Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m310270200

ISSN

1083-351X

Autores

Eva Böhm, Eva M. Sturm, Iris Weiglhofer, Hilary Sandig, Michitaka Shichijo, Anne McNamee, James E. Pease, Manfred Kollroser, Bernhard A. Peskar, Ákos Heinemann,

Tópico(s)

Mast cells and histamine

Resumo

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. 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. Accumulation of eosinophils at sites of allergic reactions, such as asthma and atopic rhinitis, is associated with tissue injury and lung dysfunction (1De Monchy J.G. Kauffman H.F. Venge P. Koeter G.H. Jansen H.M. Sluiter H.J. De Vries K. Am. Rev. Respir. Dis. 1985; 131: 373-376PubMed Google Scholar, 2Naclerio R.M. J. Allergy Clin. Immunol. 1988; 82: 927-934Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 3Bousquet J. Chanez P. Lacoste J.Y. Barneon G. Ghavanian N. Enander I. Venge P. Ahlstedt S. Simony-Lafontaine J. Godard P. Michel F.-B. N. Engl. J. Med. 1990; 323: 1033-1039Crossref PubMed Scopus (2194) Google Scholar, 4Bradley B.L. Azzawi M. Jacobson M. Assoufi B. Collins J.V. Irani A.M. Schwartz L.B. Durham S.R. Jeffery P.K. Kay A.B. J. Allergy Clin. Immunol. 1991; 88: 661-674Abstract Full Text PDF PubMed Scopus (588) Google Scholar). Several chemoattractants can mediate eosinophil recruitment, in particular the CC-chemokines acting through the chemokine receptor CCR3 (5Bochner B.S. Schleimer R.P. Immunol. Rev. 2001; 179: 5-15Crossref PubMed Scopus (148) Google Scholar, 6Rothenberg M.E. Zimmermann N. Mishra A. Brandt E. Birkenberger L.A. Hogan S.P. Foster P.S. J. Clin. Immunol. 1999; 19: 250-265Crossref PubMed Scopus (77) Google Scholar, 7Pease J.E. Williams T.J. Curr. Opin. Pharmacol. 2001; 1: 248-253Crossref PubMed Scopus (83) Google Scholar). The prostaglandin (PG) 1The 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;. 1The 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;.D2 is a major mast cell mediator released during the allergic response (8Schleimer R.P. Fox C.C. Naclerio R.M. Plaut M. Creticos P.S. Togias A.G. Warner J.A. Kagey-Sobotka A. Lichtenstein L.M. J. Allergy Clin. Immunol. 1985; 76: 369-374Abstract Full Text PDF PubMed Scopus (97) Google Scholar) 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) (9Monneret G. Gravel S. Diamond M. Rokach J. Powell W.S. Blood. 2001; 98: 1942-1948Crossref PubMed Scopus (293) Google Scholar, 10Hirai H. Tanaka K. Yoshie O. Ogawa K. Kenmotsu K. Takamori Y. Ichimasa M. Sugamura K. Nakamura M. Takano S. Nagata K. J. Exp. Med. 2001; 193: 255-261Crossref PubMed Scopus (958) Google Scholar). Moreover, CRTH2 is the most reliable marker for TH2-cells (11Cosmi L. Annunziato F. Galli M.I.G. Maggi R.M.E. Nagata K. Romagnani S. Eur. J. Immunol. 2000; 30: 2972-2979Crossref PubMed Scopus (222) Google Scholar). 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 (12Heinemann A. Schuligoi R. Sabroe I. Hartnell A. Peskar B.A. J. Immunol. 2003; 170: 4752-4758Crossref PubMed Scopus (101) Google Scholar). 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 (13Fujitani Y. Kanaoka Y. Aritake K. Uodome N. Okazaki-Hatake K. Urade Y. J. Immunol. 2002; 168: 443-449Crossref PubMed Scopus (203) Google Scholar).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 (14Devillier P. Bessard G. Fundam. Clin. Pharmacol. 1997; 11: 2-18Crossref PubMed Scopus (59) Google Scholar, 15Yamasaki M. Matsumoto T. Fukuda S. Nakayama T. Nagaya H. Ashida Y. J. Pharmacol. Exp. Ther. 1997; 280: 1471-1479PubMed Google Scholar), as it causes bronchoconstriction (16Underwood D.C. Muccitelli R.M. Luttmann M.A. Hay D.W. Torphy T.J. Wasserman M.A. J. Pharmacol. Exp. Ther. 1994; 268: 304-310PubMed Google Scholar, 17Inoue Y. Tokuyama K. Nishimura H. Arakawa H. Kato M. Mochizuki H. Morikawa A. Pharmacology. 2002; 65: 62-68Crossref PubMed Scopus (6) Google Scholar), plasma extravasation (17Inoue Y. Tokuyama K. Nishimura H. Arakawa H. Kato M. Mochizuki H. Morikawa A. Pharmacology. 