interleukin-8 and Cystic-Fibrosis

Cystic fibrosis (CF) is a genetically inherited disease, with mortality and morbidity associated with respiratory disease. The inflammatory response in CF is characterized by excessive neutrophil influx to the airways, mainly due to the increased local production and retention of interleukin-8 (IL-8), a potent neutrophil chemoattractant.. We discuss how the chemokine IL-8 dominates the inflammatory profile of the airways in CF lung disease. Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies are designed to correct the malfunctioning protein resulting from specific CFTR mutations. This review covers current evidence on the impact of CFTR impairment on levels of IL-8 and outlines the influence of effective CFTR modulation on inflammation in CF with a focus on cytokine production. Review of the literature was carried out using the PUBMED database, Google Scholar, and The Cochrane Library databases, using several appropriate generic terms.. Therapeutic interventions specifically targeting the defective CFTR protein have improved the outlook for CF. Accumulating studies on the effect of highly effective CFTR modulation on inflammation indicate an impact on IL-8 levels. Further studies are required to increase our knowledge of early onset innate inflammatory dysregulation and on anti-inflammatory mechanisms of CFTR modulators.

Topics: Anti-Inflammatory Agents; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Interleukin-8; Mutation; Respiratory System

Progressive lung disease is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). Methods of correctly predicting the future progression of lung disease in patients with CF are essential for directing aggressive treatment to prevent loss of lung function and end stage respiratory failure. Areas covered: This review addresses predictors of respiratory disease progression in patients with CF. We searched Web of Science and Medline, with no restriction on publication date, with the search terms 'cystic fibrosis' and 'disease progression', 'lung function decline', 'prognosis', 'prediction/predictive', 'prediction/prognostic scores', 'risk factors', 'outcome measures/endpoints/disease surrogate', 'longitudinal/long term', 'statistical model', and 'survival'. Expert commentary: Forced expiratory volume in 1 sec (FEV

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Disease Progression; Exhalation; Forced Expiratory Volume; Humans; Interleukin-8; Leukocyte Elastase; Lung; Prognosis; Respiratory Insufficiency; Severity of Illness Index; Spirometry; Sputum

This review explores the potential for vitamin D to favorably alter the gut microbiota, given emerging evidence of the role of vitamin D in controlling mucosal inflammation in the gut. It will focus on cystic fibrosis (CF) patients, a population with both vitamin D deficiency due to gut malabsorption and an altered gut microbiota composition. Recent evidence shows that vitamin D acts to maintain the integrity of the gut mucosal barrier by enhancement of intercellular junctions that control mucosal permeability and reduction of pro-inflammatory cytokines such as IL-8. In addition, vitamin D receptor-mediated signaling has been shown to inhibit inflammation-induced apoptosis of intestinal epithelial cells. As a result of these effects on the intestinal mucosa, maintenance of sufficient vitamin D status may be essential for the development of a healthy gut microbiota, particularly in conditions defined by chronic mucosal inflammation such as CF. We hypothesize here that high dose vitamin D may be used to favorably manipulate the aberrant mucosa seen in patients with CF. This may result in improved clinical outcomes in association with a low inflammatory environment that allows beneficial bacteria to outcompete opportunistic pathogens. Current evidence is sparse but encouraging, and additional evidence is needed to establish vitamin D as a therapeutic approach for gut microbiota modification.

Topics: Animals; Cystic Fibrosis; Dysbiosis; Epithelial Cells; Gastrointestinal Microbiome; Gastrointestinal Tract; Gene Expression Regulation; Humans; Intercellular Junctions; Interleukin-8; Intestinal Mucosa; Mice; Receptors, Calcitriol; Signal Transduction; Vitamin D; Vitamin D Deficiency

Cystic fibrosis (CF) is a lifelong, inflammatory multi-organ disease and the most common lethal, genetic condition in Caucasian populations, with a median survival rate of 41.5 years. Pulmonary disease, characterized by infective exacerbations, bronchiectasis and increasing airway insufficiency is the most serious manifestation of this disease process, currently responsible for over 80% of CF deaths. Chronic dysregulation of the innate immune and host inflammatory response has been proposed as a mechanism central to this genetic condition, primarily driven by the nuclear factor κB (NF-κB) pathway. Chronic activation of this transcription factor complex leads to the production of pro-inflammatory cytokines and mediators such as IL-6, IL-8 and TNF-α. A20 has been described as a central and inducible negative regulator of NF-κB. This intracellular molecule negatively regulates NF-κB-driven pro-inflammatory signalling upon toll-like receptor activation at the level of TRAF6 activation. Silencing of A20 increases cellular levels of p65 and induces a pro-inflammatory state. We have previously shown that A20 expression positively correlates with lung function (FEV1%) in CF. Despite improvement in survival rates in recent years, advancements in available therapies have been incremental. We demonstrate that the experimental use of naturally occurring plant diterpenes such as gibberellin on lipopolysaccharide-stimulated cell lines reduces IL-8 release in an A20-dependent manner. We discuss how the use of a novel bio-informatics gene expression connectivity-mapping technique to identify small molecule compounds that similarly mimic the action of A20 may lead to the development of new therapeutic approaches capable of reducing chronic airway inflammation in CF.

Topics: Cell Culture Techniques; Chromosome Mapping; Cystic Fibrosis; Cytokines; DNA-Binding Proteins; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Intracellular Signaling Peptides and Proteins; Neoplasm Proteins; NF-kappa B; Nuclear Proteins; Phenotype; Signal Transduction; Toll-Like Receptors; Tumor Necrosis Factor alpha-Induced Protein 3; Tumor Necrosis Factor-alpha

Interleukin-8 (IL-8) is a neutrophil chemokine that is encoded on the CXCL8 gene. Normally CXCL8 expression is repressed due to histone deacetylation, octamer-1 binding to the promoter and the inhibitory effect of nuclear factor-κB repressing factor (NRF). However, in response to a suitable stimulus, the human CXCL8 gene undergoes transcription due to its inducible promoter that is regulated by the transcription factors nuclear factor-κB (NF-κB), activating protein (AP-1), CAAT/enhancer-binding protein β (C/EBPβ, also known as NF-IL-6), C/EBP homologous protein (CHOP) and cAMP response element binding protein (CREB). CXCL8 mRNA is then stabilised by the activity of p38 mitogen-activated protein kinase (p38 MAPK). Cystic fibrosis (CF) lung disease is characterised by a neutrophil-dominated airway inflammatory response. A major factor contributing to the large number of neutrophils is the higher than normal levels of IL-8 that are present within the CF lung. Infection and inflammation, together with intrinsic alterations in CF airway cells are responsible for the abnormally high intrapulmonary levels of IL-8. Strategies to inhibit aberrantly high CXCL8 expression hold therapeutic potential for CF lung disease.

Topics: Animals; Cystic Fibrosis; Gene Expression Regulation; Humans; Interleukin-8; Lung; Molecular Targeted Therapy; Transcription, Genetic

Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa. In addition, the effects of decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA-DNA-PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent decoy molecules. Interestingly, PDP/PDP NF-kappaB decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance.

Topics: Animals; Cell Line; Cystic Fibrosis; Cytokines; DNA; Humans; Inflammation; Interleukin-8; Molecular Targeted Therapy; NF-kappa B; Oligodeoxyribonucleotides; Peptide Nucleic Acids; Pseudomonas aeruginosa

The development of drugs able to inhibit the expression of pro-inflammatory genes is of great interest in the treatment of cystic fibrosis (CF). Chronic pulmonary inflammation in the lungs of patients affected by CF is characterized by massive intra-bronchial infiltrates of neutrophils. This process is initiated upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells. Consequently, they release cytokines, the most represented being the potent neutrophilic chemokine Interleukin (IL)-8 and the pro-inflammatory cytokine IL-6. The chronic inflammatory process is crucial, since it leads to progressive tissue damage and severe respiratory insufficiency. In order to reduce the adverse effects of the excessive inflammatory response, one of the approaches leading to inhibition of IL-8 and IL-6 gene expression is the transcription factor (TF) decoy approach, based on intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of TFs and causing inhibition of binding of TF-related proteins to regulatory sequences identified in the promoters of specific genes. Since the promoters of IL-8 and IL-6 contain consensus sequences for NF-κ B and Sp1, double stranded TF "decoy" ODNs targeting NF-κB and Sp1 can be used. Alternatively, screening of drugs targeting relevant TFs can be performed using drug cocktails constituted by extracts from medicinal plants inhibiting TF/DNA interactions. Finally, virtual screening might lead to identification of putative bioactive molecules to be validated using molecular and cellular approaches. By these means, low-molecular drugs targeting NF-κB and inhibiting IL-8 gene expression are available for pre-clinical testing using experimental systems recapitulating chronic pulmonary inflammation of patients affected by CF.

Topics: Animals; Anti-Inflammatory Agents; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Molecular Weight; NF-kappa B; Oligonucleotides; Transcription Factors

Inhaled air is contaminated with pathogens and particulates that may deposit in the airways and damage the host. In response to these invaders, the airway epithelium has developed innate immune responses that provide a defence against the invaders and protect the airway structure and function. Thus, the epithelium of conducting airways becomes the "battleground" between the invaders and the host. Recent evidence suggests that airway epithelial surface signalling through the epidermal growth factor receptor (EGFR) is a convergent pathway producing innate immune responses to a variety of infectious and noninfectious noxious stimuli. In the present review, the EGFR signalling pathways leading to airway mucin production, neutrophil recruitment (via interleukin-8 production) and airway epithelial repair were examined. The importance of these findings in human airway diseases was also investigated. The current authors suggest that the exaggerated innate immune responses found in chronic inflammatory airway diseases (e.g. chronic obstructive pulmonary disease, cystic fibrosis and severe asthma) contribute to the pathogenesis or the aggravation of these diseases. Potential therapies include inhibition of the various elements of the described epidermal growth factor receptor cascade. In considering each therapeutic intervention, the potential benefits must be considered in relation to potential deleterious effects.

Topics: Animals; Asthma; Cystic Fibrosis; ErbB Receptors; Humans; Immunity, Innate; Interleukin-8; Ligands; Models, Biological; Mucins; Neutrophils; Pulmonary Disease, Chronic Obstructive; Reactive Oxygen Species; Signal Transduction

Cystic fibrosis (CF) is a progressive disease in which the lung is perceived to be normal at birth and is injured by recurrent infection. However, there is increasing evidence that the lung is functionally and structurally abnormal prior to the appearance of clinical infection. The cystic fibrosis transmembrane regulator (CFTR) is highly expressed in fetal tissues, and this review examines the role of CFTR in regulatory cascades during lung development. Early changes in the CF lung are examined from a perspective of disrupted fetal development, explaining a number of paradoxes seen with the disease.

Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Genetic Therapy; Humans; Interleukin-8; Lung; Mutation; Phenotype; Respiratory Function Tests; Respiratory Tract Infections; RNA, Messenger

Cystic fibrosis (CF) is the most common genetic autosomal recessive disease in caucasian north-american and european populations. The CF gene codes for a transmembrane glycoprotein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a chloride channel which regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells. Mutations of the CF gene lead to a dysregulation of chloride and sodium channel associated to airway mucus dehydration, neutrophil-dominated airway inflammation and chronic infection responsible for the morbidity and mortality of CF patients. Although a high number of studies has been devoted to the CFTR pleiotropic functions, the chronology of the physiopathological events leading to the airway inflammation linked to mutations of the CF gene is still an open question. The issue of whether airway inflammation takes place before infection or is a consequence of infection during CF pathogenesis is still controversial. It has been recently reported that in broncho-alveolar lavages collected in CF infants, there is an increased level of interleukin IL-8 and abnormal low level of IL-10. The decreased IL-10 production has been confirmed in peripheral blood monocytes as well as in airway cell lines. Under basal conditions, the increased expression of the pro-inflammatory IL-8 cytokine has also been recently observed in the airway liquid secreted by CF naïve humanized airway xenografts and in the supernatant culture of CF human airway epithelial cells. These results suggest that CFTR dysfunction may result in a constitutive pro-inflammatory vs anti-inflammatory imbalance in CF disease. Recent data from the literature suggest that the failure of chloride transport, the maturation defect and mistraffricking of mutated CFTR, lead to its accumulation in the endoplasmic reticulum and activation of NF-kappa B, responsible for the imbalance in the CF airway cell cytokine production.

Topics: Animals; Bacterial Infections; Bronchitis; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Humans; Interleukin-10; Interleukin-8; Mutation; Virus Diseases

Topics: Anti-Bacterial Agents; Bacterial Adhesion; Biofilms; Bronchi; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Drug Resistance, Multiple, Bacterial; Humans; Interleukin-8; Macrolides; Neutrophils; Respiratory Tract Infections; Sputum; Viscosity

Structural lung disease and neutrophil-dominated airway inflammation is present from 3 months of age in children diagnosed with cystic fibrosis after newborn screening. We hypothesised that azithromycin, given three times weekly to infants with cystic fibrosis from diagnosis until age 36 months, would reduce the extent of structural lung disease as captured on chest CT scans.. A phase three, randomised, double-blind, placebo-controlled trial was done at eight paediatric cystic fibrosis centres in Australia and New Zealand. Infants (aged 3-6 months) diagnosed with cystic fibrosis following newborn screening were eligible. Exclusion criteria included prolonged mechanical ventilation in the first 3 months of life, clinically significant medical disease or comorbidities other than cystic fibrosis, or macrolide hypersensitivity. Participants were randomly assigned (1:1) to receive either azithromycin (10 mg/kg bodyweight orally three times per week) or matched placebo until age 36 months. Randomisation was done with a permuted block strategy and an interactive web-based response system, stratified by study site. Unblinding was done once all participants completed the trial. The two primary outcomes were the proportion of children with radiologically defined bronchiectasis, and the percentage of total lung volume affected by disease. Secondary outcomes included clinical outcomes and exploratory outcomes were inflammatory markers. Analyses were done with the intention-to-treat principle. This study is registered at ClinicalTrials.gov (NCT01270074).. Between June 15, 2012, and July 10, 2017, 281 patients were screened, of whom 130 were enrolled, randomly assigned, and received first study dose. 68 participants received azithromycin and 62 received placebo. At 36 months, 88% (n=50) of the azithromycin group and 94% (n=44) of the placebo group had bronchiectasis (odds ratio 0·49, 95% CI 0·12 to 2·00; p=0·32), and total airways disease did not differ between groups (median difference -0·02%, 95% CI -0·59 to 0·56; p=0·96). Secondary outcome results included fewer days in hospital for pulmonary exacerbations (mean difference -6·3, 95% CI -10·5 to -2·1; p=0·0037) and fewer courses of inhaled or oral antibiotics (incidence rate ratio 0·88, 95% CI 0·81 to 0·97; p=0·0088) for those in the azithromycin group. For the preplanned, exploratory analysis, concentrations of airway inflammation were lower for participants receiving azithromycin, including interleukin-8 (median difference -1·2 pg/mL, 95% CI -1·9 to -0·5; p=0·0012) and neutrophil elastase activity (-0·6 μg/mL, -1·1 to -0·2; p=0·0087) at age 36 months, although no difference was noted between the groups for interleukin-8 or neutrophil elastase activity at 12 months. There was no effect of azithromycin on body-mass index at age 36 months (mean difference 0·4, 95% CI -0·1 to 0·9; p=0·12), nor any evidence of pathogen emergence with the use of azithromycin. There were few adverse outcomes with no differences between the treatment groups.. Azithromycin treatment from diagnosis of cystic fibrosis did not reduce the extent of structural lung disease at 36 months of age; however, it did reduce airway inflammation, morbidity including pulmonary exacerbations in the first year of life and hospitalisations, and improved some clinical outcomes associated with cystic fibrosis lung disease. Therefore we suggest thrice-weekly azithromycin is a strategy that could be considered for the routine early management of paediatric patients with cystic fibrosis.. Cystic Fibrosis Foundation.

Topics: Anti-Bacterial Agents; Azithromycin; Bronchiectasis; Child; Child, Preschool; Cystic Fibrosis; Double-Blind Method; Humans; Infant; Infant, Newborn; Inflammation; Interleukin-8; Leukocyte Elastase

Cystic fibrosis (CF) lung disease progresses by a combination of airway inflammation, bacterial colonization, and infection. Airway inflammation is predominantly neutrophilic and complicates airway clearance therapies through cellular debris; excessive DNA; excessive and viscous mucus; and high concentrations of neutrophils, IL-8, and related cytokines liberated along the nuclear factor-κB signaling pathway.. We conducted a preliminary, single-site, randomized, double-blind, placebo-controlled study to evaluate the effects over 28 days of two dose levels (0.05 mg and 0.1 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on safety, pharmacokinetics, and inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease.. Patients with CF 18-45 years old with any genotype combination were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure (1) the pharmacokinetics of digitoxin in sera of patients with stable CF; (2) safety indices, including ECG changes and sputum microbiology; (3) the effect of digitoxin on gene expression in nasal epithelial cells of patients with stable CF; and (4) quality-of-life scores using the Cystic Fibrosis Questionnaire-Revised.. It took several weeks to achieve a therapeutic serum level of digitoxin in subjects with CF. No safety concerns emerged during the study. Digitoxin treatment showed a trend toward reduction in sputum free neutrophil elastase and neutrophil counts, but not a reduction in sputum IL-8. Digitoxin treatment did not reach statistical significance for the primary or secondary outcome measures over the 28-day study period. However, the nasal mRNA from the group receiving 0.1 mg of digitoxin daily had a distinct distribution of global gene expression levels as compared with either the 0.05-mg dose or placebo treatment. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment, and mucus hypersecretion.. At a dose of 0.1 mg daily for 28 days, digitoxin was safe for adults with CF lung disease, but it did not achieve a significant decrease in sputum inflammatory markers. Clinical trial registered with www.clinicaltrials.gov (NCT00782288).

Topics: Adolescent; Adult; Anti-Inflammatory Agents; Biomarkers; Cystic Fibrosis; Digitoxin; Double-Blind Method; Female; Humans; Inflammation; Interleukin-8; Leukocyte Count; Lung; Male; Maryland; Neutrophils; Sputum; Young Adult

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Topics: Adolescent; Animals; Autophagy; Biomarkers; Catechin; Child; Cysteamine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Drug Therapy, Combination; Homozygote; Humans; Interleukin-8; Lung; Mice; Mice, Knockout; Mutation; Sputum; Tumor Necrosis Factor-alpha

The aim of this study was to evaluate in patients with cystic fibrosis (CF) the effect of Lactobacillus reuteri (LR) on the rate of respiratory exacerbations and of the infections of both upper respiratory and gastrointestinal tracts.. Prospective randomized, double-blind, placebo-controlled study enrolling 61 patients with CF with mild-to-moderate lung disease at the Regional Center for CF of the Department of Pediatrics, University of Rome "La Sapienza." All of the patients were not hospital inpatients at the time of the enrollment. Inclusion criteria were forced expiratory volume in the first second (FEV1) >70% predicted; no inhaled or systemic steroids, no anti-inflammatory drugs, antileukotrienes, and mast cell membrane stabilizers; and no serious organ involvement. Exclusion criteria were a history of pulmonary exacerbation or upper respiratory infection in the previous 2 months; changes in medications in the last 2 months; a history of hemoptysis in the last 2 months; and colonization with Burkholderia cepacia or mycobacteria. Patients were randomly assigned to receive LR (30 patients) in 5 drops per day (10(10) colony-forming units) or placebo (31 patients) for 6 months. Main outcomes were number of episodes of pulmonary exacerbations and hospital admissions for pulmonary exacerbations, number of gastrointestinal and upper respiratory tract infections. FEV1, fecal calprotectin, and cytokine profile in induced sputum and plasma were assessed at baseline and at the end of the trial.. Pulmonary exacerbations were significantly reduced in the LR group compared with the placebo group (P<0.01; odds ratio 0.06 [95% confidence interval {CI} 0-0.40]; number needed to treat 3 [95% CI 2-7]). Similarly, the number of upper respiratory tract infections (in our series only otitis) was significantly reduced in the LR group compared with the placebo group (P<0.05; odds ratio 0.14 [95% CI 0-0.96]; number needed to treat 6 [95% CI 3-102]). The 2 groups did not differ statistically in the mean number and duration of hospitalizations for pulmonary exacerbations and gastrointestinal infections. There was no significant statistical difference in the mean delta value of FEV1, fecal calprotectin concentration, and tested cytokines (tumor necrosis factor-α and interleukin-8) between the 2 groups.. LR reduces pulmonary exacerbations and upper respiratory tract infections in patients with CF with mild-to-moderate lung disease. LR administration may have a beneficial effect on the disease course of CF.

Topics: Adolescent; Adult; Child; Cystic Fibrosis; DNA-Binding Proteins; Double-Blind Method; Female; Forced Expiratory Volume; Gastrointestinal Diseases; Hospitalization; Humans; Interleukin-8; Leukocyte L1 Antigen Complex; Limosilactobacillus reuteri; Lung; Lung Diseases; Male; Nuclear Proteins; Numbers Needed To Treat; Probiotics; Prospective Studies; Respiratory Tract Infections; Transcription Factors; Tumor Necrosis Factor-alpha; Young Adult

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Topics: Adaptor Proteins, Signal Transducing; Administration, Oral; Adolescent; Adult; Animals; Apoptosis Regulatory Proteins; Beclin-1; Catechin; Cell Membrane; Child; Chlorides; Cystamine; Cysteamine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Homozygote; Humans; Interleukin-8; Male; Membrane Proteins; Mice; Mice, Inbred CFTR; Mice, Transgenic; Mutation; Phenotype; Pilot Projects; Sequestosome-1 Protein; Tumor Necrosis Factor-alpha; Young Adult

Low plasma concentrations of docosahexaenoic acid (DHA) are reported in unsupplemented cystic fibrosis (CF) patients. Forty-one CF patients aged from 6 to 12 years were randomized to receive high-dose DHA (100 mg/kg/day in the first month and 1g per day thereafter through a 12-month supplementation) or placebo (germ oil). Primary outcome was percentage change in plasma AA:DHA ratio. Secondary outcomes were changes in the number of pulmonary exacerbations compared to previous year, lung function, BMI, skinfold thicknesses, and body composition assessed by DXA and in serum concentrations of C-reactive protein, cytokines and vitamin (α-tocopherol and retinol). Compared to the control group plasma AA:DHA ratio decreased in the intervention group after 6 months (median percentage changes: -73% in the intervention group vs. -10% in the control group, P=0.001). No differences were detected between groups for secondary outcomes. Despite a decrease of the AA/DHA ratio, DHA supplementation for one year did not induce any significant biochemical and clinical improvement in CF patients.

Topics: Administration, Oral; alpha-Tocopherol; Body Composition; Bone Density; C-Reactive Protein; Child; Cystic Fibrosis; Docosahexaenoic Acids; Female; Humans; Interleukin-8; Male; Tumor Necrosis Factor-alpha; Vitamin A

Measurement of exhaled breath condensate (EBC) biomarkers offers a noninvasive means to assess airway disease, but the ability of EBC biomarkers to track longitudinal changes in disease severity remains unproven. EBC was collected from pediatric patients with cystic fibrosis (CF) during regular clinic visits over 1 yr. EBC biomarkers urea, adenosine (Ado), and phenylalanine (Phe) were measured by mass spectrometry, and biomarker ratios were used to control for variable dilution of airway secretions. EBC biomarker ratios were assessed relative to lung function in longitudinal, multivariate models and compared with sputum inflammatory markers and quality of life assessment (CFQ-R). EBC was successfully analyzed from 51 subjects during 184 visits (3.6 ± 0.9 visits per subject). EBC Ado/urea ratio was reproducible in duplicate samples (r = 0.62, P < 0.01, n = 20) and correlated with sputum neutrophil elastase (β = 2.5, P < 0.05). EBC Ado/urea correlated with the percentage predicted of forced expiratory volume in 1 s in longitudinal, multivariate models (β = -2.9, P < 0.01); EBC Ado/Phe performed similarly (β = -2.1, P < 0.05). In contrast, IL-8 and elastase measured in spontaneously expectorated sputum (n = 57 samples from 25 subjects) and the CFQ-R respiratory scale (n = 90 tests from 47 subjects) were not significantly correlated with lung function. EBC was readily collected in a clinic setting from a wide range of subjects. EBC Ado tracked longitudinal changes in lung function in CF, with results similar to or better than established measures.

