Mucosal LTE4, PGD2 and 15(S)-HETE as potential prognostic markers for polyp recurrence in chronic rhinosinusitis.

BACKGROUND: Altered biosynthesis of eicosanoids is linked to type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP), but their role in recalcitrant NPs is unclear. OBJECTIVES: We sought to identify endotypes that are linked to recalcitrant CRSwNP, based on eicosanoids, their biosynthetic enzymes, and receptors as well as cytokines and the presence of eosinophils and mast cells in recurrent NPs. METHODS: Mucosal tissue collected at the time of sinus surgery from 54 patients with CRSwNP and 12 non-CRS controls were analysed for leukotriene (LT) E4, prostaglandin (PG) D2, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 17 cytokines with ELISAs and Bio-Plex immunoassays. Patient subgroups were identified by cluster analysis and the probability of NP recurrence were tested with logistic regression analyses. Gene expressions were analysed with qPCR. Tryptase and eosinophil-derived neurotoxin (EDN) were measured with ELISAs as indications of the presence of mast cells and eosinophils, respectively. RESULTS: Clustering of patients showed that an inflammatory signature characterised by elevated LTE4, PGD2, 15(S)-HETE and IL-13 was associated with NP recurrence. Previous NP surgery as well as aspirin-exacerbated respiratory disease were significantly more common among these patients. Expression of cyclooxygenase 1 was the only gene associated with NP recurrence. Levels of EDN, but not tryptase, were significantly higher in patients with recurrent NPs. CONCLUSION: Distinguishing endotypes that include LTE4, PGD2, 15HETE and conventional biomarkers of type 2 inflammation could help predict recurrent nasal polyposis and thus identify cases of recalcitrant CRSwNP.

High levels of eicosanoids and docosanoids in the lungs of intubated COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID-19). While COVID-19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID-19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID-19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID-19 patients requiring mechanical ventilation. BALs from severe COVID-19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4 , LTE4 , and eoxin E4 . Monohydroxylated 15-lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID-19 involves pro- and anti-inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipid mediators.

Urinary Leukotriene E4 and Prostaglandin D2 Metabolites Increase in Adult and Childhood Severe Asthma Characterized by Type 2 Inflammation. A Clinical Observational Study.

Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE2 pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE2 metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE4 and the PGD2 metabolite 2,3-dinor-11ß-PGF2α. High concentrations of LTE4 and PGD2 metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).

The synergistic effects of clopidogrel with montelukast may be beneficial for asthma treatment.

Platelets modulate asthma pathogenesis by forming the platelet-eosinophil aggregation (PEA), which facilitates the activation of eosinophils. Platelets exhibit the purinergic receptor (P2Y12R), which responds to cysteinyl leukotriene E4 (LTE4 ). We have suggested that the combination of an antiplatelet drug (clopidogrel, [Clo]) and montelukast (Mon) would synergistically suppress asthma. BALB/c mice were intraperitoneally sensitized with ovalbumin (OVA) on days 0 and 14 and subsequently challenged on days 28-30 and 42-44. Mice were administered with Clo (10 mg/kg), Mon (10 mg/kg) or both drugs (Clo/Mon) orally 30 minutes before the OVA (1%) challenge on days 42-44. Mice were assayed for airway hyper-responsiveness (AHR) to methacholine and airway inflammation. Clopidogrel and montelukast attenuated the increased AHR; the combined treatment was more effective than a single treatment for total and eosinophil counts (all P < 0.05). Levels of interleukin (IL)-4, IL-5, IL-13, platelet factor 4, eosinophil peroxidase and LTE4 increased in the bronchoalveolar lavage fluid of asthmatic mice, but these levels decreased in mice treated with Clo/Mon (all P < 0.05). Goblet cell hyperplasia decreased in response to Clo/Mon. Mouse platelets and eosinophils were isolated and co-cultured for an in vitro assay with 10 µmol/L adenosine diphosphate (ADP), LTE4 (200 nmol/L), Mon (1 µmol/L), Clo (1 µmol/L) and Clo/Mon (1 µmol/L). Flow cytometry revealed that the increased formation of the PEA (%) was fully mediated by ADP and partly mediated by LTE4 . Clo/Mon reduced ADP-induced PEA formation and P-selectin expression (P < 0.05). In conclusion, Clo/Mon synergistically relieved asthma by inhibiting ADP-mediated PEA formation.

