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• W1969871007 abstract "Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E4 is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE4 potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE4 activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)γ, a nuclear receptor for dietary lipids. Although LTD4 is more potent than LTE4 for inducing calcium flux by the human MC sarcoma line LAD2, LTE4 is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D2 (PGD2) generation. LTE4 caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE4, but not to LTD4, was resistant to inhibitors of phosphoinositol 3-kinase. LTE4-mediated COX-2 induction, PGD2 generation, and ERK phosphorylation were all sensitive to interference by the PPARγ antagonist GW9662 and to targeted knockdown of PPARγ. Although LTE4-mediated PGD2 production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT1R), it was resistant to knockdown of this receptor. This LTE4-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE4in vivo. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E4 is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE4 potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE4 activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)γ, a nuclear receptor for dietary lipids. Although LTD4 is more potent than LTE4 for inducing calcium flux by the human MC sarcoma line LAD2, LTE4 is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D2 (PGD2) generation. LTE4 caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE4, but not to LTD4, was resistant to inhibitors of phosphoinositol 3-kinase. LTE4-mediated COX-2 induction, PGD2 generation, and ERK phosphorylation were all sensitive to interference by the PPARγ antagonist GW9662 and to targeted knockdown of PPARγ. Although LTE4-mediated PGD2 production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT1R), it was resistant to knockdown of this receptor. This LTE4-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE4in vivo. Cysteinyl leukotrienes (cys-LTs) 2The abbreviations used are: cys-LT, cysteinyl leukotriene; Ab, antibody; 5-LO, 5 lipoxygenase; AERD, aspirin-exacerbated respiratory disease; AHR, airway hyper-responsiveness; BAL, bronchoalveolar lavage; COX, cyclooxygenase; CREB, cyclic AMP-regulated-binding protein; CysLT1R, type 1 receptor for cys-LTs; CysLT2R, type 2 receptor for cys-LTs; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; FcϵRI, high-affinity Fc receptor for IgE; FLAP, 5-lipoxygenase activating protein; GPCR, G protein-coupled receptor; hMC, cord blood-derived human MC; IL, interleukin; LBD, ligand binding domain; LC-MS, liquid chromatography-mass spectroscopy; LT, leukotriene; LTC4S, leukotriene C4 synthase; MC, mast cell; MEK, mitogen-activated protein kinase kinase; MIP-1β, macrophage inflammatory protein 1β; MOX, methoxylamine; p90RSK, 90-kDa ribosomal S6 kinase; PGD2, prostaglandin D2; PGDS, PGD2 synthase; PI3K, phosphatidylinositol 3-kinase; PLA2, phospholipase A2; PPAR, peroxisome proliferator-activated receptor; PTX, pertussis toxin; RT, reverse transcriptase; SCF, stem cell factor; shRNA, short hairpin RNA; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α. (LTC4, LTD4, LTE4) are potent inflammatory mediators derived from arachidonic acid and generated by mast cells (MCs), eosinophils, basophils, and macrophages (reviewed in Ref. 1Kanaoka Y. Boyce J.A. J. Immunol. 