2002; 65: 62-68Crossref PubMed Scopus (6) Google Scholar, 18Lotvall J. Elwood W. Tokuyama K. Sakamoto T. Barnes P.J. Chung K.F. J. Appl. Physiol. 1992; 72: 2415-2419Crossref PubMed Scopus (46) Google Scholar), and the growth of smooth muscle cells (16Underwood D.C. Muccitelli R.M. Luttmann M.A. Hay D.W. Torphy T.J. Wasserman M.A. J. Pharmacol. Exp. Ther. 1994; 268: 304-310PubMed Google Scholar, 19Morinelli T.A. Zhang L.M. Newman W.H. Meier K.E. J. Biol. Chem. 1994; 269: 5693-5698Abstract Full Text PDF PubMed Google Scholar). In addition, the wide range of TXA2 biological actions includes platelet aggregation (20Ogletree M.L. Harris D.N. Greenberg R. Haslanger M.F. Nakane M. J. Pharmacol. Exp. Ther. 1985; 234: 435-441PubMed Google Scholar), vasoconstriction (21Svensson J. Fredholm B.B. Acta Physiol. Scand. 1977; 101: 366-368Crossref PubMed Scopus (30) Google Scholar), and the promotion of angiogenesis and tumor metastasis (22Nie D. Lamberti M. Zacharek A. Li L. Szekeres K. Tang K. Chen Y. Honn K.V. Biochem. Biophys. Res. Commun. 2000; 267: 245-251Crossref PubMed Scopus (180) Google Scholar). TXA2 is short lived and very rapidly transformed non-enzymatically in aqueous solution to TXB2 (23Roberts 2nd, L.J. Brash A.R. Oates J.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1982; 10: 211-225PubMed Google Scholar). 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 (24Westlund P. Kumlin M. Nordenstrom A. Granstrom E. Prostaglandins. 1986; 31: 413-443Crossref PubMed Scopus (48) Google Scholar). The dehydrogenase-catalyzing 11-dehydro-TXB2 formation is tissue bound and widely expressed, with the highest occurrence being found in lung and kidney (24Westlund P. Kumlin M. Nordenstrom A. Granstrom E. Prostaglandins. 1986; 31: 413-443Crossref PubMed Scopus (48) Google Scholar). Because of its stability, 11-dehydro-TXB2 has been used to monitor TXA2 production in vivo and in vitro (25Westlund P. Granstrom E. Kumlin M. Nordenstrom A. Prostaglandins. 1986; 31: 929-960Crossref PubMed Scopus (63) Google Scholar, 26Lupinetti M.D. Sheller J.R. Catella F. Fitzgerald G.A. Am. Rev. Respir. Dis. 1989; 140: 932-935Crossref PubMed Scopus (40) Google Scholar). In contrast to PGD2 metabolites, which retain potent TP (27Coleman R.A. Sheldrick R.L. Br. J. Pharmacol. 1989; 96: 688-692Crossref PubMed Scopus (135) Google Scholar) or CRTH2 (9Monneret G. Gravel S. Diamond M. Rokach J. Powell W.S. Blood. 2001; 98: 1942-1948Crossref PubMed Scopus (293) Google Scholar, 10Hirai H. Tanaka K. Yoshie O. Ogawa K. Kenmotsu K. Takamori Y. Ichimasa M. Sugamura K. Nakamura M. Takano S. Nagata K. J. Exp. Med. 2001; 193: 255-261Crossref PubMed Scopus (958) Google Scholar, 12Heinemann A. Schuligoi R. Sabroe I. Hartnell A. Peskar B.A. J. Immunol. 2003; 170: 4752-4758Crossref PubMed Scopus (101) Google Scholar, 28Monneret G. Li H. Vasilescu J. Rokach J. Powell W.S. J. Immunol. 2002; 168: 3563-3569Crossref PubMed Scopus (108) Google Scholar) agonistic activity, TXB2 and 11-dehydro-TXB2 have been considered as being devoid of TP agonistic activity (23Roberts 2nd, L.J. Brash A.R. Oates J.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1982; 10: 211-225PubMed Google Scholar). Considerable cross-reactivity of PGD2 in a radio-immunoassay for 11-dehydro-TXB2 has been reported (29Kumlin M. Dahlen B. Bjorck T. Zetterstrom O. Granstrom E. Dahlen S.E. Am. Rev. Respir. Dis. 1992; 146: 96-103Crossref PubMed Scopus (287) Google Scholar), 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 PROCEDURESReagents—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 (12Heinemann A. Schuligoi R. Sabroe I. Hartnell A. Peskar B.A. J. Immunol. 2003; 170: 4752-4758Crossref PubMed Scopus (101) Google Scholar, 30Stubbs V.E. Schratl P. Hartnell A. Williams T.J. Peskar B.A. Heinemann A. Sabroe I. J. Biol. Chem. 2002; 277: 26012-26020Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 31Heinemann A. Hartnell A. Stubbs V.E. Murakami K. Soler D. LaRosa G. Askenase P.W. Williams T.J. Sabroe I. J. Immunol. 