Topics: Adenosine; Adolescent; Adult; Biomarkers; Child; Cystic Fibrosis; Female; Humans; Interleukin-8; Male; Pancreatic Elastase; Quality of Life; Respiratory Function Tests; Sputum; Urea

Long-term administration of azithromycin (AZM) in children with cystic fibrosis (CF) has improved outcomes. However, the doses and schedule of administration are not very well studied in children with CF.. A randomized controlled trial was conducted to compare the effect of two doses of azithromycin (5mg/kg/day and 15mg/kg/day) on FEV(1) and pulmonary exacerbations in children with cystic fibrosis. Enrolled children were randomly allocated to receive daily azithromycin (5mg/kg/day or 15mg/kg/day) for 6months. Clinical assessment and FEV(1) measurement were performed monthly.. 56 children (28 in high dose group and 28 in low dose group) were enrolled. 47 (24 and 23 children in low and high dose groups) completed 12months of follow up. There was no difference in clinical scores, FEV(1), pulmonary exacerbation rates between two groups at baseline, 6months and at 12months. Per protocol analysis revealed that pulmonary exacerbation increased after discontinuing AZM and there was significantly more increase after 12months of enrolment in children getting high dose azithromycin. There was no improvement in FEV(1) in either group at the end of treatment period. Children tolerated daily low as well as high dose AZM well for 6months. There was no significant side effect of azithromycin.. In this randomized controlled trial, we did not find differences in the effect of 2 doses (5mg/kg/day or 15mg/kg/day) of AZM on change in percentage predicted FEV(1), clinical scores, Pseudomonas colonization rates, pulmonary exacerbations and need for antibiotics. There was increase in exacerbations after stopping azithromycin in both the groups. Our results also suggest that the decrease in the incidence of LRTI persists only till 6months after discontinuing azithromycin.

Topics: Anti-Bacterial Agents; Azithromycin; Body Weight; Candidiasis; Child; Child, Preschool; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Spirometry; Sputum; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus pneumoniae; Treatment Outcome

Cystic fibrosis (CF) is characterized by malnutrition, chronic pulmonary inflammation, and oxidative stress. Whey protein is rich in sulfhydryl groups and is recognized for its ability to increase glutathione and reduce oxidative stress. Previously, we have shown that supplementation with whey increased intracellular glutathione levels in patients with CF. We have subsequently shown that hyperbaric pressure treatment of whey protein promotes the release of novel peptides for absorption, increases intracellular glutathione in healthy subjects, and reduces in vitro production of interleukin (IL)-8. We hypothesized that pressurized whey supplementation in children and adults with CF could have significant nutritional and anti-inflammatory benefits. A pilot open-label study of 1-month dietary supplementation with pressurized whey in CF patients was undertaken to assess the effects. Twenty-seven patients with CF (nine children, 18 adults) were enrolled. The dose of pressurized whey was 20 g/day in patients less than 18 years of age and 40 g/day in older patients. Anthropometric measures, pulmonary function, serum C-reactive protein (CRP), whole blood glutathione, and whole blood IL-8 and IL-6 responses to phytohemagglutinin (PHA) stimulation were measured at baseline and at 1 month. Three adults withdrew (one with gastrointestinal side effects, two with acute infection). Both children and adults showed enhancements in nutritional status, as assessed by body mass index. Children showed improvement in lung function (forced expiratory volume in 1 second). The majority of patients with an initially elevated CRP showed a decrease. PHA-stimulated IL-8 responses tended to decrease in the adults. Whole blood glutathione levels did not change. Thus, oral supplementation with pressurized whey improves nutritional status and can have additional beneficial effects on inflammation in patients with CF.

Topics: Adolescent; Adult; Anti-Inflammatory Agents; Body Mass Index; C-Reactive Protein; Child; Cystic Fibrosis; Dietary Proteins; Dietary Supplements; Female; Forced Expiratory Volume; Glutathione; Humans; Interleukin-8; Lung; Male; Milk Proteins; Nutritional Status; Oxidative Stress; Pilot Projects; Pressure; Weight Gain; Whey Proteins; Young Adult

To obtain a balance in the fatty acid (FA) metabolism is important for the inflammatory response and of special importance in cystic fibrosis (CF), which is characterized by impaired FA metabolism, chronic inflammation, and infection in the airways. Nitric oxide (NO) has antimicrobial properties and low nasal (nNO) and exhaled NO (FENO), commonly reported in CF that may affect bacterial status. The present study investigates the effect of different FA blends on nNO and FENO and immunological markers in patients with CF.. Forty-three patients with CF and "severe" mutations were consecutively enrolled in a randomized double-blind placebo-controlled study with 3 FA blends containing mainly n-3 or n-6 FA or saturated FA acting as placebo. FENO, nNO, serum phospholipid concentrations of FA, and biomarkers of inflammation were measured before and after 3 months of supplementation.. Thirty-five patients in clinically stable condition completed the study. The serum phospholipid FA pattern changed significantly in all 3 groups. An increase of the n-6 FA, arachidonic acid, was associated with a decrease of FENO and nNO. The inflammatory biomarkers, erythrocyte sedimentation rate, and interleukin-8 decreased after supplementation with n-3 FA and erythrocyte sedimentation rate increased after supplementation with n-6 FA.. This small pilot study indicated that the composition of dietary n-3 and n-6 FA influenced the inflammatory markers in CF. FENO and nNO were influenced by changes in the arachidonic acid concentration, supporting previous studies suggesting that both the lipid abnormality and the colonization with Pseudomonas influenced NO in the airways.

Topics: Adolescent; Adult; Arachidonic Acid; Biomarkers; Blood Sedimentation; Child; Cystic Fibrosis; Dietary Supplements; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Humans; Inflammation Mediators; Interleukin-8; Male; Nasal Mucosa; Nitric Oxide; Phospholipids; Pilot Projects; Respiratory System; Young Adult

We conducted a single-centre, randomised, double-blinded, placebo-controlled phase II clinical study to test safety and efficacy of a 12-week therapy with low-dose (700 mg/daily) or high-dose (2800 mg/daily) of NAC.. Twenty-one patients (DeltaF508 homo/heterozygous, FEV1>40% pred.) were included in the study. After a 3-weeks placebo run-in phase, 11 patients received low-dose NAC, and 10 patients received high-dose NAC. Outcomes included safety and clinical parameters, inflammatory (total leukocyte numbers, cell differentials, TNF-alpha, IL-8) measures in induced sputum, and concentrations of extracellular glutathione in induced sputum and blood.. High-dose NAC was a well-tolerated and safe medication. High-dose NAC did not alter clinical or inflammatory parameters. However, extracellular glutathione in induced sputum tended to increase on high-dose NAC.. High-dose NAC is a well-tolerated and safe medication for a prolonged therapy of patients with CF with a potential to increase extracellular glutathione in CF airways.

Topics: Acetylcysteine; Adult; Cystic Fibrosis; Dose-Response Relationship, Drug; Double-Blind Method; Female; Forced Expiratory Volume; Free Radical Scavengers; Glutathione; Humans; Interleukin-8; Male; Sputum; Tumor Necrosis Factor-alpha; Young Adult

Antibiotics are largely prescribed for cystic fibrosis (CF) respiratory exacerbations. Effects of antibiotics on the inflammatory profile of the patients have been shown but remain controversial. Lipoxin A(4) (LXA(4)) is a lipid mediator, reported to play a central role in resolving airway inflammation. The aim of the study was to investigate the consequences of antibiotherapy on LXA(4) and IL-8 levels in CF patients' airways.. Eighteen CF patients (7 females, median age 20, range 8 to 47 years) consecutively admitted at the CF center of Montpellier for antibiotics during pulmonary exacerbation, were enrolled. Before and after antibiotics, all patients underwent spirometry (FEV(1) and FVC), bacterial cultures and cell counts in sputa. IL-8 and LXA(4) concentrations were determined in sputum samples by the median of immunometric assays.. As previously reported, after antibiotics therapy, FEV(1) and FVC significantly improved. While neutrophil cell counts and IL-8 levels decreased, the LXA(4) levels significantly increased after antibiotics therapy and were inversely correlated with IL-8 levels. In conclusion, we reported a correlation between antibiotics treatments and inflammatory markers in CF sputum. Our data provide evidences for a novel effect of antibiotics increasing the concentration of the anti-inflammatory lipid mediator LXA(4).

Topics: Adolescent; Adult; Anti-Bacterial Agents; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Infusions, Intravenous; Interleukin-8; Lipoxins; Male; Middle Aged; Prospective Studies; Sputum; Treatment Outcome; Vital Capacity; Young Adult

Pancreatic insufficiency and a diminished bile acid pool cause malabsorption of important essential nutrients and other dietary components in cystic fibrosis (CF). Of particular significance is the malabsorption of fat-soluble antioxidants such as carotenoids, tocopherols and coenzyme Q(10) (CoQ(10)). Despite supplementation, CF patients are often deficient in these compounds, resulting in increased oxidative stress, which may contribute to adverse health effects. This pilot study was designed to evaluate the safety of a novel micellar formulation (CF-1) of fat-soluble nutrients and antioxidants and to determine its efficacy in improving plasma levels of these compounds and reducing inflammatory markers in induced sputum.. Ten CF subjects, ages 8 to 45 years old, were given orally 10 ml of the CF-1 formulation daily for 56 days after a 21-day washout period in which subjects stopped supplemental vitamin use except for a standard multivitamin. Plasma obtained at -3, 0 (baseline), 1, 2, 4, and 8 weeks was assayed for beta-carotene, gamma-tocopherol, retinol, and CoQ(10) as well as for safety parameters (comprehensive metabolic panel and complete blood count). In addition, pulmonary function was measured and induced sputum was assayed for markers of inflammation and quantitative bacterial counts both prior and during dosing.. No serious adverse effects, laboratory abnormalities or elevated nutrient levels (above normal) were identified as related to CF-1. Supplementation with CF-1 significantly increased beta-carotene levels at all dosing time points when compared to screening and baseline. In addition, gamma-tocopherol and CoQ(10) significantly increased from baseline in all subjects. Induced sputum myeloperoxidase significantly decreased and there was a trend toward decreases in PMN elastase and total cell counts with CF-1. There was a significant inverse correlation between the antioxidant levels and induced sputum changes in IL-8 and total neutrophils. Lung function and sputum bacterial counts were unchanged.. The novel CF-1 formulation safely and effectively increased plasma levels of important fat-soluble nutrients and antioxidants. In addition, improvements in antioxidant plasma levels were associated with reductions in airway inflammation in CF patients.

Topics: Adolescent; Adult; Antioxidants; beta Carotene; Biological Availability; Child; Cystic Fibrosis; Dietary Supplements; Exocrine Pancreatic Insufficiency; gamma-Tocopherol; Humans; Interleukin-8; Micelles; Middle Aged; Pilot Projects; Sputum; Ubiquinone

Xylitol is a 5-carbon sugar that can lower the airway surface salt concentration, thus enhancing innate immunity. We tested the safety and tolerability of aerosolized iso-osmotic xylitol in subjects with cystic fibrosis.. In this pilot study, 6 subjects with cystic fibrosis and an FEV1>60% predicted underwent a baseline spirometry followed by exposures to aerosolized saline (10 ml) and 5% xylitol (10 ml). Serum osmolarity and electrolytes were measured at baseline and after xylitol exposure. Spirometry, oxygen saturation and respiratory symptom questionnaire using visual analog scale were tested at baseline and after each exposure. Sputum for cytokine analysis was collected after saline and xylitol nebulizations.. There was no change in FEV1 after xylitol exposure compared with baseline or normal saline exposure (p=0.19). Laboratory values and respiratory symptoms were not affected by xylitol inhalation. The mean IL-8 level in the sputum was similar with saline and xylitol exposures (3.5+/-0.5 vs. 3.5+/-0.6 ng/ml).. A single dose inhalation of aerosolized iso-osmotic xylitol was well tolerated by subjects with cystic fibrosis. Future studies of long term safety are required.

Topics: Adjuvants, Immunologic; Administration, Inhalation; Adult; Animals; Cystic Fibrosis; Female; Humans; Immunity, Innate; Interleukin-8; Male; Mice; Xylitol

Detecting and tracking early cystic fibrosis (CF) lung disease are difficult due to lack of sensitive markers of airway dysfunction.. The goals were to detect regional distribution of airway disease through high-resolution computed tomography, correlate abnormalities to lower airway inflammation/infection, and compare computed tomography findings before and after intravenous antibiotic therapy in children with CF younger than 4 years experiencing a pulmonary exacerbation.. High-resolution computed tomography was performed in 17 children scheduled for bronchoscopy. The radiologist identified the lobes with the "greatest" and "least" disease based on computed tomography, and bronchoalveolar lavage was performed in these areas. In 13 subjects, imaging was repeated after antibiotic completion. Modified Brody scores were assigned by two radiologists.. The lobe with greatest disease was predominantly localized to the right and had higher modified Brody scores, indicating more severe abnormalities (p < 0.01), compared with the lobe with least disease. The total modified Brody score (p < 0.01), hyperinflation subscore (p < 0.01), and bronchial dilatation/bronchiectasis subscore (p < 0.01) improved after antibiotics and intensified airway clearance. Interleukin-8 levels (p < 0.01) and % neutrophils (p = 0.04) were increased in the lobe with greatest disease compared with the lobe with least disease.. These results indicate that, in young children with CF experiencing a pulmonary exacerbation, computed tomography detects regional differences in airway inflammation, may be a sensitive outcome to evaluate therapeutic interventions, and identifies early lung disease as being more prominent on the right.

Topics: Anti-Bacterial Agents; Biomarkers; Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Female; Follow-Up Studies; Humans; Interleukin-8; Male; Reproducibility of Results; Severity of Illness Index; Tomography, X-Ray Computed; Treatment Outcome

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

Interferon gamma-1b (IFN-gamma1b) is a pleiotropic cytokine with immunomodulatory activities that could decrease bacterial burden, inflammation, and obstruction in patients with CF. Patients with CF (> or =12 years old, FEV1 > or =40% predicted) were randomly assigned to sequential dose cohorts inhaling 500 microg IFN-gamma1b, 1,000 microg IFN-gamma1b, or placebo by Respirgard II nebulizer thrice weekly for 12 weeks. Sputum bacterial density and spirometry were measured. Safety, antibiotic use, hospitalization, and sputum neutrophils, elastase, DNA, IL-8, and myeloperoxidase were also evaluated. Sixty-six patients (mean age, 24 years, with mean baseline FEV1 of 74 +/- 20 (SD) percent predicted) were studied. One patient had bronchospasm after the first dose of IFN-gamma1b; the overall withdrawal rate was 15% (5 in the placebo group, 2 in the 500-microg IFN-gamma1b group, and 3 in the 1,000 microg IFN-gamma1b group). The 500-microg IFN-gamma1b dose was well-tolerated, but the 1,000-mug dose cohort, who had a higher baseline bacterial density than placebo patients (mean difference, 1.2 log(10) CFU/g sputum, 95% confidence interval (CI), 0.1,2.8, P=0.04), had 24% more hospitalizations for exacerbation than placebo patients (95% CI, 2,45%, P=0.05). There was a 0.12-l difference between the 500-microg IFN-gamma1b and placebo groups with respect to the 12-week change in FEV1 (active group minus placebo group, 95% CI, -0.03,0.26, P=0.11), as compared to a 0.01-l difference between the 1,000-microg IFN-gamma1b and placebo groups (95% CI, -0.16,0.17, P=0.96). No effects of IFN-gamma1b were seen in sputum bacterial density or inflammatory biomarkers at 12 weeks. Aerosolized IFN-gamma1b did not improve pulmonary function, reduce sputum bacterial density, or affect inflammatory sputum markers in patients with mild-moderate lung disease.

Topics: Administration, Inhalation; Adolescent; Adult; Anti-Bacterial Agents; Antineoplastic Agents; Colony Count, Microbial; Cystic Fibrosis; Dose-Response Relationship, Drug; Double-Blind Method; Drug Administration Schedule; Dyspnea; Female; Hemoptysis; Hospitalization; Humans; Interferon-gamma; Interleukin-8; Male; Neutrophils; Pancreatic Elastase; Peroxidase; Recombinant Proteins; Respiratory Tract Infections; Sputum; Treatment Outcome

In cystic fibrosis (CF), the inflammatory process contributes to progressive lung tissue damage. Cysteinyl leukotrienes have been found in the sputum of patients with CF at high concentrations sufficient to cause potent biological effects.. To evaluate the effect of anti-inflammatory treatment with montelukast sodium in patients with CF.. Twenty-six patients aged 6 to 18 years were recruited to this 20-week, randomized, double-blind, placebo-controlled, crossover trial. Patients received montelukast or placebo for 8 weeks in addition to their regular CF treatment. Before and after treatment, findings from spirometry, whole-body plethysmography, and the clinical wheezing and cough scales were evaluated. At the same time, serum and sputum samples were obtained for the measurement of eosinophil cationic protein, interleukin 10 (IL-10), IL-8, and myeloperoxidase levels.. Twenty-three patients completed the study. Compared with placebo use, montelukast treatment significantly improved forced expiratory volume in I second, peak expiratory flow, and forced expiratory flow between 25% and 75% and significantly decreased cough and wheezing scale scores (P < .001 for all). There were no significant changes in vital capacity, thoracic gas volume, airway resistance, and residual volume after treatment. Compared with placebo use, montelukast treatment decreased serum and sputum levels of eosinophil cationic protein and IL-8, decreased sputum levels of myeloperoxidase, and increased serum and sputum levels of IL-10 (P < .001 for all).. Montelukast may have measurable anti-inflammatory properties in patients with CF.

Topics: Acetates; Adolescent; Airway Resistance; Anti-Asthmatic Agents; Child; Cough; Cross-Over Studies; Cyclopropanes; Cystic Fibrosis; Double-Blind Method; Eosinophil Cationic Protein; Female; Forced Expiratory Volume; Humans; Interleukin-10; Interleukin-8; Male; Peroxidase; Quinolines; Respiratory Sounds; Sputum; Sulfides; Treatment Outcome

Recombinant human deoxyribonuclease (rhDNase) has been shown to improve lung function and reduce the number of pulmonary exacerbations in patients with cystic fibrosis (CF), but its long-term effect on airway inflammation remains unknown. In this study, we used bronchoalveolar lavage (BAL) to investigate the long-term effect of rhDNase on inflammation in patients with CF having mild lung disease. A total of 105 patients with CF (> or =5 years of age) having normal lung function were randomized to receive rhDNase (2.5 mg/day) or no rhDNase. Patients with a normal percentage of neutrophils in BAL fluid at baseline were not randomized and served as the control group. The percentage of neutrophils in the pooled BAL sample was similar in both randomized groups at baseline. A significant increase in neutrophils was observed over the 3-year study period in both untreated patients and control subjects, whereas neutrophils remained unchanged in patients treated with rhDNase. Elastase activities and interleukin-8 concentrations also increased in untreated patients and remained stable in patients on rhDNase. We conclude that in patients with CF, an increase in neutrophilic airway inflammation is found that is positively influenced by rhDNase treatment.

Topics: Adolescent; Adult; Bronchoalveolar Lavage; Child; Child, Preschool; Cystic Fibrosis; Deoxyribonuclease I; Female; Follow-Up Studies; Humans; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Male; Neutrophils; Peroxidase; Pneumonia; Recombinant Proteins; Time Factors

To determine reproducibility of inflammatory marker concentrations in induced sputum from subjects with cystic fibrosis (CF), 15 nonexpectorating children, 6 to 13 years of age with mild CF lung disease, underwent 3 weekly sputum inductions with 3% saline. Neutrophil elastase concentration and bacterial cultures were reproducible. This study provides useful information for investigators designing trials of anti-inflammatory therapies in CF involving sputum induction.

Topics: Administration, Inhalation; Adolescent; Child; Cystic Fibrosis; Humans; Interleukin-6; Interleukin-8; Leukocyte Elastase; Nebulizers and Vaporizers; Peroxidase; Reproducibility of Results; Sodium Chloride; Sputum

Treatment strategies for cystic fibrosis (CF) lung disease include antibiotics, mucolytics, and anti-inflammatory therapies. Increasing evidence suggests that macrolide antibiotics might be beneficial in patients with CF.. To determine if an association between azithromycin use and pulmonary function exists in patients with CF.. A multicenter, randomized, double-blind, placebo-controlled trial conducted from December 15, 2000, to May 2, 2002, at 23 CF care centers in the United States.. Of the 251 screened participants with a diagnosis of CF, 185 (74%) were randomized. Eligibility criteria included age 6 years or older, infection with Pseudomonas aeruginosa for 1 or more years, and a forced expiratory volume in 1 second (FEV1) of 30% or more. Participants were stratified by FEV1 (> or =60% predicted vs <60% predicted), weight of less than 40 kg vs 40 kg or more, and CF center.. The active group (n = 87) received 250 mg (weight <40 kg) or 500 mg (weight > or =40 kg) of oral azithromycin 3 days a week for 168 days; placebo group (n = 98) received identically packaged tablets.. Change in FEV1 from day 0 to completion of therapy at day 168 and determination of safety. Secondary outcomes included pulmonary exacerbations and weight gain.. The azithromycin group had a mean 0.097-L (SD, 0.26) increase in FEV1 at day 168 compared with 0.003 L (SD, 0.23) in the placebo group (mean difference, 0.094 L; 95% confidence interval [CI], 0.023-0.165; P =.009). Nausea occurred in 17% more participants in the azithromycin group (P =.01), diarrhea in 15% more (P =.009), and wheezing in 13% more (P =.007). Participants in the azithromycin group had less risk of experiencing an exacerbation than participants in the placebo group (hazard ratio, 0.65; 95% CI, 0.44-0.95; P =.03) and weighed at the end of the study an average 0.7 kg more than participants receiving placebo (95% CI, 0.1-1.4 kg; P =.02).. Azithromycin treatment was associated with improvement in clinically relevant end points and should be considered for patients with CF who are 6 years or older and chronically infected with P aeruginosa.

Topics: Adolescent; Anti-Bacterial Agents; Azithromycin; Child; Chronic Disease; Cystic Fibrosis; Double-Blind Method; Female; Forced Expiratory Flow Rates; Hospitalization; Humans; Interleukin-8; Male; Pancreatic Elastase; Proportional Hazards Models; Pseudomonas Infections; Quality of Life; Treatment Outcome

The macrolide antibiotic azithromycin has anti-inflammatory properties potentially beneficial in cystic fibrosis. Since findings of open pilot studies seemed to show clinical benefit, we undertook a formal trial.. 41 children with cystic fibrosis, aged 8-18 years, and with a median forced expiratory volume in 1 s (FEV1) of 61% (range 33-80%) participated in a 15-month randomised double-blind, placebo-controlled crossover trial. They received either azithromycin (bodyweight < or =40 kg: 250 mg daily, >40 kg: 500 mg daily) or placebo for 6 months. After 2 months of washout, the treatments were crossed over. The primary outcome was median relative difference in FEV1 between azithromycin and placebo treatment periods. Sputum cultures, sputum interleukin 8 and neutrophil elastase, exercise testing, quality of life, antibiotic use, and pulmonary exacerbation rates were secondary outcome measures. Side-effects were assessed by pure tone audiometry and liver function tests. Analysis was by intention-to-treat.. Median relative difference in FEV1 between azithromycin and placebo was 5.4% (95% CI 0.8-10.5). 13 of 41 patients improved by more than 13% and five of 41 deteriorated by more than 13% (p=0.059). Forced vital capacity and mid-expiratory flow did not significantly change overall. 17 of 41 patients had 24 fewer oral antibiotic courses when on azithromycin than when taking placebo, and five had six extra courses (p=0.005). Sputum bacterial densities, inflammatory markers, exercise tolerance, and subjective well-being did not change. There were no noticeable side-effects.. A 4-6-month trial of azithromycin is justified in children with cystic fibrosis who do not respond to conventional treatment. The mechanism of action remains unknown.

