hata et al. (2003), however, nicely fit to the results we obtained for murine CRTH2.
As expected from studies performed in human and mouse cells, chemotaxis of rat CRTH2 transfectants was observed after PGD2 stimulation; however, the response was comparatively weak (data not shown). The fact that chemotaxis was completely abolished in the presence of ramatroban confirms the general role of CRTH2 as a chemoattractant receptor. Why primary rat cells failed to respond to CRTH2 agonists remains obscure. Either the CRTH2 receptor protein is quickly down-regulated during cell isolation or during the preincubation period in culture, or some additional unknown factors come into play.
In vivo, however, the migratory response was obvious. Based on our in vitro results performed in transfectants and on a recent report by Heinemann and colleagues, who demonstrated eosinophil recruitment from the bone marrow in the bloodstream after Δ12-PGJ2 injection into the hind limbs of guinea pigs (Heinemann et al., 2003), we challenged rats intravenously with DK-PGD2. In addition to cell recruitment from the bone marrow which accounts for approximately 50% of the total cell increase in the blood, DK-PGD2 seems to recruit leukocytes from additional, not yet identified sources of the body. Although the specificity of this CRTH2 agonist is unknown in vivo and additional effects cannot be excluded, the leukocyte recruitment observed was clearly CRTH2-mediated, because ramatroban pretreatment dose dependently blocked eosinophilia in this model (c.f. Figs. 7, 8, 9). Preliminary data obtained in our laboratory argue for a direct, IL-5-independent mechanism of eosinophil recruitment from the bone marrow via CRTH2 activation; however, further studies are required for final clarification.
Collectively, this report characterizes the CRTH2 receptor of the rat, presents evidence for an in vivo role of CRTH2 in leukocyte recruitment and confirms ramatroban as a potent small molecule antagonist and a useful tool to study CRTH2 biology.
Acknowledgments
We thank E. Takao for experimental support.
Footnotes
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↵1 These authors contributed equally to this work.
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DOI: 10.1124/jpet.103.055442.
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ABBREVIATIONS: CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; PG, prostaglandin; DP, prostaglandin D2 receptor; DK-PGD2, 13,14-dihydro-15-keto-prostaglandin D2; PTX, pertussis toxin; EST, expressed sequence tag; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-PCR; BWA868C, 3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexy-2-hydroxyethylamino)-hydantoin; BW245C, 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin.
- Received July 13, 2003.
- Accepted August 8, 2003.
- The American Society for Pharmacology and Experimental Therapeutics