leukotriene-b4 and Cystic-Fibrosis

We determined the generation and liberation of LTB4 in peripheral granulocytes and the histamine release from basophils in patients suffering from cystic fibrosis (CF, median 17.2 years of age, n = 12). We compared the data with an age matched group of healthy donors (n = 12). All patients suffered from an exacerbation of a chronic pulmonary infection caused by P. aeruginosa. Peripheral granulocytes were stimulated at different days before, during and after antiinfectious treatment with Ca-Ionophore, opsonized zymosan and arachidonic acid. The granulocytes from patients with CF as compared to the control group showed an increased omega-Oxidation of the synthesized LTB4 into 20-COOH- and 20-OH-LTB4 after stimulation with Ca-Ionophore and opsonized zymosan (Ca-Ionophore: ratio of LTB4 versus omega-oxidated products (CF): 0.77 +/- 0.007 mean +/- S.E.M., n = 12, control group: 1.07 +/- 0.1, n = 12, p less than 0.01). Stimulation of the cells with Ca-Ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group. No significant differences in the basophil counts were determined between both populations. However, the absolute histamine content per basophil was elevated in the CF group (2.4 +/- 0.3 versus 1.6 +/- 0.2 mean +/- S.E.M., n = 12/12, p less than 0.04). Stimulation of basophils with Ca-Ionophore and anti-IgE leads to a significant higher release of histamine per basophil in CF patients (Ca-Ionophore: 2.6 +/- 0.2 versus 1.3 +/- 0.8 pg/basophil, mean +/- S.E.M., p less than 0.05). These data indicate that basophils in CF may have a greater potential to release mediators. During the antiinfectious treatment a normalization of the altered pattern was observed. Within the CF-groups a strong correlation between the release of LTB4, its metabolites, the histamine release per basophil, the total histamine content and clinical (e.g. pO2 FEV1) and laboratory findings (e.g. IgE and IgG levels, CRP) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway and histamine releasability of granulocyte subpopulations which correlates with the clinical signs of inflammation.

Topics: Adolescent; Basophils; Child; Cystic Fibrosis; Granulocytes; Histamine Release; Humans; Leukotriene B4; Lipoxygenase; Oxidation-Reduction

Inflammation causes irreparable damage in the cystic fibrosis (CF) lung. Despite high standards of care and the advent of new therapies, inflammation continues to cause significant loss of lung function and morbidity. Acebilustat is a once-daily, oral molecule with anti-inflammatory activity through the inhibition of LTA4 hydrolase and modulation of LTB4. It has potential to reduce lung function decline and pulmonary exacerbations in patients with CF and is currently being tested in a Phase II multicenter, randomized, double-blind, placebo-controlled, parallel-group study (EMPIRE-CF). Strict inclusion criteria based on modeling of the Cystic Fibrosis Foundation Patient Registry data were selected to enrich the trial with patients most likely to benefit from chronic anti-inflammatory therapy that reduces lung function decline. 200 patients between 18 and 30 years of age, with an FEV

Topics: Adolescent; Adult; Anti-Inflammatory Agents; Azabicyclo Compounds; Benzoates; C-Reactive Protein; Cystic Fibrosis; Disease Progression; Double-Blind Method; Epoxide Hydrolases; Female; Forced Expiratory Volume; Humans; Leukotriene B4; Male; Young Adult

Acebilustat is a new once-daily oral antiinflammatory drug in development for treatment of cystic fibrosis (CF) and other diseases. It is an inhibitor of leukotriene A4 hydrolase; therefore, production of leukotriene B4 (LTB4) in biological fluids provides a direct measure of the pharmacodynamic (PD) response to acebilustat treatment. Here we compare the pharmacokinetics (PK) and PD between CF patients and healthy volunteers, and investigate the food effect and CYP3A4 induction in healthy volunteers. No significant differences between study populations were observed for peak plasma level (C

Topics: Administration, Oral; Azabicyclo Compounds; Benzoates; Cystic Fibrosis; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inducers; Food; Healthy Volunteers; Humans; Leukotriene B4; Sputum; Time Factors

Various anti-inflammatory therapies, including dietary omega-3 polyunsaturated fatty acids (PUFA) supplementation, have been investigated in cystic fibrosis (CF) patients. To further explore this nutritional approach, biological effects of an omega-3 PUFA oral liquid supplementation were measured in 17 CF patients in a double-blind, randomized, crossover without a washout period and placebo-controlled study.. CF patients (age: 18+/-9 year; weight: 43+/-13 kg) received a liquid dietary supplementation either enriched or not in omega-3 PUFA (390-1170 mg/day according to patient weight) during two 6-month periods.. Increase in eicosapentaenoic acid was observed in neutrophil membrane following omega-3 PUFA dietary supplementation (from 0.7+/-0.6 to 1.6+/-0.6 micromol%, P<0.01). The leukotriene B(4) (LTB(4))/leukotriene B(5) (LTB(5)) ratio was decreased (from 72+/-27 to 24+/-7, P<0.001) in CF patients taking omega-3 PUFA supplements. In contrast, omega-3 PUFA supplementation affected neither internalization of IL-8 receptors following IL-8 exposure, nor IL-8-induced neutrophil chemotaxis.. Our results show that omega-3 PUFA are incorporated in neutrophil membranes. The subsequent decrease in LTB(4)/LTB(5) ratio suggests that, in such conditions, neutrophils may produce less pro-inflammatory mediators from the acid arachidonic pathway. These data indicate that omega-3 PUFA intake may have anti-inflammatory effect that still need to be assessed by long-term studies following large groups of patients.