Topics: Adolescent; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Azithromycin; Child; Cross-Over Studies; Cystic Fibrosis; Double-Blind Method; Exercise Test; Forced Expiratory Volume; Humans; Interleukin-8; Leukocyte Elastase; Pilot Projects; Quality of Life; Sputum

Immune-mediated inflammation contributes to progressive pulmonary damage in cystic fibrosis (CF). Sputum cysteinyl leukotriene levels, eosinophil cationic protein (ECP), and interleukin-8 (IL-8) are significantly related to disease severity.. The aim of this study was to evaluate the anti-inflammatory and clinical effects of the cysteinyl leukotriene receptor antagonist montelukast in children with CF.. A double-blind, randomized, crossover design was used. Patients received montelukast (6 to < or = 14 years, 5 mg; > 14 years, 10 mg) or placebo as a once-daily tablet for 21 days and then, after a washout period of at least 4 weeks, crossed over to receive the alternative treatment. Blood and native nasal fluid were taken on days 1 and 21 of each treatment block, and WBC count, ECP, and IL-8 were analyzed using a chemiluminescent immunometric assay.. Sixteen CF patients (10 boys, 6 girls; age, 5 to 18 years, median 9.5 years) completed the trial. There was a significant (P < or = 0.02) reduction of serum ECP (median reduction: montelukast 7.7 microg/L vs placebo 0.15 microg/L) and eosinophils (P < or = 0.027; median reduction: montelukast 85/microL vs placebo 0/microL). There was no significant change in nasal ECP, IL-8, or serum IL-8 after a 21-day course of montelukast. Clinical symptom scores did not change significantly.. Montelukast reduces eosinophilic inflammation in CF patients. Multicenter trials providing more patients to create more data to prove the hypothesis that montelukast is an effective tool to cut down disease severity in CF patients are needed.

Topics: Acetates; Adolescent; Anti-Inflammatory Agents, Non-Steroidal; Blood Proteins; Child; Cross-Over Studies; Cyclopropanes; Cystic Fibrosis; Double-Blind Method; Eosinophil Granule Proteins; Eosinophilia; Humans; Inflammation; Interleukin-8; Leukocyte Count; Leukotriene Antagonists; Membrane Proteins; Pilot Projects; Quinolines; Receptors, Leukotriene; Respiratory Function Tests; Ribonucleases; Sulfides; Treatment Outcome

The aim of this study was to compare neutrophil migration in cystic fibrosis (CF) and non-CF populations and to investigate the effect of erythromycin on directed migration of neutrophils (PMNs) in CF.. PMNs were isolated and their migratory capacity in response to interleukin-8 (IL-8) or f-Met-Leu-Phe (fMLP) in the presence or absence of erythromycin (1-100 microg/ml) was assessed.. CF derived PMNs showed significantly increased migration to IL-8 but not to fMLP compared with non-CF PMNs. Erythromycin had no significant effect on migration responses to IL-8 and in vitro exposure of PMNs to erythromycin had no effect.. CF derived PMNs show higher migratory responsiveness to IL-8 but not to fMLP, suggesting that CF PMNs may be "primed" to IL-8 which is significantly increased in CF serum compared with non-CF serum. Treatment with erythromycin had no significant effect on PMN migration in vitro.

Topics: Adolescent; Anti-Bacterial Agents; Chemotaxis, Leukocyte; Child; Cystic Fibrosis; Erythromycin; Female; Humans; Interleukin-8; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

Viscous negatively charged cystic fibrosis (CF) sputum allows colonization by pathogens, inducing a chronic inflammatory response. Heparin thins sputum by decreasing the mucin molecule amino group negative charge, altering its intermolecular hydrogen bonding, and ionically shielding its polyionic moieties. It also has an anti-inflammatory effect within the lung. It may, therefore, be useful in the treatment of CF patients. In order to test this, six fully informed Burkholderia cepacia colonized stable adult CF patients, received 25,000 IU nebulized heparin sulphate daily for 7 days. Subjective sputum parameters, spirometry, platelets, coagulation parameters, and serum and sputum interleukin (IL)-6 and -8 were measured before and after treatment. All patients tolerated the heparin with no evidence of bleeding, thrombocytopenia or change in coagulation parameters. There was no change in spirometry, but a reduction in interleukins (sputum IL-6, p=0.01; sputum IL-8, p=0.002; serum IL-6, p=0.02; serum IL-8, p=0.02). Sputum was easier to expectorate (p < 0.04), with a trend towards thinner sputum (p=0.07) but no change in sputum volume. Heparin therapy was well tolerated and had an anti-inflammatory effect, with subjective sputum mucolysis. Further studies are necessary to define the role of heparin in the treatment of cystic fibrosis patients.

Topics: Adult; Aerosols; Anti-Inflammatory Agents; Blood Coagulation; Burkholderia cepacia; Cystic Fibrosis; Expectorants; Female; Heparin; Humans; Interleukin-6; Interleukin-8; Male; Pilot Projects; Sputum; Viscosity

Cystic fibrosis (CF) patients continue to have reservoirs of Pseudomonas aeruginosa infection in their sinuses and trachea after transplantation, and studies indicate that nontransplanted CF patients have high bronchoalveolar lavage (BAL) levels of proinflammatory factors, interleukin-8 (IL-8), and elastase and decreased airway lavage levels of IL-10. The aims of our study were to measure the IL-8 and IL-10 levels and elastase activity in the BAL of lung transplant patients, with correlation to microbiologic and pathologic findings, and to identify any differences in the findings between CF and non-CF patients. Fifty serial BAL samples were collected from 38 lung transplant recipients over 8 months. The BAL supernatant fluid was cultured for bacterial, viral, and fungal organisms. Histologic tissue analysis was performed as indicated. The fluid IL-10 and IL-8 levels were measured in duplicate using ELISA techniques. Elastase activity was measured using a colorimetric assay system. The mean IL-8, IL-10, and elastase levels for the group studied were 1894 pg/ml, 394 pg/ml, and 4.2 U/ml, respectively. The CF patients had significantly higher levels of IL-8, with a mean value of 4093 pg/ml (p < 0.02), and lower IL-10, mean 217 pg/ml (n = 9). Elastase activity correlated strongly with IL-8 level (p < 0.04). Pseudomonas growth was associated with higher elastase and IL-8 concentrations (p < 0.02). There was no association between allograft rejection and the markers studied. CF transplanted patients have higher airway lavage concentrations of IL-8 and elastase correlated to the presence of Pseudomonas in the lower airway. They also have lower BAL levels of anti-inflammatory cytokine IL-10.

Topics: Adult; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-10; Interleukin-8; Leukocyte Elastase; Lung Transplantation; Maxillary Sinus; Middle Aged; Pseudomonas Infections; Trachea

Controlling lung inflammation may be the key to improving morbidity and mortality in cystic fibrosis.. To assess the effects of inhaled corticosteroids on lung inflammation in cystic fibrosis.. Double blind placebo controlled randomised sequence crossover trial. Fluticasone propionate (400 micrograms/day) was given as a dry powder inhaler for six weeks with a four week washout period before crossover.. Sputum inflammatory markers (interleukin-8, tumour necrosis factor-alpha (TNF-alpha) and neutrophil elastase-both free and bound to alpha 1-antiprotease), sputum interleukin-10, lung function, and symptomatology.. Twenty three children from a regional cystic fibrosis centre were enrolled into the study, with mean age 10.3 years (range 7 to 17 years) and mean baseline forced expiratory volume in one second (FEV1) of 64% (range 21% to 102%) predicted for sex and height. One patient was excluded for non-compliance to the study protocol.. No significant benefit was shown for the use of fluticasone propionate in any of the outcomes. For sputum interleukin-8 there was an estimated true treatment median difference of 142 pg/ml (95% confidence interval (CI) 8 to 2866 pg/ml) in favour of placebo; while for maximal expiratory flow at 25% (MEF25%) remaining forced vital capacity predicted for sex and height there was a 15 percentage points (pp) (95% CI 4 to 26 pp) mean treatment difference in favour of placebo. Sputum interleukin-10 was undetected in any samples and unaffected by fluticasone propionate. Neither atopic status, baseline FEV1, nor concomitant DNase therapy had any effect on response to treatment.. Lack of benefit from fluticasone propionate was most likely due to failure of the drug to penetrate the viscid mucus lining the airways. It is suggested a large multicentre trial with higher doses given for a longer time by a different delivery system is required to assess efficacy.

Topics: Administration, Inhalation; Adolescent; Androstadienes; Anti-Inflammatory Agents; Biomarkers; Child; Cross-Over Studies; Cystic Fibrosis; Double-Blind Method; Female; Fluticasone; Humans; Inflammation; Interleukin-10; Interleukin-8; Leukocyte Elastase; Lung; Male; Neutrophils; Sputum; Treatment Outcome; Tumor Necrosis Factor-alpha

DNA released by degenerating inflammatory neutrophils contributes to mucous plugging of airways in patients with cystic fibrosis. Neutrophil elastase, a major effector of tissue destruction in the lungs of patients with cystic fibrosis, is a highly cationic molecule which is bound and inhibited by negatively charged polyanions such as mucin and DNA in purulent sputum. Thus, the solubilisation of DNA in the airways by aerosolised recombinant DNase may remove a source of neutrophil elastase inhibition, effectively increasing elastase load. The aim of this study was to assess the effect of rhDNase therapy on neutrophil elastase load in patients with cystic fibrosis.. Blood and sputum were collected from 15 patients with cystic fibrosis before initiation of nebulised DNase therapy and at 12 weeks following therapy. The long term effects of continuous rhDNase administration were evaluated at 52 weeks for 11 of these patients. Plasma was analysed for neutrophil elastase, interleukin (IL)-8 and neutrophil elastase in complex with alpha 1-protease inhibitor (alpha 1PI). Sputum was assessed for neutrophil elastase, IL-8, and active elastase. At each visit spirometric measurements were carried out.. Sputum elastase activity decreased at 12 weeks and was maintained at 52 weeks when a decline in total plasma elastase was also observed. Although, as expected, there was a correlation between plasma levels of total elastase and neutrophil elastase/alpha 1PI complex, the decrease in the levels of the complex at 52 weeks did not reach statistical significance.. This study indicates that prolonged daily administration of rhDNase results in a reduction in elastase load in patients with cystic fibrosis.

Topics: Adolescent; Adult; Aerosols; Cystic Fibrosis; Deoxyribonuclease I; Drug Administration Schedule; Female; Forced Expiratory Volume; Humans; Interleukin-8; Leukocyte Elastase; Male; Pancreatic Elastase; Recombinant Proteins; Sputum; Vital Capacity

Cystic fibrosis (CF) is a multisystem disease with progressive deterioration. Recently, CF transmembrane conductance regulator (CFTR) modulator therapies were introduced that repair underlying protein defects. Objective of this study was to determine the impact of elexacaftor-tezacaftor-ivacaftor (ETI) on clinical parameters and inflammatory responses in people with CF (pwCF).. Lung function (FEV. Sample size was 48 pwCF, 28 (58.3%) males with a mean age of 28.8 ± 10.7 years. Significant increases in %predicted FEV. In pwCF, ETI significantly improved clinical outcomes, reduced systemic pro-inflammatory cytokines, and restored circulating immune cell composition after 6 months of therapy.

Topics: Adolescent; Adult; Aminophenols; Benzodioxoles; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Female; Humans; Interleukin-17; Interleukin-8; Male; Mutation; Young Adult

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

Topics: Adult; Cystic Fibrosis; Epithelium; Humans; Interleukin-8; Pseudomonas Infections; Quality of Life

The variable response to fat-soluble vitamin supplementation in young children with cystic fibrosis (CF), and factors contributing to this variability, remain under-investigated.. To determine if recommended supplement doses normalize serum vitamins A (retinol), D (25-hydroxy-vitamin D, 25OHD), and E (α-tocopherol), and identify factors predictive of achieving sufficiency, in children with CF in the first 3 years of life.. We studied 144 infants born during 2012-2017 and diagnosed with CF through newborn screening. Serum retinol, 25OHD, α-tocopherol and plasma cytokines interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α were measured in early infancy and yearly thereafter. Vitamin supplement intakes and respiratory microbiology were assessed every 1-2 months in infancy and quarterly thereafter.. The prevalence of vitamin D insufficiency (<30 ng/ml) at all ages combined was significantly higher (22%) compared to vitamin A (<200 ng/ml, 3%) and vitamin E (<5 µg/ml, 5%). All children were vitamin A sufficient by age 2 years. Vitamin E insufficiency was rare. Only 42% were early responders of vitamin D and 17% remain insufficient despite high supplement intakes. IL-6 was positively correlated, while IL-8, IL-10, and TNF-α were negatively correlated, with retinol and 25OHD. Multiple regression analysis revealed that supplement dose, season, α-tocopherol, pancreatic insufficiency, respiratory infections and IL-10 were significant predictors of 25OHD.. Diagnosis through newborn screening coupled with supplementation normalized serum retinol and α-tocopherol in almost all infants with CF by age 3 years. However, response to vitamin D supplements in young children with CF occurred later and variably despite early and sustained supplementation.

Topics: alpha-Tocopherol; Child; Child, Preschool; Cystic Fibrosis; Dietary Supplements; Humans; Infant; Infant, Newborn; Interleukin-10; Interleukin-8; Vitamin A; Vitamin D; Vitamin E; Vitamins

Airway nitric oxide (NO) deficiency is a hallmark of cystic fibrosis (CF), but the reasons for the reduced NO production in CF airways are unclear. Interleukin (IL)-1 pathway activation plays a role in early CF lung disease and is also involved in the regulation of NO synthase activity. Treatment of CF patients with the CFTR-targeting drug ivacaftor, among other beneficial effects, results in an increase in airway NO levels. In this longitudinal observational trial, we show that ivacaftor therapy leads to a significant reduction in sputum IL-1β concentration but not in other IL-1- or Th17-associated cytokines. IL-1β concentrations were closely linked to improvement in pulmonary function, measures of NO metabolism in sputum and exhaled NO. These data therefore suggest a potential interaction between transepithelial chloride conductance, IL-1β and airway NO production.

Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Interleukin-1beta; Interleukin-8; Lung; Nitric Oxide

Highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have led to dramatic improvements in lung function in many people with cystic fibrosis (PwCF). However, the efficacy of CFTR modulators may be hindered by persistent airway inflammation. The cytokine transforming growth factor-beta1 (TGF-β1) is associated with worse pulmonary disease in PwCF and can diminish modulator efficacy. Thus, strategies to augment the CFTR response to modulators in an inflammatory environment are needed. Here, we tested whether the CFTR amplifier nesolicaftor (or PTI-428) could rescue the effects of TGF-β1 on CFTR function and ciliary beating in primary human CF bronchial epithelial (CFBE) cells. CFBE cells homozygous for F508del were treated with the combination of elexacaftor/tezacaftor/ivacaftor (ETI) and TGF-β1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-β1-induced reductions in CFTR conductance by increasing the expression of

Topics: Benzodioxoles; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Interleukin-6; Interleukin-8; MicroRNAs; Mutation; RNA, Messenger; Transforming Growth Factor beta1

Cytokines play a crucial role in the immune system's regulation by mediating protective responses to infections. anti-inflammatory and pro-inflammatory cytokines are in equilibrium. Therefore, any alteration in cytokine production or cytokine receptor expression might result in pathological illnesses and health issues. Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane regulator (CFTR) gene. Lung infection in these patients is related to chronic bacterial airway infection and inflammation, which is triggered by some inflammatory cytokines. Our goal was to compare the cytokine patterns in CF patient's serum and PBMCs caused by microbial pathogens that colonized their airways to controls.. ELISA and Real-time PCR were used to determine the levels of IL-10, IFN-γ, IL-4, TGF-β, IL-8, and IL-17 in serum and PBMC cells. Blood parameters in both patients and healthy people were studied.. An increase in IL-10, IFN-γ, IL-4 (p-v = 0.03, 0.024 and 0.003) levels and a decrease in IL-17 (p-v = 0.004) was found in Pseudomonas aeruginosa positive patients. There were no different in TGF-β and IL-8 (p-value = 0.778 and 0.903) in this patients. IL-10, IFN-γ, and IL-4 (p-value = 0.023, 0.001 and 0.002) levels were high in Staphylococcus aureus positive patients and TGF-β, IL-17, and IL-8 (p-value = 0.085, 0.167 and 0.362) were not significantly different in the patient and control groups. IFN-γ and IL-4 levels were higher in patients without infection who had normal microbiota (p-v = 0.002 and 0.024). In patients with P. aeruginosa, WBC and platelets increased, and MCH and MCV decreased. Patients with normal microbiota had less MCV.. According to our research, patients with P. aeruginosa, S. aureus, and normal microbiota are exposed to cytokine alterations and changes in blood factors. The link between the CF patient's airway microbiota and the kind of generated cytokines might lead to the modulation of inflammatory cytokines alone or in combination with antibiotics, reducing disease-causing effects while avoiding drug resistance.

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Humans; Interleukin-10; Interleukin-17; Interleukin-4; Interleukin-8; Leukocytes, Mononuclear; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Cytokine; Staphylococcus aureus; Transforming Growth Factor beta

In cystic fibrosis (CF), acute respiratory exacerbations critically enhance pulmonary destruction. Since these mainly occur outside regular appointments, they remain unexplored. We previously elaborated a protocol for home-based upper airway (UAW) sampling obtaining nasal-lavage fluid (NLF), which, in contrast to sputum, does not require immediate processing. The aim of this study was to compare UAW inflammation and pathogen colonization during stable phases and exacerbations in CF patients and healthy controls.. Initially, we obtained NLF by rinsing 10 ml of isotonic saline/nostril during stable phases. During exacerbations, subjects regularly collected NLF at home. CF patients directly submitted one aliquot for microbiological cultures. The remaining samples were immediately frozen until transfer on ice to our clinic, where PCR analyses were performed and interleukin (IL)-1β/IL-6/IL-8, neutrophil elastase (NE), matrix metalloproteinase (MMP)-9, and tissue inhibitor of metalloproteinase (TIMP)-1 were assessed.. Non-invasive and partially home-based UAW sampling opens new windows for the assessment of inflammation and pathogen colonization in the unified airway system.

Topics: Anti-Bacterial Agents; Azithromycin; Cystic Fibrosis; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 9; Multiplex Polymerase Chain Reaction; Nasal Lavage

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Survival; Colistin; Cystic Fibrosis; Drug Combinations; Humans; Ibuprofen; Inflammation; Interleukin-8; Lipopolysaccharides; Pseudomonas aeruginosa; Tobramycin

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

Topics: Anesthetics, Inhalation; Animals; Cell Line, Tumor; Cystic Fibrosis; Epithelial Cells; Female; Flagellin; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Isoflurane; Lung; Male; Mice; Molecular Docking Simulation; Neutrophils; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Retrospective Studies; Sevoflurane; Toll-Like Receptor 5

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD-CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD-CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm-forming bacterial strains. Forty-one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C-reactive protein (CRP) and NLR (neutrophil-lymphocyte ratio) were examined as representative blood-based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil-derived defence proteins. Isolated sputum bacteria were identified and their biofilm-forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm-forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)-8 and low concentrations of IL-6 and IL-10. The low concentration of sputum IL-6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL-6, IL-8 and IL-10.

Topics: Adolescent; Adult; Biofilms; Biomarkers; C-Reactive Protein; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Inflammation Mediators; Interleukin-10; Interleukin-6; Interleukin-8; Leukocyte Elastase; Lymphocyte Count; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Sputum; Young Adult

Cystic fibrosis (CF) is a multi-system disease that is characterized by lung disease due to recurrent airway infection and inflammation. Endocrine complications, such as CF bone disease (CFBD), are increasingly identified as patients are living longer. The cause of CFBD is multifactorial with chronic systemic inflammation theorized to be a contributing factor. Thus, we attempted to identify inflammatory biomarkers that are associated with CFBD. We conducted a retrospective observational study of 56 adult patients with CF with an average percentage predictive forced expiratory volume in one second (ppFEV

Topics: Adolescent; Adult; Bone Density; Bone Remodeling; Cystic Fibrosis; Femur Neck; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Osteoporosis; Tumor Necrosis Factor-alpha; Young Adult

Respiratory infections are a leading cause of global morbidity and mortality and are of significant concern for individuals with chronic inflammatory lung diseases. There is an urgent need for novel antimicrobials. Antimicrobial peptides (AMPs) are naturally occurring innate immune response peptides with therapeutic potential. However, therapeutic development has been hindered by issues with stability and cytotoxicity. Availing of direct drug delivery to the affected site, for example the lung, can reduce unwanted systemic side effects and lower the required dose. As cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lungs typically exhibit elevated protease levels, the aim of this study was to assess their impact on snake-derived AMPs. Peptide cleavage was determined using SDS-PAGE and antimicrobial and anti-inflammatory activities of neutrophil elastase (NE)-incubated peptides were assessed using a radial diffusion assay (RDA) and an in vitro LPS-induced inflammation model, respectively. Although the snake-derived AMPs were found to be susceptible to cleavage by lung proteases including NE, several retained their function following NE-incubation. This facilitated the design of novel truncated derivatives that retained functionality following NE incubation. Snake-derived AMPs are tractable candidate treatments for use in environments that feature elevated NE levels, such as the CF airways.

Topics: Animals; Anti-Infective Agents; Anti-Inflammatory Agents; Cystic Fibrosis; Humans; Immunity, Innate; Inflammation; Inhibitory Concentration 50; Interleukin-6; Interleukin-8; Leukocyte Elastase; Lipopolysaccharides; Lung; Macrophages; Monocytes; Peptide Hydrolases; Peptides; Pore Forming Cytotoxic Proteins; Protein Structure, Secondary; Pseudomonas aeruginosa; Pulmonary Disease, Chronic Obstructive; Snakes; THP-1 Cells

Topics: Adaptor Proteins, Signal Transducing; Anti-Bacterial Agents; Autophagy; Azithromycin; Case-Control Studies; Chemokine CCL4; Chloroquine; Cystic Fibrosis; Gene Expression Regulation; Host-Pathogen Interactions; Humans; Immunosuppressive Agents; Interleukin-8; Mycobacterium abscessus; Neutrophils; Phagocytosis; Primary Cell Culture; Reactive Oxygen Species; Signal Transduction; Sirolimus; Wortmannin

Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.

Topics: A549 Cells; Aryldialkylphosphatase; beta-Defensins; Cystic Fibrosis; Endoplasmic Reticulum Stress; Epithelial Cells; Host-Pathogen Interactions; Humans; Immunity, Innate; Interleukin-8; Mitochondria; Pioglitazone; PPAR gamma; Pseudomonas aeruginosa; Pseudomonas Infections; Transcription Factor CHOP; Unfolded Protein Response

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.

Topics: Bronchi; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; NF-kappa B; Prevotella; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Toll-Like Receptors

Topics: Adolescent; Adult; Aftercare; Biomarkers; Cohort Studies; Cystic Fibrosis; Disease Progression; Exercise; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Male; Middle Aged; Prospective Studies; Respiratory Function Tests; Tumor Necrosis Factor-alpha; Wearable Electronic Devices; Young Adult

Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear.. Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR. All lines maintained CFTR mRNA production and formation of tight junctions. CFTR. Together, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.