2004; 173: 1503-1510Crossref PubMed Scopus (297) Google Scholar). Arachidonic acid is liberated from nuclear membrane phospholipids by a cytosolic phospholipase A2 (2Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar) and converted by 5-lipoxygenase (5-LO) and its molecular partner, 5-LO-activating protein (FLAP), to the unstable intermediate LTA4 at the nuclear envelope (3Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (655) Google Scholar, 4Malaviya R. Malaviya R. Jakschik B.A. J. Biol. Chem. 1993; 268: 4939-4944Abstract Full Text PDF PubMed Google Scholar). LTA4 is then conjugated to reduced glutathione by an integral nuclear membrane protein, leukotriene C4 synthase (LTC4S) (5Lam B.K. Penrose J.F. Freeman G.J. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7663-7667Crossref PubMed Scopus (251) Google Scholar, 6Nicholson D.W. Ali A. Vaillancourt J.P. Calaycay J.R. Mumford R.A. Zamboni R.J. Ford-Hutchinson A.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2015-2019Crossref PubMed Scopus (110) Google Scholar), forming LTC4. After transport to the extracellular space by multidrug resistance protein-1 (7Leier I. Jedlitschky G. Buchholz U. Cole S.P. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar), LTC4 is converted extracellularly to LTD4 by a γ-glutamyl leukotrienase (8Carter B.Z. Shi Z.Z. Barrios R. Lieberman M.W. J. Biol. Chem. 1998; 273: 28277-28285Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and then to the terminal product LTE4 by a dipeptidase (9Lee C.W. Lewis R.A. Corey E.J. Austen K.F. Immunology. 1983; 48: 27-35PubMed Google Scholar). This rapid conversion ensures that LTC4 and LTD4 are very short-lived in vivo. In contrast, LTE4 is stable, being the only cys-LT detected in biologic fluids and excreted in the urine without further modification (10Drazen J.M. O'Brien J. Sparrow D. Weiss S.T. Martins M.A. Israel E. Fanta C.H. Am. Rev. Respir. Dis. 1992; 146: 104-108Crossref PubMed Scopus (174) Google Scholar). Cys-LTs are the most potent known bronchoconstrictors (11Davidson A.B. Lee T.H. Scanlon P.D. Solway J. McFadden Jr., E.R. Ingram Jr., R.H. Corey E.J. Austen K.F. Drazen J.M. Am. Rev. Respir. Dis. 1987; 135: 333-337PubMed Google Scholar, 12Drazen J.M. Austen K.F. Am. Rev. Respir. Dis. 1987; 136: 985-998Crossref PubMed Scopus (256) Google Scholar), and they also potentiate airway hyperresponsiveness (AHR) to histamine when they are administered by inhalation to human subjects (13Christie P.E. Hawksworth R. Spur B.W. Lee T.H. Am. Rev. Respir. Dis. 1992; 146: 1506-1510Crossref PubMed Scopus (28) Google Scholar). Bronchoalveolar lavage (BAL) fluids collected from allergen-challenged atopic asthmatic individuals contain high levels of cys-LTs (14Wenzel S.E. Larsen G.L. Johnston K. Voelkel N.F. Westcott J.Y. Am. Rev. Respir. Dis. 1990; 142: 112-119Crossref PubMed Scopus (287) Google Scholar), and levels of LTE4 are elevated in urine samples from patients during spontaneous asthmatic exacerbations (10Drazen J.M. O'Brien J. Sparrow D. Weiss S.T. Martins M.A. Israel E. Fanta C.H. Am. Rev. Respir. Dis. 1992; 146: 104-108Crossref PubMed Scopus (174) Google Scholar). Drugs that block the type 1 receptor for cys-LTs (CysLT1R) (15Altman L.C. Munk Z. Seltzer J. Noonan N. Shingo S. Zhang J. Reiss T.F. J. Allergy Clin. Immunol. 1998; 102: 50-56Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Hamilton A. Faiferman I. Stober P. Watson R.M. O'Byrne P.M. J. Allergy Clin. Immunol. 1998; 102: 177-183Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) or that interfere with cys-LT synthesis (17Israel E. Cohn J. Dube L. Drazen J.M. J. Am. Med. Assoc. 1996; 275: 931-936Crossref PubMed Google Scholar) are clinically efficacious in asthma. Studies with mice lacking LTC4S and/or cys-LT receptors suggest additional prominent functions for these mediators in adaptive immunity and fibrosis (18Beller T.C. Friend D.S. Maekawa A. Lam B.K. Austen K.F. Kanaoka Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3047-3052Crossref PubMed Scopus (127) Google Scholar, 19Beller T.C. Maekawa A. Friend D.S. Austen K.F. Kanaoka Y. J. Biol. Chem. 2004; 279: 46129-46134Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 20Kim D.C. Hsu F.I. Barrett N.A. Friend D.S. Grenningloh R. Ho I.C. Al-Garawi A. Lora J.M. Lam B.K. Austen K.F. Kanaoka Y. J. Immunol. 