CysLT Release Research Articles

Leptin and exercise-induced bronchoconstriction in obese asthmatic children

Exercise-induced bronchoconstriction (EIB) in asthmatic children has been associated with obesity. Leptin, the proinflammatory adipokine, is typically increased in obesity, whereas cysteinyl leukotrienes (cysLTs) are the main inflammatory mediators implicated in the pathogenesis of EIB. The aim of this work was to study the possible impact of obesity on the severity of EIB in asthmatic children and the possible association with the adipokine leptin. The study included eighty pre-pubertal asthmatic children divided according to their body mass index (BMI) and response to exercise into four groups; obese exercise-responders (n=20), normal-weight exercise-responders (n=20), obese exercise non-responders (n=20), and normal-weight exercise non-responders (n=20). A baseline spirometry test and a standardized exercise challenge test (ECT) were performed. The severity of EIB was assessed by the maximum percentage fall in forced expiratory volume in 1 second (MF%FEV1) after exercise. The level of fasting serum leptin and the release of cysLTs during exercise were compared between the 4 groups. The MF%FEV1 during exercise was significantly greater in obese responder compared to normal-weight responder patients (p=0.004) and MF%FEV1 was positively correlated with cysLT release during exercise, BMI z-score, waist circumference and serum leptin. CysLT release during exercise was positively correlated with the level of serum leptin (r =0.514, p=0.001). This study reports that the severity of EIB is significantly greater in obese compared to normal-weight asthmatic children and suggests an association between leptin and airway hyperresponsiveness to exercise in obese asthmatic children through a mechanism related to cysLT release

Suppression of aspirin-mediated eosinophil activation by prostaglandin E2: Relevance to aspirin and nonsteroidal anti-inflammatory drug hypersensitivity

BackgroundAspirin-exacerbated respiratory disease (AERD) is characterized by severe, sometimes life-threatening reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). Mechanisms driving the disease include overproduction of leukotrienes and loss of anti-inflammatory prostaglandin E2 (PGE2) production. Many cell types contribute to the disease; however, eosinophils are markedly elevated and are important drivers of pathologic findings. ObjectiveTo investigate the capacity of aspirin and NSAIDs to drive eosinophil activation and the ability of PGE2 to inhibit this activation. MethodsEosinophils were purified from blood of healthy individuals without AERD and stimulated with lysine aspirin, ketorolac, or sodium salicylate. The role of PGE2 in altering activation was determined by incubating eosinophils with increasing doses of PGE2 before lysine aspirin stimulation. Specific PGE2 receptor use was determined by incubating eosinophils with receptor agonists and antagonists before aspirin stimulation. Cysteinyl leukotrienes (CysLTs), leukotriene B4 (LTB4), and eosinophil-derived neurotoxin (EDN) were quantified by enzyme-linked immunosorbent assay. ResultsStimulation of eosinophils with lysine aspirin, ketorolac, or sodium salicylate resulted in secretion of CysLTs and LTB4 in the absence of EDN release. Low doses of PGE2 inhibited LTB4 and CysLT release, an effect lost at higher PGE2 concentrations. Use of butaprost, an EP2 receptor agonist, suppressed lysine aspirin stimulation. This mechanism was supported by blocking activity of the EP1 and EP3 receptors. ConclusionEosinophils can be directly activated by NSAIDs via cyclooxygenase-independent pathways to produce CysLTs and LTB4. This effect can be inhibited by PGE2 acting through the EP2 receptor. The recognized loss of EP2 receptor expression combined with low PGE2 levels explains in part the sensitivity to NSAIDs.