Topics: Caco-2 Cells; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression Regulation; Gene Regulatory Networks; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) results from deficient CF transmembrane conductance regulator (CFTR) protein activity leading to defective epithelial ion transport. Pulmonary degradation due to excessive inflammation is the main cause of morbidity and mortality in CF patients. By analysing miRNAs (small RNAseq) in human primary air-liquid interface cell cultures, we measured the overexpression of miR-636 in CF patients compared to non-CF controls. We validated these results in explant biopsies and determined that the mechanism underlying miR-636 overexpression is linked to inflammation. To identify specific targets, we used bioinformatics analysis to predict whether miR-636 targets the 3'-UTR mRNA regions of

Topics: Cells, Cultured; Cystic Fibrosis; Humans; I-kappa B Kinase; Interleukin-6; Interleukin-8; MicroRNAs; NF-kappa B; Pneumonia; Receptor Activator of Nuclear Factor-kappa B; Receptors, Interleukin-1 Type I; Signal Transduction

Children with CF are insulin deficient from infancy but very little is known about the impact of glucose abnormalities in early life. We aimed to identify and describe interstitial glucose levels in CF children <6 years and to evaluate the association with pulmonary infection and inflammation.. We assessed 18 children (5 females) with median age of 3.2 years (range 0·9-5.5) with Continuous Glucose Monitoring for 3 days. Bronchoalveolar lavage (BAL) fluid was cultured for known pathogenic microbial agents and assessed for total white blood cells, percentage of neutrophils and IL-8 level.. Children with CF frequently demonstrate elevated SG levels before age 6 years, which are associated with increased pulmonary inflammation and Pseudomonas aeruginosa infection. Transient SG elevations into the diabetic range (≥11.1 mmol/L) were identified in children from 1 year of age.

Topics: Australia; Blood Glucose; Bronchoalveolar Lavage Fluid; Child, Preschool; Correlation of Data; Cystic Fibrosis; Female; Humans; Hyperglycemia; Infant; Interleukin-8; Leukocyte Count; Male; Monitoring, Physiologic; Neutrophils; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections

Bronchoalveolar lavage (BAL) is a potentially useful outcome measure for clinical trials in children with CF but its use is limited by variations in approach internationally. We sought to determine if pooling adversely affected the diagnostic properties of BAL.. Children undergoing bronchoscopy for clinical reasons were included. A multi-step study protocol ensured BAL was collected and analysed both separately and as a pooled fluid.. Eighty-five children (53 CF, 32 control) were recruited. There was a high level of concordance between pooled and non-pooled samples in terms of organism identification (76%). There was good agreement (Bland Altman) between the two methods in terms of detection of inflammation independent of centre, microbiological concordance or disease status. Bi-directional variability in IL-8 levels between pooled and non-pooled samples was seen. Free neutrophil elastase (NE) was detected in 4 cases in pooled lavage when absent in non-pooled lavage. Levels of interleukin-8 (IL-8) were similar between the two groups with pooled samples showing a greater spread of values.. Pooling of BAL in children does not negatively impact on either the detection of pulmonary infection or inflammation or the observed relationship between infection and inflammation. Intra-patient variability in BAL IL-8 levels suggests regional differences in inflammation.

Topics: Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child; Child, Preschool; Correlation of Data; Cystic Fibrosis; Female; Humans; Interleukin-8; Male; Pneumonia; Respiratory Tract Infections

N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation.. TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid.. We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins.. Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

Topics: Anti-Inflammatory Agents; Cell Line; Cell Survival; Chemokine CCL5; Cystic Fibrosis; Glutathione Peroxidase; HEK293 Cells; Humans; Inflammation; Interleukin-8; Isopentenyladenosine; NF-kappa B; Signal Transduction; STAT3 Transcription Factor; Thioredoxin-Disulfide Reductase; Tumor Necrosis Factor-alpha

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Topics: Aminophenols; Aminopyridines; Benzodioxoles; Bronchi; Cells, Cultured; Chloride Channel Agonists; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-8; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones

Neutrophil elastase (NE) is a key risk factor for severity of cystic fibrosis (CF) lung disease. Recent studies identified increased NE activity on the surface of airway neutrophils from CF-like mice and patients with CF. However, the role of surface-bound NE in CF lung disease remains unknown. We determined the relationship between surface-bound NE activity and severity of lung disease in CF.Surface-bound NE activity was measured on sputum neutrophils from 35 CF patients and eight healthy controls using novel lipidated Förster resonance energy transfer reporters and correlated with free NE activity, neutrophil counts, interleukin-8, myeloperoxidase and antiproteases in sputum supernatant, and with lung function parameters.Surface-bound NE activity was increased in CF compared to healthy controls (p<0.01) and correlated with free NE activity (p<0.05) and other inflammation markers (p<0.001). Surface-bound and free NE activity correlated with forced expiratory volume in 1 s % predicted (p<0.01 and p<0.05), but only surface-bound NE activity correlated with plethysmographic functional residual capacity % pred (p<0.01) in patients with CF.We demonstrate that surface-bound NE activity on airway neutrophils correlates with severity of lung disease in patients with CF. Our results suggest that surface-bound NE activity may play an important role in the pathogenesis and serve as novel biomarker in CF lung disease.

Topics: Adult; Cystic Fibrosis; Female; Humans; Interleukin-8; Leukocyte Elastase; Lung Diseases; Male; Middle Aged; Neutrophils; Peroxidase; Respiratory Function Tests; Risk Factors; Severity of Illness Index; Spirometry; Sputum; Young Adult

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-β3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca

Topics: Bronchi; Calcium Signaling; Cell Line; Computer Simulation; Cystic Fibrosis; Humans; Interleukin-8; Mucus; Mutation; Phospholipase C beta; Pseudomonas aeruginosa; Serine; Structure-Activity Relationship

In children with cystic fibrosis (CF) the associations between oropharyngeal swabs (OPSs) for detection of

Topics: Australia; Biomarkers; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child, Preschool; Cystic Fibrosis; Disease Progression; Female; Humans; Infant; Interleukin-8; Leukocyte Elastase; Male; Oropharynx; Predictive Value of Tests; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; ROC Curve; Severity of Illness Index; Tomography, X-Ray Computed

Topics: Animals; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Molecular Targeted Therapy; Neutrophil Infiltration

Cystic fibrosis (CF) is the most common lethal genetic disease, caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. CF is characterized by an ionic imbalance and thickened mucus, which impair mucociliary clearance and promote bacterial colonization and the establishment of infection/inflammation cycles. However, the origin of this inflammation remains unclear, although microRNAs (miRNAs) are suspected to be involved. MiRNAs are small non-coding RNAs that bind to the 3'-untranslated regions (UTRs) of target gene mRNA, thereby repressing their translation and/or inducing their degradation. The goal of this study was to investigate the role of microRNAs associated with pulmonary inflammation in CF patients. Through the analysis of all miRNAs (miRNome) in human primary air-liquid interface cultures, we demonstrated that miR-199a-3p is the only miRNA downregulated in CF patients compared to controls. Moreover, through RNA sequencing (transcriptome) analysis, we showed that 50% of all deregulated mRNAs are linked directly or indirectly to the NF-κB pathway. To identify a specific target, we used bioinformatics analysis to predict whether miR-199a-3p targets the 3'-UTR of IKBKB, which encodes IKKβ, a major protein in the NF-κB pathway. Subsequently, we used bronchial explants from CF patients to show that miR-199a-3p expression is downregulated compared to controls and inversely correlated with increases in expression of IKKβ and IL-8. Through functional studies, we showed that miR-199a-3p modulates the expression of IKBKB through a direct interaction at its 3'-UTR in bronchial epithelial cells from CF patients. In miR-199a-3p overexpression experiments, we demonstrated that for CF cells, miR-199a-3p reduced IKKβ protein expression, NF-κB activity, and IL-8 secretion. Taken together, our findings show that miR-199a-3p plays a negative regulatory role in the NF-κB signalling pathway and that its low expression in CF patients contributes to chronic pulmonary inflammation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: 3' Untranslated Regions; Binding Sites; Case-Control Studies; Cells, Cultured; Cystic Fibrosis; Down-Regulation; Gene Expression Profiling; Humans; I-kappa B Kinase; Interleukin-8; Lung; MicroRNAs; NF-kappa B; Pneumonia; RNA, Messenger; Sequence Analysis, RNA; Signal Transduction; Tissue Culture Techniques

Cystic fibrosis (CF) lung infection is a complex condition where opportunistic pathogens and defective immune system cooperate in developing a constant cycle of infection and inflammation. The major pathogen, Pseudomonas aeruginosa, secretes a multitude of virulence factors involved in host immune response and lung tissue damage. In this study, we examined the possible anti-inflammatory effects of molecules inhibiting P. aeruginosa virulence factors.. Pyocyanin, pyoverdine and proteases were measured in bacterial culture supernatant from different P. aeruginosa strains. Inhibition of virulence factors by sub-inhibitory concentrations of clarithromycin and by protease inhibitors was evaluated. Lung inflammatory response was monitored by in vivo bioluminescence imaging in wild-type and CFTR-knockout mice expressing a luciferase gene under the control of a bovine IL-8 promoter.. The amount of proteases, pyocyanin and pyoverdine secreted by P. aeruginosa strains was reduced after growth in the presence of a sub-inhibitory dose of clarithromycin. Intratracheal challenge with culture supernatant containing bacteria-released products induced a strong IL-8-mediated response in mouse lungs while lack of virulence factors corresponded to a reduction in bioluminescence emission. Particularly, sole inactivation of proteases by inhibitors Ilomastat and Marimastat also resulted in decreased lung inflammation.. Our data support the assumption that virulence factors are involved in P. aeruginosa pro-inflammatory action in CF lungs; particularly, proteases seem to play an important role. Inhibition of virulence factors production and activity resulted in decreased lung inflammation; thus, clarithromycin and protease inhibitors potentially represent additional therapeutic therapies for P. aeruginosa-infected patients.

Topics: Animals; Bacterial Proteins; Clarithromycin; Cystic Fibrosis; Disease Models, Animal; Female; Humans; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pseudomonas aeruginosa; Pseudomonas Infections; Virulence Factors

Mesenchymal stromal/stem cells (MSCs) are multi-potent non-hematopoietic stem cells, residing in most tissues including the lung. MSCs have been used in therapy of chronic inflammatory lung diseases such as Cystic Fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) but the main beneficial effects reside in the anti-inflammatory potential of the released extracellular vesicles (EVs). Recent reports demonstrate that EVs are effective in animal model of asthma, E.coli pneumonia, lung ischemia-reperfusion, and virus airway infection among others. Despite this growing literature, the EVs effects on CF are largely unexplored.. We treated IB3-1 cells, an in vitro human model of CF, with EVs derived from human lung MSCs under basal and inflammatory conditions (TNFα stimulation).. We demonstrated here that treatment of IB3-1 CF cell line with EVs, down-regulates transcription and protein expression of pro-inflammatory cytokines such as IL-1β, IL-8, IL-6 under TNFα - stimulated conditions. EVs treatment upregulates the mRNA expression of PPARγ, a transcription factor controlling anti-inflammatory and antioxidant mechanisms via NF-kB and HO-1. Accordingly, NF-kB nuclear translocation is reduced resulting in impairment of the downstream inflammation cascade. In addition, the mRNA of HO-1 is enhanced together with the antioxidant defensive response of the cells.. We conclude that the anti-inflammatory and anti-oxidant efficacy of EVs derived from lung MSCs could be mediated by up-regulation of the PPARγ axis, whose down-stream effectors (NF-kB and HO-1) are well-known modulators of these pathways.. EVs could be a novel strategy to control the hyper-inflamed condition in Cystic Fibrosis.

Topics: Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Extracellular Vesicles; Heme Oxygenase-1; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Lung; Mesenchymal Stem Cells; NF-kappa B; PPAR gamma; Tumor Necrosis Factor-alpha

We sought to address whether CF macrophages have a primary functional defect as a consequence of CFTR loss and thus contribute to the onset of infection and inflammation observed in CF lung disease.. Monocyte derived macrophages (MDMs) were prepared from newborn CF and non-CF pigs. CFTR mRNA expression was quantified by rtPCR and anion channel function was determined using whole cell patch clamp analysis. IL8 and TNFα release from MDMs in response to lipopolysaccharide stimulation was measured by ELISA.. CFTR was expressed in MDMs by Q-rtPCR at a lower level than in epithelial cells. MDMs exhibited functional CFTR current at the cell membrane and this current was absent in CF MDMs. CF MDMs demonstrated an exaggerated response to lipopolysaccharide stimulation.. In the absence of CFTR function, macrophages from newborn CF pigs exhibit an increased inflammatory response to a lipopolysaccharide challenge. This may contribute to the onset and progression of CF lung disease.

Topics: Animals; Animals, Newborn; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Immunization; Inflammation; Interleukin-8; Lipopolysaccharides; Macrophages; Patch-Clamp Techniques; Swine; Tumor Necrosis Factor-alpha

The underlying defect in the cystic fibrosis (CF) airway leads to defective mucociliary clearance and impaired bacterial killing, resulting in endobronchial infection and inflammation that contributes to progressive lung disease. Little is known about the respiratory microbiota in the early CF airway and its relationship to inflammation.. To examine the bacterial microbiota and inflammatory profiles in bronchoalveolar lavage fluid and oropharyngeal secretions in infants with CF.. Infants with CF from U.S. and Australian centers were enrolled in a prospective, observational study examining the bacterial microbiota and inflammatory profiles of the respiratory tract. Bacterial diversity and density (load) were measured. Lavage samples were analyzed for inflammatory markers (interleukin 8, unbound neutrophil elastase, and absolute neutrophil count) in the epithelial lining fluid.. Thirty-two infants (mean age, 4.7 months) underwent bronchoalveolar lavage and oropharyngeal sampling. Shannon diversity strongly correlated between upper and lower airway samples from a given subject, although community compositions differed. Microbial diversity was lower in younger subjects and in those receiving daily antistaphylococcal antibiotic prophylaxis. In lavage samples, reduced diversity correlated with lower interleukin 8 concentration and absolute neutrophil count.. In infants with CF, reduced bacterial diversity in the upper and lower airways was strongly associated with the use of prophylactic antibiotics and younger age at the time of sampling; less diversity in the lower airway correlated with lower inflammation on bronchoalveolar lavage. Our findings suggest modification of the respiratory microbiome in infants with CF may influence airway inflammation.

Topics: Antibiotic Prophylaxis; Australia; Bacteria; Biomarkers; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Female; Humans; Infant; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Linear Models; Male; Microbiota; Missouri; Neutrophils; Prospective Studies; Respiratory System

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β

Topics: Antigens, CD; beta-Arrestin 2; Cadherins; Cell Proliferation; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Endothelial Cells; Homeostasis; Human Umbilical Vein Endothelial Cells; Humans; Insulin; Interleukin-8; Nitric Oxide Synthase Type III; Nitrogen Oxides; Phosphorylation; Pulmonary Artery

Bronchiectasis is a heterogeneous entity, taking into account clinical characteristics, inflammatory response, effectiveness of treatment and frequency of exacerbations. In stable state non-cystic fibrosis (non-CF) bronchiectasis, little is known about non-invasive techniques used for evaluating airway inflammation in obstructive airway diseases.. We sought to evaluate the associations between induced sputum and clinical/radiologic characteristics, and the differences between biomarkers expressing Th1 and Th2 response in patients with non-CF bronchiectasis and to compare our findings with a previously studied population of patients with asthma and COPD.. We evaluated prospectively collected data from subjects with bronchiectasis. Comparisons were made between clinical, radiographic and physiologic characteristics, as well as induced sputum markers using appropriate statistical tools. We compared the levels of sputum markers with those of a previously studied cohort of asthma and COPD patients.. We enrolled 40 subjects (21men, mean age 63.5yrs) with bronchiectasis. Fifteen subjects (37.5%) had a neutrophilic phenotype, 7 (17.5%) had an eosinophilic phenotype, 3 (12.5%) had a mixed neutrophilic-eosinophilic phenotype and 15 (37.5%) had a paucigranulocytic phenotype. Subjects with sputum neutrophilia had more severe bronchiectasis in HRCT and higher levels of IL-8 in sputum, whereas subjects with eosinophilia had higher levels of FeNO, greater bronchodilator reversibility and higher sputum IL-13. Sputum IL-8 levels were higher in subjects exhibiting frequent exacerbations and correlated with neutrophils in sputum (r=0.799), the extent of bronchiectasis in HRCT (r=0.765) and post-bronchodilator FEV. Sputum cell counts and IL-8 and IL-13 correlate with distinct clinical and functional measurements of disease severity and therefore may have a role for non-invasively assessing inflammation in non-cystic fibrosis bronchiectasis.

Topics: Bronchiectasis; Cell Count; Cystic Fibrosis; Demography; Female; Humans; Inflammation; Interleukin-13; Interleukin-8; Male; Middle Aged; Neutrophils; Phenotype; Pseudomonas aeruginosa; Respiratory Function Tests; Severity of Illness Index; Sputum

Chronic inflammation is a hallmark of cystic fibrosis (CF) and associated with increased production of transforming growth factor (TGF) β and interleukin (IL)-8. α-klotho (KL), a transmembrane or soluble protein, functions as a co-receptor for Fibroblast Growth Factor (FGF) 23, a known pro-inflammatory, prognostic marker in chronic kidney disease. KL is downregulated in airways from COPD patients. We hypothesized that both KL and FGF23 signaling modulate TGF β-induced IL-8 secretion in CF bronchial epithelia. Thus, FGF23 and soluble KL levels were measured in plasma from 48 CF patients and in primary CF bronchial epithelial cells (CF-HBEC). CF patients showed increased FGF23 plasma levels, but KL levels were not different. In CF-HBEC, TGF-β increased KL secretion and upregulated FGF receptor (FGFR) 1. Despite increases in KL, TGF-β also increased IL-8 secretion via activation of FGFR1 and Smad 3 signaling. However, KL excess via overexpression or supplementation decreased IL-8 secretion by inhibiting Smad 3 phosphorylation. Here, we identify a novel signaling pathway contributing to IL-8 secretion in the CF bronchial epithelium with KL functioning as an endocrine and local anti-inflammatory mediator that antagonizes pro-inflammatory actions of FGF23 and TGF-β.

Topics: Animals; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Epithelium; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glucuronidase; Humans; Inflammation; Interleukin-8; Klotho Proteins; Male; Mice; Rats; Respiratory Mucosa; Signal Transduction; Transforming Growth Factor beta

Endoplasmic reticulum (ER) stress has been recognized to play an important role in chronic inflammatory diseases such as cystic fibrosis (CF), and targeting ER stress may be useful for alleviating damaging neutrophilic inflammation in CF airways. Cellular models were used in conjunction with data from a recent CF genome-wide association study (GWAS) meta-analysis to determine modulators of ER stress-mediated inflammation. Surprisingly, cells undergoing ER stress during inflammatory stimulation showed reduced interleukin 8 (IL-8) and CXCL1 secretion (P < .001). Neutralization of CXCL1 and IL-8 reduced neutrophil chemotaxis >50% to supernatants from IL-1β-stimulated CF airway epithelial cells (P < .01). The clinical importance of these chemokines was validated by association of CXCL1 and IL8 polymorphisms with changes in lung disease severity in patients with CF (n = 6365; IL8, P = .001; CXCL1, P = .001), confirming that targeting these chemokine pathways could help improve lung disease. We determined that production of these chemokines was partially controlled by ER stress in a signal transducer and activator of transcription 3 (STAT3)-dependent manner, whereby ER stress inhibited STAT3 activation. Our findings support a role for CXCL1 and IL-8 in CF lung disease severity and identify STAT3 as a modulating pathway. Targeting these pathways may help improve health outcomes in CF.

Topics: Adult; Cell Line; Chemokine CXCL1; Cystic Fibrosis; Endoplasmic Reticulum Stress; Epithelial Cells; Female; Gene Expression Profiling; Humans; Interleukin-8; STAT3 Transcription Factor

Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P. aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.

Topics: 4-Butyrolactone; Antioxidant Response Elements; Biofilms; Cell Line; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Heme Oxygenase-1; Homoserine; Humans; Interleukin-8; Lung; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing

Discovery of novel antimicrobial agents against Pseudomonas aeruginosa able to inhibit bacterial growth as well as the resulting inflammatory response is a key goal in cystic fibrosis research. We report in this paper that a peptide nucleic acid (PNA3969) targeting the translation initiation region of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably, no effect on PAO1 induction of VEGF, GM-CSF and IL-17 was observed. Analogous experiments using a two base mis-match control PNA did not show such inhibition. Furthermore, no significant effects of the PNAs were seen on cell growth, apoptosis or secretome profile in uninfected IB3-1 cells (with the exception of a PNA-mediated up-regulation of PDGF, IL-17 and GM-CSF). Thus, we conclude that in cell culture an antimicrobial PNA against P. aeruginosa can inhibit the expression of pro-inflammatory cytokines otherwise induced by the infection. In particular, the effects of PNA-3969 on IL-8 gene expression are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection.

Topics: Apoptosis; Cell Line; Cell Proliferation; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Oligonucleotides, Antisense; Peptide Nucleic Acids; Pseudomonas aeruginosa; Up-Regulation

Lung disease in patients with both primary ciliary dyskinesia (PCD) or cystic fibrosis (CF) is associated with impaired mucociliary clearance; however, clinical outcomes are typically worse in CF patients. We assessed whether CF and PCD patients differ in inflammatory response in the airways during pulmonary exacerbation.We first studied clinically stable PCD patients with a spectrum of bacterial pathogens to assess inflammatory response to different pathogens. Subsequently, PCD and CF patients with similar bacterial pathogens were studied at the time of a pulmonary exacerbation and after 21 days of antibiotics treatment. Qualitative and quantitative microbiology, cell counts, interleukin-8 concentrations, and neutrophil elastase activity were assessed in sputum samples obtained before and after treatment.In stable PCD patients, no significant differences were found in sputum inflammatory markers between individuals colonised with different bacterial pathogens. Pulmonary exacerbation severity assessed by a pulmonary exacerbation score and lung function decline from their previous baseline did not differ between CF and PCD patients. Bacterial density for Staphylococcus aureus and Haemophilus influenzae was higher in CF versus PCD (p<0.05), but absolute neutrophil counts were higher in PCD patients (p=0.02). While sputum elastase activity was similar in PCD and CF at the time of exacerbation, it decreased with antibiotic therapy in PCD (p<0.05) but not CF patients.PCD patients differ from those with CF in their responses to treatment of pulmonary exacerbations, with higher neutrophil elastase activity persisting in the CF airways at the end of treatment.

Topics: Adolescent; Biomarkers; Child; Cystic Fibrosis; Disease Progression; Female; Haemophilus influenzae; Humans; Inflammation; Interleukin-8; Kartagener Syndrome; Lung; Male; Neutrophils; Ontario; Respiratory Function Tests; Sputum; Staphylococcus aureus

Rhinoviruses (RV) replicate in both upper and lower airway epithelial cells. We evaluated the possibility of using nasal epithelial cells (NEC) as surrogate of bronchial epithelial cells (BEC) for RV pathogenesis cell culture studies.. We used primary paired NEC and BEC cultures established from healthy subjects and compared the replication of RV belonging to the major (RV16) and minor (RV1B) group, and the cellular antiviral and proinflammatory cytokine responses towards these viruses. We related antiviral and pro-inflammatory responses of NEC isolated from CF and COPD patients with those of BEC.. RV16 replication and major group surface receptor (ICAM-1) expression were higher in healthy NEC compared with BEC (P < 0.05); RV1B replication and minor group surface receptor (LDLR) expression were similar. Healthy NEC and BEC produced similar levels of IFN-β and IFN-λ2/3 upon RV infection or after simulation with poly(IC). IL-8 production was similar between healthy NEC and BEC. IL-6 release at baseline (P < 0.01) and upon infection with RV16 (P < 0.05) and poly(IC) stimulation (P < 0.05) was higher in NEC. RV1B viral load in NEC was related to RV1B viral load in BEC (r = 0.49, P = 0.01). There was a good correlation of IFN levels between NEC and BEC (r = 0.66, P = 0.0004 after RV1B infection). IL-8 production in NEC was related to IL-8 production in BEC (r = 0.48, P = 0.02 after RV1B infection).. NEC are a suitable alternative cellular system to BEC to study the pathophysiology of RV infections and particularly to investigate IFN responses induced by RV infection.