2006; 176: 4440-4448Crossref PubMed Scopus (121) Google Scholar). Thus, mechanisms that control cys-LT-dependent biologic responses are of considerable pathobiologic and clinical interest in both allergic and nonallergic disease. CysLT1R and CysLT2R are the two known G protein-coupled receptors (GPCRs) selective for cys-LTs (21Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar, 22Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (892) Google Scholar). CysLT1R is expressed prominently by smooth muscle and leukocytes (22Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (892) Google Scholar, 23Figueroa D.J. Borish L. Baramki D. Philip G. Austin C.P. Evans J.F. Clin. Exp. Allergy. 2003; 33: 1380-1388Crossref PubMed Scopus (113) Google Scholar), while CysLT2R is expressed by cardiac Purkinje cells, endothelium, brain, and leukocytes (21Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). A third receptor, GPR17, recognizes both LTD4 and uracil nucleotides and is expressed primarily in the brain (24Ciana P. Fumagalli M. Trincavelli M.L. Verderio C. Rosa P. Lecca D. Ferrario S. Parravicini C. Capra V. Gelosa P. Guerrini U. Belcredito S. Cimino M. Sironi L. Tremoli E. Rovati G.E. Martini C. Abbracchio M.P. EMBO J. 2006; 25: 4615-4627Crossref PubMed Scopus (354) Google Scholar). CysLT1R binds LTD4 with higher affinity than LTC4 (EC50 values for binding of 10–9m and 10–8m, respectively) (22Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (892) Google Scholar), whereas CysLT2R has equal affinity for LTD4 and LTC4 (EC50 of 10–8m for each) (21Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). LTE4 is a weak, partial agonist for CysLT1R and CysLT2R, binding each with 1–2-log fold lower affinity than do LTC4 and LTD4 (21Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar, 23Figueroa D.J. Borish L. Baramki D. Philip G. Austin C.P. Evans J.F. Clin. Exp. Allergy. 2003; 33: 1380-1388Crossref PubMed Scopus (113) Google Scholar). Although it is a modest bronchoconstrictor relative to LTD4 (25Gauvreau G.M. Parameswaran K.N. Watson R.M. O'Byrne P.M. Am. J. Respir. Crit. Care Med. 2001; 164: 1495-1500Crossref PubMed Scopus (123) Google Scholar), LTE4 nonetheless elicits biologic responses than are distinct from those induced by its precursors. After inhalation by human subjects, LTE4 (but not LTD4) causes significant increases in the numbers of eosinophils, basophils, and MCs in sputum over several hours (25Gauvreau G.M. Parameswaran K.N. Watson R.M. O'Byrne P.M. Am. J. Respir. Crit. Care Med. 2001; 164: 1495-1500Crossref PubMed Scopus (123) Google Scholar, 26Laitinen L.A. Laitinen A. Haahtela T. Vilkka V. Spur B.W. Lee T.H. Lancet. 1993; 341: 989-990Abstract PubMed Scopus (486) Google Scholar). Humans with aspirin-exacerbated respiratory disease (AERD), a variant of asthma characterized by markedly elevated baseline generation of cys-LTs, exhibit bronchoconstrictor responses to inhaled LTE4 that are disproportionate relative to their responses to histamine (27Arm J.P. O'Hickey S.P. Hawksworth R.J. Fong C.Y. Crea A.E. Spur B.W. Lee T.H. Am. Rev. Respir. Dis. 1990; 142: 1112-1118Crossref PubMed Scopus (73) Google Scholar), LTC4, or LTD4 (28Christie P.E. Schmitz-Schumann M. Spur B.W. Lee T.H. Eur. Respir. J. 1993; 6: 1468-1473PubMed Google Scholar). Prior inhalation of LTE4 by humans with asthma potentiates AHR to histamine; this response can be blocked by pretreatment of the subjects with the cyclooxygenase (COX) inhibitor indomethacin (29O'Hickey S.P. Hawksworth R.J. Fong C.Y. Arm J.P. Spur B.W. Lee T.H. Am. Rev. Respir. Dis. 1991; 144: 1053-1057Crossref PubMed Scopus (98) Google Scholar). Likewise, LTE4 (but not LTC4 or LTD4) potentiates contraction of guinea pig tracheal rings to histamine in an indomethacin-sensitive fashion (30Lee T.H. Austen K.F. Corey E.J. Drazen J.