Topics: Adolescent; Adult; Cell Membrane; Chemotaxis, Leukocyte; Child; Cross-Over Studies; Cystic Fibrosis; Dietary Supplements; Eicosapentaenoic Acid; Fatty Acids, Omega-3; Humans; Interleukin-8; Leukotriene B4; Neutrophils; Placebos; Receptors, Interleukin

The substitution of omega-3 (n-3) fatty acids for omega-6 (n-6) fatty acids generates eicosanoids with diminished inflammatory effects. As the lungs of patients with cystic fibrosis (CF) are in a state of chronic inflammation in which increased amounts of eicosanoids are found, n-3 supplementation may reduce this level of inflammation and result in clinical improvement. The absorption and clinical effects of n-3 vs. n-6 fatty acids in CF were measured in a prospective, randomized, double-blind, crossover study in which 14 patients with CF (age: 6-16 years, mean 10.5 years; baseline Shwachman-Brasfield scores: 41-88, mean 76.7) received 6 weeks of n-3 ethyl ester concentrate from menhaden oil (100-131 mg/kg/day, mean 112.8) or n-6 fatty acids from safflower oil (102-132 mg/kg/day, mean 113.3), followed by a washout period of 6 weeks, and then 6 weeks of the other supplement. Analysis by gas chromatography showed that n-3 supplementation resulted in increased eicosapentaenoic acid (20:5n-3) in platelet phospholipids, from 0.14 to 2.16%, P < 0.05 and in increased docosahexaenoic acid (22:6n-3), from 1.33 to 3.72%, P < 0.05. Clinical effects were evaluated at weeks 0, 6, 12, and 18, and analyzed for differences among the n-3, n-6, and washout periods. No adverse effects were reported or observed. No statistically significant differences were found (ANOVA, P > 0.05) in Shwachman-Brasfield scores, sweat test, weight change, or forced expiratory volume and flow (FEV1, FEF25-75%, and FVC) percentiles. Tumor necrosis factor was not measurable in any serum sample. Serum leukotriene B4 (LTB4) levels were significantly reduced by n-3 fatty acids, mean reduction (-177 pg/mL) compared to n-6 fatty acids (+63 pg/mL) P < 0.05. These results show that both n-3 fatty acids are absorbed and incorporated into platelet phospholipids in patients with CF and reduced serum LTB4. No significant clinical differences or adverse effects were found.

Topics: Absorption; Adolescent; Analysis of Variance; Blood Platelets; Child; Cross-Over Studies; Cystic Fibrosis; Dietary Fats, Unsaturated; Docosahexaenoic Acids; Double-Blind Method; Eicosapentaenoic Acid; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Female; Food, Fortified; Humans; Leukotriene B4; Male; Phospholipids; Prospective Studies; Respiratory Function Tests

Much of the lung damage that limits the life of young adults with cystic fibrosis is due to proteases and oxygen metabolites generated by neutrophils, which are recruited into the airway by the interaction between Pseudomonas aeruginosa and pulmonary macrophages. Leukotriene B4 (LTB4) has been proposed as a local mediator of this process; its production is susceptible to specific modulation with dietary eicosapentaenoic acid (EPA). We carried out a placebo-controlled trial of EPA (2.7 g daily for 6 weeks) to assess its effects on markers of clinical state, peripheral neutrophil function, and lung inflammation in sixteen patients with cystic fibrosis colonised with P aeruginosa. EPA was well tolerated and resulted in a significant reduction in sputum volume (median change with EPA -10 mL/day, placebo 0; p = 0.015), and improvements in Schwachman score (EPA 5%, placebo 0; p = 0.034), forced expiratory volume in 1 s (EPA 0.25 L, placebo -0.1 L; p = 0.006), and vital capacity (EPA 0.6 L, placebo 0; p = 0.011). Relative chemotaxis of circulating neutrophils to LTB4 increased from a subnormal baseline of 4 (median; range 0-10) microns/30 min before treatment, to a near normal value of 11 (5-18) microns/30 min after EPA. Relative chemotaxis to LTB4 of patients taking placebo did not change: the difference in response was highly significant (p = 0.001). Specific reduction of neutrophil chemotaxis to LTB4 is a sensitive assay of chronic in-vivo exposure to LTB4. Our results suggest that LTB4 has a pathogenetic role in the lung damage of cystic fibrosis. Longer-term clinical trials of EPA are warranted in a larger number of cystic fibrosis patients.

