Production Of CysLTs Research Articles

Exercise but not mannitol provocation increases urinary Clara cell protein (CC16) in elite swimmers

Elite swimmers have an increased risk of developing asthma, and exposure to chloramine is believed to be an important trigger factor. The aim of the present study was to explore pathophysiological mechanisms behind induced bronchoconstriction in swimmers exposed to chloramine, before and after swim exercise provocation as well as mannitol provocation. Urinary Clara cell protein (CC16) was used as a possible marker for epithelial stress. 101 elite aspiring swim athletes were investigated and urinary samples were collected before and 1h after completed exercise and mannitol challenge. CC16, 11β-prostaglandin (PG)F(2α) and leukotriene E(4) (LTE(4)) were measured. Urinary levels of CC16 were clearly increased after exercise challenge, while no reaction was seen after mannitol challenge. Similar to CC16, the level of 11β-PGF(2α) was increased after exercise challenge, but not after mannitol challenge, while LTE(4) was reduced after exercise. There was no significant difference in urinary response between those with a negative compared to positive challenge, but a tendency of increased baseline levels of 11β-PGF(2α) and LTE(4) in individuals with a positive mannitol challenge. The uniform increase of CC16 after swim exercise indicates that CC16 is of importance in epithelial stress, and may as such be an important pathogenic factor behind asthma development in swimmers. The changes seen in urinary levels of 11β-PGF(2α) and LTE(4) indicate a pathophysiological role in both mannitol and exercise challenge.

Comparison of the inhibitory effects of resveratrol and tranilast on IgE, 48/80 and substance P dependent-mast cell activation

.Resveratrol and tranilast blocked cytokine formation,reducing substance P-induced TNF production (65%;P=0.04 and 46%; P=0.09, respectively), but not MCP-1production. Furthermore, resveratrol inhibited Fcepsi-lonRI mediated production of cysLT by 31% compared tocontrol, whereas tranilast had no effect. The effects ofresveratrol on degranulation and release of cysLT weremore marked in human primary mast cells (HuMC)(64% and 90% inhibition, respectively; P<0.05), and thepolyphenol was found to be significantly more efficaciousthan tranilast in these cells.ConclusionsResveratrol inhibited mast cell function at the level ofdegranulation, and cytokine and cysLT production, andwas comparable, and in some cases, more potent thanthe anti-allergy drug tranilast. Thus resveratrol may bean effective therapeutic agent for the treatment of aller-gic disease.

Treatment With the Leukotriene Inhibitor Montelukast for 10 Days Attenuates Portal Hypertension in Rat Liver Cirrhosis

The mechanisms underlying intrahepatic vasoconstriction are not fully elucidated. Here we investigated the Kupffer cell (KC)-dependent increase in portal pressure by way of actions of vasoconstrictive cysteinyl leukotrienes (Cys-LTs). Liver cirrhosis was induced in rats by bile duct ligation (BDL for 4 weeks; controls: sham-operation) and thioacetamide application (18 weeks). Infusion of leukotriene (LT) C(4) or LTD(4) in isolated perfused livers (20 nM, BDL and sham) demonstrated that LTC(4) is a more relevant vasoconstrictor. In BDL animals the Cys-LT(1) receptor inhibitor montelukast (1 microM) reduced the maximal portal perfusion pressure following LTC(4) or LTD(4) infusion. The infusion of LTC(4) or D(4) in vivo (15 microg/kg b.w.) confirmed LTC(4) as the more relevant vasoconstrictor. Activation of KCs with zymosan (150 microg/mL) in isolated perfused BDL livers increased the portal perfusion pressure markedly, which was attenuated by LT receptor blockade (Ly171883, 20 microM). Cys-LTs in the effluent perfusate increased with KC activation but less with additional blockade of KCs with gadolinium chloride (10 mg/kg body weight, 48 and 24 hours pretreatment). KCs were isolated from normal rat livers and activated with zymosan or lipopolysaccharide at different timepoints. This resulted in an increase in Cys-LT production that was not influenced by preincubation with montelukast (1 microM). Infusion of LTC(4) (20 nM) and the thromboxane analog U46619 (0.1 microM) further enhanced portal pressure, indicating additive effects. Treatment with montelukast for 10 days resulted in an impressive reduction in the basal portal pressure and an attenuation of the KC-dependent increase in portal pressure. Activation of isolated KCs produced Cys-LTs. Infusion of Cys-LTs increased portal pressure and, vice versa, treatment with montelukast reduced portal pressure in rat liver cirrhosis. Therefore, montelukast may be of therapeutic benefit for patients with portal hypertension.