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Cysteinyl leukotrienes(cysLTs) facilitate type 2 immunity by incompletely understood mechanisms. Aspirin exacerbated respiratory disease(AERD) is typified by marked cysLT-overproduction, accompanied by cysLT-driven reactions to aspirin and exaggerated type 2 respiratory inflammation. We sought to determine the receptors and mechanisms by which cysLTs contribute to type 2 immunopathology. We studied the effects of each cysLT and their receptors in two different models: a model in which exogenous cysLTs amplify pulmonary inflammation in ovalbumin-sensitized mice, and another in which endogenous cysLT generation is dysregulated by deletion of microsomal-prostaglandinE2 synthase(ptges-/-mice), resulting in an AERD-like phenotype. Intranasally administered cysLTs increase OVA-induced airway eosinophilia in wild-type mice with a rank order of LTC4>LTE4>>LTD4. The effects of LTC4 were mimicked by a CysLT2R-selective agonist. Cysltr2-/- mice showed dramatically blunted responses to LTC4. Although all three cysLTs expanded intrapulmonary ILC2s, only LTC4 substantially induced IL-33 expression. LTC4 also caused platelet-dependent release of high-mobility-group-box 1(HMGB1), which drove platelet activation, thromboxane production, and endothelial adhesion receptor expression downstream of CysLT2R-driven IL-33. Deletion or blockade of CysLT2R in ptges-/- mice suppressed dust mite-induced pulmonary eosinophilia, IL-33 expression and lung-ILC2s expansion. Blockade of CysLT2R or neutralization of HMGB1 prevented all features of reactions to aspirin challenges in ptges-/- mice, including cysLT release, platelet and mast cell activation. CysLT2R controls IL-33 induction and downstream effects on platelets(through HMGB1) and ILC2s expansion, respectively, both essential for type 2 immunopathology and AERD-like physiology. Drugs that target CysLT2R may show efficacy in AERD in particular and type 2 diseases in general.

Bisphenol A at concentrations relevant to human exposure enhances histamine and cysteinyl leukotriene release from bone marrow-derived mast cells

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxide resin, acts as an endocrine-active compound and has been shown to enhance the inflammatory response to allergen challenge. Previous reports in rodents have demonstrated that perinatal BPA exposure alters airway inflammation following sensitization and challenge to ovalbumin in juvenile and adult offspring. Additionally, a high concentration of BPA has been shown to enhance mediator release in mast cell lines. This study aimed to determine if short-term BPA exposure, at levels relevant to human exposure, enhances mast cell release of histamine and cysteinyl leukotrienes (CysLTs). Primary murine bone marrow-derived mast cells (BMMC) produced from the femurs of female C57BL/6 mice were stimulated with BPA or estradiol (E2) in vitro. It was observed that both BPA and E2 increased BMMC histamine release over a range of nanomolar concentrations (1–1000 nM). The estrogen receptor (ER) antagonist ICI 182,780 partially blocked the ability of E2, but not BPA, to elevate histamine release. BPA also increased CysLT release, which was not abrogated by ER inhibition. It was also observed that the ability of BPA to enhance histamine and CysLT release was inhibited by blocking the extracellular signal-regulated kinase (ERK) pathway with U0126 or by chelating extracellular calcium (Ca2+) using EGTA. In summary, these experiments are the first to demonstrate that acute BPA exposure enhances mast cell histamine and CysLT release in vitro—an effect that is not dependent on an ER-mediated mechanism. Instead, BPA-induced mast cell histamine and CysLT release may be mediated, in part, by the ERK pathway and extracellular Ca2+ concentrations. These data suggest that exposure to BPA at levels relevant to human exposure may provoke an acute inflammatory response in atopic individuals via enhanced mast cell activation.

Different potency of bacterial antigens TLR2 and TLR4 ligands in stimulating mature mast cells to cysteinyl leukotriene synthesis