Topics: Adolescent; Adult; Chemotaxis, Leukocyte; Child; Cystic Fibrosis; Dietary Fats; Double-Blind Method; Eicosapentaenoic Acid; Female; Forced Expiratory Volume; Humans; Leukocyte Count; Leukotriene B4; Male; Neutrophils; Pilot Projects; Placebos; Pseudomonas aeruginosa; Sputum; Vital Capacity

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Topics: Animals; Azabicyclo Compounds; Benzoates; Blood Cells; Bone Marrow Cells; Cells, Cultured; Chemotaxis, Leukocyte; Culture Media, Conditioned; Cystic Fibrosis; Exocytosis; Flow Cytometry; Glycolysis; Humans; Leukocyte Elastase; Leukotriene B4; Lipopolysaccharides; Metformin; Mice; Neutrophil Activation; Neutrophils; Oxygen Consumption; Pinocytosis; Pseudomonas aeruginosa; Respiratory System; Sputum; Transendothelial and Transepithelial Migration

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Antigens, Human Platelet; Cell Communication; Cell Line; Chemotaxis; Coculture Techniques; Cystic Fibrosis; Cytosol; Humans; Leukotriene B4; Mice; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Tomography, Optical Coherence; Transendothelial and Transepithelial Migration

Airway disease in cystic fibrosis (CF) is characterised by impaired mucociliary clearance, persistent bacterial infection and neutrophilic inflammation. Lipoxin A4 (LXA4) initiates the active resolution of inflammation and promotes airway surface hydration in CF models. 15-Lipoxygenase (LO) plays a central role in the "class switch" of eicosanoid mediator biosynthesis from leukotrienes to lipoxins, initiating the active resolution of inflammation. We hypothesised that defective eicosanoid mediator class switching contributes to the failure to resolve inflammation in CF lung disease. Using bronchoalveolar lavage (BAL) samples from 46 children with CF and 19 paediatric controls we demonstrate that the ratio of LXA4 to leukotriene B4 (LTB4) is depressed in CF BAL (p<0.01), even in the absence of infection (p<0.001). Furthermore, 15-LO2 transcripts were significantly less abundant in CF BAL samples (p<0.05). In control BAL, there were positive relationships between 15-LO2 transcript abundance and LXA4/LTB4 ratio (p=0.01, r=0.66) and with percentage macrophage composition of the BAL fluid (p<0.001, r=0.82), which were absent in CF. Impoverished 15-LO2 expression and depression of the LXA4/LTB4 ratio are observed in paediatric CF BAL. These observations provide mechanistic insights into the failure to resolve inflammation in the CF lung.

Topics: Anti-Bacterial Agents; Arachidonate 15-Lipoxygenase; Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Cystic Fibrosis; Female; Humans; Inflammation; Leukotriene A4; Leukotriene B4; Lipoxins; Longitudinal Studies; Lung; Lung Diseases; Macrophages, Alveolar; Male; Neutrophils

Leukotriene B(4) (LTB(4)) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB(4) production of neutrophils within minutes, a process called "priming". In this study, we demonstrate that GM-CSF also limits the production of LTB(4) by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh(-/-)) mice demonstrated increased LTB(4) production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB(4) upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.

Topics: Animals; Blotting, Western; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukotriene B4; Mice; Mice, Knockout; Microscopy, Confocal; Neutrophils; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; Transcription Factors

Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) are inflammatory mediators involved in the neutrophil response to pulmonary bacterial colonization in cystic fibrosis (CF). The aim of this study was to investigate whether the LTB(4) and IL-8 levels in exhaled breath condensate (EBC) could be related to the type of bacterial colonization in CF patients. The pH level in EBC was analyzed as an estimate of airway acidification. Forty children were evaluated: 10 CF patients with P. aeruginosa, 10 CF patients with S. aureus, 10 not colonized CF patients, and 10 healthy children. LTB(4) and IL-8 in EBC were analyzed by specific enzyme immunoassay kits (EIA). The pH of EBC was measured with a pH-meter after deareation by bubbling with argon. Exhaled LTB(4) was higher in CF children with P. aeruginosa compared to those with S. aureus (P < 0.01), not colonized (P < 0.001), and healthy children (P < 0.01). Exhaled IL-8 was elevated in CF patients colonized by P. aeruginosa compared with other subgroups (vs. not colonized, P < 0.05; vs. healthy children, P < 0.001). IL-8 levels were higher in CF children with S. aureus than in healthy children (P < 0.05). There was an increase in IL-8 levels in not colonized CF patients compared with healthy children (P < 0.05). EBC pH was higher in healthy children compared to CF patients not colonized (P < 0.05). Our data suggest that EBC is suitable for evaluating neutrophil inflammatory mediators (LTB(4), IL-8, and pH) involved in the response to pulmonary bacterial colonization in CF children.