Topics: Adolescent; Aged; Cells, Cultured; Child; Child, Preschool; Cystic Fibrosis; Epithelial Cells; Female; Humans; Immunity, Innate; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Male; Middle Aged; Picornaviridae Infections; Pulmonary Disease, Chronic Obstructive; Respiratory System; Rhinovirus

The effects of cigarette smoke extract (CSE) on airway epithelial cells (AECs) from cystic fibrosis (CF) and non-cystic fibrosis (non-CF) individuals are not fully understood. It has been suggested that CSE modulates inflammatory cytokine release from AECs by modulating the epidermal growth factor receptor (EGFR) pathway; these pathways could reveal novel therapeutic targets. We compared the effect of CSE pre-incubation on IL-8 release from CF and non-CF bronchial epithelial cell lines, and separately, with primary nasal epithelial cells (NECs) retrieved from CF and non-CF individuals. We also determined if the EGFR pathway regulates IL-8 release by LPS or cytomix in non-CF and CF AECs at baseline and following CSE exposure. CF and non-CF cell lines, NECs derived from both CF patients (R117H heterozygous and F508del homozygous), and from healthy subjects, were cultured in the presence or absence of CSE, and subsequently exposed to inflammatory stimuli. In cell lines CSE significantly reduced IL-8 release following inflammatory challenge. Conversely, CSE pre-treatment was pro-inflammatory in primary NECs. In NECs from control subjects, CSE increased cytomix and LPS induced IL-8 release, and for the R117H heterozygous NEC cultures, CSE enhanced basal IL-8 release. Cytomix and LPS induced IL-8 release from F508del homozygous NEC cultures was further heightened following CSE pre-treatment. EGFR inhibition mitigated IL-8 release from immortalised and primary non-CF and CF AECs, suggesting that constitutive and CSE elicited IL-8 release from AECs is partly regulated via the EGFR pathway. This study demonstrates the importance of the EGFR cascade in the regulation of constitutive and CSE induced inflammatory mediator release from immortalised and primary AECs. Moreover, it clearly highlights the significance of using primary cells to confirm results obtained from immortalised cell studies, as these model systems may respond very differently to the stimuli under investigation.

Topics: Bronchi; Cell Line; Cigarette Smoking; Cystic Fibrosis; Epithelial Cells; ErbB Receptors; Humans; Inflammation; Interleukin-8; Nasal Mucosa; Signal Transduction; Smoke

The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), is required for degradation of sulfated glycosaminoglycans (GAGs) which accumulate in cystic fibrosis. ARSB is reduced in cystic fibrosis cells and increases when defective CFTR is repaired by insertion of the normal gene. This study was undertaken to determine if modification of CFTR by small molecule correctors or potentiators could also increase ARSB and reduce the accumulation of chondroitin 4-sulfate (C4S).. CF bronchial epithelial cells homozygous for the F508 deletion (ACD#14071) and normal human bronchial epithelial cells (BEC) were grown and differentiated following an established protocol. Cells were treated with either VRT-532, a CFTR potentiator, or VRT-534, a CFTR corrector, or vehicle control. The impact on ARSB activity, protein and mRNA expression, C4S and total sulfated glycosaminoglycan content, Interleukin-8 and Interleukin-6 secretion, and neutrophil chemotaxis was determined by specific assays.. The CFTR potentiator, but not the corrector, increased ARSB activity and expression to the level in the normal bronchial epithelial cells (BEC). Concomitantly, total sulfated glycosaminoglycans and C4S declined, secreted IL-8 increased, secreted IL-6 declined, and neutrophil chemotaxis to the spent media obtained from the potentiator-treated CF cells increased.. The CFTR potentiator increased ARSB activity and expression and associated effects. This suggests that a critical interaction between CFTR and ARSB is related to CFTR function in regulation of a ligand-gated anion channel at the cell membrane, rather than to CFTR processing and intracellular trafficking.

Topics: Bronchi; Cell Line; Chemotaxis, Leukocyte; Chondroitin Sulfates; Cresols; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Glycosaminoglycans; Humans; Interleukin-6; Interleukin-8; N-Acetylgalactosamine-4-Sulfatase; Pyrazoles; Respiratory Mucosa

The severity of cystic fibrosis (CF) is associated with classes of mutations in the CFTR gene (cystic fibrosis transmembrane regulator), physical environment and modifier genes interaction. The IL8 gene (interleukin 8), according to its respective polymorphisms, influences inflammatory responses. This study analyzed IL8 gene polymorphisms (rs4073, rs2227306 and rs2227307), by means of PCR/RFLP, and their association with pulmonary function markers and clinical severity scores in 186 patients with CF, considering the CFTR genotype. There was an association between rs2227307 and precocity of the disease. The severity of lung disease was associated with the following markers: transcutaneous arterial hemoglobin oxygen saturation (SaO2) (regardless of CFTR genotype, for the polymorphisms rs4073, rs2227306 and rs2227307); mucoid Pseudomonas aeruginosa (regardless of CFTR genotype, for the polymorphisms rs2227306 and rs2227307). Pulmonary function markers (SaO2 and spirometric variables) and clinical severity scores were also associated with IL8 gene polymorphisms. This study identified the IL8 gene, represented by rs4073 and rs2227306 polymorphisms, and particularly the rs2227307 polymorphism, as potentiating factors for the degree of variability in the severity of CF, especially in pulmonary clinical manifestation correlated with increased morbidity and mortality.

Topics: Adolescent; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Genetic Association Studies; Genotype; Hemoglobins; Humans; Inflammation; Interleukin-8; Lung Diseases; Male; Oxygen; Polymorphism, Single Nucleotide; Pseudomonas aeruginosa; Pseudomonas Infections; Severity of Illness Index

Airway epithelial cells in cystic fibrosis (CF) overexpress Interleukin 8 (CXCL8) through poorly defined mechanisms. CXCL8 transcription is dependent on coordinated binding of CCAAT/enhancer binding protein (C/EBP)β, nuclear factor (NF)-κB, and activator protein (AP)-1 to the promoter. Here we show abnormal epigenetic regulation is responsible for CXCL8 overexpression in CF cells. Under basal conditions CF cells had increased bromodomain (Brd)3 and Brd4 recruitment and enhanced NF-κB and C/EBPβ binding to the CXCL8 promoter compared to non-CF cells due to trimethylation of histone H3 at lysine 4 (H3K4me3) and DNA hypomethylation at CpG6. IL-1β increased NF-κB, C/EBPβ and Brd4 binding. Furthermore, inhibitors of bromodomain and extra-terminal domain family (BET) proteins reduced CXCL8 production in CF cells suggesting a therapeutic target for the BET pathway.

Topics: Azepines; CCAAT-Enhancer-Binding Protein-beta; Cell Cycle Proteins; Cells, Cultured; Cystic Fibrosis; DNA Methylation; Epigenesis, Genetic; Epithelial Cells; Humans; Interleukin-1beta; Interleukin-8; Mutation; NF-kappa B; Nuclear Proteins; Promoter Regions, Genetic; Transcription Factor AP-1; Transcription Factors; Triazoles

Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o

Topics: A549 Cells; Adult; Bronchi; Calcium Channels; Cystic Fibrosis; Cytokines; Epithelial Cells; Female; Gene Silencing; Humans; Interleukin-8; Lung; Male; Middle Aged; Models, Biological; Nerve Tissue Proteins; Pneumonia; Pseudomonas aeruginosa; RNA, Messenger; Tissue Donors; Transcription, Genetic; Transient Receptor Potential Channels; TRPA1 Cation Channel; Young Adult

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.

Topics: Anti-Inflammatory Agents; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; Lactams; NF-kappa B; Quercetin; Respiratory Mucosa; Transcriptome; Tumor Necrosis Factor alpha-Induced Protein 3

Endoplasmic reticulum (ER) stress is associated with chronic pulmonary inflammatory diseases. We hypothesized that the combined activation of both Toll-like receptor (TLR) signaling and ER stress might increase inflammatory reactions in otherwise tolerant airway epithelial cells. Indeed, ER stress resulted in an increased response of BEAS-2B and human primary bronchial epithelial cells to pathogen-associated molecular pattern stimulation with respect to IL6 and IL8 production. ER stress elevated p38 and ERK MAP kinase activation, and pharmacological inhibition of these kinases could inhibit the boosting effect. Knockdown of unfolded protein response signaling indicated that mainly PERK and ATF6 were responsible for the synergistic activity. Specifically, PERK and ATF6 mediated increased MAPK activation, which is needed for effective cytokine secretion. We conclude that within airway epithelial cells the combined activation of TLR signaling and ER stress-mediated MAPK activation results in synergistic proinflammatory activity. We speculate that ER stress, present in various chronic pulmonary diseases, boosts TLR signaling and therefore proinflammatory cytokine production, thus acting as a costimulatory danger signal.

Topics: Activating Transcription Factor 6; Bronchi; Cell Line; Cystic Fibrosis; eIF-2 Kinase; Endoplasmic Reticulum Stress; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Immunity, Innate; Inflammation; Interleukin-6; Interleukin-8; p38 Mitogen-Activated Protein Kinases; RNA, Small Interfering; Signal Transduction; Toll-Like Receptors; Unfolded Protein Response

Activation of the transcription factor nuclear factor-kappa B (NF-κB) signaling pathway is associated with enhanced secretion of pro-inflammatory mediators and is thought to play a critical role in diseases hallmarked by inflammation, including cystic fibrosis (CF). Small nucleic acids that interfere with gene expression have been proposed as promising therapeutics for a number of diseases. However, applications have been limited by low cellular penetration and a lack of stability. Nano-sized carrier systems have been suggested as a means of improving the effectiveness of nucleic acid-based treatments. In this study, we successfully coated polysialic acid-N-trimethyl chitosan (PSA-TMC) nanoparticles with NF-κΒ decoy oligonucleotides (ODNs). To demonstrate anti-inflammatory activity, the decoy ODN-coated PSA-TMC nanoparticles were administered to an in vitro model of CF generated via interleukin-1β or P. aeruginosa lipopolysaccharides stimulation of IB3-1 bronchial epithelial cells. While free ODN and PSA-TMC nanoparticles coated with scrambled ODNs did not have substantial impacts on the inflammatory response, the decoy ODN-coated PSA-TMC nanoparticles were able to reduce the secretion of interleukin-6 and interleukin-8, pro-inflammatory mediators of CF, by the epithelial cells, particularly at longer time points. In general, the results suggest that NF-κB decoy ODN-coated TMC-PSA nanoparticles may serve as an effective method of altering the pro-inflammatory environment associated with CF.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Chitosan; Coated Materials, Biocompatible; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Genes, Reporter; Humans; Immunomodulation; Interleukin-6; Interleukin-8; Luciferases; Nanoparticles; NF-kappa B; Oligodeoxyribonucleotides; Polysaccharides; Rats; Sialic Acids; Signal Transduction

Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is demonstrating promise as an inflammatory biomarker of acute infection in various pulmonary conditions; including community acquired pneumonia, ventilator associated pneumonia and non-tuberculous mycobacterial infection.. The expression of sTREM-1 has been poorly studied in all forms of bronchiectasis, both in the context of cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis.. Induced sputum samples were collected for sTREM-1 determination in children with HIV-associated bronchiectasis and CF-bronchiectasis. The presence or absence of an exacerbation was noted at study entry. Lung function parameters (FEV1, FVC, FEV1 /FVC, FEF(25-75)) were measured using the Viasys SpiroPro Jaeger Spirometer (Hoechberg, Germany).. A total of twenty-six children with HIV-associated bronchiectasis and seventeen with CF were included. With respect to sTREM-1, the levels were readily detected in both groups, but were significantly higher in children with HIV-associated bronchiectasis (1244.0 pg/ml (iqr 194.5; 3755.3 pg/ml) and 204.9  pg/ml (iqr 66.9; 653.6 pg/ml) P = 0.003. There was a positive correlation between sTREM-1 and IL-8 as well as sputum neutrophil elastase in the HIV-bronchiectasis group (r = 0.715 and r = 0.630), respectively both P < 0.005. sTREM-1 was not further increased in subjects presenting with an acute pulmonary exacerbation in the HIV-associated bronchiectasis and in CF participants (P = 0.971 and P = 0.481), respectively. In the CF group sTREM-1 strongly correlated with FVC% predicted and FEV1 % predicted (r = 0.950 and r = 0.954), both P < 0.005.. The pulmonary innate immune functions are over-active in HIV-associated bronchiectasis, with readily detected sTREM-1 values, which were higher than those in CF. sTREM-1 does not correlate with markers of HIV-disease activity but does correlate with markers of neutrophilic inflammation. In CF sTREM-1 has a negative correlation with pulmonary function parameters.

Topics: Adolescent; Biomarkers; Bronchiectasis; Child; Cystic Fibrosis; Female; HIV Infections; Humans; Interleukin-8; Leukocyte Elastase; Membrane Glycoproteins; Receptors, Immunologic; Sputum; Triggering Receptor Expressed on Myeloid Cells-1

The chemokine interleukin-8 (CXCL8) is a key mediator of inflammation in airways of patients with cystic fibrosis (CF). Glycosaminoglycans (GAGs) possess the ability to influence the chemokine profile of the CF lung by binding CXCL8 and protecting it from proteolytic degradation. CXCL8 is maintained in an active state by this glycan interaction thus increasing infiltration of immune cells such as neutrophils into the lungs. As the CXCL8-based decoy PA401 displays no chemotactic activity, yet demonstrates glycan binding affinity, the aim of this study was to investigate the anti-inflammatory effect of PA401 on CXCL8 levels, and activity, in CF airway samples in vitro.. Bronchoalveolar lavage fluid (BALF) was collected from patients with CF homozygous for the ΔF508 mutation (n=13). CXCL8 in CF BALF pre and post exposure to PA401 was quantified by ELISA. Western blot analysis was used to determine PA401 degradation in CF BALF. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post exposure to PA401 by use of a Boyden chamber-based motility assay.. Exposure of CF BALF to increasing concentrations of PA401 (50-1000pg/ml) over a time course of 2-12h in vitro, significantly reduced the level of detectable CXCL8 (P<0.05). Interestingly, PA401 engendered release of CXCL8 from GAGs exposing the chemokine susceptible to proteolysis. Subsequently, a loss of PA401 was observed (P<0.05) due to proteolytic degradation by elastase like proteases. A 25% decrease in neutrophil chemotactic efficiency towards CF BALF samples incubated with PA401 was also observed (P<0.05).. PA401 can disrupt CXCL8:GAG complexes, rendering the chemokine susceptible to proteolytic degradation. Clinical application of a CXCL8 decoy, such as PA401, may serve to decrease the inflammatory burden in the CF lung in vivo.

Topics: Bronchoalveolar Lavage Fluid; Chemotaxis; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Humans; Interleukin-8; Neutrophils; Proteolysis; Recombinant Proteins; Young Adult

Bacterial tyrosine kinases and their cognate protein tyrosine phosphatases are best known for regulating the biosynthesis of polysaccharides. Moreover, their roles in the stress response, DNA metabolism, cell division, and virulence have also been documented. The aim of this study was to investigate the pathogenicity and potential mechanisms of virulence dependent on the tyrosine kinase BceF and phosphotyrosine phosphatase BceD of the cystic fibrosis opportunistic pathogen Burkholderia contaminans IST408. The insertion mutants bceD::Tp and bceF::Tp showed similar attenuation of adhesion and invasion of the cystic fibrosis lung epithelial cell line CFBE41o- compared to the parental strain B. contaminans IST408. In the absence of bceD or bceF genes, B. contaminans also showed a reduction in the ability to translocate across polarized epithelial cell monolayers, demonstrated by a higher transepithelial electrical resistance, reduced flux of fluorescein isothiocyanate-labeled bovine serum albumin, and higher levels of tight junction proteins ZO-1, occludin, and claudin-1 present in monolayers exposed to these bacterial mutants. Furthermore, bceD::Tp and bceF::Tp mutants induced lower levels of interleukin-6 (IL-6) and IL-8 release than the parental strain. In conclusion, although the mechanisms of pathogenicity dependent on BceD and BceF are not understood, these proteins contribute to the virulence of Burkholderia by enhancement of cell attachment and invasion, disruption of epithelial integrity, and modulation of the proinflammatory response.

Topics: Albumins; Anti-Bacterial Agents; Bacterial Adhesion; Burkholderia cepacia complex; Burkholderia Infections; Cell Line; Ciprofloxacin; Claudin-1; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Impedance; Humans; Inflammation; Interleukin-6; Interleukin-8; Lung; Membrane Potentials; Mutation; Occludin; Protein Transport; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Respiratory Mucosa; Tight Junctions; Virulence Factors; Zonula Occludens-1 Protein

Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.. Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.. These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.. These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

Topics: Animals; Cell Death; Cell Line; Ceramides; Cystic Fibrosis; Cytochromes c; HL-60 Cells; Humans; Interleukin-8; Jurkat Cells; Liver; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Rats; Reactive Oxygen Species; Sphingomyelin Phosphodiesterase

Cystic fibrosis (CF) is a hereditary, chronic disease of the exocrine glands, characterized by the production of viscid mucus that obstructs the pancreatic ducts and bronchi, leading to infection and fibrosis. ω3 fatty acid supplementations are known to improve the essential fatty acid deficiency as well as reduce inflammation in CF. The objective of this study was to determine the effects of docosahexaenoic acid monoacylglyceride (MAG-DHA) on mucin overproduction and resolution of airway inflammation in two in vitro models related to CF. Isolated human bronchi reverse permeabilized with CF transmembrane conductance regulator (CFTR) silencing (si) RNA and stable Calu3 cells expressing a short hairpin (sh) RNA directed against CFTR (shCFTR) were used. Lipid analyses revealed that MAG-DHA increased DHA/arachidonic acid (AA) ratio in shCFTR Calu-3 cells. MAG-DHA treatments, moreover, resulted in a decreased activation of Pseudomonas aeruginosa LPS-induced NF-κB in CF and non-CF Calu-3 cells. Data also revealed a reduction in MUC5AC, IL-6, and IL-8 expression levels in MAG-DHA-treated shCFTR cells stimulated, or not, with LPS. Antiinflammatory properties of MAG-DHA were also investigated in a reverse-permeabilized human bronchi model with CFTR siRNA. After MAG-DHA treatments, messenger RNA transcript levels for MUC5AC, IL-6, and IL-8 were markedly reduced in LPS-treated CFTR siRNA bronchi. MAG-DHA displays antiinflammatory properties and reduces mucin overexpression in Calu-3 cells and human bronchi untreated or treated with P. aeruginosa LPS, a finding consistent with the effects of resolvinD1, a known antiinflammatory mediator.

Topics: Anti-Inflammatory Agents; Bronchi; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Evaluation, Preclinical; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lung; Monoglycerides; Mucin 5AC; NF-kappa B; Signal Transduction

The hypoxic environment of cystic fibrosis airways allows the persistence of facultative anaerobic bacteria, which can produce short-chain fatty acids (SCFAs) through fermentation. However, the relevance of SCFAs in cystic fibrosis lung disease is unknown. We show that SCFAs are present in sputum samples from cystic fibrosis patients in millimolar concentrations (mean±sem 1.99±0.36 mM).SCFAs positively correlated with sputum neutrophil count and higher SCFAs were predictive for impaired nitric oxide production. We studied the effects of the SCFAs acetate, propionate and butyrate on airway inflammatory responses using epithelial cell lines and primary cell cultures. SCFAs in concentrations present in cystic fibrosis airways (0.5-2.5 mM) affected the release of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin (IL)-6. SCFAs also resulted in higher IL-8 release from stimulated cystic fibrosis transmembrane conductance regulator (CFTR) F508del-mutant compared to wild-type CFTR-corrected bronchial epithelial cells. At 25 mM propionate reduced IL-8 release in control but not primary cystic fibrosis epithelial cells. Low (0.5-2.5 mM) SCFA concentrations increased, while high (25-50 mM) concentrations decreased inducible nitric oxide synthase expression. In addition, SCFAs affected the growth of Pseudomonas aeruginosa in a concentration- and pH-dependent manner.Thus, our data suggest that SCFAs contribute to cystic fibrosis-specific alterations of responses to airway infection and inflammation.

Topics: Acetates; Adolescent; Bacterial Infections; Butyrates; Child; Chromatography, Gas; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fatty Acids, Volatile; Female; Fermentation; Forced Expiratory Volume; Gene Expression Regulation; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Hypoxia; Inflammation; Interleukin-6; Interleukin-8; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Propionates; Pseudomonas aeruginosa; Sputum

Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca(2+) concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca(2+) in CF bronchial epithelial cells have not been evaluated.. We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca(2+)concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide.. GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca(2+) concentration.. These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca(2+) concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

Topics: Anti-Inflammatory Agents; Apoptosis; Bronchi; Budesonide; Calcium; Cell Line; Cells, Cultured; Cystic Fibrosis; Dexamethasone; Epithelial Cells; Epithelium; Glucocorticoids; Humans; Interleukin-6; Interleukin-8; Pseudomonas aeruginosa; Pseudomonas Infections

Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

Topics: Adult; Animals; Bronchi; Bronchoalveolar Lavage Fluid; Cell Count; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Female; Humans; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Middle Aged; Neutrophil Infiltration; Young Adult

There is current interest in vitamin D as a potential anti-inflammatory treatment for chronic inflammatory lung disease, including cystic fibrosis (CF). Vitamin D transcriptionally up-regulates the anti-inflammatory gene DUSP1, which partly controls production of the inflammatory chemokine IL-8. IL-8 is overabundant in CF airways, potentially due to hyperinflammatory responses of CF macrophages. We tested the ability of vitamin D metabolites to down-regulate IL-8 production in CF macrophages.. CF and healthy monocyte-derived macrophages (MDM) were treated with two vitamin D metabolites, 25-hydroxyvitamin D3 (25OHD3 ) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), or paricalcitol, synthetic analogue of 1,25(OH)2 D3 . 25OHD3 was tested at doses of 25-150 nM, whereas 1,25(OH)2 D3 and paricalcitol at doses of up to 100 nM. IL-8 was stimulated by bacterial virulence factors. As potential anti-inflammatory mechanism of vitamin D metabolites, we assessed up-regulation of DUSP1.. MDM from patients with CF and some healthy donors showed excessive production of stimulated IL-8, highlighting their hyperinflammatory phenotype. Vitamin D metabolites down-regulated stimulated IL-8 only in those hyperinflammatory MDM, and only when used at high doses (>100 nM for 25OHD3 , or >1 nM for 1,25(OH)2 D3 and paricalcitol). The magnitude of IL-8 down-regulation by vitamin D metabolites or paricalcitol was moderate (∼30% vs. >70% by low-dose dexamethasone). Transcriptional up-regulation of DUSP1 by vitamin D metabolites was seen in all tested MDM, regardless of IL-8 down-regulation.. Vitamin D metabolites and their analogues moderately down-regulate IL-8 in hyperinflammatory macrophages, including those from CF. This down-regulation appears to go through DUSP1-independent mechanisms.

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Adult; Calcifediol; Cells, Cultured; Cystic Fibrosis; Down-Regulation; Dual Specificity Phosphatase 1; Ergocalciferols; Gene Expression Regulation; Humans; Interleukin-8; Macrophages; Receptors, Calcitriol; Up-Regulation; Vitamin D; Vitamin D3 24-Hydroxylase; Young Adult

Monitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring.. To measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically.. Whole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians' assessments were also collected from patients with cystic fibrosis on the day of saliva collection.. Similar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1β (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV1 and disease severity (as evaluated by clinicians) with both platforms (P < 0.05).. Significant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.