Pathogenic Importance of Cysteinyl Leukotrienes in Development of Gastric Lesions Induced by Ischemia/Reperfusion in Mice

We examined the role of cysteinyl leukotrienes (CysLTs) in the gastric ulcerogenic response to ischemia/reperfusion (I/R) in mice. Experiments were performed in male C57BL/6J mice after 18-h fasting. Under urethane anesthesia, the celiac artery was clamped for 30 min, and then reperfusion was achieved by removing the clamp. The stomach was examined for lesions 60 min thereafter. The severity of I/R-induced gastric damage was reduced by prior administration of pranlukast [CysLT receptor type 1 (CysLT(1)R) antagonist] as well as 1-[[5'-(3''-methoxy-4''-ethoxycarbonyl-oxyphenyl)-2',4'-pentadienoyl]aminoethyl]-4-diphenylmethoxypiperidine [TMK688; 5-lipoxygenase (5-LOX) inhibitor]. On the contrary, these lesions were markedly worsened by pretreatment with indomethacin, and this response was abrogated by the coadministration of TMK688 or pranlukast. The gene expression of CysLT(1)R but not 5-LOX was up-regulated in the stomach after I/R, but both expressions were increased under I/R in the presence of indomethacin. I/R slightly increased the mucosal CysLT content of the stomach, yet this increase was markedly enhanced when the animals were pretreated with indomethacin. The increased CysLT biosynthetic response to indomethacin during I/R was attenuated by TMK688. Indomethacin alone caused a slight increase of CysLT(1)R expression and markedly up-regulated 5-LOX expression in the stomach. We concluded that I/R up-regulated the expression of CysLT(1)R in the stomach; CysLTs play a role in the pathogenesis of I/R-induced gastric damage through the activation of CysLT(1)R; and the aggravation by indomethacin of these lesions may be brought about by the increase of CysLT production and the up-regulation of 5-LOX expression, in addition to the decreased prostaglandin production.

Leukotriene pathway polymorphisms are associated with altered cysteinyl leukotriene production in children with acute asthma

Cysteinyl leukotrienes (cysLTs) are pro-inflammatory mediators with increasing evidence for a role in childhood acute asthma. This study examined the influence of polymorphisms in cysLT pathway genes on urinary leukotriene E 4 (uLTE 4) levels and clinical status in acute asthmatic children. Children aged 2–16 years were recruited during an asthma attack ( n=205). Where possible, asthma severity scores were assigned, ALOX5AP G-336A, ALOX5 G-1708A, LTC4S A-444C and G-1072A, GPX4 C718T, and CYSTLTR1 T927C genotypes were determined and uLTE 4 was measured in acute and convalescent samples. uLTE 4 levels were higher acutely compared with convalescence (acute GM: 115.7 pg/mg creatinine; 95% CI 88.6–151.1, convalescence GM: 66.4 pg/mg creatinine; 95% CI 51.5–85.6; n=50 paired samples, p=0.003) and paired sample analysis showed genotype-specific effects with significantly increased uLTE 4 for LTC 4S -444AA (acute GM: 127.9 pg/mg creatinine; 95% CI 91.8–178.3, convalescence GM: 68.2 pg/mg creatinine; 95% CI 50.5–92.0; n=32, p=0.002), LTC 4S -1072 GG (acute GM: 126.7 pg/mg creatinine; 95% CI 95.4–168.3, convalescence GM: 78.9 pg/mg creatinine; 95% CI 59.7–104.1; n=39, p=0.019) and CYSLTR1 927 TT/T_ (acute GM: 96.8 pg/mg creatinine; 95% CI 73.8–126.9, convalescence GM: 62.4 pg/mg creatinine; 95% CI 46.8–83.3; n=28, p=0.036) but not AC/CC, GA/AA, or TC/CC/C_, respectively. When we compared the allele frequencies of the CYSLTR1 SNP between asthmatics and non-asthmatics, the 927C allele was found to be a risk allele for asthma (OR=2.13, 95% CI: 1.06–4.26, p=0.033). Genotypes were not associated with acute or convalescent uLTE 4 levels alone and neither the SNPs nor uLTE 4 correlated with acute asthma severity. Leukotriene pathway gene polymorphisms may influence the magnitude of cysLT production during an attack, yet their influence alone may not be substantial enough to alter the severity of exacerbations.

