Leukotriene E4 activates peroxisome proliferator-activated receptor gamma and induces prostaglandin D2 generation by human mast cells

Abstract

Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E(4) is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE(4) potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE(4) activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)gamma, a nuclear receptor for dietary lipids. Although LTD(4) is more potent than LTE(4) for inducing calcium flux by the human MC sarcoma line LAD2, LTE(4) is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D(2) (PGD(2)) generation. LTE(4) caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE(4), but not to LTD(4), was resistant to inhibitors of phosphoinositol 3-kinase. LTE(4)-mediated COX-2 induction, PGD(2) generation, and ERK phosphorylation were all sensitive to interference by the PPARgamma antagonist GW9662 and to targeted knockdown of PPARgamma. Although LTE(4)-mediated PGD(2) production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT(1)R), it was resistant to knockdown of this receptor. This LTE(4)-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE(4) in vivo.

FIGURE 1.

cys-LT receptor expression and calcium signaling by LTD₄ and LTE₄ in LAD2 cells. A, dose-dependent effects of LTC₄, LTD₄, and LTE₄ on the accumulation of intracellular calcium in LAD2 cells. LAD2 cells were loaded with Fura-2-AM and stimulated with the indicated concentrations of LTs in various orders. B, effect of treatment with MK571 (1 μm, 5 min) on calcium flux by Fura-2-AM-loaded LAD2 cells stimulated with the indicated LTs (500 nm each). C, real-time PCR analysis of CysLT₁R, CysLT₂R, and GPR17 transcripts expressed by LAD2 cells. D, flow cytometry analysis of CysLT₁R, CysLT₂R, and GPR17 proteins. CysLT₁R and CysLT₂R were detected after permeabilization of LAD2 cells and staining with anti-C terminus Abs. GPR17 staining was performed with an anti-N terminus Ab both with (+perm) and without (–perm) permeabilization. Shaded curves are staining with nonspecific rabbit IgG. Results depicted are from single experiments, representative of at least three performed for each assay.

FIGURE 2.

Effect of cys-LTs on proliferation and Kit internalization in LAD2 cells. A, dose-dependent effect of LTD₄ and LTE₄ on thymidine incorporation by LAD2 cells stimulated for 48 h in the absence of SCF. B, effect of MK571 added 30 min before the addition of the cys-LTs on cell proliferation. Results are expressed as the mean ± S.D. of triplicate samples in a single experiment representative of the three performed. C, flow cytometric analysis of surface Kit expression by LAD2 cells stimulated with SCF (as a positive control for internalization), LTD₄, or LTE₄ for 1 h before staining with an Ab specific for Kit receptor or an isotype-matched control. MFI, mean fluorescence intensity; Iso ctl., isotype control (mouse IgG1). D, effect of cys-LTs on Kit surface staining expressed as net MFI. Data represent mean ± S.D. from the three experiments performed. * indicates p < 0.05, relative to the control, and ** reflects p < 0.01 compared with the LTD₄-treated samples at the same doses.

FIGURE 3.

Cytokine mRNA induction and MIP-1β generation by LAD2 cells stimulated with cys-LTs. A, real-time PCR showing relative levels of MIP-1β, MCP-1, IL-5, IL-8, and TNFα transcript expression by LAD2 cells stimulated with 100 nm LTD₄ or LTE₄ for 2 h. Data are mean ± S.E. from three experiments. B, dose-dependent effect of LTE₄ on MIP-1β secretion by LAD2 cells stimulated for 6 h with the indicated concentrations of cys-LTs. Results are mean ± S.E. from three experiments. C, MIP-1β concentrations were measured in supernatants collected from cells after 6 h of stimulation with LTD₄ (100 nm), LTE₄ (100 nm), or anti-IgE (1 μg/ml) as a positive control. D, effect of pretreatment with MK571 (1 μm) on MIP-1β generation by cells stimulated with LTD₄ (100 nm), or LTE₄ (100 nm) for 6 h. Results are expressed as the mean ± S.D. from the three experiments performed. * and ** indicate p < 0.05 and <0.01, respectively, relative to the control in B and C and relative to the samples without MK571 in D.

FIGURE 4.

Phosphorylation of signaling intermediates by LAD2 cells in response to cys-LTs. A, SDS-PAGE immunoblotting was performed on cell lysates obtained after 15 min of cell stimulation with LTD₄ (100 nm) and LTE₄ (100 nm), using Abs specific for total and phosphorylated (phospho) ERK, MEK, p90RSK, and CREB (top). Representative blots are from a single experiment of three performed. The bottom panel indicates the quantitative densitometry where phosphorylation is the measure of phosphorylated protein compared with the total protein and is expressed as fold change compared with control, where the control is set to 1. Data are expressed as mean ± S.D. from three experiments. B, effect of treatment of the cells with the PI3K inhibitor LY294002 (10 μm)(LY) for 30 min on ERK phosphorylation in response to stimulation with 100 nm cys-LTs. The bottom panel represents quantitative densitometry of ERK phosphorylation compared with total ERK from three separate experiments. * indicates p < 0.05 relative to the LTD₄-stimulated sample not treated with LY294002.

FIGURE 5.