Cysteinyl leukotriene E4 activates human group 2 innate lymphoid cells and enhances the effect of prostaglandin D2 and epithelial cytokines.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood. OBJECTIVES: We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s. METHODS: For ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants. RESULTS: Human ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25. CONCLUSION: CysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Platelet activation markers overexpressed specifically in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by respiratory reactions on ingestion of COX-1 inhibitors and cysteinyl leukotriene overproduction. The hypersensitivity reaction is induced by low doses of aspirin that inhibit COX-1 in platelets. OBJECTIVE: We sought to explore the role of platelets in the pathogenesis of AERD in patients under stable conditions and during an aspirin challenge test. METHODS: Stable patients with AERD (n = 30), aspirin-tolerant asthma (ATA; n = 21), or idiopathic chronic eosinophilic pneumonia (n = 10) were enrolled. Platelet activation was estimated based on expression levels of P-selectin (CD62P), CD63, CD69, and GPIIb/IIIa (PAC-1) in peripheral platelets; percentages of circulating platelet-adherent leukocytes; and plasma levels of soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L). RESULTS: In the stable condition, expression of all surface markers on platelets, the percentage of platelet-adherent eosinophils, and the plasma levels of sP-selectin and sCD40L were significantly higher in patients with AERD compared with those in patients with ATA. P-selectin and CD63 expression on platelets and plasma sP-selectin and sCD40L levels were positively correlated with the percentage of platelet-adherent eosinophils. Among these markers, P-selectin expression and plasma sP-selectin levels positively correlated with urinary concentrations of leukotriene E4. Additionally, plasma sP-selectin and sCD40L levels were negatively correlated with lung function. In contrast, platelet activation markers in patients with AERD did not change during the aspirin challenge test. CONCLUSION: Peripheral platelets were activated more in patients with stable AERD compared with those in patients with stable ATA, patients with idiopathic chronic eosinophilic pneumonia, and control subjects. Platelet activation was involved in cysteinyl leukotriene overproduction and persistent airflow limitations in patients with AERD.

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.

Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4), a conjugation product of glutathione and eicosatetraenoic acid, and its metabolites, LTD4 and LTE4, are lipid mediators of smooth muscle constriction and inflammation in asthma. LTD4 is the most potent ligand for the type 1 cys-LT receptor (CysLT1R), and LTC4 and LTD4 have similar lesser potency for CysLT2R, whereas LTE4 has little potency for either receptor. Cysltr1/Cysltr2(-/-) mice, lacking the two defined receptors, exhibited a comparable dose-dependent vascular leak to intradermal injection of LTC4 or LTD4 and an augmented response to LTE4 as compared with WT mice. As LTE4 retains a cysteine residue and might provide recognition via a dicarboxylic acid structure, we screened cDNAs within the P2Y nucleotide receptor family containing CysLTRs and dicarboxylic acid receptors with trans-activator reporter gene assays. GPR99, previously described as an oxoglutarate receptor (Oxgr1), showed both a functional and a binding response to LTE4 in these transfectants. We generated Gpr99(-/-) and Gpr99/Cysltr1/Cysltr2(-/-) mice for comparison with WT and Cysltr1/Cysltr2(-/-) mice. Strikingly, GPR99 deficiency in the Cysltr1/Cysltr2(-/-) mice virtually eliminated the vascular leak in response to the cys-LT ligands, indicating GPR99 as a potential CysLT3R active in the Cysltr1/Cysltr2(-/-) mice. Importantly, the Gpr99(-/-) mice showed a dose-dependent loss of LTE4-mediated vascular permeability, but not to LTC4 or LTD4, revealing a preference of GPR99 for LTE4 even when CysLT1R is present. As LTE4 is the predominant cys-LT species in inflamed tissues, GPR99 may provide a new therapeutic target.

Arachidonic acid metabolites and enzyme transcripts in asthma are altered by cigarette smoking.