The aim of study was to compare the potency of different bacterial antigens to induce rat mature mast cell to cysteinyl leukotriene (cysLT) generation. We examined Toll-like receptor (TLR)2 agonists, i.e. lipoteichoic acid (LTA) Staphylococcus faecalis, Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus, lipoarabinomannan (LAM) Mycobacterium smegmatis, peptydoglican (PGN) Staphylococcus aureus, as well as TLR4 agonists, i.e. lipopolysaccharide (LPS) Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Pophyromonas gingivalis and Escherichia coli. We also estimated the effect of tumor necrosis factor (TNF)-, interleukin (IL)-6-, CCL5-, and IL-10-priming on mast cell cysLT synthesis following bacterial antigen activation. We found that all bacterial antigens activated mast cells to cysLT generation; however, the extent of cysLT release in response to stimulation varied. Out of the examined antigens LPS P. gingivalis exhibited the highest potency, as it induced cysLT generation acting at a very low concentration (10(-4) ng/mL). Other LPSs affected mast cells at higher (up to 10(5) -fold) concentrations. LTAs were the most effective at concentrations of 5 × 10(2) ng/mL, while LAM and PGN stimulated mast cells to maximal cysLT generation at concentrations as high as 10(5) ng/mL. Anti-TLR2 and anti-TLR4 antibodies, as well as nuclear factor κB (NF-κB) inhibitor significantly diminished cysLT generation in response to bacterial antigen stimulation. Priming with TNF, IL-6 and CCL5 did not affect bacterial antigen-induced cysLT generation, while IL-10-pretreatment caused significant decrease in cysLT synthesis by mast cells. These observations might have a great pathophysiological importance; inasmuch cysLTs strongly influence the development and intensity of inflammation during bacterial infection.

Montelukast during primary infection prevents airway hyperresponsiveness and inflammation after reinfection with respiratory syncytial virus.

Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway. To determine the effects of montelukast (MK), a cysteinyl leukotriene (cysLT) receptor antagonist, in primary and secondary RSV-infected newborn and adult mice. BALB/c mice were infected with RSV at 1 week (neonate) or 6 to 8 weeks (adult) of age and reinfected 5 weeks later. MK was administered 1 day before the initial infection and through Day 6 after infection. Seven days after primary or secondary infection, airway function was assessed by lung resistance to increasing doses of inhaled methacholine; lung inflammation, goblet cell metaplasia, and cytokine levels in bronchoalveolar lavage fluid were monitored. RSV infection induced cysLT release in bronchoalveolar lavage fluid. MK decreased RSV-induced AHR, airway inflammation, and increased IFN-gamma production in primary infected adult and neonatal mice. MK, administered during initial infection of neonates but not during secondary infection, prevented subsequent enhancement of AHR, airway eosinophilia, and mucus hyperproduction upon reinfection. MK attenuated the initial responses to primary RSV infection in both age groups and altered the consequences of RSV reinfection in mice initially infected as neonates. These data support an important role for cysLT in RSV-induced AHR and inflammation.

P2X7Receptor Activation in Rat Brain Cultured Astrocytes Increases the Biosynthetic Release of Cysteinyl Leukotrienes

Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured astrocytes P2X7ATP receptors may participate in the control of CysLT release thus further supporting a role for extracellular ATP as an integral component of the inflammatory brain response.

Roflumilast Inhibition of Pulmonary Leukotriene Production and Bronchoconstriction in Ovalbumin-Sensitized and -Challenged Guinea Pigs

This study investigated the effects of roflumilast, a PDE4 inhibitor, on slow-reacting substance of anaphylaxis (SRS-A)-mediated bronchoconstriction and pulmonary leukotriene (LT) release in ovalbumin (OVA)-sensitized and -challenged guinea pigs. Animals were treated with roflumilast orally (0.04, 0.12, 0.4, or 4 mg/kg) or placebo 1 hour before OVA challenge. Bronchoconstriction was quantified by measuring airway conductance (Gaw) and dynamic lung compliance (Cdyn). Roflumilast significantly attenuated the decrease in Gaw (50% inhibitory dose [ID50] = 0.33 mg/kg) and Cdyn (ID50 = 0.25 mg/kg) in a dose-dependent manner and significantly inhibited Cys-LT (ID50 = 0.06 mg/kg) and LTB4 (ID50 = 0.05 mg/kg) release versus placebo-treated animals. Roflumilast did not affect LTD4-induced bronchoconstriction. These findings support the role of roflumilast as an anti-inflammatory treatment for asthma.