Topics: Adolescent; Adult; Aged; Biomarkers; Chemokine CXCL10; Child; Cross-Sectional Studies; Cystic Fibrosis; Epidermal Growth Factor; Female; Humans; Immunoassay; Interleukin-8; Longitudinal Studies; Male; Matrix Metalloproteinase 9; Middle Aged; Protein Array Analysis; Respiratory Function Tests; Saliva; Spirometry; Vascular Endothelial Growth Factor A

A hallmark of cystic fibrosis (CF) lung disease is neutrophilic airway inflammation. Elevated neutrophil counts have been associated with decreased forced expiratory volume in 1 second and poor clinical measures in patients with CF. Interleukin 8 (IL-8), epithelial neutrophil activating protein 78 (ENA-78), tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) contribute to neutrophil activation and disease pathogenesis in the airways of patients with CF. Drugs that modify the production of these chemokines in the airways could potentially benefit CF patients. Thus, we determined the effects of fenofibrate on their production in cell populations obtained from the airways. Human small airway epithelial cells and CF bronchial epithelial cells were treated with IL-1β to induce inflammation. We cotreated the cells with fenofibrate at concentrations ranging from 10 to 50 μM to determine if this drug could attenuate the inflammation. IL-8, ENA-78, TNF-α, GM-CSF, and G-CSF production were measured from the cell culture supernates by ELISA. ANOVA statistical testing was conducted using SPSS 17.0. IL-1β increased the production of each of the chemokines by several fold. Fenofibrate reduced IL-1β induced production of each of these neutrophilic chemokines at the concentrations used. IL-1β increases the production of neutrophilic chemokines in airway epithelial cells. Cotreatment with fenofibrate blunts these processes. Fenofibrate should be explored as a therapeutic option to modulate the abundant neutrophilic inflammation observed in CF.

Topics: Bronchi; Cells, Cultured; Chemokine CXCL5; Chemokines; Cystic Fibrosis; Dose-Response Relationship, Drug; Drug Repositioning; Epithelial Cells; Fenofibrate; Forced Expiratory Volume; Gene Expression Profiling; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-8; Neutrophils; Tumor Necrosis Factor-alpha

Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation.. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells.. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion.. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Topics: Cell Line; Cell Movement; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression; Genetic Therapy; Genetic Vectors; Genome; Humans; Interleukin-8; Lentivirus; Respiratory System; RNA Interference; RNA, Small Interfering

Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

Topics: Biofilms; Burkholderia cepacia complex; Burkholderia Infections; Cell Death; Cell Line; Cystic Fibrosis; Gene Expression Profiling; Humans; Interleukin-8; Microbial Viability; Models, Biological; Neutrophils

Measures of ventilation distribution are promising for monitoring early lung disease in cystic fibrosis (CF). This study describes the cross-sectional and longitudinal impacts of pulmonary inflammation and infection on ventilation homogeneity in infants with CF.Infants diagnosed with CF underwent multiple breath washout (MBW) testing and bronchoalveolar lavage at three time points during the first 2 years of life.Measures were obtained for 108 infants on 156 occasions. Infants with a significant pulmonary infection at the time of MBW showed increases in lung clearance index (LCI) of 0.400 units (95% CI 0.150-0.648; p=0.002). The impact was long lasting, with previous pulmonary infection leading to increased ventilation inhomogeneity over time compared to those who remained free of infection (p<0.05). Infection with Haemophilus influenzae was particularly detrimental to the longitudinal lung function in young children with CF where LCI was increased by 1.069 units for each year of life (95% CI 0.484-1.612; p<0.001).Pulmonary infection during the first year of life is detrimental to later lung function. Therefore, strategies aimed at prevention, surveillance and eradication of pulmonary pathogens are paramount to preserve lung function in infants with CF.

Topics: Breath Tests; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Disease Progression; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Infant, Newborn; Interleukin-8; Longitudinal Studies; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Aspergillosis; Pulmonary Ventilation; Staphylococcal Infections; Staphylococcus aureus

This study aimed to evaluate the ability of the forced oscillation technique (FOT) to detect underlying lung disease in preschool children with cystic fibrosis (CF) diagnosed following newborn screening.184 children (aged 3-6 years) with CF underwent lung function testing on 422 occasions using the FOT to assess respiratory resistance and reactance at the time of their annual bronchoalveolar lavage collection and chest computed tomography scan. We examined associations between FOT outcomes and the presence and progression of respiratory inflammation, infection and structural lung disease.Children with CF who had pronounced respiratory disease, including free neutrophil elastase activity, infection with pro-inflammatory pathogens and structural lung abnormalities had similar FOT outcomes to those children without detectable lung disease. In addition, the progression of lung disease over 1 year was not associated with worsening FOT outcomes.We conclude that the forced oscillation technique is relatively insensitive to detect underlying lung disease in preschool children with CF. However, FOT may still be of value in improving our understanding of the physiological changes associated with early CF lung disease.

Topics: Airway Resistance; Bronchoalveolar Lavage; Child; Child, Preschool; Cohort Studies; Cross-Sectional Studies; Cystic Fibrosis; Female; Humans; Interleukin-8; Leukocyte Count; Longitudinal Studies; Lung; Lung Diseases; Male; Neutrophils; Respiratory Function Tests; Tomography, X-Ray Computed

Lung disease in cystic fibrosis (CF) is often exacerbated following acute upper respiratory tract infections caused by the human rhinovirus (HRV). Pathophysiology of these exacerbations is presently unclear and may involve deficient innate antiviral or exaggerated inflammatory responses in CF airway epithelial cells. Furthermore, responses of CF cells to HRV may be adversely affected by pre-exposure to virulence factors of Pseudomonas (P.) aeruginosa, the microorganism that frequently colonizes CF airways. Here we examined production of antiviral cytokine interferon-β and inflammatory chemokine interleukin-8, expression of the interferon-responsive antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1), and intracellular virus RNA load in primary CF (delF508 CFTR) and healthy airway epithelial cells following inoculation with HRV16. Parallel cells were exposed to virulence factors of P. aeruginosa prior to and during HRV16 inoculation. CF cells exhibited production of interferon-β and interleukin-8, and expression of OAS1 at levels comparable to those in healthy cells, yet significantly higher HRV16 RNA load during early hours post-inoculation with HRV16. In line with this, HRV16 RNA load was higher in the CFBE41o- dF cell line overexpessing delF508 CFTR, compared with the isogenic control CFBE41o- WT (wild-type CFTR). Pre-exposure to virulence factors of P. aeruginosa did not affect OAS1 expression or HRV16 RNA load, but potentiated interleukin-8 production. In conclusion, CF cells demonstrate elevated HRV RNA load despite preserved interferon-β and OAS1 responses. High HRV load in CF airway epithelial cells appears to be due to deficiencies manifesting early during HRV infection, and may not be related to interferon-β.

Topics: 2',5'-Oligoadenylate Synthetase; Adult; Bronchi; Cell Line; Cystic Fibrosis; Epithelial Cells; Female; Genotype; Humans; Interferon-beta; Interleukin-8; Lung Diseases; Male; Primary Cell Culture; Pseudomonas aeruginosa; Recombinant Proteins; Rhinovirus; RNA, Viral; Viral Load; Virulence; Young Adult

Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage.. Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined.. IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B.. These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Topics: Adult; Antibodies, Bacterial; Biomarkers; Cathepsin B; Cathepsins; Cystic Fibrosis; Female; Humans; Immunoglobulin A; Immunoglobulin G; Interleukin-6; Interleukin-8; Kaplan-Meier Estimate; Leukocyte Elastase; Male; Multivariate Analysis; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Tumor Necrosis Factor, Type I; Sputum; Tumor Necrosis Factor-alpha; Young Adult

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.. The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.. We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.. The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.. Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.

Topics: Animals; Anti-Inflammatory Agents; Antifungal Agents; Blotting, Western; Cell Proliferation; Ceramides; Chromatography, Liquid; Cystic Fibrosis; Drug Carriers; Enzyme-Linked Immunosorbent Assay; Fatty Acids, Monounsaturated; Female; Humans; Interleukin-6; Interleukin-8; Male; Mice; Mice, Inbred CFTR; Nanoparticles; Pseudomonas aeruginosa; Pseudomonas Infections; Real-Time Polymerase Chain Reaction; Respiratory Mucosa; Respiratory Tract Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids

The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated.. Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared.. AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species.. The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Topics: Animals; Biofilms; Cell Line; Cystic Fibrosis; Epidemics; Epithelial Cells; Humans; Interleukin-8; Larva; Moths; Pancreatic Elastase; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Streptococcal Infections; Streptococcus; Virulence; Virulence Factors

Bio-active trace metals have been identified in respiratory tract secretions of subjects with lung disease and may potentially influence bacterial virulence, inflammation and disease severity. We measured a diverse range of metal ions in sputum samples from subjects with CF and non-CF bronchiectasis (NCFB) compared to healthy controls and examined their relationship to airway inflammation, disease severity and the presence of bacterial pathogens.. We studied 45 subjects with CF, 8 with NCFB and 8 healthy controls. Metal concentrations were measured in sputum supernatant by inductively-coupled plasma mass spectrometry and correlated with sputum inflammatory cell counts, lactate dehydrogenase (LDH) and interleukin (IL)-8 concentrations, atmospheric particulate matter, lung function, clinical status and participant demographics.. Sputum from subjects with CF and NCFB contained increased concentrations of magnesium, calcium, iron and zinc. Metal ion concentrations correlated positively with LDH levels. The concentrations of magnesium, iron and zinc positively correlated with IL-8. A sub-group of CF subjects with severe lung disease demonstrated increased sputum molybdenum concentrations.. Elevated concentrations of sputum metal ions appear to be associated with cell/tissue necrosis and inflammation in subjects with CF and NCFB. Sputum molybdenum concentrations may be a biomarker of severe CF airway disease.

Topics: Adolescent; Adult; Aged; Biomarkers; Cross-Sectional Studies; Cystic Fibrosis; Female; Humans; Interleukin-8; Iron; L-Lactate Dehydrogenase; Magnesium; Male; Middle Aged; Molybdenum; Respiratory Function Tests; Severity of Illness Index; Sputum; Young Adult; Zinc

Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized.. Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CF patients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays.. NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage.. NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided.

Topics: Adolescent; Adult; Aged; Biomarkers; Case-Control Studies; Centrifugation; Child; Child, Preschool; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Freezing; Humans; Inflammation; Interleukin-1; Interleukin-6; Interleukin-8; Leukocyte Elastase; Male; Middle Aged; Nasal Lavage Fluid; Peroxidase; Protease Inhibitors; Specimen Handling; Tumor Necrosis Factor-alpha

Eleven decoctions, obtained from indian plants widely used in ayurvedic medicine, have been investigated as a possible source of molecules exhibiting biological activity on the interaction between DNA and NF-kB, a transcription factor involved in the expression of proinflammatory genes. Cystic fibrosis (CF) cell line stimulated by TNF-α has been used as inflammatory cellular model to determinate interleukin-8 (IL-8), one of the most relevant pro-inflammatory mediator in CF regulated by the NF-kB. The chemical characterization of these 11 decoctions by spectrophotometric analysis and NMR fingerprinting highlighted that sugars and polyphenols seemed to be the main compounds. Our results demonstrated that Azadirachta indica, Terminalia bellerica, Terminalia chebula, Hemidesmus indicus, Emblica officinalis and Swertia chirata are the most active decoctions in inhibiting NF-kB/DNA interactions by EMSA assay and in reducing pro-inflammatory IL- 8 expression in CF cells at IC50 concentrations by Real-Time and Bio-plex analyses. Finally, we observed the increase of all inhibitory activities with the rise of total polyphenols, procyanidins and flavonoids, except for the levels of IL-8 mRNA accumulation, that were as high as flavonoid content grown up by the statistical multivariate analyses. In conclusion, these six decoctions might be interesting to explore new anti-inflammatory treatments for diseases, such as CF.

Topics: Azadirachta; Cell Line; Cystic Fibrosis; Hemidesmus; Humans; Interleukin-8; Medicine, Ayurvedic; NF-kappa B; Phyllanthus emblica; Plant Extracts; Polyphenols; Swertia; Terminalia; Tumor Necrosis Factor-alpha

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

Topics: 3' Untranslated Regions; Bronchi; Cell Line; Cystic Fibrosis; Down-Regulation; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; MicroRNAs; Minichromosome Maintenance Complex Component 7; NF-kappa B; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Messenger; Transcription, Genetic; Up-Regulation

Environmental and genetic factors may modify or contribute to the phenotypic differences observed in multigenic and monogenic diseases, such as cystic fibrosis (CF). An analysis of modifier genes can be helpful for estimating patient prognosis and directing preventive care. The aim of this study is to determine the association between seven genetic variants of four modifier genes and CF by comparing their corresponding allelic and genotypic frequencies in CF patients (n = 81) and control subjects (n = 104). Genetic variants of MBL2 exon 1 (A, B, C and D), the IL-8 promoter (-251 A/T), the TNFα promoter (TNF1/TNF2), and SERPINA1 (PI*Z and PI*S) were tested in CF patients and control subjects from northeastern Mexico by PCR-RFLP.. The TNF2 allele (P = 0.012, OR 3.43, 95% CI 1.25-9.38) was significantly associated with CF under the dominant and additive models but was not associated with CF under the recessive model. This association remained statistically significant after adjusting for multiple tests using the Bonferroni correction (P = 0.0482). The other tested variants and genotypes did not show any association with the disease.. An analysis of seven genetic variants of four modifier genes showed that one variant, the TNF2 allele, appears to be significantly associated with CF in Mexican patients.

Topics: Alleles; alpha 1-Antitrypsin; Case-Control Studies; Cystic Fibrosis; Female; Gene Frequency; Genes, Modifier; Genotype; Humans; Interleukin-8; Male; Mannose-Binding Lectin; Mexico; Models, Genetic; Polymorphism, Genetic; Promoter Regions, Genetic; Tumor Necrosis Factor-alpha

The search for modifier genes to explain inconsistencies in cystic fibrosis (CF) genotype-phenotype relationships has yielded mixed results. In a previous cross-sectional study from our centre the clinical effect (as described by FEV1, BMI z-score, admitted days and NIH score) of single nucleotide polymorphisms (SNPs) of four cytokine genes (IL-8, TNF-α, IL-1β and IL-10) was examined in 158 children with CF. No association between cytokine genotype and any biological outcome measure was found. In this present study a cross-sectional and longitudinal examination of this relationship was undertaken to test the hypothesis that pro-inflammatory SNPs would affect longitudinal changes in CF lung disease.. Using the cohort examined in our earlier study we performed both longitudinal and cross-sectional data analyses examining the relationship between SNPs (TNF-α, IL-8, IL-10 and IL-1β) and clinical outcome measurements. In the first part of this current study, lung function data (annual decline of FEV1 percent predicted) was compared with the cytokine genotype over a 13 year period. In the second part of this current study multiple regression was used to assess associations between clinical outcomes (best FEV1 percent predicted and BMI at the age of 10 years) and alleles of cytokine genes, adjusting for gender, CF genotype and lung infection status.. A total of 152 patients with CF were analysed in the longitudinal study and data from 130 patients at the age of 10 years were analysed in the cross-sectional study. There was evidence for an association between pro-inflammatory SNPs of the IL-8, IL-10 and IL-1β genes and more severe lung disease. Multiple regression of the longitudinal data with a total of 10,956 lung function measurements showed an additional annual decline of the percentage predicted FEV1 of -1.15 (IL-8, p<0.001), -0.24 (IL-10, p=0.049) and -0.41 (IL-1β, p<0.001) for patients with any of the pro-inflammatory alleles. None of the cross-sectional data showed a significant association between the cytokine genotypes and the clinical outcomes.. Pro-inflammatory alleles of three cytokine genotypes, IL-8, IL-10 and IL-1β, appear to be associated with slightly more severe lung disease in patients with CF over a 13 year period. Further studies are required to exclude influence of confounders on the severity of lung disease.

Topics: Body Mass Index; Child; Cross-Sectional Studies; Cystic Fibrosis; Female; Forced Expiratory Volume; Genotype; Humans; Interleukin-10; Interleukin-1beta; Interleukin-8; Longitudinal Studies; Male; Outcome Assessment, Health Care; Polymorphism, Single Nucleotide; Severity of Illness Index; Tumor Necrosis Factor-alpha

In cystic fibrosis (CF) patients, the upper airways display the same ion channel defect as evident in the lungs, resulting in chronic inflammation and infection. Recognition of the sinonasal area as a site of first and persistent infection with pathogens, such as Pseudomonas aeruginosa, reinforces the "one-airway" hypothesis. Therefore, we assessed the effect of systemic antibiotics against pulmonary pathogens on sinonasal inflammation.. Nasal lavage fluid (NLF) from 17 CF patients was longitudinally collected prior to and during elective intravenous (i.v.) antibiotic treatment to reduce pathogen burden and resulting inflammation (median treatment time at time of analysis: 6 days). Samples were assessed microbiologically and cytologically. Cytokine and chemokine expression was measured by Cytometric Bead Array and ELISA (interleukin (IL)-1β, IL-6, IL-8, MPO, MMP9, RANTES and NE). Findings were compared with inflammatory markers from NLF obtained from 52 healthy controls.. Initially, the total cell count of the NLF was significantly higher in CF patients than in controls. However after i.v. antibiotic treatment it decreased to a normal level. Compared with controls, detection frequencies and absolute concentrations of MPO, IL-8, IL-6 and IL-1β were also significantly higher in CF patients. The detection frequency of TNF was also higher. Furthermore, during i.v. therapy sinonasal concentrations of IL-6 decreased significantly (P = 0.0059), while RANTES and MMP9 levels decreased 10-fold and two-fold, respectively. PMN-Elastase, assessed for the first time in NFL, did not change during therapy.. Analysis of NLF inflammatory markers revealed considerable differences between controls and CF patients, with significant changes during systemic i.v. AB treatment within just 6 days. Thus, our data support further investigation into the collection of samples from the epithelial surface of the upper airways by nasal lavage as a potential diagnostic and research tool.

Topics: Administration, Intravenous; Adolescent; Adult; Anti-Bacterial Agents; Case-Control Studies; Child; Cystic Fibrosis; Cytokines; Female; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Leukocyte Elastase; Longitudinal Studies; Male; Monitoring, Physiologic; Nasal Lavage Fluid; Reference Values; Risk Assessment; Severity of Illness Index; Statistics, Nonparametric; Treatment Outcome; Young Adult

The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation.. Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files.. Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration.. This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject.

Topics: Adult; Biomarkers; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Flagellin; Humans; Interleukin-8; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Insufficiency; Sampling Studies; Severity of Illness Index; Sputum

Cystic fibrosis (CF), the most common autosomal recessive disorder in Western countries, is characterized by chronic pulmonary inflammation, reduced mucociliary clearance, and increased susceptibility to infection. Our studies using Cftr-deficient mice and human CF specimens showed that ceramide accumulates in CF lungs and mediates increased cell death, susceptibility to infections, and inflammation.. We used Cftr-deficient and syngenic wildtype mice as well as Cftr-deficient mice heterozygous for the acid sphingomyelinase. We determined activation and topology of inflammasome components as well as expression of tight junction proteins by confocal microscopy, western blotting and ELISA.. We demonstrate an upregulation and membrane recruitment of the adapter protein apoptosis-associated speck-like protein (Asc), a major component of the inflammasome, and caspase 1, an activation of Jun N-terminal kinase as well as an altered distribution and a degradation of the tight junction proteins ZO-1, ZO-2 and Occludin in lungs of CF mice. All of these events are abrogated in CF mice that are heterozygous for the acid sphingomyelinase and, therefore, show normal levels of ceramide in their lungs. These alterations indicate an activation of the inflammasome by ceramide in the lungs of CF mice. Consistent with this notion, we observe a normalization of the increased levels of the cytokines IL-1β and KC/IL-8 in lungs of CF mice upon treatment with caspase 1 inhibitors.. Our data suggest a signaling cascade from ceramide via the inflammasome to caspase 1, the release of cytokines and an alteration of tight junction proteins in CF epithelia.

Topics: Animals; Apoptosis Regulatory Proteins; Caspase 1; Ceramides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Humans; Inflammasomes; Interleukin-1beta; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lung; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Sphingomyelin Phosphodiesterase; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein

Cystic fibrosis (CF) lung disease is characterised by chronic Pseudomonas aeruginosa infection and leukocyte infiltration. Chemokines recruit leukocytes to sites of infection. Gene expression analysis identified the chemokine CCL18 as upregulated in CF leukocytes. We hypothesised that CCL18 characterises infection and inflammation in patients with CF lung disease. Therefore, we quantified CCL18 protein levels in the serum and airway fluids of CF patients and healthy controls, and studied CCL18 protein production by airway cells ex vivo. These studies demonstrated that CCL18 levels were increased in the serum and airway fluids from CF patients compared with healthy controls. Within CF patients, CCL18 levels were increased in P. aeruginosa-infected CF patients. CCL18 levels in the airways, but not in serum, correlated with severity of pulmonary obstruction in CF. Airway cells isolated from P. aeruginosa-infected CF patients produced significantly higher amounts of CCL18 protein compared with airway cells from CF patients without P. aeruginosa infection or healthy controls. Collectively, these studies show that CCL18 levels characterise chronic P. aeruginosa infection and pulmonary obstruction in patients with CF. CCL18 may, thus, serve as a potential biomarker and therapeutic target in CF lung disease.

Topics: Adolescent; Adult; Case-Control Studies; Chemokine CXCL1; Chemokine CXCL2; Chemokines, CC; Child; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Leukocytes; Male; Pseudomonas Infections; Sputum; Young Adult

The bronchial brushing technique presents an opportunity to establish a gold standard in vitro model of Cystic Fibrosis (CF) airway disease. However, unique obstacles exist when establishing CF airway epithelial cells (pAECCF). We aimed to identify determinants of culture success through retrospective analysis of a program of routinely brushing children with CF.. Anaesthetised children (CF and non-CF) had airway samples taken which were immediately processed for cell culture. Airway data for the CF cohort was obtained from clinical records and the AREST CF database.. Of 260 brushings processed for culture, 114 (43.8%) pAECCF successfully cultured to passage one (P1) and 63 (24.2% of total) progressed to passage two (P2). However, >80% of non-CF specimens (pAECnon-CF) cultured to P2 from similar cell numbers. Within the CF cohort, specimens successfully cultured to P2 had a higher initial cell count and lower proportion of severe CF mutation phenotype than those that did not proliferate beyond initial seeding. Elevated airway IL-8 concentration was also negatively associated with culture establishment. Contamination by opportunistic pathogens was observed in 81 (31.2% of total) cultures and brushings from children with lower respiratory tract infections were more likely to co-culture contaminating flora.. Lower passage rates of pAECCF cultures uniquely contrasts with pAECnon-CF despite similar cell numbers. An equivalent establishment rate of CF nasal epithelium reported elsewhere, significant associations to CFTR mutation phenotype, elevated airway IL-8 and opportunistic pathogens all suggest this is likely related to the CF disease milieu.

Topics: Bronchoalveolar Lavage Fluid; Cell Culture Techniques; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytological Techniques; Female; Humans; Infant; Inflammation; Interleukin-8; Leukocyte Elastase; Male; Mutation; Respiratory Mucosa; Retrospective Studies; Specimen Handling

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.