BACKGROUND: Arachidonic acid metabolites are implicated in the pathogenesis of asthma although only limited information is available on the impact of current smoking history on these metabolites. The aim of the study was to examine the effect of smoking status on urinary, sputum, and plasma eicosanoid concentrations and relevant enzyme transcripts in asthma. METHODS: In 108 smokers and never smokers with asthma and 45 healthy controls [smokers and never smokers], we measured urinary tetranor prostaglandin (PG)D2 (PGDM) and leukotriene (LT)E4 , induced sputum fluid LTB4 , LTE4 , PGD2 , and PGE2 , plasma secretory phospholipase A2 (sPLA2 ), and 11ß prostaglandin F2α (11ßPGF2α ), and, in a subgroup with severe asthma, airway leukocyte and epithelial cell mRNA expression levels of arachidonic acid metabolic enzymes. RESULTS: Smokers with asthma had higher urinary LTE4 ; 83 (59, 130) vs 59 (40, 90) pg/mg creatinine, P = 0.008, and PGDM; 60 (35, 100) vs 41 (28, 59) ng/mg creatinine, P = 0.012 concentrations, respectively, and lower sputum PGE2 concentrations 80 (46, 157) vs 192 (91, 301) pg/ml, P = 0.001 than never smokers with asthma. Sputum LTB4 (P = 0.013), and plasma 11ßPGF2α (P = 0.032), concentrations, respectively, were increased in smokers with asthma compared with healthy smokers. Asthma-specific and smoking-related increases (>1.5-fold expression) in arachidonate 15-lipoxygenase and gamma-glutamyltransferase transcripts were demonstrated. CONCLUSIONS: Several arachidonic acid metabolites and enzyme transcripts involving both lipoxygenase and cyclooxygenase pathways are increased in smokers with asthma and differ from never smokers with asthma. Possibly targeting specific lipoxygenase and cyclooxygenase pathways that are activated by asthma and cigarette smoking may optimize therapeutic responses.

Increased Excretion of Mast Cell Mediator Metabolites During Mast Cell Activation Syndrome.

BACKGROUND: One requirement for diagnosing mast cell activation syndrome (MCAS) is an increase, above an established baseline level, in serum tryptase by 20% plus 2 ng/mL. However, there is no consensus of what constitutes excretion of a substantial increase in metabolites from prostaglandin D2, histamine, or leukotriene E4 in MCAS. OBJECTIVE: Ratios of acute/baseline levels for each urinary metabolite that accompanied tryptase increases of 20% plus 2 ng/mL were determined. METHODS: Mayo Clinic databases of patients with systemic mastocytosis with or without MCAS were reviewed. Patients with the requisite increase in serum tryptase during MCAS were examined for those who also had acute/baseline measurements of urinary mediator metabolite(s). RESULTS: Ratios of acute/baseline levels for tryptase and for each urinary metabolite were calculated. For all patients, the average acute/baseline ratio (SD) for tryptase was 4.88 (3.77). Average ratios of urinary mediator metabolites were: leukotriene E4: 35.98 (50.59), 2,3-dinor-11ß-prostaglandin F2α: 7.28 (6.89), and N-methyl histamine: 3.2 (2.31). The lowest acute-baseline ratios for each of the three metabolites accompanying a tryptase increase of 20% plus 2 ng/mL were similar, with values of about 1.3. CONCLUSIONS: To the author's knowledge, this is the largest series of mast cell mediator metabolite measurements during episodes of MCAS that were verified by the requisite tryptase increase above baseline. Unexpectedly, leukotriene E4 showed the greatest average increase. Acute/baseline increase of 1.3 or greater in any of these mediators could be useful for corroborating a diagnosis of MCAS.

Metabolomics of human intestinal transplant rejection.

Surveillance endoscopy with biopsy is the standard method to monitor intestinal transplant recipients but it is invasive, costly and prone to sampling error. Early noninvasive biomarkers of intestinal rejection are needed. In this pilot study we applied metabolomics to characterize the metabolomic profile of intestinal allograft rejection. Fifty-six samples of ileostomy fluid or stool from 11 rejection and 45 nonrejection episodes were analyzed by ultraperformance liquid chromatography in conjunction with Quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). The data were acquired in duplicate for each sample in positive ionization mode and preprocessed using XCMS (Scripps) followed by multivariate data analysis. We detected a total of 2541 metabolites in the positive ionization mode (mass 50-850 Daltons). A significant interclass separation was found between rejection and nonrejection. The proinflammatory mediator leukotriene E4 was the metabolite with the highest fold change in the rejection group compared to nonrejection. Water-soluble vitamins B2, B5, B6, and taurocholate were also detected with high fold change in rejection. The metabolomic profile of rejection was more heterogeneous than nonrejection. Although larger studies are needed, metabolomics appears to be a promising tool to characterize the pathophysiologic mechanisms involved in intestinal allograft rejection and potentially to identify noninvasive biomarkers.