2000; 165: 7224-7233Crossref PubMed Scopus (68) Google Scholar). 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 (12Heinemann A. Schuligoi R. Sabroe I. Hartnell A. Peskar B.A. J. Immunol. 2003; 170: 4752-4758Crossref PubMed Scopus (101) Google Scholar, 31Heinemann A. Hartnell A. Stubbs V.E. Murakami K. Soler D. LaRosa G. Askenase P.W. Williams T.J. Sabroe I. J. Immunol. 2000; 165: 7224-7233Crossref PubMed Scopus (68) Google Scholar). 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 × 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 (31Heinemann A. Hartnell A. Stubbs V.E. Murakami K. Soler D. LaRosa G. Askenase P.W. Williams T.J. Sabroe I. J. Immunol. 2000; 165: 7224-7233Crossref PubMed Scopus (68) Google Scholar, 32Olweus J. BitMansour A. Warnke R. Thompson P.A. Carballido J. Picker L.J. Lund-Johansen F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12551-12556Crossref PubMed Scopus (390) Google Scholar). 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 (31Heinemann A. Hartnell A. Stubbs V.E. Murakami K. Soler D. LaRosa G. Askenase P.W. Williams T.J. Sabroe I. J. Immunol. 2000; 165: 7224-7233Crossref PubMed Scopus (68) Google Scholar). 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 (33Sugimoto H. Shichijo M. Iino T. Manabe Y. Watanabe A. Shimazaki M. Gantner F. Bacon K.B. J. Pharmacol. Exp. Ther. 2003; 305: 347-352Crossref PubMed Scopus (152) Google Scholar), SQ29,548, a TP receptor antagonist (20Ogletree M.L. Harris D.N. Greenberg R. Haslanger M.F. Nakane M. J. Pharmacol. Exp. Ther. 1985; 234: 435-441PubMed Google Scholar), 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 (30Stubbs V.E. Schratl P. Hartnell A. Williams T.J. Peskar B.A. Heinemann A. Sabroe I. J. Biol. Chem. 2002; 277: 26012-26020Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 34Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar) 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 (12Heinemann A. Schuligoi R. Sabroe I. Hartnell A. Peskar B.A. J. Immunol. 2003; 170: 4752-4758Crossref PubMed Scopus (101) Google Scholar, 28Monneret G. Li H. Vasilescu J. Rokach J. Powell W.S. J. Immunol. 2002; 168: 3563-3569Crossref PubMed Scopus (108) Google Scholar, 35Heinemann A. Ofner M. Amann R. Peskar B.A. Pharmacology. 2003; 67: 49-54Crossref PubMed Scopus (26) Google Scholar). 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 × 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 (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 × 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 (36Martinelli R. Sabroe I. LaRosa G. Williams T.J. Pease J.E. J. Biol. Chem. 2001; 276: 42957-42964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). 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 × 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 (30Stubbs V.E. Schratl P. Hartnell A. Williams T.J. Peskar B.A. Heinemann A. Sabroe I. J. Biol. Chem. 2002; 277: 26012-26020Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).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 (36Martinelli R. Sabroe I. LaRosa G. Williams T.J. Pease J.E. J. Biol. Chem. 2001; 276: 42957-42964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). 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 (37Scheurlen M. Kirchner M. Clemens M.R. Jaschonek K. Biochem. Pharmacol. 1993; 46: 245-249Crossref PubMed Scopus (22) Google Scholar). 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.RESULTS11-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).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 (10 μm; data not shown, n = 4).Fig. 2Calcium 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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) (33Sugimoto H. Shichijo M. Iino T. Manabe Y. Watanabe A. Shimazaki M. Gantner F. Bacon K.B. J. Pharmacol. Exp. Ther. 2003; 305: 347-352Crossref PubMed Scopus (152) Google Scholar), 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).Fig. 311-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,

Referência(s)