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  • br Rationale Recently we have shown that the nasal

    2019-12-03


    Rationale Recently we have shown that the nasal CysLT2 receptor localized exclusively in blood vessels and the expression level of the CysLT2 receptor in patients with nasal allergy was higher than that in patients with non-allergic rhinitis (Shirasaki et al. Allergol Int 2013). We hypothesized that Th2 cytokines could regulate CysLT2 receptor expression and its function in vascular endothelial cells.
    Methods Human umbilical vein endothelial Cathepsin K Activity Fluorometric Assay Kit (HUVECs) were stimulated with interleukin (IL) -4 or IL-13 for 48 hours, and the levels of CysLT2 receptor expression were evaluated by western blot analysis. HUVECs in 96-well plates were loaded with Ca2+ indicator Fluo-4, and agonist-induced Ca2+ influx was measured by using fluorescence plate reader.
    Results CysLT2 receptor protein was augmented about 2-fold after treatment with IL-4 (10ng/ml) and IL-13 (10ng/ml) at 48 hours of incubation. In non-pretreated HUVECs, any significant changes in intracellular Ca2+ levels were not observed by the stimulation with CysLTs. On the other hand, cellular responses to leukotriene C4 and leukotriene D4 were occurred in Th2 cytokine-pretreated HUVECs.
    Conclusions CysLT2 receptor expression in vascular endothelial cells can be regulated by Th2 cytokines at protein level. And Th2 cytokines may activate the functions of vascular CysLT receptors.
    Cysteinyl leukotrienes (cys-LTs) are potent stimulants of signaling events and proliferation of mast cells (MCs). Despite the presence of CysLT receptors on MCs, these responses can be largely blocked by antagonists of CysLT receptors. We studied functional responses of human MCs to exogenous cys-LTs following short-hairpin (sh)RNA-based manipulation of CysLT and CysLT receptor expression. In parallel, we studied these responses in MCs from mice lacking each receptor. We used biochemical and imaging techniques to identify receptor-receptor interactions. CysLT and CysLT receptors co-precipitate from extracts of a human MC line, LAD2, and from primary cord blood-derived human MCs (hMCs), and strikingly co-localized at the surfaces of LAD2 cells and hMCs, as determined by confocal imaging. Antibody-based fluorescent lifetime imaging (FLIM) confirmed the formation of heterodimers based on fluorescence energy transfer (FRET). Knock-down of CysLT receptors using shRNA abrogated both cys-LT-dependent proliferation and cytokine generation, whereas cells subjected to CysLT knock-down displayed enhanced responses; these were abrogated by treatment of the cells with the CysLT receptor antagonist, MK571. Cys-LT-mediated c-Kit transactivation, ERK phosphorylation, and cytokine generation were abrogated and enhanced, respectively, by the lack of CysLT and CysLT receptors in mouse mBMMCs. CysLT receptors negatively regulate cys-LT-induced MC functions, possibly through formation of heterodimers that dampen signaling.
    Cysteinyl leukotrienes (cysLTs) are formed by the action of 5-lipoxygenase and its activating protein on arachidonic acid released from cell membrane phospholipids by the enzyme phospholipase A. This produces leukotriene (LT) A, which is hydrolyzed in neutrophils and monocytes to form LTB and conjugated to glutathione in mast cells, basophils, eosinophils, and structural cells such as epithelial cells by the enzyme LTC synthase to form LTC, and then enzymatically converted to LTD and LTE. The cysLTs LTC, LTD, and LTE likely contribute to the pathogenesis of chronic rhinosinusitis through their effects on microvascular leakage, epithelial cell activation, elevated mucus secretion, and mucosal inflammation., , , , The cysLTs exert their actions on target cells through specific receptors. So far, 2 G-protein–coupled receptors for the cysLTs, termed cysLT type 1 receptor (cysLT) and cysLT type 2 receptor (cysLT), have been cloned., Both receptors bind to LTC, LTD, and LTE with affinity order LTD ≫ LTC>LTE (cysLT) and LTD=LTC ≫ LTE (cysLT), and both have been described to be expressed, at least at the level of mRNA, in various tissues (cysLT, lung including smooth muscle, small intestine, pancreas; cysLT, cardiac and vascular smooth muscle, adrenal medulla, central nervous system) as well as inflammatory leukocytes. Potent cysLT antagonists have been shown to ameliorate the clinical symptoms of chronic rhinosinusitis,, suggesting that at least some of the disease regulatory actions of the cysLTs are mediated through cysLT. In contrast, the precise function of cysLT in both health and disease is unknown. Furthermore, there have been no detailed studies of its tissue distribution in allergic disease.