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.

Eoxins: a new inflammatory pathway in childhood asthma.

BACKGROUND: Increased levels of leukotrienes (LTs) in exhaled breath condensate (EBC) are associated with asthma and bronchial hyperresponsiveness (BHR), whereas eicosanoids generated through the 15-lipoxygenase (LO) pathway (15-hydroxyeicosatetraenoic acid [HETE] and eoxins) have been less studied. OBJECTIVE: We investigated whether metabolites of the 5- and 15-LO pathways in EBC are associated with childhood asthma, asthma severity, and clinical parameters. METHODS: The present study included 131 school-aged children (27 children with problematic severe asthma, 80 children with mild-to-moderate asthma, and 24 healthy children) from the Severe Asthma Recognized in Childhood study and 19 children with other nonasthmatic chronic lung diseases. Clinical work-up included spirometry, fractional exhaled nitric oxide measurements, skin prick testing, and methacholine challenge. Eicosanoids were analyzed in EBC by using mass spectrometry and are reported as concentrations (in picograms per milliliter) and eicosanoid/palmitic acid (PA) ratios. RESULTS: Eoxin C4/PA, eoxin D4/PA, eoxin E4/PA, 15-HETE/PA, and LTC4/PA ratios were significantly increased in asthmatic versus healthy children. Eoxin D4/PA and LTE4/PA ratios were also significantly higher in children with BHR. A nonsignificant trend was observed toward higher eoxin/PA ratios with increasing asthma severity. In contrast to asthma, children with chronic lung disease had the highest 15-HETE/PA, LTC4/PA, LTE4/PA, and LTB4/PA ratios. CONCLUSION: The results point to increased activity of the 15-LO inflammatory pathway in childhood asthma. Mass spectrometric analyses of EBC demonstrate that increased eoxin levels not only accompany the increased 5-LO product LTC4 but are also associated with BHR. These markers might represent a new therapeutic target for asthma treatment.

Epoxyeicosatrienoates are the dominant eicosanoids in human lungs upon microbial challenge.

Lipoxygenase, cyclo-oxygenase and cytochrome P450 (CYP) products of arachidonic acid (AA) are implicated in pulmonary vasoregulation. The CYP-mediated epoxyeicosatrienoates (EETs) have been described previously as the predominant eicosanoids in human lungs upon stimulation with the Ca(2+) ionophore A23187. In this study, we challenged perfused human lungs with two microbial agents: Escherichia coli haemolysin (ECH) and formyl-methionyl-leucyl-phenylalanine (fMLP). Both stimuli elicited pronounced generation of leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), prostanoids (PTs) and EETs/dihydroxyeicosatrienoic acids (DHETs), as assessed by liquid chromatography-mass spectrometry, paralleled by pulmonary artery pressor response and lung oedema formation. The maximum buffer concentrations of EETs/DHETs surpassed those of LTs plus HETEs and PTs by a factor of four (ECH) or three (AA/fMLP). Dual 5-lipoxygenase/cyclo-oxygenase inhibition caused pronounced reduction of AA/fMLP-induced LT/PT synthesis and oedema formation but only limited attenuation of pulmonary vasoconstriction, while inhibition of CYP epoxygenase clearly attenuated AA/fMLP-induced EET/DHET synthesis and vasoconstriction but not oedema formation, suggesting a major contribution of LTs/PTs to vascular leakage and of EETs/DHETs to pressor response. Consequently, generation of EETs/DHETs is greater than that of LTs plus HETEs and PTs in ex vivo perfused human lungs upon microbial challenge suggesting a substantial contribution of these mediators to inflammatory-infectious pulmonary injury.

Increased leukotriene E4 excretion in systemic mastocytosis.