Topics: Cell Line; Cell Membrane; Ceramides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Evaluation, Preclinical; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Humans; Immediate-Early Proteins; Interleukin-8; Nedd4 Ubiquitin Protein Ligases; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Stability; Protein Transport; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Sequence Deletion; Signal Transduction; Structure-Activity Relationship; Ubiquitin-Protein Ligases

CF patients are often treated with proton pump inhibitors (PPIs) to reduce acidic gastro-esophageal reflux (GER) and bronchial aspiration of duodeno-gastric contents is common in CF. We have previously demonstrated that gastric juice (GJ) from patients "on" PPI can induce interleukin-8 (IL-8) production by bronchial epithelial cells in culture. We hypothesized that such effect would be more pronounced in CF patients known to have high inflammatory susceptibility. We aimed to evaluate the effect of GJ on IL-8 production by primary bronchial epithelial cells (PBEC), derived from a CF patient and a healthy subject.. PBEC obtained from one donor (normal PBEC) and one receptor (CF-PBEC) for lung transplantation were stimulated with GJ from patients "off" and "on" PPI. IL-8 levels were measured in the supernatant.. GJ from patients "on" PPI provoked a significant higher IL-8 production compared to GJ from patients "off" PPI, both in normal PBEC [462 (200-1468) vs. 11 (4-28) pg/ml, p=0.0001] as in CF-PBEC [1468 (841-2449) vs. 85 (26-131) pg/ml, p<0.0001]. Exposure of the cells to GJ "off" PPI and "on" PPI provoked significantly higher IL-8 production in the CF-PBEC compared to the normal PBEC ["off" PPI 85 (26-131) vs. 11 (4-28) pg/ml, p=0.01; "on" PPI 1468 (841-2449) vs. 462 (200-1468) pg/ml, p=0.01]. Filtration (0.20 μm) of the GJ "on" PPI, to eliminate large particles and bacterial sub-products, resulted in a significant decrease of IL-8 production.. Patients with CF, treated with PPIs, have GJ with high pH and high endotoxin levels. These patients often have GER and bronchial aspiration. The aspirated material (GJ "on" PPI) has a significantly enhanced inflammatory effect on CF bronchial epithelial cells in culture. As chronic PPI treatment in CF may result in a paradoxically increased inflammatory effect in the airways, alternative anti-reflux therapies should be considered in CF.

Topics: Bronchi; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Gastric Juice; Humans; Interleukin-8; Proton Pump Inhibitors

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

Topics: Adenosine Triphosphate; Calcium Signaling; Cell Size; Cells, Cultured; Chelating Agents; Connexins; Cystic Fibrosis; Epithelial Cells; Humans; Inflammation Mediators; Interleukin-8; Mucins; Mucociliary Clearance; Nerve Tissue Proteins; Nucleotide Transport Proteins; Osmotic Pressure; Pneumonia; Primary Cell Culture; Respiratory Mucosa; Secretory Vesicles; Time Factors

Treatment of acute and chronic pulmonary infections caused by opportunistic pathogen Pseudomonas aeruginosa is limited by the increasing frequency of multidrug bacterial resistance. Here, we describe a novel adjunctive therapy in which administration of a mix of simple sugars-mannose, fucose, and galactose-inhibits bacterial attachment, limits lung damage, and potentiates conventional antibiotic therapy. The sugar mixture inhibits adhesion of nonmucoid and mucoid P. aeruginosa strains to bronchial epithelial cells in vitro. In a murine model of acute pneumonia, treatment with the sugar mixture alone diminishes lung damage, bacterial dissemination to the subpleural alveoli, and neutrophil- and IL-8-driven inflammatory responses. Remarkably, the sugars act synergistically with anti-Pseudomonas antibiotics, including β-lactams and quinolones, to further reduce bacterial lung colonization and damage. To probe the mechanism, we examined the effects of sugars in the presence or absence of antibiotics during growth in liquid culture and in an ex vivo infection model utilizing freshly dissected mouse tracheas and lungs. We demonstrate that the sugar mixture induces rapid but reversible formation of bacterial clusters that exhibited enhanced susceptibility to antibiotics compared with individual bacteria. Our findings reveal that sugar inhalation, an inexpensive and safe therapeutic, could be used in combination with conventional antibiotic therapy to more effectively treat P. aeruginosa lung infections.

Topics: Animals; Anti-Bacterial Agents; Bacterial Adhesion; Bronchi; Carbohydrates; Cells, Cultured; Cystic Fibrosis; Fucose; Galactose; Humans; Interleukin-8; Lung Injury; Male; Mannose; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Pneumonia, Bacterial; Polysaccharides; Pseudomonas aeruginosa; Pseudomonas Infections; Trachea

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.

Topics: Blotting, Western; Bronchi; Cell Line; Chromatography, High Pressure Liquid; Cystic Fibrosis; Down-Regulation; Electrophoretic Mobility Shift Assay; gamma-Glutamyltransferase; Humans; In Vitro Techniques; Interleukin-8; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; S-Nitrosoglutathione

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Morbidity is mainly due to lung disease, which is characterized by chronic neutrophilic inflammation. Deregulation of inflammatory pathways is observed in the airways of CF patients, as evidenced by exaggerated NF-κB activity, causing an increase in the local release of pro-inflammatory cytokines such as IL-8. COMMD1, a pleiotropic protein, was recently shown to interact with CFTR and to promote CFTR cell surface expression. The effect of COMMD1 on the NF-κB pathway was assessed in CF and non-CF bronchial epithelial cells by knockdown and overexpression experiments. Results showed that (i) COMMD1 knockdown induced NF-κB-dependent transcription, (ii) COMMD1 overexpression inhibited NF-κB activity and was associated with a decrease in IL-8 transcript level and protein secretion. These data demonstrate the anti-inflammatory properties of COMMD1 in bronchial epithelial cells and open new therapeutic avenues in CF.

Topics: Adaptor Proteins, Signal Transducing; Bronchi; Cell Line; Cystic Fibrosis; Down-Regulation; Epithelial Cells; Humans; Inflammation; Interleukin-8; Models, Biological; NF-kappa B; Promoter Regions, Genetic; Transcription Factor AP-1; Transcription, Genetic

High-producer TGFβ1 genotypes are associated with severe lung disease in cystic fibrosis (CF), but studies combining IL-8, TNFα-, and TGFβ1(+genotype) levels and their impact on CF lung disease are scarce.. Assessing the relationship between TGF β 1, IL-8, and TNF- α and lung disease in CF in an exacerbation-free interval.. Twenty four patients delta F508 homozygous (median age 20.5 y, Shwachman score 75, FEV1(%) 83) and 8 controls (median age 27.5 y) were examined. TGF β 1 was assessed in serum and induced sputum (IS) by ELISA, for IL-8 and TNF- α by chemiluminescence in IS and whole blood. Genotyping was performed for TGF β 1 C-509T and T+869C utilizing RFLP.. TGF β 1 in IS (CF/controls median 76.5/59.1 pg/mL, P < 0.074) was higher in CF. There was a negative correlation between TGF β 1 in serum and lung function (LF) (FEV1 (r = -0.488, P = 0.025), MEF 25 (r = -0.425, P = 0.055), and VC (r = -0.572, P = 0.007)). Genotypes had no impact on TGF β 1 in IS, serum, and LF. In IS TGF β 1 correlated with IL-8 (r = 0.593, P < 0.007) and TNF- α (r = 0.536, P < 0.018) in patients colonized by bacteria with flagellin.. TGF β 1 in serum not in IS correlates with LF. In patients colonized by bacteria with flagellin, TGF β 1 correlates with IL-8 and TNF- α in IS.

Topics: Adolescent; Adult; Bacteria; Cell Differentiation; Child; Cystic Fibrosis; Disease-Free Survival; Female; Flagellin; Gene Expression Regulation; Genotype; Homozygote; Humans; Interleukin-8; Male; Spirometry; Sputum; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Young Adult

A20 is a lipopolysaccharide (LPS)-inducible, cytoplasmic zinc finger protein, which inhibits Toll-like receptor-activated nuclear factor (NF)-κB signalling by deubiquitinating tumour necrosis factor receptor-associated factor (TRAF)-6. The action of A20 is facilitated by complex formation with ring finger protein (RNF)-11, Itch and TAX-1 binding protein-1 (TAX1BP1). This study investigated whether the expression of A20 is altered in the chronically inflamed cystic fibrosis (CF) airway epithelium. Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-κB subunits were analysed. Formation of the A20 ubiquitin editing complex was also investigated. In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-. The expression of A20 is significantly altered in CF, and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-κB-driven inflammation.

Topics: Bronchi; Cell Line; Cystic Fibrosis; DNA-Binding Proteins; Epithelial Cells; Epithelium; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; NF-kappa B; Nuclear Proteins; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Signal Transduction; Tumor Necrosis Factor alpha-Induced Protein 3

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.

Topics: Case-Control Studies; Chemokine CXCL2; Cohort Studies; Cystic Fibrosis; Humans; Immediate-Early Proteins; Immunity, Innate; Interleukin-8; Lung; Neutrophils; Polymorphism, Single Nucleotide; Reactive Oxygen Species

Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity.. (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.

Topics: Anti-Inflammatory Agents; Antioxidants; Cell Line; Cystic Fibrosis; Epithelial Cells; Humans; Inflammation; Interleukin-8; Milk Proteins; Peptides; Pressure; Protein Binding; Protein Hydrolysates; Respiratory Mucosa; Toll-Like Receptor 4; Whey Proteins

Cystic fibrosis (CF) is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause a massively proinflammatory phenotype in the CF airway. The chemical basis of the inflammation is hyperproduction of interleukin-8 (IL-8) by CF airway epithelial cells, based on both an intrinsic mutation-dependent mechanism and by infection. In infection-free, cultured CF lung epithelial cells, high levels of the microRNA (miR), miR-155, is responsible for hyperexpression of IL-8. However, whether infection-induced IL-8 expression in CF cells is also mediated by miR-155 is not known. We have hypothesized that miR-155 might be a general mediator of enhanced IL-8 expression in CF cells, either in response to other cytokine/chemokine mediators of inflammation, or after exposure to infectious agents. Here we find that a reduction in miR-155 accompanies suppression of IL-8 by either the anti-inflammatory cytokine IL-10 or by inhibition of ambient IL-1β with a neutralizing antibody. However, attempts to elevate IL-8 levels with either intact bacteria [viz. a mucoid strain of Pseudomonas aeruginosa (PA)], or lipopolysaccharide were unable to elevate miR-155 above its intrinsically high level in the absence of these agents. Instead, in response to PA infection, the CF cells modestly suppress the expression of miR-155, and express a novel set of miRs, including miR-215. We find that ex vivo CF lung epithelial cells also express high levels of both miR-155 and miR-215. The predicted module of infection-induced mRNA targets focuses on activation of the NFκB-signaling pathway, and on the proapoptotic p53-signaling pathway. We interpret these data to suggest that that CF lung epithelial cells respond to PA or bacterial cell products with a novel miR program that may carry with it serious challenges to survival.

Topics: Cell Line; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Interleukin-10; Interleukin-1beta; Interleukin-8; Lung; MicroRNAs; Pseudomonas aeruginosa; Pseudomonas Infections

Cystic fibrosis is a hereditary disease caused by a mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene that encodes a chloride (Cl(-)) channel. Cystic fibrosis pulmonary pathophysiology is characterised by chronic inflammation and bacterial infections. Azithromycin, a macrolide antibiotic, has shown promising anti-inflammatory properties in some inflammatory pulmonary diseases. Moreover, all clinical studies have presented an improvement of the respiratory condition of cystic fibrosis patients, but the molecular and cellular mechanisms remain unknown. The aim of this study was to investigate, in bronchial epithelial cells, the effects of azithromycin on inflammatory pathways involved in cystic fibrosis. We have analysed the effects of azithromycin on cystic fibrosis and non-cystic fibrosis bronchial epithelial cell lines but also in non-immortalized non-cystic fibrosis human glandular cells. To create an inflammatory context, cells were treated with Tumor Necrosis Factor (TNF)-α or Interleukin (IL)1-β. Activation of the NF-κB pathway was investigated by luciferase assay, western blotting, and by Förster Resonance Energy Transfer imaging, allowing the detection of the interaction between the transcription factor and its inhibitor in live cells. In all conditions tested, azithromycin did not have an anti-inflammatory effect on the cystic fibrosis human bronchial epithelial cells and on CFTR-inhibited primary human bronchial glandular cells. More, our data showed no effect of azithromycin on IL-1β- or TNF-α-induced IL-8 secretion and NF-κB pathway activation. Taken together, these data show that azithromycin is unable to decrease in vitro inflammation in cystic fibrosis cells from airways.

Topics: Azithromycin; Bronchi; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Inflammation; Interleukin-1beta; Interleukin-8; NF-kappa B; Signal Transduction; Tumor Necrosis Factor-alpha

Topics: Cystic Fibrosis; Humans; Interleukin-8; Saline Solution, Hypertonic; Sputum

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However, CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-κB and MAPK pathways through direct interaction with AP-1 and NF-κB in the nucleus. Conversely, MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.

Topics: Anisomycin; Anti-Inflammatory Agents; Bronchioles; Cell Line; Cystic Fibrosis; Dexamethasone; Enzyme Induction; Epithelial Cells; Glucocorticoids; Humans; Inflammation; Interleukin-8; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Transport; Receptors, Glucocorticoid; Signal Transduction; Transcription Factor AP-1; Transcriptional Activation

Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response.

Topics: Bacterial Proteins; Bacteriological Techniques; Cell Line; Culture Media, Conditioned; Cystic Fibrosis; Gene Expression; Humans; Inflammation Mediators; Interleukin-8; Metalloproteases; Neutrophils; Proteomics; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Interleukin-8A; RNA, Messenger; Systems Biology; Virulence

Neutrophil elastase (NE)-mediated inflammation contributes to lung damage in cystic fibrosis (CF). We investigated if DX-890, a small-protein NE inhibitor, could reduce neutrophil trans-epithelial migration and reduce activity released from neutrophils and NE-induced cytokine expression in airway epithelial cells.. Activated blood neutrophils (CF and healthy) treated ±DX-890 were assayed for NE activity. Transmigration of calcein-labeled neutrophils was studied using a 16HBE14o(-) epithelial monolayer. IL-8 release from primary nasal epithelial monolayers (CF and healthy) was measured after treatment ±DX-890 and NE or CF sputum.. DX-890 reduced NE activity from neutrophils (CF and healthy) and reduced neutrophil transmigration. DX-890 pre-treatment reduced IL-8 release from epithelial cells of healthy or CF subjects after stimulation with NE and CF sputum sol. All improvements with DX-890 were statistically significant (p<0.05).. DX-890 reduces NE-mediated transmigration and inflammation. NE inhibition could be useful in managing neutrophilic airway inflammation in CF.

Topics: Anti-Inflammatory Agents; Cell Movement; Cells, Cultured; Cystic Fibrosis; Fluoresceins; Humans; In Vitro Techniques; Interleukin-8; Nasal Mucosa; Neutrophils; Peptides; Proteinase Inhibitory Proteins, Secretory

Cystic fibrosis (CF) lung disease is characterized by structural changes and remodeling in airway architecture and lung parenchyma. Neutrophilic inflammation and infection lead to injury and breakdown of airway matrix constituents, including elastin. The non-invasive measurement of urinary desmosine (UDes), a breakdown product of elastin, may be reflective of ongoing lung injury and may serve as a biomarker of active short-term damage during pulmonary exacerbation. Our objectives were to measure desmosine in the urine of CF patients hospitalized for treatment of a pulmonary exacerbation and to explore the correlation between desmosine concentration and other markers of clinical improvement, including lung function and inflammatory mediators.. Urine and blood samples plus lung function measurements were collected at up to three points during hospitalization for treatment of a CF pulmonary exacerbation. We used a repeated measures model, adjusted for age and time between measurements, to compare log transformed urine desmosine concentrations across multiple time points and to correlate those concentrations with related clinical variables. Change in UDes concentration was investigated using a statistical model that incorporated normalization factors to account for variations in urinary concentration.. Desmosine was measured by radioimmunoassay (RIA) in 155 spot urine samples from 53 CF patients hospitalized for 63 pulmonary exacerbations (range of results: 0-235 pmol Des/ml). Specific gravity (SG) adjusted UDes concentration decreased significantly during admission for CF pulmonary exacerbation, P < 0.01 (average length of stay = 11 days). No correlation was observed between UDes concentration and lung function or inflammatory markers.. UDes decreased significantly following treatment for an acute pulmonary exacerbation and may be a useful biomarker of short-term injury to the CF lung. Further investigation is needed to evaluate the utility of UDes concentration in the long-term progression of CF lung disease.

Topics: Airway Remodeling; Biomarkers; C-Reactive Protein; Cohort Studies; Cystic Fibrosis; Desmosine; Disease Progression; Elastin; Female; Humans; Interleukin-8; Lung Injury; Male; Pneumonia; Prospective Studies; Respiratory Function Tests

Recently, Achromobacter xylosoxidans has been related to chronic lung diseases in patients suffering from cystic fibrosis (CF), but its involvement has not been elucidated. Some virulence properties of A. xylosoxidans isolated from Brazilian patients with CF were revealed in this work.. This study examined the production of a cytotoxic factor of A. xylosoxidans capable of stimulating the secretion of inflammatory cytokines (IL-6 and IL-8) from lung mucoepidermoid carcinoma cells (NCI-H292). The cytokines were measured using enzyme-linked immunosorbent (ELISA) assays. To investigate whether the cytotoxic factors may be endotoxins, they were treated with polymyxin B.. The culture supernatants of all A. xylosoxidans produced a heat stable, active cytotoxin in NCI-H292 cells capable of leading to intracellular vacuoles and subsequent cell contact loss, chromatin condensation, a picnotic nucleus and cell death. There was a higher concentration of proinflammatory cytokines in the NCI-H292 cells after 24 h of incubation, with the fraction greater than 50 kDa from the culture supernatant. The cytotoxin activity remained even after treatment with polymyxin B, which suggested that the release of IL-6 and IL-8 was not stimulated by lipopolysaccharide (LPS).. The cytotoxic factor produced by A. xylosoxidans may represent an important virulence factor, which when associated with CF chronic lung inflammation, may cause tissue damage and decline of lung function.

Topics: Achromobacter denitrificans; Brazil; Carcinoma, Mucoepidermoid; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Cystic Fibrosis; Endotoxins; Gram-Negative Bacterial Infections; Humans; In Vitro Techniques; Interleukin-6; Interleukin-8; Pneumonia; Sputum; Virulence

Cystic fibrosis (CF) is the most common lethal inherited disorder and is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The CF lung expresses a profound proinflammatory phenotype that appears to be related to a constitutive hypersecretion of interleukin (IL)-8 from airway epithelial cells in response to microbial infection. Since overproduction of IL-8 in CF contributes to massive bronchial infiltrates of neutrophils, identification of the pathways underlying IL-8 induction could provide novel drug targets for treatment of neutrophil-dominated inflammatory diseases such as CF. Here, we show that IL-17A synergistically increases IL-8 production induced by a toll-like receptor (TLR) 2 agonist, peptidoglycan (PGN), or TLR4 agonist, lipopolysaccharide (LPS), in a human CF bronchial epithelial cell line (CFBE41o-). A strong synergism was also observed in primary human CF bronchial epithelial cells, but not in human non-CF cell lines and primary cells. Notably, despite the induction of nuclear factor-κB and MAP kinases during TLR2 or TLR4 activation in CFBE41o-, IL-17A-dependent synergism appears to be the result of enhanced PGN- or LPS-induced phosphorylation of p38. Taken together, these studies provide evidence that IL-17A is a critical factor in increasing IL-8 expression in bacteria-infected CF airways via a pathway that regulates p38 phosphorylation.

Topics: Cell Line; Cystic Fibrosis; Dose-Response Relationship, Immunologic; Epithelial Cells; Gene Expression Regulation; Humans; Interleukin-17; Interleukin-8; Respiratory Mucosa; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

Topics: Adolescent; Cell Membrane; Child; Child, Preschool; Chloride Channels; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Female; Glutamic Acid; Humans; Interleukin-10; Interleukin-8; Ligands; Lymphocytes; Male; Mutation; Phosphoproteins; Receptors, Metabotropic Glutamate; Sodium-Hydrogen Exchangers; T-Lymphocytes

Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose), a gallotannin identified in several plants, including Phyllanthus urinaria, has been shown to exhibit versatile medicinal activities. As far as possible anti-inflammatory effects of corilagin, only few reports are available, and the potential use of corilagin as possible therapeutic molecule for cystic fibrosis has not been evaluated. In the present paper we report experiments aimed at determining the activity of corilagin on nuclear factor kappaB (NF-kappaB) binding to DNA target and on the expression of the major pro-inflammatory gene involved in cystic fibrosis, interleukin-8 (IL-8). Both IL-8 mRNA content and IL-8 protein secretion were analyzed in cystic fibrosis bronchial IB3-1 cells stimulated by tumor necrosis factor-alpha (TNF-alpha), one of the most potent pro-inflammatory agents. The data obtained demonstrate that corilagin binds to NF-kappaB, inhibits NF-kappaB/DNA interactions and affects IL-8 gene expression in TNF-alpha treated IB3-1 cells. In addition, corilagin inhibits TNF-alpha induced secretion of MCP-1 and RANTES, exhibiting low or no effect on the release of G-CSF, IL-6 and VEGF. Therefore, corilagin might be of interest for experimental anti-inflammatory therapy of cystic fibrosis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Base Sequence; Cell Line; Chemokine CCL2; Chemokine CCL5; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Glucosides; Humans; Hydrolyzable Tannins; Interleukin-8; Mutation; NF-kappa B; RNA, Messenger; Tumor Necrosis Factor-alpha

It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis.. Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry.. ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment.. Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Antifungal Agents; Apoptosis; Biomarkers; Child; Cystic Fibrosis; Drug Monitoring; Female; Humans; Interleukin-8; Male; Neutrophils; Pilot Projects; Pneumonia, Bacterial; Pulmonary Aspergillosis; Reactive Oxygen Species; Young Adult

Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1β) is a key inflammatory mediator. Secretion of biologically active IL-1β involves inflammasome-mediated processing. Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.. Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators. This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.. Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function.

Topics: Analysis of Variance; Caspases; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Humans; Immunoblotting; Inflammasomes; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections

A hallmark of cystic fibrosis is the massive recruitment of neutrophils into the lung compartment in response to chronic Pseudomonas aeruginosa infection. The overexuberant neutrophilic response results in release of proteases (e.g. neutrophil elastase and matrix metalloproteinase-9) leading to matrix breakdown, airway remodeling, and progressive loss of lung function. Doxycycline is used clinically for the management of periodontitis due to its potent direct inhibition of matrix metalloproteinases; however, little is known regarding its potential anti-inflammatory properties and clinical utility in the context of cystic fibrosis airway disease. CF (IB3-1) and corrected (S9) bronchial epithelial cell lines were used to determine the cytotoxicity and anti-inflammatory effects of doxycycline in-vitro. Exposure to doxycycline, at low concentrations, resulted in minimal cell death and dose dependent reductions in release of CXCL-8 and MMP-9 protein. To confirm these findings, mechanistic analysis revealed ERK 1/2, p38, and JNK, but not NF-κB p65 dependent cell signaling inhibition with doxycycline treatment. These findings indicate that doxycycline exhibits anti-inflammatory activity in CF lung epithelial cells at concentrations below the cytotoxic potential. These data are encouraging and indicate in-vivo studies are warranted.

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents; Bronchi; Cells, Cultured; Cystic Fibrosis; Doxycycline; Epithelial Cells; Humans; Interleukin-8; Matrix Metalloproteinase 9; Signal Transduction

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa (P. aeruginosa) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Epithelial Cells; Humans; Interleukin-8; Lung; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Topics: Adult; Amiloride; Cells, Cultured; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Sodium Channel Blockers; Female; Humans; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Male; Nasal Lavage Fluid; Nasal Mucosa; Tumor Necrosis Factor-alpha; Uridine Triphosphate; Young Adult

The airway surface liquid (ASL) of Cystic Fibrosis (CF) patients contains a lower concentration of reduced glutathione (GSH) with respect to healthy people. It is not known whether this defect may favor lung colonization by opportunistic pathogens.. We have analyzed the effects of extracellular GSH on the ability of Burkholderia cenocepacia to penetrate and multiply in epithelial respiratory cells. Extracellular GSH proved to be able to drastically reduce the pathogen ability to adhere and invade airway epithelial cells. This effect is correlated to a GSH-dependent increase in the number of free thiols on the surface of epithelial cells, suggestive of a change in the oxidoreductive status of membrane proteins involved in B. cenocepacia recognition. Moreover, treatments with GSH led to a consistent reduction of the expression of IL-8, TNF-α and IL-1β in response to B. cenocepacia infection.. Extracellular GSH modulates the interaction between B. cenocepacia and epithelial respiratory cells and inhibits the bacterial invasion into these cells. This suggests that therapies aimed at restoring normal levels of GSH in the ASL might be beneficial to control CF lung infections.