Topics: Biomarkers; Breath Tests; Child; Cystic Fibrosis; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Interleukin-8; Leukotriene B4; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Sputum; Staphylococcal Infections; Staphylococcus aureus

The noninvasive assessment and monitoring of airway inflammation could be important in respiratory disease. The pH of exhaled breath condensate (EBC) is a promising marker. Although pH has been measured in the EBC of adults with inflammatory airway diseases, no study has measured this in children.. This study aimed to assess whether there is a change in pH in the EBC of children with cystic fibrosis (CF) and asthma, and to try to determine whether pH could be used as a marker of airway inflammation. Furthermore, the relationships among EBC pH, severity of disease, and oxidative stress were studied.. We studied 20 children with CF (mean [+/- SEM] age, 7 +/- 3 years), 20 children with asthma (mean age, 7 +/- 2 years), and 15 age-matched healthy children (mean age, 7 +/- 2 years). The pH of EBC was measured using a pH meter.. Lower pH values were observed in the EBC of children with CF and asthma compared to control subjects (mean pH, 7.23 +/- 0.03 and 7.42 +/- 0.01 vs 7.85 +/- 0.02, respectively). Furthermore, relationships among EBC pH, severity of asthma, and the presence of an infective exacerbation of CF was found. There was a negative correlation between exhaled pH and exhaled leukotriene B(4) concentrations (r = -0.5; p < 0.005).. We conclude that the measurement of EBC pH may be useful in the evaluation of airway inflammation in children with asthma and CF.

Topics: Asthma; Breath Tests; Case-Control Studies; Child; Child, Preschool; Cystic Fibrosis; F2-Isoprostanes; Female; Humans; Hydrogen-Ion Concentration; Inflammation Mediators; Leukotriene B4; Male; Predictive Value of Tests; Probability; Prognosis; Prospective Studies; Reference Values; Respiratory Function Tests; Risk Assessment; Sensitivity and Specificity; Severity of Illness Index; Statistics, Nonparametric

Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-alpha, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa.

Topics: Animals; Chemokine CXCL1; Chemokine CXCL2; Chemokines; Chemokines, CXC; Chronic Disease; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Dinoprostone; Female; Genotype; Interleukin-1; Interleukin-6; Leukocyte Count; Leukotriene B4; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Tumor Necrosis Factor-alpha; Weight Loss

Chronic neutrophilic airway inflammation is an important feature of cystic fibrosis (CF). Noninvasive inflammatory markers may be useful in monitoring CF. Leukotriene B4 (LTB4) and interleukin (IL)-6 are inflammatory mediators that are increased in chronic neutrophilic inflammation. The aim of this study was to assess whether LTB4 and IL-6 were increased in exhaled breath condensate of CF patients and whether they could be used to monitor inflammation. Twenty patients with CF (13 males, age of 28 +/- 9 years) were recruited together with 15 age-matched healthy subjects (8 males, age 35 +/- 7 years). LTB4 and IL-6 levels were markedly elevated in patients with acute exacerbations (28.8 +/- 4.3 and 8.7 +/- 0.4 pg/ml) compared with control subjects (6.8 +/- 0.7 and 2.6 +/- 0.1 pg/ml, p < 0.0001). We also observed a decrease of exhaled LTB4 and IL-6 concentrations after antibiotic treatment in six patients who were followed until clinically stable (31.1 +/- 4.4 and 9.5 +/- 0.4 pg/ml vs. 18.8 +/- 0.8 and 6.4 +/- 0.2 pg/ml, respectively) and an increase in 15 CF patients infected with Pseudomonas aeruginosa (34.3 +/- 5.0 and 9.3 +/- 0.3 pg/m) compared with those infected with other bacteria (18.3 +/- 0.7 and 6.9 +/- 0.5 pg/ml). These findings suggest that LTB4 and IL-6 levels are increased in exhaled breath condensate of patients with CF during exacerbation and could be used to monitor airway inflammation in these patients.

Topics: Adult; Breath Tests; Cystic Fibrosis; Female; Humans; Interleukin-6; Leukotriene B4; Male; Nitric Oxide

Topics: alpha 1-Antitrypsin; Bronchoalveolar Lavage Fluid; Cell Count; Chemistry, Clinical; Cystic Fibrosis; Humans; Interleukin-8; Leukotriene B4; Neutrophils; Pancreatic Elastase