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4922-4925Crossref PubMed Scopus (102) Google Scholar). Thus, LTE4-induced pulmonary responses in vivo are dissimilar to those caused by LTC4 and LTD4, are not explained by the pharmacology of the established GPCRs for cys-LTs, and may be mediated by induced prostanoids. MCs are stem cell factor (SCF)-dependent hematopoietic cells that are ubiquitously distributed at interfaces with the external environment (reviewed in Ref. 31Gurish M.F. Boyce J.A. J. Allergy Clin. Immunol. 2006; 117: 1285-1291Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 32Wedemeyer J. Tsai M. Galli S.J. Curr. Opin. Immunol. 2000; 12: 624-631Crossref PubMed Scopus (286) Google Scholar) and abound in human airways. MCs trigger exacerbations of asthma through the elaboration of soluble mediators. Among these are especially large quantities of prostaglandin D2 (PGD2), a COX product that is a bronchoconstrictor and chemoattractant for eosinophils, basophils, and Th2 cells. MCs express both CysLT1R and CysLT2R (33Mellor E.A. Frank N. Soler D. Hodge M.R. Lora J.M. Austen K.F. Boyce J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11589-11593Crossref PubMed Scopus (136) Google Scholar, 34Mellor E.A. Maekawa A. Austen K.F. Boyce J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7964-7969Crossref PubMed Scopus (159) Google Scholar), which form heteromeric complexes on these cells (35Jiang Y. Borrelli L.A. Kanaoka Y. Bacskai B.J. Boyce J.A. Blood. 2007; 110: 3263-3270Crossref PubMed Scopus (119) Google Scholar). Stimulation of primary human MCs derived in vitro from cord blood progenitors (hMCs) with LTD4 potently induces calcium flux (32Wedemeyer J. Tsai M. Galli S.J. Curr. Opin. Immunol. 2000; 12: 624-631Crossref PubMed Scopus (286) Google Scholar), extracellular signal-regulated kinase (ERK) phosphorylation, and cytokine generation (36Mellor E.A. Austen K.F. Boyce J.A. J. Exp. Med. 2002; 195: 583-592Crossref PubMed Scopus (138) Google Scholar). Based on RNA interference and/or pharmacologic antagonism with MK571, a drug that blocks CysLT1R but not CysLT2R, each of these responses requires CysLT1R. In a model of allergen-induced pulmonary inflammation, LTC4S–/– mice showed a striking deficit in the number of MCs in the tracheal epithelium (20Kim D.C. Hsu F.I. Barrett N.A. Friend D.S. Grenningloh R. Ho I.C. Al-Garawi A. Lora J.M. Lam B.K. Austen K.F. Kanaoka Y. J. Immunol. 2006; 176: 4440-4448Crossref PubMed Scopus (121) Google Scholar). In a separate study, exogenous LTD4 induced the proliferation of hMCs by causing transactivation of c-Kit, the receptor for SCF, through CysLT1R (37Jiang Y. Kanaoka Y. Feng C. Nocka K. Rao S. Boyce J.A. J. Immunol. 2006; 177: 2755-2759Crossref PubMed Scopus (56) Google Scholar), while CysLT2R counter-regulates these responses (35Jiang Y. Borrelli L.A. Kanaoka Y. Bacskai B.J. Boyce J.A. Blood. 2007; 110: 3263-3270Crossref PubMed Scopus (119) Google Scholar). Unexpectedly, despite its weak activity at CysLT1R and CysLT2R, LTE4 increased the numbers of MCs arising from liquid culture of cord blood mononuclear cells more potently than LTC4 or LTD4 (37Jiang Y. Kanaoka Y. Feng C. Nocka K. Rao S. Boyce J.A. J. Immunol. 