Autocrine production of cysteinyl leukotrienes activates human mast cells (139.13)

Abstract Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid mediators which are derived from 5-lipoxygenase activity. CysLTs mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability, increased mucus secretion, decreased mucociliary clearance, eosinophil migration, and increased eosinophil survival. We now report that peripheral blood CD34+ progenitor-derived human mast cells and the human mast cell line (LAD2) express both CysLT receptors (CysLT1R and CysLT2R), the G protein-coupled receptors that bind CysLTs. LTC4, LTD4 and LTE4 activated LAD2 to produce MIP-1β and MCP-1 and production of these chemokines was inhibited by the CysLT1R inhibitor, monelukast. LAD2 activated by IgE/anti-IgE produced CysLT (980 + 55 pg/million cells) but LAD2 activated by substance P, a neuropeptide that also binds a G protein-coupled receptor, did not produce CysLT. Montelukast had no effect on SP-mediated LAD2 degranulation. CysLT did not induce human mast cell degranulation but montelukast inhibited FcεRI-mediated human mast cell degranulation by approximately 20% suggesting that autocrine production of CysLT potentiated FcεRI-dependent degranulation. IL-4 (10 ng/mL) in the presence of stem cell factor (SCF; 100 ng/mL) significantly upregulated expression of both CysLT1R and CysLT2R (16% and 32% respectively) and potentiated mast cell responses to LTE4 by up to 25%. Overall, these results show that human mast cell activation and degranulation is subject to autocrine CysLT-induced signaling which can be blocked by CysLT1R inhibitors such as montelukast. Funded by the Investigator Initiated Studies Program (IISP) from Merck and Company, Inc.

Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells

House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRgamma-/- mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRgamma/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.

Role of leukotrienes in exercise-induced bronchoconstriction

Exercise-induced bronchoconstriction (EIB) refers to acute airflow obstruction that is triggered by a period of physical exertion. EIB occurs mainly in individuals with other features of asthma but is especially prominent in a subset of asthmatics with pronounced indirect airway hyperresponsiveness. Leukotrienes (LTs) play a critical role in the pathophysiology of EIB. Asthmatics who are susceptible to EIB have increased levels of cysteinyl LTs (cysLTs [ie, LTs C4, D4, and E4]) in induced sputum and exhaled breath condensate. Exercise challenge in individuals susceptible to this disorder initiates the sustained increase in cysLTs in the airways and an increase in the ratio of cysLTs to prostaglandin E(2). The effects of cysLTs leading to secreted mucin release and smooth muscle constriction may be mediated in part through activation of sensory nerves. Therapies that block cysLT production or the cysLT(1) receptor effectively reduce the severity of EIB.

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D 4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT 1R and CysLT 2R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF- α), and leukotriene D 4 (LTD 4), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC 4 synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF- α and LTD 4, to a lesser extent, up-regulate the CysLT 1R levels. Interestingly, TNF- α also reduced CysLT 2R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.

Expression of kinin receptors on eosinophils: comparison of asthmatic patients and healthy subjects

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B(1) and B(2) receptors (B(1)R and B(2)R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca(2+) was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg(9)]-BK were assessed. Eosinophils expressed kinin B(1)R and B(2)R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B(1)R and B(2)R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B(1)R and B(2)R mRNA expression did not differ significantly between these groups. Expression of kinin B(1)R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B(1) and B(2) agonists up-regulated expression of their respective receptors but did not increase intracellular Ca(2+) or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B(1)R.