Progressive neutrophil-mediated lung damage causes much of the morbidity and mortality in cystic fibrosis (CF). Neutrophil chemoattractants implicated in CF include interleukin (IL-)8, tumour necrosis factor (TNFalpha) and leukotriene (LT)B4, but growth-related protein alpha (GROalpha), a highly potent neutrophil chemokine, has not been investigated. Atopic status has been considered to contribute to the marked heterogeneity of pulmonary disease in CF. We hypothesized that GROalpha may be produced in biologically-significant amounts in the CF lung, and that enhanced production of GROalpha, IL-8 or LTB4 may contribute to the poorer lung function seen in atopic CF patients compared to non-atopic CF patients. GROalpha, IL-8 and LTB4 levels in the sputum of atopic and non-atopic CF patients were assessed by immunoassays, and GROalpha and IL-8 levels were also assessed in the plasma of CF patients and normal controls. As expected, there were high levels of IL-8 and LTB4 in most CF sputum samples, and IL-8 levels were higher in CF plasma than in control plasma (P=0.02). In contrast, GROalpha was undetectable (< 5 pg ml(-1)) in the sputum of 21 out of 25 CF patients, with low levels (range 144-825 pg ml(-1)) in the remainder, and median levels of GROalpha in CF plasma (33 pg ml(-1), n=24) were not significantly different from controls (34 pg ml(-1), n=25). Lung function [forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC)] was significantly poorer in atopic CF compared to non-atopic CF patients (P<0.02), but sputum levels of GROalpha, IL-8 and LTB4 were not different between the subgroups. Our results suggest that unlike LTB4 and IL-8, GROalpha does not contribute to neutrophilic inflammation in the CF lung, and other factors must determine the impaired lung function observed in atopic CF patients. These results may have important implications in the development of chemokine receptor antagonists as novel anti-inflammatory agents in CF.

Topics: Adolescent; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Child; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Forced Expiratory Volume; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Leukotriene B4; Neutrophils; Sputum; Vital Capacity

To investigate the effects of glucocorticoids on leukotriene (LT) generation in patients with cystic fibrosis (CF), we evaluated calcium ionophore A23187-induced LTB4 and LTC4 production by leukocytes with and without pretreatment with dexamethasone. The CF patients were in good condition and did not have acute infection. There were no significant differences in LTB4 and LTC4 production without dexamethasone pretreatment between the CF patients and controls. However, the ratios of LTB4 and LTC4 production by leukocytes preincubated with dexamethasone to those of leukocytes without dexamethasone pretreatment were significantly higher in the CF patients than in the controls (both p < 0.05). Our data suggest that the response of LTB4 and LTC4 production to dexamethasone is disturbed in patients with CF. The generation of LTs may be enhanced due to a disturbance in glucocorticoid suppression.

Topics: Adolescent; Adult; Calcimycin; Child; Child, Preschool; Cystic Fibrosis; Dexamethasone; Female; Gene Expression Regulation; Humans; Leukocytes; Leukotriene B4; Leukotriene C4; Male

Persistent neutrophil infiltration into the airways of patients with cystic fibrosis (CF) results in lung destruction. Eicosanoid lipid mediators, particularly leukotriene B4 (LTB4), may play a role in neutrophil influx and activation. We compared the eicosanoid content of epithelial lining fluid (ELF) obtained by bronchoalveolar lavage (BAL) from 17 patients with CF and 10 healthy subjects. LTB4 was the predominant eicosanoid in the CF airway (16.7 +/- 9.1 ng/ml ELF in CF versus 0.5 +/- 0.1 ng/ml ELF in healthy subjects). Prostaglandins (PG) and thromboxane (TX) were also elevated in CF (PGE2, 8.5 +/- 2.2; PGF2 alpha, 6.0 +/- 2.0; and TXB2, 14.0 +/- 3.0 ng/ml ELF) compared with healthy subjects (PGE2, 0.4 +/- 0.2; PGF2 alpha, 0.5 +/- 0.2; and TXB2, 1.2 +/- 0.4 ng/ml ELF). We also developed a protocol for the storage and subsequent analysis of BAL fluid that assures accurate and reproducible measurements of these eicosanoids. BAL samples stored for up to 8 months retain greater than 80% of their original eicosanoid content if the BAL fluid is immediately treated with methanol, concentrated, and stored at -70 degrees C without further purification. These data suggest that CF airways contain sufficient amounts of LTB4 both to recruit additional neutrophils into the airways and to stimulate neutrophils to release their injurious products. Therapies aimed at interfering with the production or action of LTB4 may be beneficial in CF and other lung diseases with a significant neutrophil response.

Topics: Adolescent; Adult; Bronchoalveolar Lavage Fluid; Child; Chromatography, High Pressure Liquid; Cystic Fibrosis; Epithelium; Female; Humans; Immunoenzyme Techniques; Leukotriene B4; Male; Specimen Handling; Spectrophotometry, Ultraviolet

It is postulated that a vigorous host inflammatory response in the cystic fibrosis lung contributes to lung injury. Tumour necrosis factor-alpha (TNF-alpha) may play a part in that process and in the generation of leukotrienes. Therefore, the relationships between sputum TNF-alpha, leukotriene concentration, and lung function abnormalities in 16 children with cystic fibrosis were investigated. Each subject provided sputum samples and performed spirometry. TNF-alpha was measured by enzyme linked immunosorbent assay; individual leukotrienes were separated using high performance liquid chromatography and quantified by radioimmunoassay. The geometric mean concentration of TNF-alpha was 129.7 pg/ml and 95% confidence interval 48.2 to 348.3. Mean (SEM) leukotriene B4 (LTB4) was 97.8 (22.9) pmol/g and total cysteinyl leukotrienes were 60.9 (14.8) pmol/g. Mean (SD) forced expiratory volume in one second (FEV1) of the group was 53 (15)% of predicted and forced vital capacity (FVC) was 65 (14)% of predicted. There was a significant positive correlation between TNF-alpha and both LTB4 and the total cysteinyl leukotriene sputum content. An inverse relationship existed between TNF-alpha and FEV1 and FVC. Moreover, a negative correlation was observed between sputum LTB4 and FEV1 and FVC. These results suggest that TNF-alpha and the leukotrienes may participate in the airways inflammation and airflow obstruction observed in cystic fibrosis subjects and support the hypothesis that TNF-alpha upregulates the 5-lipoxygenase pathway in vivo.

