Concentration-Dependent Noncysteinyl Leukotriene Type 1 Receptor-Mediated Inhibitory Activity of Leukotriene Receptor Antagonists

BACKGROUND: The use of leukotriene antagonists (LTRAs) for asthma therapy has been associated with a significant degree of inter-patient variability in response to treatment. Some of that variability may be attributable to non-cysteinyl leukotriene type 1 receptor (CysLT(1)) mediated inhibitory mechanisms that have been demonstrated for this group of drugs. OBJECTIVE: We have used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and CysLT(1)-independent anti-inflammatory activity of two chemically different, clinically relevant, LTRAs (montelukast and zafirlukast). RESULTS: Using receptor desensitization experiments in monocytes and CysLT(1) transfected HEK293 cells, and IL-10 and CysLT(1) siRNA induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists, LTD(4) and uridine diphosphate (UDP), signal through calcium mobilization, acting on separate receptors and that both pathways were inhibited by montelukast and zafirlukast. However, 3 logs higher concentrations of LTRAs were required for inhibition of UDP induced signaling. In monocytes, UDP, but not LTD(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. Both LTRAs, at low micromolar concentrations, also inhibited calcium ionophore induced leukotriene (LTB(4) and LTC(4)) production, indicating 5-lipoxygenase inhibitory activities. CONCLUSION: We report here that montelukast and zafirlukast, acting in a concentration dependent manner, can inhibit non-CysLT(1) mediated, proinflammatory reactions, suggesting activities potentially relevant for inter-patient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma

Cytosolic phospholipase A2α activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells

Cytosolic phospholipase A2α (cPLA2α) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA2α in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA2α inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA2α, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA2α in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA2α activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA2α and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA2α activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung

Functional Characterization of Human Cysteinyl Leukotriene 1 Receptor Gene Structure

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT(1)R). The cysLT(1)R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT(1)R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT(1)R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT(1)R promoter region. IL-4 stimulation increased cysLT(1)R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD(4) and LTC(4)) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT(1)R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT(1)R/cysLT(2)R inhibitor BAY-u9773, implying a cysLT(1)R-mediated mechanism. Thus, cysLTs signaling through cysLT(1)R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.</p

IFN-γ Induces Cysteinyl Leukotriene Receptor 2 Expression and Enhances the Responsiveness of Human Endothelial Cells to Cysteinyl Leukotrienes

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-gamma in human primary endothelial cells. IFN-gamma increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-gamma signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-gamma. To determine mechanisms of IFN-gamma-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t(1/2) assays in response to IFN-gamma, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-gamma response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to G(q/11), activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-gamma stimulation. Thus, IFN-gamma induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.</p