2006; 177: 2755-2759Crossref PubMed Scopus (56) Google Scholar). We now report that LTE4 signals though a distinct, MK571-sensitive pathway independent of CysLT1R and CysLT2R, thereby linking extracellular LTE4 to peroxisome proliferator-activated receptor γ (PPARγ)-dependent ERK activation, inducible expression of COX-2, and generation of PGD2. These findings support the possible existence of a LTE4-activated GPCR that accounts for the distinct effects of LTE4in vivo. Reagents—LTD4, LTE4, PGJ2, GW9662, NS398, MK571, and anti-COX-2 and PPARγ Abs were purchased from Cayman Chemical. Fura-2 AM was from Molecular Probes, and all primers were from SuperArray. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and all phosphospecific Abs were from Cell Signaling. A second PPARγ Ab was from UBI. The siRNA for PPARγ was from Dharmacon, pertussis toxin (PTX) was from Sigma, and PD98059 was from Chemicon. Cell Culture—The LAD2 line (38Kirshenbaum A.S. Akin C. Wu Y. Rottem M. Goff J.P. Beaven M.A. Rao V.K. Metcalfe D.D. Leuk. Res. 2003; 27: 677-682Crossref PubMed Scopus (421) Google Scholar) isolated from the bone marrow of a patient with MC leukemia was a kind gift of Dr. Arnold Kirshenbaum (NIH). These cells were cultured in Stem-pro 34™ (Invitrogen) supplemented with 2 mm l-glutamine (Invitrogen), Pen-strep (100 international units/ml) (Invitrogen), and SCF (Endogen) (100 ng/ml). Cell culture medium was hemi-depleted every week with fresh medium and 100 ng/ml SCF. Primary hMCs were derived from cord blood mononuclear cells cultured for 6–9 weeks in RPMI supplemented with SCF, interleukin IL-6, and IL-10 (39Ochi H. Hirani W.M. Yuan Q. Friend D. Austen K.F. Boyce J.A. J. Exp. Med. 1999; 190: 267-280Crossref PubMed Scopus (309) Google Scholar). Calcium Flux—LAD2 cells (0.5–1 × 106/sample) were washed and labeled with fura 2-AM for 30 min at 37 °C. Cells were stimulated with the indicated concentrations of LTC4, LTD4, and LTE4, and changes in intracellular calcium concentration were measured using excitation at 340 and 380 nm in a fluorescence spectrophotometer (Hitachi F-4500) (34Mellor E.A. Maekawa A. Austen K.F. Boyce J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7964-7969Crossref PubMed Scopus (159) Google Scholar). The relative ratios of fluorescence emitted at 510 nm were recorded and displayed as a reflection of intracellular calcium concentration. In some experiments, cells were preincubated with the CysLT1R antagonist MK571 (1 μm) for 5 min before the stimulation. Flow Cytometry—The expressions of Kit, CysLT1R, CysLT2R, GPR17, and PPARγ in LAD2 cells were determined by flow cytometry. Briefly, LAD2 cells (2 × 105) were washed in fluorescence-activated cell sorting (FACS) buffer (1% bovine serum albumin, 0.2 mm EDTA in phosphate-buffered saline), fixed with 4% paraformaldehyde, and incubated with mouse anti-human IgG1 against Kit (BIOSOURCE International) or with custom-generated Abs against extracellular domains of the human CysLT1R (RB34) (35Jiang Y. Borrelli L.A. Kanaoka Y. Bacskai B.J. Boyce J.A. Blood. 2007; 110: 3263-3270Crossref PubMed Scopus (119) Google Scholar) and CysLT2R (RB19) (Orbigen). In some experiments, polyclonal Abs against the C termini of human CysLT1R and CysLT2R (Cayman) were used. For experiments with the latter Abs, as well as those used to detect intracellular PPARγ, the cells were permeabilized with 0.5% saponin before staining, followed by a fluorescein isothiocyanate-conjugated secondary Ab for another 30 min. Staining for GPR17 was done using a polyclonal Ab raised against the extracellular N terminus (Novus) with and without permeabilization. Nonspecific rabbit IgG and mouse IgG1 (BioSource International) were used as respective negative controls. Cells were washed with FACS buffer three times, and flow cytometric analyses were performed with a Becton-Dickinson FACScan flow cytometer. Real-time Quantitative PCR—The expressions of CysLT1R, CysLT2R, macrophage inflammatory protein-1β (MIP-1β), MCP-1, IL-5, IL-8, COX-1, COX-2, phospholipase