Placenta Growth Factor Induces 5-Lipoxygenase-Activating Protein Via Hypoxia-Inducible Factor-1α and Contributes to Increased Leukotrienes in Sickle Cell Disease.

Subjects with sickle cell disease (SCD) have a chronic baseline inflammation and higher incidence of lung disease – airway hyper-reactivity predominates in children and restrictive lung disease in adults. The degree of inflammation, reflected in leukocytosis and leukocyte activation, correlates with higher mortality (Platt, NEJM, 1994). Stuart and colleagues have shown high plasma and urinary leukotriene (LT)-B4 at steady state, and even higher levels during acute sickle events and acute chest syndrome (J Lab Clin Med 2002). DeBaun and colleagues have shown high levels of urinary LT-E4 in this patient population (Am J Hematol, 2008). LT-B4 is one of the most potent activator of neutrophils, while cysteinyl leukotrienes (cys-LT), LT-C4, LT-D4 and LT-E4, promote bronchial smooth muscle constriction and asthma. LT are produced from arachidonic acid through a series of metabolic steps, with two critical steps catalyzed by five-lipoxygenase (5LO) and 5-lipoxygenase activating protein (FLAP). We have previously shown that that placenta growth factor (PLGF) is produced at high levels from the hyperplastic erythroid compartment in SCD and thalassemia and induces a strong proinflammatory cytochemokine response from monocytes (Perelman et al, Blood 2003; Selvaraj et al, Blood 2003). PLGF levels were high in subjects with SCD, and associated with activated mononuclear cells with augmented expression of pro-inflammatory cytochemokines (Perelman et al, Blood 2003). We now show PLGF induces FLAP expression. FLAP mRNA was significantly higher in peripheral blood mononuclear cells (PBM) of subjects with SCD at steady state, compared to healthy controls. PLGF activated PBM to increase FLAP expression and LT-B4 formation. It also increased Cys-LT production from PBM and the THP-1 monocytic cell line. PLGF-mediated increased FLAP expression was attenuated by PI-3 kinase inhibitor and PTEN; and by NADPH-oxidase inhibitor and p47phox siRNA in THP-1 cells. An in silico analysis of the FLAP promoter showed presence of hypoxia response elements, CEBP and NFκB sites. We found that FLAP induction by PLGF was mediated via HIF-1α. PLGF induced HIF-1α mRNA expression. Silencing with HIF-1α siRNA attenuated PLGF mediated FLAP expression, while over expression of HIF-1α had the converse effect. PLGF augmented FLAP-promoter luciferase activity by several folds, and this effect was abrogated upon mutation of hypoxia-response element (HRE), but not the NF-κB binding site in the FLAP proximal promoter. These studies indicate the role of HIF-1α motif in inducing FLAP expression, which was confirmed by electrophoretic mobility shift assays. Chromatin immunoprecipitation (ChIP) analysis further confirmed the role of HIF-1a in regulating PLGF-mediated FLAP expression. Our studies identify, for the first time, HIF-1α as a hypoxia-independent target of PLGF, which then upregulates FLAP and increases LT formation. Our studies define a mechanism of increased LT in SCD. The intrinsic elevated levels of PLGF seen in SCD could explain the increased leukotriene levels observed in patients with SCD, thereby contributing to the baseline inflammation and pulmonary pathology present in this disease. LT inhibitors are FDA approved in asthma, and could be used as a new modality to ameliorate inflammation and lung complications in SCD.

Increased levels of cysteinyl-leukotrienes in saliva, induced sputum, urine and blood from patients with aspirin-intolerant asthma

Background:A diagnosis of aspirin-intolerant asthma requires aspirin provocation in specialist clinics. Urinary leukotriene E4 (LTE4) is increased in aspirin-intolerant asthma. A study was undertaken to investigate new biomarkers of aspirin...

