Surfactant protein D alleviates eosinophil-mediated airway inflammation and remodeling in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Surfactant protein D (SPD) is a member of the collectin family that lines the airway epithelial cells with host defense. However, the role of SPD in the pathogenesis of aspirin-exacerbated respiratory disease (AERD) is still unclear. METHODS: The serum SPD level was measured in patients with AERD (n = 336), those with aspirin-tolerant asthma (ATA, n = 442), and healthy controls (HC, n = 104). Polymorphisms of SFTPD in the study subjects were analyzed. The effect of LTE4 on SPD production through eosinophil infiltration was investigated in BALB/c mice. The protective function of SPD against eosinophils inducing inflammation and remodeling was assessed in vitro/vivo. The potential efficacy of nintedanib against airway remodeling through the production of SPD was evaluated. RESULTS: The serum SPD level was significantly lower (P < .001) in AERD compared with ATA patients, and negatively correlated with fall in FEV1 (%) after lysine-aspirin bronchoprovocation test and/or the urinary LTE4 level. In addition, polymorphism of SFTPD at rs721917 was significantly different in the study subjects (odds ratio, 1.310; 95% confidence intervals, 2.124-3.446; P = .002). LTE4-exposed mice showed an increased eosinophil count with a decreased SPD level in bronchoalveolar lavage fluid. Eosinophils increased α-smooth muscle actin expression in airway epithelial cells, which was attenuated by SPD treatment. Furthermore, nintedanib protected the airway epithelial cells against eosinophils by enhancing the production of SPD. CONCLUSION: The decreased level of SPD in AERD was associated with airway inflammation/remodeling under the eosinophilic condition, suggesting that modulation of SPD may provide a potential benefit in AERD.

Leukotriene E4 elicits respiratory epithelial cell mucin release through the G-protein-coupled receptor, GPR99.

Cysteinyl leukotrienes (cysLTs), leukotriene C4 (LTC4), LTD4, and LTE4 are proinflammatory lipid mediators with pathobiologic function in asthma. LTE4, the stable cysLT, is a weak agonist for the type 1 and type 2 cysLT receptors (CysLTRs), which constrict airway smooth muscle, but elicits airflow obstruction and pulmonary inflammation in patients with asthma. We recently identified GPR99 as a high-affinity receptor for LTE4 that mediates cutaneous vascular permeability. Here we demonstrate that a single intranasal exposure to extract from the respiratory pathogen Alternaria alternata elicits profound epithelial cell (EpC) mucin release and submucosal swelling in the nasal mucosa of mice that depends on cysLTs, as it is absent in mice deficient in the terminal enzyme for cysLT biosynthesis, LTC4 synthase (LTC4S). These mucosal changes are associated with mast cell (MC) activation and absent in MC-deficient mice, suggesting a role for MCs in control of EpC function. Of the three CysLTRs, only GPR99-deficient mice are fully protected from EpC mucin release and swelling elicited by Alternaria or by intranasal LTE4 GPR99 expression is detected on lung and nasal EpCs, which release mucin to doses of LTE4 one log lower than that required to elicit submucosal swelling. Finally, mice deficient in MCs, LTC4S, or GPR99 have reduced baseline numbers of goblet cells, indicating an additional function in regulating EpC homeostasis. These results demonstrate a novel role for GPR99 among CysLTRs in control of respiratory EpC function and suggest that inhibition of LTE4 and of GPR99 may have therapeutic benefits in asthma.

Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression.

Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.

Leukotriene E4 induces MUC5AC release from human airway epithelial NCI-H292 cells.

BACKGROUND: Hypersecretion of mucin in the airway epithelium is an important feature of allergic airway diseases. Of the 3 cysteinyl leukotrienes (CysLTs; LTC4 LTD4 and LTE4), only LTE4 is sufficiently stable to be detectable in extracellular fluids. However, LTE4 has received little attention because it binds poorly to the CysLT1 and CysLT2 receptors; therefore, little is known about the effects of LTE4 on mucous secretion. Recently, studies have focused on the P2Y12 receptor as a potential receptor for LTE4, because this receptor is required for LTE4-mediated pulmonary inflammation. In our previous study, we confirmed the expression of P2Y12 receptor in human airway epithelial cells. To clarify the roles of LTE4 in airway epithelial cells, we investigated mucus secretion by LTE4 in vitro. METHODS: Confluent NCI-H292 cells were stimulated with LTE4 (0.01-1 µM) for 24 h. The release and production of MUC5AC protein, a gel-forming mucin, were evaluated with an enzyme-linked immunosorbent assay. RESULTS: Western blot analysis revealed that NCI-H292 cells expressed P2Y12 receptor protein. LTE4 significantly induced the release of MUC5AC mucin in a dose-dependent manner. Th2 cytokines such as IL-4 (10 ng/mL) and IL-13 (10 ng/mL) accelerated the LTE4-induced release of MUC5AC protein. MRS2935, a P2Y12 receptor antagonist, partially inhibited the LTE4-induced release of MUC5AC protein in the airway. In contrast, MK571, a CysLT1 receptor antagonist, did not affect the release of MUC5AC protein elicited by LTE4. CONCLUSIONS: These results suggest that LTE4 may play some important roles in allergic mucus secretion partially via activation of P2Y12 receptor.

Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk.

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.

Biological effects of leukotriene E4 on eosinophils.

Studies demonstrate the existence of novel receptors for cysteinyl leukotrienes (CysLTs) that are responsive to leukotriene (LT) E4 and might be pathogenic in asthma. Given the eosinophilic infiltration in this disorder, we investigated eosinophil expression of P2Y12 and gpr99 and their capacity to respond to LTE4. Receptor transcript expression was investigated via quantitative PCR and surface protein expression via flow cytometry. We investigated LTE4 influences on eosinophils including Ca(+2) flux, cAMP induction, modulation of adhesion molecule expression, apoptosis and degranulation. Eosinophils displayed both transcript and surface protein expression of P2Y12 and gpr99. We could not find evidence of LTE4 activation of eosinophils, however, LTE4 induced cAMP expression, and preincubation of eosinophils with LTE4 inhibited degranulation. Even though eosinophils are an important source of CysLTs in AERD, eosinophils are not themselves the pro-inflammatory biological target and, in contrast, LTE4 via cAMP primarily elicits anti-inflammatory responses.

Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway.

Extremely low-frequency electromagnetic fields (ELF-EMF) causes various biological effects through altering intracellular calcium homeostasis. The role of high voltage-gated (HVA) calcium channels in ELF-EMF induced effects has been extensively studied. However, the effect of ELF-EMF on low-voltage-gated (LVA) T-type calcium channels has not been reported. In this study, we test the effect of ELF-EMF (50Hz) on human T-type calcium channels transfected in HEK293 cells. Conversely to its stimulant effects on HVA channels, ELF-EMF exposure inhibited all T-type (Cav3.1, Cav3.2 and Cav3.3) channels. Neither the protein expression nor the steady-state activation and inactivation kinetics of Cav3.2 channels were altered by ELF-EMF (50Hz, 0.2mT) exposure. Exposure to ELF-EMF increased both arachidonic acid (AA) and leukotriene E4 (LTE4) levels in HEK293 cells. CAY10502 and bestatin, which block the increase of AA and LTE4 respectively, abrogated the ELF-EMF inhibitory effect on Cav3.2 channels. Exogenous LTE4 mimicked the ELF-EMF inhibition of T-type calcium channels. ELF-EMF (50Hz) inhibits native T-type calcium channels in primary cultured mouse cortical neurons via LTE4. We conclude that 50Hz ELF-EMF inhibits T-type calcium channels through AA/LTE4 signaling pathway.

Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Differential regulation of cysteinyl leukotriene receptor signaling by protein kinase C in human mast cells.

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1ß generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1ß generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.

Leukotriene-induced contraction is mediated by cysteinyl leukotriene receptor CysLT1 in guinea pig fundus but by CysLT1 and CysLT2 in antrum.

AIMS: Leukotriene D(4) (LTD(4)) causes contraction of the stomach through unclear receptors. The aim of the present study is to characterize the cysteinyl leukotriene receptor (CysLT) mediating leukotriene-induced muscle contraction in the stomach. MAIN METHODS: We measured contraction of gastric muscle strips isolated from the guinea pig fundus and antrum caused by cysteinyl leukotrienes, including LTC(4), LTD(4) and LTE(4), as well as the dihydroxy leukotriene LTB(4) in vitro. KEY FINDINGS: In both fundic and antral muscle strips, LTC(4) and LTD(4) caused marked whereas LTE(4) caused moderate, concentration-dependent contractions. In contrast, LTB(4) caused only small contraction. The relative potencies for cysteinyl leukotrienes to cause contraction in both fundus and antrum were LTC(4)=LTD(4)>LTE(4). The LTD(4)-induced contraction was not affected by tetrodotoxin or atropine, suggesting that the action is not neurally mediated. The LTD(4)-induced contraction in the fundus was almost abolished by the CysLT(1) selective antagonist montelukast. In contrast, the LTD(4)-induced contraction in the antrum was only partially inhibited by montelukast or the dual CysLT(1) and CysLT(2) antagonist BAY u9773. This antral contraction was almost abolished by the combination of montelukast and BAY u9773, indicating enhancement of inhibition. SIGNIFICANCE: The results of the present study demonstrate that cysteinyl leukotrienes LTC(4), LTD(4) and LTE(4) cause moderate to marked whereas the dihydroxy leukotriene LTB(4) causes small muscle contraction in the stomach in vitro. The leukotriene-induced contraction is mediated by CysLT(1) in fundus but by CysLT(1) and CysLT(2) in antrum.

Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors.

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C(4), which then undergoes extracellular metabolism to LTD(4) and LTE(4). LTE(4), the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT(1)R and CysLT(2)R, respectively). We had recognized a greater potency for LTE(4) than LTC(4) or LTD(4) in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE(4) in mice lacking both the CysLT(1)R and CysLT(2)R could establish the existence of a separate LTE(4) receptor. We now report that the intradermal injection of LTE(4) into the ear of mice deficient in both CysLT(1)R and CysLT(2)R elicits a vascular leak that exceeds the response to intradermal injection of LTC(4) or LTD(4), and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE(4) is approximately 64-fold more potent in the CysLT(1)R/CysLT(2)R double-deficient mice than in sufficient mice. The administration of a CysLT(1)R antagonist augmented the permeability response of the CysLT(1)R/CysLT(2)R double-deficient mice to LTC(4), LTD(4), and LTE(4). Our findings establish the existence of a third receptor, CysLT(E)R, that responds preferentially to LTE(4), the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

Eoxins are proinflammatory arachidonic acid metabolites produced via the 15-lipoxygenase-1 pathway in human eosinophils and mast cells.

Human eosinophils contain abundant amounts of 15-lipoxygenase (LO)-1. The biological role of 15-LO-1 in humans, however, is unclear. Incubation of eosinophils with arachidonic acid led to formation of a product with a UV absorbance maximum at 282 nm and shorter retention time than leukotriene (LT)C4 in reverse-phase HPLC. Analysis with positive-ion electrospray tandem MS identified this eosinophil metabolite as 14,15-LTC4. This metabolite could be metabolized to 14,15-LTD4 and 14,15-LTE4 in eosinophils. Because eosinophils are such an abundant source of these metabolites and to avoid confusion with 5-LO-derived LTs, we suggest the names eoxin (EX)C4, -D4, and -E4 instead of 14,15-LTC4, -D4, and -E4, respectively. Cord blood-derived mast cells and surgically removed nasal polyps from allergic subjects also produced EXC4. Incubation of eosinophils with arachidonic acid favored the production of EXC4, whereas challenge with calcium ionophore led to exclusive formation of LTC4. Eosinophils produced EXC4 after challenge with the proinflammatory agents LTC4, prostaglandin D2, and IL-5, demonstrating that EXC4 can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayer in vitro, indicating that EXs can modulate and enhance vascular permeability, a hallmark of inflammation. In this model system, EXs were 100 times more potent than histamine and almost as potent as LTC4 and LTD4. Taken together, this article describes the formation of proinflammatory EXs, in particular in human eosinophils but also in human mast cells and nasal polyps.

Cysteinyl leukotrienes induce monocyte chemoattractant protein 1 in human monocytes/macrophages.

BACKGROUND: Monocytes/macrophages have a cysteinyl leukotriene 1 (CysLT1) receptor, but its function is poorly understood. Objective To elucidate the biological function of the CysLT1 receptor of human monocytes/macrophages. METHODS: We examined the production of TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and eotaxin induced by CysLTs (leukotriene (LT)C4, -D4, and -E4) in THP-1 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages. Moreover, we examined the effect of CysLTs on the expression of beta-chemokine receptor 2B (CCR2B) as the receptor of MCP-1 by Western blot analysis. RESULTS: ELISA revealed that CysLTs induced MCP-1 in THP-1 cells and peripheral blood CD14+ monocytes/macrophages, but not other cytokines. PCR demonstrated that CysLTs increased MCP-1 mRNA expression in THP-1 cells, and Western blotting showed that CysLTs increased the expression of CCR2B in THP-1 cells. Moreover, we demonstrated that pranlukast, a CysLT1 receptor antagonist, blocked MCP-1 production by CysLTs in THP-1 cells almost completely, and partially inhibited MCP-1 release by CysLTs in peripheral blood CD14+ monocytes/macrophages and CCR2B expression by CysLTs in THP-1 cells. CONCLUSION: CysLTs induce MCP-1 and increase CCR2B expression in human monocytes/macrophages.

Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods.

Endogenous ligands acting on a human P2Y12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca2+ assay using the CHO cells stably expressing P2Y12-G16alpha fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y12 receptor as agonists with the EC50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y12, P2Y13, P2Y14, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions.

Effects of cysteinyl leukotrienes in small human bronchus and antagonist activity of montelukast and its metabolites.

BACKGROUND: Evidence suggests that small airways contribute to clinically significant processes in asthma. Cysteinyl leukotrienes (CysLTs) are considered to be pivotal mediators in the pathogenesis of asthma. Montelukast (MK), a specific CysLT1 receptor antagonist, is metabolized in two main hydroxylated metabolites (termed M5 and M6, respectively). OBJECTIVES: The aims of this study were to compare the responsiveness of small and large human bronchi to the three CysLTs, to evaluate the antagonist activity of MK, M5 and M6 in these preparations of human bronchi, and to characterize the CysLT receptors involved in the contractile response. METHODS AND RESULTS: In isolated small bronchus (i.d. 0.5-2 mm), the potencies (-log molar EC50) of LTC4, LTD4 and LTE4 were 9.3 (n=11), 9.1 (n=30) and 8.4 (n=14), respectively. The three CysLTs were about 30-fold more potent in small bronchi than in larger bronchi (i.d. 4-6 mm). In small bronchi, MK significantly shifted to the right the CysLT concentration-effect curves with pA2 values against LTC4, LTD4 and LTE4 of 9.1 (n=3), 9.0 (n=11) and 8.7 (n=5), respectively. The antagonist potencies of M6 and M5 were similar to MK and fivefold lower, respectively. A similar activity of MK against the three CysLTs suggested that CysLT1 receptors are involved in the contraction of human bronchus. Analysis by RT-PCR also indicated that human bronchus mainly expressed CysLT1 receptors. CONCLUSION: MK exerts a potent antagonist activity against the particularly potent constricting effects of CysLTs in isolated human small bronchi, which only expressed the CysLT1 receptor subtype. The metabolites of MK are also potent in vitro antagonists, but may not participate in the therapeutic activity of MK due to their low plasma concentrations in patients treated with the recommended dose of MK.

Pranlukast inhibits NF-kappa B activation in human monocytes/macrophages and T cells.

BACKGROUND: Pranlukast is a leukotriene 1 (LT1) receptor antagonist and is effective against bronchial asthma. Pranlukast inhibits contraction of the tracheal muscle, and thereby antagonizes the binding of LTC4, LTD4 and LTE4. However, the action of pranlukast on monocytes/macrophages and T cells is unknown. OBJECTIVE: We examined whether or not pranlukast inhibits TNF-alpha-induced activation of nuclear transcription factor NF-kappa B, a factor that is essential for the expression of proinflammatory cytokines, on human monocytic 1.3% dimethylsulphoxide (DMSO)-differentiated U-937 cells, which have cysteinyl LT1 (CysLT1) receptors on their membranes, and T cells (Jurkat), which do not. METHODS: We examined whether or not LTC4, LTD4 or LTE4 induced NF-kappa B activation in 1.3% DMSO-differentiated U-937 cells by Western blotting. The inhibitory effects of pranlukast and MK-571, which is an LTD4 receptor-selective antagonist, on TNF-alpha-induced NF-kappa B activation was evaluated by Western blotting and flow cytometry, and those on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production in peripheral blood mononuclear cells (PBMC) were evaluated by enzyme-linked immunosorbent assaying. RESULTS: LTC4, LTD4 or LTE4 did not induce NF-kappa B activation in 1.3% DMSO-differentiated U-937 cells. Western blotting demonstrated that 10-5 M pranlukast inhibits NF-kappa B activation in 1.3% DMSO-differentiated U-937 and Jurkat cells by about 40% & 30%, respectively. Flow cytometry demonstrated that pranlukast and MK-571 inhibit NF-kappa B activation in 1.3% DMSO-differentiated U-937 and Jurkat cells in a dose-related manner. Moreover, 10-5 M pranlukast and MK-571 inhibited LPS-induced IL-6 production in PBMC by about 65% and 15%, respectively. CONCLUSION: Pranlukast and MK-571 partially inhibited NF-kappa B activation in 1.3% DMSO-differentiated U-937 and Jurkat cells, and IL-6 release in PBMC. These findings are consistent with the idea that, independently of CysLT1 receptor antagonism, micromolar concentrations of pranlukast suppress the production of proinflammatory cytokines via inhibition of NF-kappa B activation in monocytes/macrophages and T cells, but the contribution of this effect to the anti-inflammatory activity of pranlukast at oral therapeutic doses in asthmatic patients is unclear.