A2 (PLA2) (groups IIA, IVA, V, and X), hematopoietic PGD2 synthase (PGDS), and tumor necrosis factor α (TNF-α) mRNAs were determined with real-time PCR performed on an ABI PRISM 7700 Sequence detection system (Applied Biosystems). LAD2 cells were growth factor-starved overnight and stimulated with LTD4 or LTE4 (100 nm) or with medium alone for 2 h at 37 °C. RNA was isolated with an RNAeasy minikit (Qiagen) and was treated with RNase-free DNase (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized from 1 μgof RNA with Superscript II RNase H-RT (Invitrogen). Reverse transcription (RT) was performed using TaqMan RT reagents. All primers and FAM-labeled PCR mix were purchased from Superarray. Short Hairpin RNA (shRNA) and Small Interfering RNA (siRNA) Knockdowns—shRNA constructs targeting human CysLT1R and CysLT2R were purchased from Open Biosystems. The constructs were cloned into a lentiviral vector (pLKo1, Open Biosystems) and used to generate infectious particles with a lentiviral packaging mix (Virapower, Invitrogen) according to the manufacturer's protocol. The transfections were carried out as described previously (35Jiang Y. Borrelli L.A. Kanaoka Y. Bacskai B.J. Boyce J.A. Blood. 2007; 110: 3263-3270Crossref PubMed Scopus (119) Google Scholar). FACs analysis was used to confirm the knockdowns. siRNA against PPARγ and scrambled double-stranded RNA controls were purchased from Dharmacon in the form of a SMART pool. Cells were transfected with 50 nm PPARγ and scrambled siRNAs using Lipofectamine according to the manufacturer's instructions. At 48 h, knockdowns were confirmed by Western blotting, and the cells were used for the indicated assays. Cell Activation—LAD2 cells and primary hMCs either were stimulated with the indicated concentrations of LTD4 or LTE4 or were passively sensitized with human myeloma IgE (2 μg/ml; Chemicon) overnight and stimulated with rabbit anti-human anti-IgE (Chemicon, 1 μg/ml), SCF (100 ng/ml), PGJ2 (20 μg/ml), or rosiglitazone (10 μm), a PPARγ agonist. To determine the contribution of various signaling events in agonist-mediated responses, cells were stimulated after preincubation with PTX (100 ng/ml) for 18 h; with the PPARγ antagonist GW9662 (10 μm) for 1 h; or with MK571 (1 μm), the mitogen-activated protein kinase kinase (MEK) inhibitor PD98058 (50 μm), the PI3K inhibitor LY294002 (10 μm), the cytosolic PLA2 (cPLA2) inhibitor Shinogi 1 (5 μm), or the COX-2 inhibitor NS398 (10 μm) for 30 min. Cells were stimulated with the agonists for 15 min for ERK phosphorylation, 2 h for PCR analysis, 6 h for the measurement of cytokine and PGD2 generation, and 18 h for the PPARγ ligand-binding domain (LBD) assay (40Zarini S. Gijon M.A. Folco G. Murphy R.C. J. Biol. Chem. 2006; 281: 10134-10146Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The concentration of MIP-1β was measured by an ELISA (Endogen). PGD2 was detected using a PGD2-methoxylamine hydrochloride (PGD2-MOX) assay (Cayman). The PGD2 values detected with this assay were similar to those identified in the supernatants of cys-LT-stimulated primary hMCs and LAD2 cells by metabolite separation and analysis by reversed-phase HPLC and electrospray ionization-mass spectrometry (LC-MS) (40Zarini S. Gijon M.A. Folco G. Murphy R.C. J. Biol. Chem. 2006; 281: 10134-10146Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Cell Lysates and Western Blotting—After stimulation with the respective agonists, LAD2 cells and primary hMCs (0.5 × 106) were lysed with lysis buffer (BD Bioscience) supplemented with protease inhibitor mixture (Roche Applied Science) and sodium vanadate (1 mm). Lysates were subjected to 4–12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with Abs against phospho- and total