Effect of secondhand cigarette smoke, RSV bronchiolitis and parental asthma on urinary cysteinyl LTE4

Cysteinyl leukotrienes promote airway inflammation, bronchoconstriction and mucus hypersecretion. Cigarette smoking and respiratory syncytial virus (RSV) bronchiolitis are known to increase urinary cysteinyl leukotriene E4 (uLTE4), the end product of the cysteinyl leukotriene biosynthetic pathway. We tested the following hypotheses: (1) Secondhand smoke (SHS) exposure increases uLTE4 in well infants and in those hospitalized for RSV bronchiolitis; (2) Length of hospital stay for those with RSV bronchiolitis correlates with uLTE4; and (3) Infants with parent(s) with asthma will have higher uLTE4. Parental asthma for infants hospitalized with RSV bronchiolitis (n = 79) and Well babies (n = 31) was determined by questionnaire. Urine was analyzed for LTE4, cotinine, and creatinine. SHS exposure was determined by cotinine to creatinine ratio. Chi square, or t-tests were used to determine significant differences between two groups. A three-way analysis of variance compared the effects of SHS exposure and parental asthma on uLTE4 in Well versus RSV babies. Independent variables predicting length of hospital stay were determined by stepwise multiple regression. High SHS exposure and RSV significantly increased uLTE4. The SHS induced increase in uLTE4 was seen in infants with no parental asthma but not in those with parental asthma. Length of hospital stay positively correlated with uLTE4. We concluded that SHS exposure may increase the severity of bronchiolitis in RSV-infected infants by enhancing production of cysLTs in infants with no parental asthma.

Corticosteroids as inhibitors of cysteinyl leukotriene metabolic and signaling pathways

Corticosteroids (CCSs) do not influence secretion of cysteinyl leukotrienes (CysLTs) that occurs on cellular activation during allergic reactions nor do they modulate bronchospastic responses to inhalation challenges with leukotrienes (LTs). We speculated that CCSs might modulate pathways responsible for CysLT production and diminish the ability of cellular activation to cause their release. Similarly, CCSs could reduce expression of CysLT receptor 1 (CysLTR1) and CysLT receptor 2 (CysLT2R) and modulate their responsiveness. We investigated influences of fluticasone on expression of mRNA for LTC(4) synthase (LTC(4)S), CysLT1R, and CysLT2R within T lymphocytes, monocytes, and eosinophils by means of quantitative PCR. Effects on receptor protein expression were evaluated by means of flow cytometry. Circulating immune cells (T cells, monocytes, and eosinophils) express low levels of LTC(4)S mRNA, and this was not influenced by CCSs. However, IL-4 induced transcripts in T lymphocytes, and this was prevented by fluticasone. Paradoxically, CCSs synergized with IL-4 to increase LTC(4)S expression in monocytes. Although not influencing basal or IL-4-stimulated CysLT1R expression, fluticasone inhibited basal CysLT2R transcript expression on monocytes and IL-4-induced expression in all 3 cell types. In addition to not blocking the acute release of CysLTs on cellular activation, CCSs do not diminish the capacity of cells to synthesize these compounds. CCSs do not diminish CysLT1R expression, consistent with their lack of influence on bronchospasm, which is mediated through this receptor.

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures

Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling. BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed. Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2). Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.

Dendritic Cell Production of Cysteinyl Leukotrienes in Response to Allergens

The cysteinyl leukotrienes (cys-LTs) are powerful inflammatory mediators that can be produced by both murine and human dendritic cells (DCs) in response to extracts from house dust mite, but the molecular mechanism is unknown. We have evaluated the requirements for cys-LT generation by murine bone marrow-derived DCs (BMDCs) in response to allergen extracts from the fungus Aspergillus fumigatus (Af), and the house mites Dermatophagoides pteronyssinus (Dp), and Dermatophagoides farinae (Df). BMDCs were generated with granulocyte macrophage colony stimulating factor and evaluated between day 8 and 10. BMDCs produced cys-LTs to extracts from Af, Dp, and Df that peaked by 30 min. There was no response to ovalbumin with or without lipopolysaccharide, to TLR2 agonists, or to NOD agonists. Treatment of the Df extract with heat, nucleases, or proteases did not diminish its activity, and after chloroform-methanol extraction the activity was retained in the aqueous phase. Recombinant Df did not elicit cys-LT production. These results demonstrate that DCs recognize a highly polar, non-proteinous agonist(s) in several common allergens that elicits a rapid and robust production of cys-LTs.