The effect of cysteinyl leukotrienes on growth of eosinophil progenitors from peripheral blood and bone marrow of atopic subjects.

BACKGROUND: The accumulation of eosinophils into the peripheral blood and airways of asthmatic subjects is, in part, dependent on cysteinyl leukotrienes (cysLTs). However, the effect of cysLTs on peripheral blood and bone marrow eosinophil pro-genitor cells in allergic subjects is not known. OBJECTIVE: The purpose of this study was to evaluate the effects of leukotriene (LT) D(4) and LTE(4) and the cysLT(1) receptor antagonist montelukast on peripheral blood and bone marrow eosinophil-basophil progenitor growth and development in atopic subjects. METHODS: Semisolid methylcellulose cultures for peripheral blood and bone marrow eosinophil-basophil colonies were counted after incubation with or without addition of LTD(4), LTE(4), and montelukast in the presence of suboptimal concentrations of GM-CSF, IL-3, and IL-5. RESULTS: Peripheral blood eosinophil-basophil colony-forming unit cultures grown in the presence of GM-CSF and bone marrow eosinophil-basophil colony-forming units grown in the presence of IL-5 were significantly increased by the addition of LTD(4) (0.1 micromol/L). This increase was suppressed by montelukast (1 micromol/L). CONCLUSION: This study has demonstrated that the cysLT LTD(4) can stimulate proliferation of eosinophil hematopoietic progenitor cells in the presence of eosinophilopoietic cytokines. The suppressive effect by montelukast demonstrates that this is a cysLT(1) receptor-mediated effect.

Cysteinyl leukotrienes induce IL-4 release from cord blood-derived human eosinophils.

BACKGROUND: Eosinophils contain preformed stores of IL-4 within their cytoplasmic granules, but physiologic stimuli to release IL-4 from eosinophils are not yet defined. OBJECTIVE: We evaluated whether cysteinyl leukotrienes (CysLTs) could elicit IL-4 release from eosinophils. METHODS: We used a dual-antibody capture and detection assay (EliCell) for IL-4 release and used eosinophils differentiated in vitro from human cord blood-derived progenitors. RESULTS: Leukotriene (LT) C4, LTD4, and LTE4 each elicited the rapid, vesicular transport-mediated, dose- and time-dependent release of IL-4 from eosinophils. Both LTD4 and LTE4 evoked similar and earlier IL-4 release than LTC4. LTC4 did not act directly but only after conversion to LTD4 because an inhibitor of gamma-glutamyl transpeptidase, acivicin, blocked LTC4-induced IL-4 release. MK571 and LY171833, receptor antagonists for CysLT1 and not CysLT2, and pertussis toxin inhibited LTC4-, LTD4-, and LTE4-induced IL-4 release. Cord blood-differentiated eosinophils contained CysLT1 protein detectable by means of immunoblotting. CONCLUSION: CysLTs acting through G(i) protein-coupled and MK571- and LY171833-inhibitable receptors on cord blood-derived human eosinophils can act as autocrine or paracrine mediators to stimulate the rapid, nonexocytotic release of preformed IL-4.

Studies of receptors and modulatory mechanisms in functional responses to cysteinyl-leukotrienes in smooth muscle.

Cysteinyl-leukotrienes, i.e. leukotriene (LT) C4, D4 and E4, are inflammatory mediators and potent airway- and vasoconstrictors. Two different cysteinyl-leukotriene receptors have been cloned, CysLT1 and CysLT2. This report reviews recent data on CysLT receptor characterisation as well as studies of modulatory mechanisms involved in cysteinyl-leukotriene-induced responses. On the basis of functional studies in isolated smooth muscle preparations, the existence of an additional receptor for cysteinyl-leukotrienes is suggested. In addition, cysteinyl-leukotriene responses in pulmonary vessels were regulated by the release of modulatory factors, of which cyclooxygenase products dominated in the arteries and nitric oxide was the main modulator in porcine pulmonary veins. Moreover, the interconversion between LTC4 and LTD4 and the metabolism into LTE4 may represent a major modulatory mechanism in the guinea-pig trachea by deciding which CysLT receptor is activated by the cysteinyl-leukotrienes.