ERK, MEK, 90 kDa ribosomal s6 kinase (p90RSK), and cyclic AMP-regulated-binding protein (CREB) (Cell Signaling Technologies) in 1× phosphate-buffered saline, 5% dry milk, 0.1% Tween-20 (1:1000) overnight at 4 °C on shaker, and then with secondary Ab (peroxidase-conjugated anti-rabbit or anti-mouse). Bands were visualized using enhanced chemiluminescence (Pierce). PPARγ LBD Assay—Bovine aortic endothelial cells, and CHO cells stably transfected with human CysLT1R or CysLT2R were plated in 24-well plates and transiently transfected in 1% delipidated plasma (DLP)/DMEM per the manufacturer's instructions (Fugene HD, Roche Applied Science). Briefly, cells were co-transfected with constructs for the human PPARγ-LBD GAL4 fusion, the GAL4-responsive luciferase reporter pUASX4TK-luc, and β-galactosidase. Cells were stimulated with the indicated reagents for 18–24 h before the PPARγ LBD-GAL4 assays were performed. Luciferase counts, normalized to β-galactosidase activity, were obtained using luciferase substrates (BD Pharmingen); chlorophenol red-β-d-galactopyranoside was used for β-galactosidase activity assays (Roche Diagnostics) (41Ziouzenkova O. Perrey S. Asatryan L. Hwang J. MacNaul K.L. Moller D.E. Rader D.J. Sevanian A. Zechner R. Hoefler G. Plutzky J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2730-2735Crossref PubMed Scopus (198) Google Scholar). Cell Proliferation—Mitogenic assays were performed in triplicate on cells suspended in fresh medium at a concentration of 0.5 × 106/ml with or without LTD4 and LTE4 (0.01–0.1 μm) in the absence of SCF. In some experiments, MK571 (1 μm) or GW9662 (10 μm) was added at the same time as the mitogens. At 48 h, the cells were pulsed overnight with [3H]thymidine (Amersham Biosciences), and counts were analyzed by β-counting. The radioactivity incorporated was measured in triplicate, and the results are expressed as mean ± S.D. Statistics—Data are expressed as mean ± S.D. from at least three experiments except where otherwise indicated. Data were converted to a percentage of control for each experiment where indicated. The significance was determined with the Student's t test. Rank Order of cys-LTs for Inducing Calcium Flux in LAD2 Cells—To determine the potency of LTE4 for calcium flux relative to the other cys-LTs, we stimulated Fura-2-loaded LAD2 cells with various doses of each cys-LT and performed cross-desensitizations. LTD4 was the most potent agonist among the cys-LTs for eliciting calcium flux and completely desensitized the LAD2 cells to the calcium fluxes induced by both LTC4 and LTE4 (Fig. 1A). LTE4 caused calcium flux at doses as low as 1 nm that was not attenuated by prior stimulation of the cells with an equal amount of LTC4. LTC4 did not induce a calcium flux at concentrations below 100 nm. LTE4 partly desensitized LAD2 cells to LTD4 and completely desensitized these cells to LTC4 (Fig. 1B). Regardless of the cys-LT used to stimulate the LAD2 cells, the calcium responses were totally blocked by pretreatment of the cells with MK571 (Fig. 1B), which competitively antagonizes CysLT1R but not CysLT2R. Thus, although LAD2 cells express CysLT2R mRNA (Fig. 1C) and protein (Fig. 1D), all cys-LT-induced calcium flux in these cells is mediated by MK571-sensitive receptors. GPR17 was detected intracellularly but not on the surfaces of the LAD2 cells. cys-LT-mediated Proliferation and Kit Internalization—We compared the effects of LTD4 with those of LTE4 for inducing proliferation of LAD2 cells. Unlike primary hMCs, LAD2 cells do not depend on exogenous SCF for their survival (38Kirshenbaum A.S. Akin C. Wu Y. Rottem M. Goff J.P. Beaven M.A. Rao V.K. Metcalfe D.D. Leuk. Res. 2003; 27: 677-682Crossref PubMe" @default.
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