Cysteinyl leukotrienes such as LTE(4) are produced by mast cells, neutrophils, eosinophils, and macrophages. LTE(4) levels have not been reported in systemic mastocytosis, a disorder with a large increase in mast cell numbers. Urinary LTE(4) from patients referred for symptoms potentially due to mast cell degranulation or systemic mastocytosis was measured by a commercial cysteinyl leukotriene enzyme immunoassay kit. The diagnosis of systemic mastocytosis was established using current World Health Organization criteria. Compared with a control group of patients with various potential mast cell-related symptoms (e.g., "spells"), patients with systemic mastocytosis had a significant (P=.01) increase in urinary LTE(4) excretion, whether expressed as LTE(4) ng/g creatinine or as LTE(4) ng/24h. There was a moderate correlation of LTE(4) ng/24h with excretion of N-methyl histamine and serum tryptase but not with urinary 11beta-prostaglandin F(2alpha) (11beta-PGF(2alpha)) excretion. LTE(4) excretion is increased in patients with systemic mastocytosis and potentially contributes to clinical symptoms.

The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications.

The intracellular parent of the cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), is formed by conjugation of LTA(4) and reduced glutathione by LTC(4) synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC(4) is converted to LTD(4) and LTE(4) through sequential enzymatic removal of glutamic acid and then glycine. Only LTE(4) is sufficiently stable to be prominent in biologic fluids, such as urine or bronchoalveolar lavage fluid, of asthmatic individuals and at sites of inflammation in animal models. LTE(4) has received little attention because it binds poorly to the classical type 1 and 2 cysLT receptors and is much less active on normal airways than LTC(4) or LTD(4). However, early studies indicated that LTE(4) caused skin swelling in human subjects as potently as LTC(4) and LTD(4), that airways of asthmatic subjects (particularly those that were aspirin sensitive) were selectively hyperresponsive to LTE(4), and that a potential distinct LTE(4) receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE(4): P2Y(12), an adenosine diphosphate receptor, and CysLT(E)R, which was observed functionally in the skin of mice lacking the type 1 and 2 cysLT receptors. These findings prompt a renewed focus on LTE(4) receptors as therapeutic targets that are not currently addressed by available receptor antagonists.

Identification of cysteinyl-leukotriene-receptor 1 antagonists as ligands for the bile acid receptor GPBAR1.

The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.

The enteropathy of prostaglandin deficiency.

BACKGROUND: Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. METHODS: Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. RESULTS: We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45-year-old man who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls (P<0.006), and serum 12-HETE was 1.3% of controls (P<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore-activated leukocytes was only 3% of controls, and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-alpha (cPLA2-alpha), which regulates cyclooxygenase- and lipoxygenase-mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-alpha cDNA demonstrated two heterozygous nonsynonymous single-base-pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known single nucleotide polymorphism (SNP), Lys651Arg (K651R). CONCLUSIONS: Characterization of this cPLA2-alpha deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers.

Changes in the proteome of human bronchial epithelial cells following stimulation with leucotriene E4 and transforming growth factor-beta1.

BACKGROUND AND OBJECTIVE: Activated bronchial epithelial cells exert considerable potential to maintain a microenvironment in the airway wall that promotes airway inflammation and remodelling. Cysteinyl leucotrienes (CysLT) and transforming growth factor-beta(1) (TGF-beta(1)) are both increased in asthmatic airways and may influence the pathophysiology of disease. However, the consequences of activation of bronchial epithelial cells by these mediators are not fully understood. A proteomic-based approach was used to characterize the inflammatory pathways in bronchial epithelial cells after stimulation with CysLT and TGF-beta(1). METHODS: Human bronchial epithelial cells (BEAS-2B) were stimulated with 1 ng/mL TGF-beta(1) and 50 nmol/L leucotriene E(4) (LTE(4)) for 48 h and whole-cell lysates were subjected to two-dimensional gel electrophoresis. Proteins showing statistically significant differential expression were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. RESULTS: Stimulation with LTE(4) increased the expression of three proteins and five proteins showed decreased expression. Of the latter group, two were definitively identified as heat shock protein (Hsp90 alpha) and stress-70 protein. Hsp90 alpha forms a heterocomplex with the glucocorticoid receptor (GR) and a significant decrease in GR following LTE(4) stimulation was confirmed. TGF-beta(1) downregulated 18 intracellular proteins, including lamin A/C, glyceraldehyde-3-phosphate dehydrogenase, protein DJ-1, voltage-dependent calcium channel gamma-7 subunit, heterogeneous nuclear ribonucleoprotein A2/B1 and stress-70 protein. CONCLUSIONS: The current findings suggest that by downregulating GR and Hsp90 alpha, CysLT may interfere with the action of glucocorticoids. Overall, the results confirm the complex role of bronchial epithelium in aspects of airway inflammation and remodelling.