Topics: Adolescent; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Leukotriene B4; Leukotrienes; Male; Sputum; Tumor Necrosis Factor-alpha; Vital Capacity

Evidence that leukotriene B4 (LTB4) is a significant inflammatory mediator in chronic pseudomonal respiratory disease was sought in adolescents and young adults with cystic fibrosis. Specific chemotaxis of peripheral blood polymorphonuclear leucocytes (PMN) was used as an indirect measure of remote in vivo exposure to LTB4. PMN from 17 patients showed a significant decrease in chemotaxis to 10(-7)-10(-9) M LTB4, but normal responses to 10(-8) M n-formyl-methionyl-leucyl-phenylalanine and 4 mg/ml casein, when compared with 17 healthy age- and sex-matched controls. This result is consistent with chronic production of LTB4, and specific deactivation of circulating PMN receptors for LTB4 in patients with cystic fibrosis. Pharmacologic inhibition of LTB4 production in vivo may help elucidate its role in the pathogenesis of lung damage in cystic fibrosis.

Topics: Adolescent; Adult; Chemotaxis, Leukocyte; Child; Cystic Fibrosis; Humans; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mucoid and nonmucoid (NM) P. aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aeruginosa (MEP). In this regard the MEP (alginate) significantly reduced the zymosan-induced leukotriene B4 (LTB4) formation (from 40 +/- 7 to 2 +/- 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were precoated with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aeruginosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate binding [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our results indicate that the MEP represents an important component which modulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacteria, e.g. the chemiluminescence response, LTB4 generation, and the interaction with components (G proteins, PKC) of the signal transduction pathway.

Topics: Alginates; Cystic Fibrosis; Glucuronic Acid; Glycosaminoglycans; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hexuronic Acids; Humans; Leukotriene B4; Luminescent Measurements; Neutrophils; Polysaccharides, Bacterial; Protein Kinase C; Pseudomonas aeruginosa; Signal Transduction; Tetradecanoylphorbol Acetate

1. Leukotrienes (LTs) are potent pro-inflammatory mediators with actions relevant to the pathophysiology of cystic fibrosis (CF), including increased mucus production, bronchoconstriction, leucocyte chemotaxis, and increased vascular permeability. We have therefore investigated the potential role of LTs in children with CF. Leukotriene E4 levels were assessed in the urine of 30 normal (N) children (aged 1.3-12.7 years) and 30 CF children (1.6-14.3 years). Sputum from 13 of the CF children was analysed from LTB4, LTC4, LTD4, and LTE4. LTs were separated by reversed-phase h.p.l.c. and quantitated by radioimmunoassay. 2. Urinary LTE4 levels were log normally distributed, with geometric mean values (95% confidence intervals) of N: 88.4 (71.3-111) pmol mmol-1 creatinine (n = 30), and CF: 112 (70.6-177) pmol mmol-1 creatinine (n = 30; P greater than 0.05). Of the CF subjects, 33% had urinary LTE4 levels above 200 pmol mmol-1 creatinine, compared with 3.3% of the N children. 3. In sputum, mean (+/- s.e. mean) LT concentrations were (pmol g-1), LTB4: 44.3 +/- 10.8, LTC4: 4.9 +/- 1.3, LTD4: 1.8 +/- 0.9, and LTE4: 67.7 +/- 18.9 (n = 13). 4. Urinary LTE4 levels correlated significantly with sputum LTE4 levels (r = 0.673, P = 0.012), and with sputum levels of total cysteinyl-LTs (r = 0.660, P = 0.014). 5. In conclusion, total cysteinyl-LT content in sputum is 10-fold higher than previously reported, consisting primarily (91%) of LTE4. The high levels of LTE4 and LTB4 in sputum suggest involvement of LTs in the pathophysiology of CF. Urinary LTE4 levels may prove useful as a marker for cysteinyl-LT production in sputum.