Autocrine activation of eosinophil transmigration with cyteinyl leukotrienes

Rationale We have previously reported that airway epithelial cells promote eosinophil transmigration by utilizing a novel transmigration system and that CCR3 ligands and extracellular matrix proteins derived from the epithelial cells support migration. To further analyze the interaction of eosinophils and airway epithelial cells, we investigated possible involvement of cysteinyl leukotrienes in this system. Methods A three-dimensional eosinophil transmigration system consisted of cultured airway epithelial cells and type I collagen matrix (Kato Y et al. Clin Exp Allergy 2002;32:889) was used. Eosinophils were preincubated with a CysLT1 receptor antagonist, montelukast, before being subjected to transmigration experiments and eosinophil migration was evaluated. Epithelial supernatants from the system were then added to eosinophils and gene expression of LTC4 synthase and CysLT production from the eosinophils were measured with real-time RT-PCR and ELISA, respectively. Cytokines in the epithelial supernatants were measured with a beads microarray system and gene expression from the cells were quantified with real-time RT-PCR. Results Montelukast inhibited epithelial cell-promoted eosinophil transmigration in a concentration dependent manner. The epithelial supernatants induced LTC4 synthase gene expression and CysLT release from eosinophils. To identify factors induce CysLT production, a panel of cytokines in the supernatants were quantified with a beads microarray and real-time PCR. GM-CSF was found to be a major cytokine and anti-GM-CSF blocked epithelial supernatant-induced eosinophil CysLT production. Conclusions Airway epithelial cells induce cysLT production from eosinophils by releasing GM-CSF, consequently enhance eosinophil migration through autocrine activation mechanism. CysLT1R antagonists may interfere with these interactions.

In-vitro-Leukotrienproduktion gemessen im CAST-ELISA über einen Beobachtungszeitraum von 5 Monaten

Background: The Cellular Antigen Stimulation Test (CAST; Buhlmann Laboratory AG, CH) is a diagnostic tool to quantify the In-vitro-Cystinly-Leukotriene production (= CysLT: LTC 4 , LTD 4 , LTE 4 ) from isolated leukocytes.The leukotrienes are responsible for the early inflammatory response with mucosal edema and smooth muscle contraction, clinically resulting in allergic diseases such as asthma and rhinitis. Objective: To evaluate seasonal effects of the in-vitro CysLT release. Population and methods: Blood samples were taken monthly from 9 children and young adults (7 - 18 years; 3 without allergic sensitization, 2 with sensitization to house dust mite and 4 with grasspollen sensitization) from February to June. Leukocytes were separated using Dextran sedimentation and stimulated either with medium alone (negative control), or with an Anti-IgE-receptor-antibody (Anti-IgE-rAK; 0.5 μg/mL; positive control) or with grass-pollen allergen (Six-Gras-MIX, GX1, 20 ng/ml). The CysLT release (LTC 4 , LTD 4 , LTE 4 ) was quantified using ELISA technique (CAST-ELISA, Buhlmann Laboratory AG, Schonenbuch, Switzerland). Results: After stimulation with Anti-IgE-rAK in-vitro CysLT-production increased from March (range: 1163 und 6187 pg/ml) to May (range: 2048 and 7620 pg/ml; median increase 63%; range: 35% -114%; p = 0.012). In response to grasspollen allergen we observed a similar increase from March (158 und 208 pg/ml) to May (337 und 7057 pg/ml; median increase 49.9%; range: 38 - 133%; p = 0.004). The median in-vitro CysLT-concentrations after stimulation with grasspollen was 6137 pg/ml (range: 4249-7620) in grass-pollen sensitized persons and 2130 pg/ml (2048 - 5421) in persons, who are not allergic to grasspollen. Conclusion: We observed a significant higher in-vitro CysLT production in May compared to March. Seasonal effects of the CysLT release must be considered performing longitudinal studies.

Cysteinyl leukotrienes modulate angiotensin II constrictor effects on aortas from streptozotocin-induced diabetic rats.