Aspirin sensitivity and eicosanoids

Aspirin sensitive respiratory disease (ASRD) was first described in 1922 by the French physician Widal.1 It is characterised by asthma, chronic rhinosinusitis and nasal polyps on a background of aspirin...

Cysteinyl Leukotrienes Are Autocrine and Paracrine Regulators of Fibrocyte Function

Pulmonary fibrosis is characterized by the accumulation of fibroblasts and myofibroblasts. These cells may accumulate from three potential sources: the expansion of resident lung fibroblasts, the process of epithelial-mesenchymal transition, or the recruitment and differentiation of circulating mesenchymal precursors known as fibrocytes. We have previously demonstrated that fibrocytes participate in lung fibrogenesis following administration of FITC to mice. We now demonstrate that leukotriene-deficient 5-LO(-/-) mice are protected from FITC-induced fibrosis. Both murine and human fibrocytes express both cysteinyl leukotriene receptor (CysLT) 1 and CysLT2. In addition, fibrocytes are capable of producing CysLTs and can be regulated via the autocrine or paracrine secretion of these lipid mediators. Exogenous administration of leukotriene (LT) D(4), but not LTC(4) induces proliferation of both murine and human fibrocytes in a dose-dependent manner. Consistent with this result, CysLT1 receptor antagonists are able to block the mitogenic effects of exogenous LTD(4) on fibrocytes. Endogenous production of CysLTs contributes to basal fibrocyte proliferation, but does not alter fibrocyte responses to basic fibroblast growth factor. Although CysLTs can induce the migration of fibrocytes in vitro, they do not appear to be essential for fibrocyte recruitment to the lung in vivo, possibly due to compensatory chemokine-mediated recruitment signals. However, CysLTs do appear to regulate the proliferation of fibrocytes once they are recruited to the lung. These data provide mechanistic insight into the therapeutic benefit of leukotriene synthesis inhibitors and CysLT1 receptor antagonists in animal models of fibrosis.

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen–glucose deprivation

We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT(1) and CysLT(2) receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24-72 h after recovery. Inhibition of phospholipase A(2) and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.

Comparative study to elucidate the mechanism underlying the difference in airway hyperresponsiveness between two mouse strains

The mechanism underlying airway hyperresponsiveness (AHR), a characteristic feature of asthma, remains obscure. We attempted to elucidate the mechanism responsible for the different degrees of AHR in two mouse strains, BALB/c and C57BL/6, following exposure to an anaphylactic trigger. When ovalbumin (OVA)-sensitized mice were challenged daily with OVA for up to three consecutive days, the BALB/c mice showed a higher degree of airway responsiveness to methacholine than did C57BL/6. Following the OVA challenge, eosinophils and macrophages in bronchoalveolar lavage fluid (BALF) from BALB/c increased significantly in number compared to those from C57BL/6. BALB/c mice also exhibited a higher serum IgE level than that of C57BL/6 after OVA challenge. The enhanced AHR and eosinophilic infiltration in BALF were significantly reduced by pretreatment with a selective cysteinyl-leukotriene type 1 receptor (cysLT 1R) antagonist, montelukast. In the in vitro study, cysLT production was significantly lower in the dissected lung tissue from BALB/c than in tissue from C57BL/6 when both groups were stimulated with saline. The lungs from BALB/c generated significantly larger amounts of cysLTs on incubation with OVA rather than with saline, while the lungs from C57BL/6 did not show any significant increase in cysLTs with antigen stimulation. Significant upregulation of cysLT 1R and cysLT 2R mRNA expression was induced by OVA challenge in the lungs of BALB/c, but not in those of C57BL/6. It is suggested that, after an anaphylactic reaction, the degree of AHR is dependent on the genetic background and that cysLTs play an important role in the mechanism involved.