Topics: Adolescent; Child; Child, Preschool; Chromatography, High Pressure Liquid; Cystic Fibrosis; Humans; Infant; Leukotriene B4; Leukotriene E4; Leukotrienes; Radioimmunoassay; Sensitivity and Specificity; Sputum; SRS-A

We studied the generation and metabolism of lipoxygenase products in peripheral granulocytes from children suffering from cystic fibrosis (CF). Peripheral granulocytes were stimulated at different times (days) before and during anti-infectious treatment with the Ca ionophore (7.5 microM, 5 and 20 min), opsonized zymosan (2 mg) and arachidonic acid (50 microM); the amount of lipoxygenase products in the cell supernatants was determined by high performance liquid chromatography. Granulocytes from patients with CF, compared to an age-matched control group, showed an increased omega-oxidation of the synthesized leukotriene (LTB4) into 20-OH- and 20-COOH-LTB4 after stimulation with the Ca ionophore (ratio of LTB4 versus omega-oxidated products in patients with CF: 0.77 +/- 0.07, mean +/- SEM, n = 11; control group: 1.07 +/- 0.1, n = 11, p less than 0.01) whereas the combined amounts of LTB4 and its omega-oxidated products did not differ significantly. A comparable profile was observed with opsonized zymosan. Stimulation of the cells with the Ca ionophore combined with arachidonic acid led to a significantly increased formation of lipoxygenase products in the patient group, whereas only a slight enhancement was observed in the control group. During the 14-day anti-infectious treatment a normalization of the altered pattern was observed. 12-Hydroxyeicosatetraenoic acid (12-HETE) production from platelets within the granulocyte fraction was significantly depressed in the CF group compared to the controls (38.5 +/- 12.5 versus 339 +/- 93 ng/5 +/- 10(6) cells, p less than 0.005). Within the CF group a strong correlation between the release of LTB4 and its metabolites, the production of 12-HETE and clinical (e.g. pO2, FEV1) and laboratory findings (e.g. IgE and IgG levels, C-reactive protein) was established. Our data suggest that the inflammatory process in patients with CF is associated with an alteration of the lipoxygenase pathway of granulocytes which correlates with the clinical signs of inflammation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adolescent; Adult; Arachidonic Acid; Arachidonic Acids; Child; Cystic Fibrosis; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukocyte Count; Leukotriene B4; Leukotrienes; SRS-A

Chronic pulmonary infection in cystic fibrosis (CF) results in an inflammatory response with persistent neutrophil influx, which contributes to lung damage. Attenuating the response with antiinflammatory agents might delay progression of lung disease. We investigated the effects of the nonsteroidal antiinflammatory agent, ibuprofen, in a rat model of chronic Pseudomonas endobronchial infection and inflammation. The areal percentage of lung inflammation 14 days after animal inoculation with Pseudomonas-containing agarose beads was significantly less in animals treated with ibuprofen (35 mg/kg orally twice daily) (39 +/- 26% SD) compared to animals given placebo (55 +/- 25% SD) (p less than 0.05). Ibuprofen did not increase the pulmonary burden of Pseudomonas, and the ibuprofen-treated infected animals gained weight better than placebo-treated controls. The administered dose of ibuprofen provides plasma concentrations sufficient to inhibit the release of leukotriene B4 (LTB4) from rat neutrophils in vitro. Since LTB4 is a potent pro-inflammatory product that promotes neutrophil adherence, aggregation, migration, degranulation, and superoxide release, inhibition of its production by ibuprofen could inhibit inflammatory damage to the lung in this model. These data in an animal model, taken together with the success of a preliminary trial of alternate-day steroid therapy in mildly affected patients with CF, suggest that antiinflammatory therapy with ibuprofen should be considered for a new therapeutic strategy in CF.

Topics: Animals; Body Weight; Chronic Disease; Cystic Fibrosis; Dinoprostone; Humans; Ibuprofen; Leukotriene B4; Lung; Lung Diseases; Neutrophils; Pseudomonas Infections; Rats

In order to investigate the possible role of arachidonic acid metabolites as lipid mediators in cystic fibrosis and chronic obstructive pulmonary disease (COPD), sputum from patients with cystic fibrosis, chronic bronchitis, or bronchiectasis was analyzed for various eicosanoids using a combination of radioimmunoassay and bioassay. Leukotriene (LT) B4, cysteinyl-containing LTs, and prostaglandins (PGs) E2, F2 alpha, 6-oxo-PGF1 alpha, and thromboxane B2 were found in all sputum samples. Saliva, which can contaminate sputum, contained low concentrations of prostanoids but not LTs. Inflammatory cells, including polymorphonuclear leukocytes (PMNs) are present in sputum. Because LTB4 generated by these cells is chemotactic for PMNs, it is suggested that this dihydroxy acid contributes to the inflammation of cystic fibrosis and other diseases characterized by airway obstruction. The source of the cysteinyl-containing LTs is less clear; these LTs may constrict respiratory smooth muscle and/or stimulate mucus formation.