Involvement of PPARγ in cys-LT-induced responses in LAD2 cells. A, flow cytometric analysis on permeabilized LAD2 cells using an Ab specific for PPARγ or an isotype-matched control (shaded curve). Data in a second experiment were identical. B and C, ERK phosphorylation by LAD2 cells in response to stimulation with LTD₄ (100 nm), LTE₄ (100 nm), PGJ₂ (20 μg/ml), or SCF (100 ng/ml) for 15 min. The samples were preincubated in the presence or absence of GW9662 (10 μm) (GW) for 1 h before stimulation with the indicated agonists. Representative blots from the three experiments are shown. D, densitometric analysis (mean ± S.D. of three separate experiments) showing the effects of GW9662. E, siRNA-mediated knockdown of PPARγ. Immunoblotting was performed using two different Abs from the indicated sources. F, effect of the PPARγ knockdown or treatment with scrambled siRNA control assessed by phosphorylation of ERK in response to the indicated agonists for 15 min. Data are from a single experiment representative of three. G, MIP-1β concentrations were measured in supernatants collected after cells were stimulated for 6 h with LTD₄ (100 nm) or LTE₄ (100 nm). Some of the cells were pretreated with GW9662 (10 μm) for 1 h. Data are the mean ± S.D. of three independent experiments. * and ** indicate p < 0.05 and <0.01, respectively.

FIGURE 6.

PGD₂ production and up-regulation of COX-2 expression by LAD2 cells in response to cys-LTs and the involvement of PPARγ in mediating these effects. A, dose response of cys-LT-mediated PGD₂ generation in LAD2 cells stimulated with the indicated concentrations of cys-LTs for 6 h. B, time course of PGD₂ generation by LAD2 cells stimulated for the indicated intervals with LTD₄ (500 nm) or LTE₄ (500 nm). C, real-time PCR showing relative levels of COX-2 transcript expression by LAD2 cells stimulated with 100 nm LTD₄ or LTE₄ for 2 h. Data in A–D are the mean ± S.E. from three experiments each. D, time course of COX-2 protein induction in cells stimulated with LTD₄ (100 nm) and LTE₄ (100 nm) for the indicated periods of time. Data are from a single experiment representative of the three performed. E, effect of GW9662 (10 μm, 1 h) on COX-2 protein up-regulation in response to LTD₄ and LTE₄ (100 nm for 4 h each,). Data are from a single experiment representative of three performed. F, effect of pretreatment with GW9662 (10 μm, 1h), PD98059 (50 μm, 30 min), and NS398 (10 μm, 30 min) on LTE₄-induced generation of PGD₂ (measured 6 h after stimulation) by LAD2 cells. Data depicted are the mean ± S.D. of three independent experiments. PGD₂ was quantitated with a PGD₂-MOX assay.

FIGURE 7.

Effect of LTD₄ and LTE₄ on PPARγ-dependent ERK activation and PGD₂ generation by primary hMCs. A, cord blood-derived hMCs (6-week-old) were stimulated for 10 min with LTD₄ or LTE₄ (500 nm) in the absence or presence of GW9662 (10 μm) or MK571 (1 μm). Results are from a single experiment representative of three separate experiments performed. B, PGD₂ generation induced by stimulation of primary hMCs with LTD₄ or LTE₄ at the indicated concentrations. Results are the mean ± ½ range for two experiments. C, effects of GW9662 (10 μm) or MK571 (1 μm) on cys-LT-induced PGD₂ generation by primary hMCs. Cells were stimulated with 100 nm of the indicated cys-LT for 6 h. Results were measured with the PGD₂-MOX assay and are the mean ± S.E. of three independent experiments. * indicates p < 0.05.

FIGURE 8.

Lack of direct stimulation of PPARγ by LTE₄ in heterologous cell systems. A, PPARγ-specific LBD assay in bovine endothelial cells in response to stimulation with rosiglitazone (rosi), LTD₄, or LTE₄. Results are expressed as relative light units corrected for perβ-galactosidase activity (RLU/β-gal). B, phosphorylation of ERK by CHO cells with or without transduced expression of CysLT₁R or CysLT₂R in response to the indicated cys-LTs, with or without MK571 or GW9662. C, activation of PPARγ specific LBD in CHO cells expressing CysLT₁R or CysLT₂R. Results in A, B, and C are each from single experiments repeated a minimum of twice. A includes triplicate samples. * indicates p < 0.05 relative to control.

FIGURE 9.

Effect of shRNA-mediated knockdown of CysLT₁R and CysLT₂R on LTE₄-mediated PGD₂ generation by LAD2 cells. A, FACS analysis of nonpermeabilized LAD2 cells showing the effect of the knockdowns after 48 h of treatment with lentivirus containing shRNA directed to the indicated receptors, or with empty virus (Mock). Results are from a single experiment representative of the three separate experiments performed. B, MIP-1β generation by LAD2 cells stimulated for 6 h with the indicated concentrations of LTE₄ or LTD₄ after treatment with CysLT₁R- and CysLT₂R-specific shRNA constructs packaged in lentivirus, or with control vector. C, PGD₂ generation by LAD2 cells treated with the same lentiviral vectors. Results in B and C are expressed as the mean ± S.E. of the same three experiments, including the one depicted in A. * indicates p < 0.05.

FIGURE 10.

Hypothetical mechanism(s) responsible for PPARγ-dependent ERK activation and PGD₂ generation by MCs. LTE₄-mediated responses are MK571-sensitive and may involve both CysLT₁R and an unidentified GPCR, whereas LTD₄ responses are regulated by respectively positive and negative signals induced through the CysLT₁R/CysLT₂R heterodimer. PPRE, PPAR response element.