Angiotensin II (Ang II) is a vasopressor peptide involved in the pathogenesis of cardiovascular diseases associated with diabetes mellitus. We have previously reported that the 5-lipoxygenase-derived products, particularly the cysteinyl leukotrienes (CysLTs), are involved in Ang II-induced contraction. In this study, we demonstrated that CysLTs contribute to the contraction elicited by Ang II in isolated aortas from streptozotocin-induced diabetic (SS) rats but not from insulin-treated diabetic rats, fructose-fed rats, or control rats. In an organ bath, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/L) reduced by 37.6+/-8.2% and 30.1+/-10.9% the Ang II-induced contractions in intact and endothelium-denuded aortic rings, respectively, from SS rats. In contrast, the CysLT(1) receptor antagonist (MK571, 1 micromol/L) or the dual CysLT(1)/CysLT(2) receptor antagonist (BAY-u9773, 0.1 micromol/L) did not affect Ang II-induced contraction. In addition, Ang II induced a 6.2+/-1.5-fold increase in CysLT release through the stimulation of the Ang II type 1 receptor. Furthermore, the urinary excretion of leukotriene E(4) was increased in SS rats (leukotriene E(4), 13.7+/-2.9 ng/24 h [SS rats, n=10] versus 1.5+/-0.5 ng/24 h [control rats, n=6]; P<0.0004). These data suggest the activation of the 5-lipoxygenase pathway in SS rats and the involvement of 5-lipoxygenase-derived products, particularly the CysLTs, in Ang II-induced contraction in aortas from SS rats through stimulation of CysLT receptors different from the well-characterized CysLT(1) or CysLT(2) receptor.

Cysteinyl-Leukotriene Levels in Intracerebral Hemorrhage:An Edema-Promoting Factor?

In 12 operatively (stereotactic aspiration and recombinant tissue-type plasminogen activator, rTPA) and 5 conservatively treated patients with spontaneous intracerebral hemorrhage the amounts of cysteinyl-leukotriene (cys-LT) released by blood-brain cell contact were measured by the urinary excretion of their metabolites during treatment. The mean cys-LT release before treatment was 14.51 ± 1.13 pg/mg creatinine/ml hematoma volume. The urinary cys-LT excretion at the end of the measurements was significantly lower in the operatively treated group than in the patients with conservative therapy (p < 0.05). We also found a significant correlation between the perifocal edema volume and the amount of cys-LT measured in patients’ urine (p < 0.01). In an additional animal experiment using dissociated rat brain cells plasmin was excluded as an activator for cerebral cys-LT formation, which emphasizes that rTPA did not influence cys-LT formation.

Allergen-induced late-phase airways obstruction in the pig: mediator release and eosinophil recruitment.

The aim of this study was to develop a novel model for studies of mediator mechanisms involved in the late asthmatic reaction in the lower airways, by using the sensitized pig. The release of histamine and cysteinyl-containing leukotrienes (cys-LTs), as well as the levels of inflammatory cells in blood and bronchoalveolar lavage fluid, were determined and their relationship to plasma cortisol levels and pulmonary airways obstruction was noted. Specific-pathogen free pigs were actively sensitized with Ascaris suum allergen, and one group of animals was treated with a cortisol-synthesis inhibitor (metyrapone) by constant intravenous infusion. Ascaris suum allergen was nebulized into the lower airways and total lung resistance, blood leucocyte count and urinary levels of methylhistamine and leukotriene E4 (LTE4) were followed for 8 h, whereafter bronchoalveolar lavage was performed for analysis of leucocytes. An increase in urinary methylhistamine and LTE4 was seen during the acute allergic reaction in both groups of pigs. Metyrapone treatment prolonged the acute release of histamine, and this was seen together with a prolonged acute bronchoconstrictor response. In metyrapone-treated pigs, a continuous release over 8 h was seen for cys-LTs, but not for histamine. A late blood eosinophilia was also seen in metyrapone-treated animals, starting 4-6 h after allergen challenge. Late cys-LT release and eosinophilia were absent in non-metyrapone-treated animals. These results suggest that allergen-induced late release of cys-LTs as well as blood eosinophilia occur simultaneously with late-phase airways obstruction in the pig, and that all these reactions are prevented by high levels of endogenous cortisol.