Topics: Adult; Arachidonic Acid; Arachidonic Acids; Cystic Fibrosis; Eicosanoic Acids; Female; Humans; Leukotriene B4; Lung Diseases, Obstructive; Male; Middle Aged; Prostaglandins; Radioimmunoassay; Sputum

We have measured arachidonic acid (AA) metabolites, leukotrienes (LTs) and prostanoids (Ps), in sputum of patients with cystic fibrosis (CF) and in normal saliva using bioassay and radioimmunoassay (RIA). Almost three times as much LTB4 is present in CF extracts compared with slow reacting substances (SRSs). Leukotrienes were not detected in normal saliva. In CF sputum there is a three-fold increase in the level of the vasodilator prostanoid prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-oxo PGF1 alpha compared with the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TxB2), a hydrolysis product of thromboxane A2. Experiments with BW755c (25 micrograms ml-1, n = 3) indicated that the majority of this activity was not produced during the extraction procedure. The detection of LTs and Ps in sputum of CF patients shows that these substances are present at biologically active concentrations and may contribute to the pathophysiology of this disease.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Adolescent; Adult; Arachidonic Acids; Biological Assay; Child; Cystic Fibrosis; Female; Humans; Leukotriene B4; Male; Prostaglandins; Pyrazoles; Radioimmunoassay; Saliva; Sputum; SRS-A

Sputum samples from patients with bronchial asthma, chronic bronchitis and cystic fibrosis were examined for the presence of leukotrienes B4, C4 and D4. Following ethanol extraction and purification on Amberlite XAD-8, leukotrienes were identified by high pressure liquid chromatography (HPLC) using the appropriate markers. Fractions from HLPC were also tested for biological activity using both the Boyden chemotaxis assay and FPL 55712 inhibitable contraction of the isolated guinea-pig ileum. LTB4 was detected in the HPLC fractionated sputa from bronchial asthma (seven of seven), chronic bronchitis (four of four) and cystic fibrosis (four of four). In contrast, bioassay on the guinea-pig ileum failed to detect LTC4 or LTD4 in 17 asthmatic sputa, although they were detected in one of five bronchitics and 16 of 25 patients with cystic fibrosis. The activity in eight of these cystic fibrosis sputa was further characterized by HPLC and shown to be LTC4 and/or LTD4. Sputum from 11 of 17 asthmatics, four of 25 patients with cystic fibrosis and two of five bronchitics contained an anaphylatoxin like substance. The majority of sputum samples containing LTB4 also possessed an activity with physical and biological characteristics of the 5(S), 12(S), 6-trans LTB4 isomer. These studies indicate that lipoxygenase products of arachidonic acid metabolism are present in the sputum in various forms of obstructive airways disease. The failure to detect the 'SRS-A' leukotrienes in sputum from bronchial asthma may be attributable to either losses during extraction, the insensitivity of the assay procedure or to more rapid catabolism of LTC4 and LTD4 by bronchial secretions in asthma than in cystic fibrosis.

Topics: Asthma; Biological Assay; Bronchitis; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Cystic Fibrosis; Humans; Leukotriene B4; Lung Diseases, Obstructive; Muscle Contraction; Neutrophils; Sputum; SRS-A

The recent definition of the pathways of generation and structures of diverse products of the lipoxygenation of arachidonic acid has established the identity of a new family of mediators of hypersensitivity and inflammation. Studies of the effects of these mediators have shown that leukotrienes C, D, and E, the constitutents of the slow-reacting substance of anaphylaxis (SRS-A), are extremely potent smooth muscle contractile and vasoactive factors. Leukotriene B is a highly active stimulus of neutrophil and eosinophil functions and suppresses the immunological capabilities of T lymphocytes. The development of specific and sensitive radioimmunoassays has permitted the detection of elevated concentrations of leukotrienes in tissues or exudates in several diseases, including asthma, diverse allergic states, adult respiratory distress syndrome, psoriasis, spondyloarthritis, and gout. The application of selective inhibitors and antagonists of leukotrienes will clarify their pathogenetic contributions in human diseases and may yield new therapeutic approaches.

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Arthritis; Asthma; Cystic Fibrosis; Humans; Hypersensitivity; Leukotriene A4; Leukotriene B4; Leukotriene E4; Lipoxygenase; Psoriasis; SRS-A; Tears

Sputum extracts from 16 of 25 adult patients with CF produced a slow, sustained contraction of isolated guinea pig ileum which was partially inhibitable by the SRS-A antagonist FPL 55712. Following progressive purification and HPLC, the major ileum-contracting substance was identified as LTD4. LTC4 was also present but in smaller amounts. The sputum contained other SRS-A-like activity which was not inhibited by FPL 55712; this substance was of a smaller molecular size than LTD4. The presence of LTB4 was also established using HPLC and an assay of neutrophil chemotaxis. These studies raise the possibility that leukotrienes and other lipoxygenase products may contribute to the progressive lung disease characteristics of CF.

Topics: Adolescent; Adult; Animals; Arachidonic Acids; Biological Assay; Chemotaxis; Chromones; Cystic Fibrosis; Female; Guinea Pigs; Humans; Ileum; Leukotriene B4; Male; Muscle Contraction; Neutrophils; Sputum; SRS-A

Topics: Airway Obstruction; Arachidonic Acids; Chromatography, High Pressure Liquid; Cystic Fibrosis; Humans; Leukotriene B4; Respiratory Hypersensitivity; Sputum; SRS-A