Topics: Bronchi; Burkholderia cenocepacia; Burkholderia Infections; Cell Line; Cystic Fibrosis; Epithelial Cells; Glutathione; Host-Pathogen Interactions; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Oxidation-Reduction; Primary Cell Culture; Respiratory Mucosa; Trachea; Tumor Necrosis Factor-alpha

Topics: Contraceptives, Oral, Hormonal; Cystic Fibrosis; Estradiol; Estrogen Receptor beta; Female; Humans; Interleukin-8; Menstrual Cycle; Pseudomonas aeruginosa; Respiratory Mucosa; Secretory Leukocyte Peptidase Inhibitor; Up-Regulation

Burkholderia cepacia complex (Bcc) is a group of Gram-negative pulmonary pathogens associated with life-threatening infections in patients with cystic fibrosis (CF). The airway epithelium plays a crucial role in the initiation and modulation of inflammatory responses to these pathogens. Interleukin (IL)-8 released from epithelial cells is a potent chemoattractant for neutrophils. The aims of this study were to compare the IL-8 response to Bcc infection in different epithelial cell types and to investigate the impact of IL-8 on Bcc growth and intracellular survival. To compare epithelial cell IL-8 responses, 4 human epithelial cell lines were used in the study; A549 cells, an alveolar epithelial cell line, Calu-3 cells, a sub-bronchial epithelial cell line, 16HBE14o- cells, and CFBE41o- cells, which are CFTR-positive and CFTR-negative bronchial epithelial cell lines, respectively. Two B. multivorans and 2 B. cenocepacia strains all induced a significant IL-8 response by 12 h and further increased in all cell lines at 24 h. Furthermore, the levels of IL-8 from Calu-3 and A549 cells were approximately 3 times that of 16HBE14o- or CFBE41o- cells. In 2 of the cell lines examined (16HBE14o- and CFBE41o-), B. cenocepacia LMG 16656 (J2315), an epidemic strain, induced greater levels of IL-8 (P<0.01) compared to other Bcc strains tested. The CFTR-positive and -negative cell lines secreted similar levels of IL-8 indicating a CFTR-independent induction of IL-8. However, the CFTR-negative cells did secrete constitutive levels of IL-8 greater than that of CFTR-positive cells. An investigation of the effect of IL-8 on Bcc extracellular and intracellular growth found that at low concentrations (<10 ng/ml) of recombinant human (rh) IL-8, the growth of B. cenocepacia LMG 16656 and B. multivorans LMG 13010 was enhanced, whereas at higher concentrations (10 ng/ml), growth of both strains was significantly reduced. Growth of both non-CF Bcc strains remained unchanged in the presence of rhIL-8. In contrast to extracellular growth, higher concentrations (10ng/ml) of rhIL-8 enhance the intracellular growth and survival of both LMG 16656 and LMG 13010 in 16HBE14o- and CFBE41o- cell lines. Although LMG 13010 uptake by epithelial cells was higher than LMG 16656 (P<0.01), the intracellular growth of LMG 16656 is greater than LMG 13010 (P<0.05). These studies demonstrated that the type of epithelial cells encountered by Bcc strains determines the extent of the IL-8 responses trigger

Topics: Burkholderia cepacia complex; Cell Line; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; Lung; Microbial Viability; Respiratory Mucosa

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

Topics: Cystic Fibrosis; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation; Humans; Interleukin-8; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 9; p38 Mitogen-Activated Protein Kinases; Phenotype; RNA, Messenger; Tristetraprolin

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p=0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm(-1) compared with 9 cells·mm(-1) basement membrane, p=0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

Topics: Cells, Cultured; Cystic Fibrosis; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-17; Interleukin-6; Interleukin-8; Lung; Lung Transplantation; Neutrophils; Pneumonia, Bacterial; Sputum

Gastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL) fluid has recently been suggested to be a reliable indicator of aspiration. The prevalence of pulmonary aspiration in a group of children with CF was assessed and its association with lung inflammation investigated.. This was a cross-sectional case-control study. BAL fluid was collected from individuals with CF (n=31) and healthy controls (n=7). Interleukin-8 (IL-8), pepsin, neutrophil numbers and neutrophil elastase activity levels were measured in all samples. Clinical, microbiological and lung function data were collected from medical notes.. The pepsin concentration in BAL fluid was higher in the CF group than in controls (mean (SD) 24.4 (27.4) ng/ml vs 4.3 (4.0) ng/ml, p=0.03). Those with CF who had raised pepsin concentrations had higher levels of IL-8 in the BAL fluid than those with a concentration comparable to controls (3.7 (2.7) ng/ml vs 1.4 (0.9) ng/ml, p=0.004). Within the CF group there was a moderate positive correlation between pepsin concentration and IL-8 in BAL fluid (r=0.48, p=0.04). There was no association between BAL fluid pepsin concentrations and age, sex, body mass index z score, forced expiratory volume in 1 s or Pseudomonas aeruginosa colonisation status.. Many children with CF have increased levels of pepsin in the BAL fluid compared with normal controls. Increased pepsin levels were associated with higher IL-8 concentrations in BAL fluid. These data suggest that aspiration of gastric contents occurs in a subset of patients with CF and is associated with more pronounced lung inflammation.

Topics: Adolescent; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Child; Child, Preschool; Cystic Fibrosis; Female; Humans; Infant; Interleukin-8; Male; Pepsin A; Respiratory Aspiration

Understanding the mechanisms underlying bacterial-driven inflammation and neutrophil recruitment is important to design better therapies for CF. CXCL8 is an important chemokine found elevated in the airways of CF patients that recruits neutrophil to sites of the inflammation.. Airway epithelial cells (AECs) expressing wild-type CFTR or CFTR∆F508 were challenged with Pseudomonas aeruginosa diffusible material (PsaDM) and the synthesis of CXCL8 was measured by quantitative real-time PCR and ELISA in absence or presence of MAPK inhibitors, TLR antagonists, glutathione and a NADPH oxidase inhibitor.. CFTR∆F508 AECs secrete more CXCL8 in response to PsaDM than their wild type counterpart, which can be reversed by addition of extracellular glutathione or incubating AECs at 27°C to favour folding and expression of CFTR at the cell membrane. Moreover, in CFTR∆F508 AECs, TLR2, TLR4 and TLR5 act redundantly to drive CXCL8 synthesis via the activation of NADPH oxidase.. These results demonstrate that NADPH oxidase is necessary for CXCL8 synthesis in response to TLRs activation by P. aeruginosa.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; Interleukin-8; MAP Kinase Signaling System; NADPH Oxidases; Pseudomonas aeruginosa; Pseudomonas Infections; Reactive Oxygen Species; Respiratory Mucosa; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptors

A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, micro-bleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from ΔF508 CFBE41o(-) bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, α(1)-antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o(-) cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.

Topics: Bronchoalveolar Lavage Fluid; Cell Line; Cystic Fibrosis; Epithelial Cells; ErbB Receptors; Heme; Hemoglobins; Humans; Interleukin-10; Interleukin-8; Leukocyte Elastase; Metalloendopeptidases; Myeloblastin; Peptide Hydrolases; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Serine Endopeptidases; Signal Transduction; Toll-Like Receptors

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ΔF508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Inositol Polyphosphate 5-Phosphatases; Interleukin-8; Lung; MicroRNAs; Phosphatidylinositol 3-Kinases; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoric Monoester Hydrolases; Respiratory Mucosa; RNA Stability; RNA, Messenger; Signal Transduction

Intermittent viral exacerbations in patients with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with the majority of viral-related exacerbations in CF and require prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known.. Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). The need for dual oxidase 2, a component of NADPH oxidase, in RV-induced generation of H2O2 in CF cells was assessed using gene-specific siRNA.. Superinfection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2 in CF cells was sufficient for dispersal of planktonic bacteria from the biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and the interleukin-8 response in MPA- and RV-infected cells.. This study shows that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations.

Topics: Biofilms; Cell Differentiation; Cells, Cultured; Chemokines; Cystic Fibrosis; Epithelial Cells; Humans; Hydrogen Peroxide; Interleukin-8; Microscopy, Electron, Scanning; Picornaviridae Infections; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Rhinovirus; Superinfection; Viral Load

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Neutrophil-dominated inflammation and chronic bacterial infection are still considered the primary cause of bronchioectasis, respiratory failure and consequent death in CF patients. Activation of nuclear factor (NF)-κB is responsible for overproduction of cytokines, such as interleukin (IL)-6 and IL-8, in airways of CF patients. Thus, decoy oligodeoxynucleotides against NF-κB (dec-ODN) may limit lung inflammation in CF. In the present study, we studied the effects of dec-ODN delivered through biodegradable and respirable poly(D,L-lactide-co-glycolide) large porous particles (LPP) on IL-6 and IL-8 mRNA expression as well as NF-κB/DNA binding activity in cystic fibrosis cells stimulated with lipopolysaccharide (LPS) from Pseudomonas aeruginosa.. dec-ODN LPP were prepared by a modified double emulsion technique and characterized in terms of size, morphology, tapped density and dec-ODN loading. Human epithelial bronchial IB3-1 (CFTR-mutated) as well as S9 (CFTR-corrected) were stimulated with LPS from P. aeruginosa for 24 and 72 h in the absence or presence of naked dec-ODN or dec-ODN LPP.. Stimulation of cells with LPS from P. aeruginosa caused an increase of IL-6 and IL-8 mRNA levels, which were significantly inhibited by dec-ODN LPP at 24 and 72 h, whereas naked dec-ODN inhibited those only at 24 h. Similar effects were exhibited by dec-ODN LPP or naked dec-ODN on NF-κB/DNA binding activity.. Our observations indicate that respirable biodegradable dec-ODN LPP may represent a promising strategy for inhibiting NF-κB transcriptional activity and related gene expression and, thus, reduce lung chronic inflammation in CF patients.

Topics: Analysis of Variance; Bronchi; Cells, Cultured; Cystic Fibrosis; DNA Primers; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; NF-kappa B; Oligodeoxyribonucleotides; Pseudomonas aeruginosa; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors

Inflammation within the cystic fibrosis (CF) lung is mediated by inflammatory chemokines, such as IL-8. IL-8 is protected from proteolytic degradation in the airways by binding to glycosaminoglycans, while remaining active. Evidence that increased hypertonicity of airway secretions induced by hypertonic saline treatment alters levels of IL-8 is lacking.. To investigate the antiinflammatory effect of hypertonic saline (HTS) treatment within the CF lung by focusing on IL-8.. Degradation of IL-8 in CF lung secretions after treatment with glycosaminoglycan lyases and HTS was analyzed by Western blot analysis and ELISA. The ex vivo chemotactic activity of purified neutrophils in response to CF airway secretions was evaluated post nebulization of HTS (7% saline).. In vivo CF bronchoalveolar lavage fluid (BALF) IL-8 levels were significantly higher than the control group (P < 0.05). Digesting glycosaminoglycans in CF BALF displaced IL-8 from glycosaminoglycan matrices, rendering the chemokine susceptible to proteolytic cleavage. High sodium concentrations also liberate IL-8 in CF BALF in vitro, and in vivo in CF sputum from patients receiving aerosolized HTS, resulting in degradation of IL-8 and decreased neutrophil chemotactic efficiency.. Glycosaminoglycans possess the ability to influence the chemokine profile of the CF lung by binding and stabilizing IL-8, which promotes neutrophil chemotaxis and activation. Nebulized hypertonic saline treatment disrupts the interaction between glycosaminoglycans and IL-8, rendering IL-8 susceptible to proteolytic degradation with subsequent decrease in neutrophil chemotaxis, thereby facilitating resolution of inflammation.

Topics: Administration, Inhalation; Adult; Blotting, Western; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Humans; Inflammation; Interleukin-8; Middle Aged; Nebulizers and Vaporizers; Saline Solution, Hypertonic; Sputum; Young Adult

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel in the plasma membrane of epithelia whose mutation is the cause of the genetic disease cystic fibrosis (CF). The most frequent CFTR mutation is deletion of Phe(508) and this mutant protein (delF508CFTR) does not readily translocate to the plasma membrane and is rapidly degraded within the cell. We hypothesized that treating epithelial cells with resveratrol, a natural polyphenolic, phyto-ooestrogenic compound from grapes, could modulate both the expression and localization of CFTR.. Cells endogenously expressing CFTR (MDCK1 and CAPAN1 cells) or delF508CFTR (CFPAC1 and airway epithelial cells, deriving from human bronchial biopsies) were treated with resveratrol for 2 or 18 h. The effect of this treatment on CFTR and delF508CFTR expression and localization was evaluated using RT-PCR, Western blot and immunocytochemistry. Halide efflux was measured with a fluorescent dye and with halide-sensitive electrodes. Production of interleukin-8 by these cells was assayed by ELISA.. Resveratrol treatment increased CFTR expression or maturation in immunoblotting experiments in MDCK1 cells or in CFPAC1 cells. Indirect immunofluorescence experiments showed a shift of delF508CFTR localization towards the (peri)-membrane area in CFPAC1 cells and in human airway epithelial cells. A cAMP-dependent increase in membrane permeability to halide was detected in resveratrol-treated CFPAC1 cells, and was inhibited by a selective inhibitor of CFTR.. These results show that resveratrol modulated CFTR expression and localization and could rescue cAMP-dependent chloride transport in delF508CFTR cells.

Topics: Animals; Anion Transport Proteins; Biological Transport; Cell Line; Cell Line, Tumor; Cell Membrane; Chloride Channels; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dogs; Epithelial Cells; Humans; Interleukin-8; Mutation; Resveratrol; Stilbenes

Respiratory insufficiency is the major cause of morbidity and mortality in patients affected by cystic fibrosis (CF). An excessive neutrophilic inflammation, mainly orchestrated by the release of IL-8 from bronchial epithelial cells and amplified by chronic bacterial infection with Pseudomonas aeruginosa, leads to progressive tissue destruction. The anti-inflammatory drugs presently used in CF patients have several limitations, indicating the need for identifying novel molecular targets. To address this issue, we preliminarily studied the association of 721 single nucleotide polymorphisms from 135 genes potentially involved in signal transduction implicated in neutrophil recruitment in a cohort of F508del homozygous CF patients with either severe or mild progression of lung disease. The top ranking association was found for a nonsynonymous polymorphism of the phospholipase C-β3 (PLCB3) gene. Studies in bronchial epithelial cells exposed to P. aeruginosa revealed that PLCB3 is implicated in extracellular nucleotide-dependent intracellular calcium signaling, leading to activation of the protein kinase Cα and Cβ and of the nuclear transcription factor NF-κB p65. The proinflammatory pathway regulated by PLCB3 acts by potentiating the Toll-like Receptors' signaling cascade and represents an interesting molecular target to attenuate the excessive recruitment of neutrophils without completely abolishing the inflammatory response.

Topics: Adenosine Triphosphate; Calcium; Cell Line, Transformed; Cystic Fibrosis; Enzyme Activation; Epithelial Cells; Gene Expression; Gene Frequency; Genotype; Green Fluorescent Proteins; Host-Pathogen Interactions; Humans; Interleukin-8; Isoenzymes; Lung Diseases; Microscopy, Fluorescence; Phospholipase C beta; Polymorphism, Single Nucleotide; Protein Kinase C; Protein Kinase C beta; Pseudomonas aeruginosa; RNA Interference; Toll-Like Receptors; Transcription Factor RelA

The clinical consequence of chronic Pseudomonas aeruginosa colonization in cystic fibrosis (CF) varies between individuals for unknown reasons. Auto-antibodies against bactericidal/permeability increasing protein (BPI-ANCA) are associated with poor prognosis in CF. We hypothesize that there is a correlation between the presence of BPI-ANCA, the properties of the colonizing bacteria and the clinical conditions of the host. We compared isolates of P. aeruginosa from BPI-ANCA positive CF patients who have deteriorating lung disease with BPI-ANCA negative CF patients who are in stable clinical conditions. Epithelial cells (A549) and isolated polymorphonuclear granulocytes (PMNs) were stimulated with the isolates and cell death was analyzed with flow cytometry. We found that the ANCA associated strains in most cases showed pyocyanin negative phenotypes. These strains also induced less inflammatory response than the non-ANCA associated strains as shown by apoptosis and necrosis of epithelial cells and neutrophils. Our results suggest that colonization with strains of P. aeruginosa that induce a weak inflammatory response is associated with unfavorable outcome in CF. We speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying PMNs in the airways, contributing to progression in CF lung disease.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antimicrobial Cationic Peptides; Blood Proteins; Cell Death; Cell Line, Tumor; Cystic Fibrosis; Disease Progression; Humans; Immunoglobulin A; Interleukin-8; Lung Neoplasms; Necrosis; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Respiratory Mucosa

Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1β) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (Citrus bergamia Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology.. The extracts were chemically characterized by 1H-NMR (nuclear magnetic resonance), GC-FID (gas chromatography-flame ionization detector), GC-MS (gas chromatography-mass spectrometry) and HPLC (high pressure liquid chromatography). Both bergamot extracts and main detected chemical constituents were assayed for their biological activity measuring (a) cytokines and chemokines in culture supernatants released from cystic fibrosis IB3-1 cells treated with TNF-α by Bio-Plex cytokine assay; (b) accumulation of IL-8 mRNA by real-time PCR.. The extracts obtained from bergamot (Citrus bergamia Risso) epicarps contain components displaying an inhibitory activity on IL-8. Particularly, the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed to heat-inactivated Pseudomonas aeruginosa.. These obtained results clearly indicate that bergapten and citropten are strong inhibitors of IL-8 expression and could be proposed for further studies to verify possible anti-inflammatory properties to reduce lung inflammation in CF patients.

Topics: Bronchi; Cell Line; Chromatography, High Pressure Liquid; Citrus; Cystic Fibrosis; Epithelial Cells; Fruit; Gene Expression Regulation; Humans; Interleukin-8; Magnetic Resonance Spectroscopy; Phytotherapy; Plant Extracts; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response. mtGSH depletion was confirmed in colonic epithelium of CFTR-null mice and in CFTR-mutated human epithelial cells. GSH uptake experiments performed on isolated mitochondria suggest that mtGSH depletion is not due to a defective GSH transport capacity by CF mitochondria, despite the decreased expression of two mtGSH carriers, oxoglutarate carrier and dicarboxylate carrier. CM-H(2)DCFDA [5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] fluorescence and aconitase activity showed an increase in reactive oxygen species levels in CFTR-defective cells and a pro-oxidative environment within CF mitochondria. The activities of respiratory chain complexes were further examined. Results showed a selective loss of Complex I (CI) function in CF models associated with an altered mitochondrial membrane potential (Δψ(m)). CI analysis showed normal expression but an overoxidation of its NADH-ubiquinone oxidoreductase Fe-S protein 1 subunit. GSH monoethyl ester (GSH-EE) significantly enhanced mtGSH levels in the IB3-1/C38 model and reversed CI inhibition, suggesting that mtGSH depletion is responsible for the loss of CI activity. Furthermore, GSH-EE attenuated Δψ(m) depolarization and restored normal IL-8 secretion by CFTR-defective cells. These studies provide evidence for a critical role of a mtGSH defect in mitochondrial dysfunction and abnormal IL-8 secretion in CF cells and reveal the therapeutic potential of mitochondria-targeted antioxidants in CF.

Topics: Animals; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dicarboxylic Acid Transporters; Electron Transport Complex I; Glutathione; Interleukin-8; Male; Membrane Potential, Mitochondrial; Membrane Transport Proteins; Mice; Mice, Inbred CFTR; Mice, Knockout; Mitochondria; Mutation; Radiation-Protective Agents; Recovery of Function

Adenosine (ADO) is an extracellular signaling molecule that is an important regulator of innate lung defense. On binding ADO, the A2B receptor (A2BR) stimulates cAMP production to activate the CFTR Cl(-) channel, increase ciliary beating, and initiate cytokine secretion. We tested the hypothesis that CFTR served as a positive regulator of the A2BRs. We found that A2BR and CFTR coimmunoprecipitated. They also underwent ADO-dependent Förster resonance energy transfer (FRET), which increased from 5% in the absence of agonist to 18% with 100 μM ADO (EC₅₀ 1.7 μM), suggesting that they dynamically associate in the plasma membrane. In contrast, despite colocalization, no FRET was observed between CFTR and GAP43. The interaction between A2BR and CFTR had some specificity: A2BR-stimulated but not forskolin-stimulated cAMP production was ~50% greater in the presence of CFTR, due to a CFTR-dependent increase in plasma membrane A2BR levels. These CFTR-dependent increases in A2BR levels and cAMP production resulted in significantly enhanced ciliary beating and increased cytokine secretion in normal compared to cystic fibrosis airway epithelia. Thus, we hypothesize that CFTR regulates A2BR levels in the plasma membrane to modulate cell signaling and to enhance selective components of the innate lung defense system.

Topics: Adenosine; Animals; Cell Communication; Cells, Cultured; Cilia; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorescence Resonance Energy Transfer; Humans; Interleukin-8; Receptor, Adenosine A2B; Respiratory Mucosa; Signal Transduction

Topics: Animals; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Mice; Nebulizers and Vaporizers; Saline Solution, Hypertonic; Sputum

ABSTRACT As part of the innate and adaptive immune system, airway epithelial cells secrete proinflammatory cytokines after activation of Toll-like receptors (TLRs) by pathogens. Nevertheless, cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. The authors have shown that in CF bronchial epithelial cells, a reduced surface expression of TLR-4 causes a diminished interleukin (IL)-8 and IL-6 response upon lipopolysaccharide (LPS) stimulation. However, there is no information regarding activation of the MyD88 (myeloid differentiation primary response gene 88)-independent TLR-4 signaling pathway by LPS, which results in the activation of adaptive immune responses by secretion of the T cell-recruiting chemokine interferon-γ-inducible protein (IP)-10. Therefore, the authors investigated the induction of IP-10 in CF bronchial epithelial cell line CFBE41o- and its CFTR-corrected isotype under well-differentiating conditions. TLR-4 surface expression was significantly reduced in CFBE41o- by a factor of 2, compared to the CFTR-corrected cells. In CFTR-corrected cells, stimulation with LPS increased IP-10 secretion. Incubating cells with siRNA directed against TLR-4 inhibited the LPS stimulated increase of IP-10 in CFTR-corrected cells. The reduced TLR-4 surface expression in CF cells causes the loss of induction of IP-10 by LPS. This could compromise adaptive immune responses in CF due to a reduced T-cell recruitment.

Topics: Cell Line; Chemokine CXCL10; Cystic Fibrosis; Endoplasmic Reticulum; Epithelial Cells; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Lipopolysaccharides; Myeloid Differentiation Factor 88; Respiratory Mucosa; Respiratory System; RNA, Small Interfering; Signal Transduction; T-Lymphocytes; Toll-Like Receptor 4

1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF.. CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3).. Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18/LL-37 mRNA and protein levels.. Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.

Topics: Calcitriol; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Receptors, Calcitriol; Respiratory Mucosa; Vitamins

Early in life, patients with cystic fibrosis (CF) are infected with microorganisms including bacteria and fungi, particularly Pseudomonas aeruginosa and Aspergillus fumigatus. Since recent research has identified the anti-inflammatory properties of statins (besides their lipid-lowering effects), we investigated the effect of fluvastatin on the production of the potent neutrophil chemoattractant chemokine, IL-8, in whole blood from CF patients, stimulated by Pseudomonas aeruginosa (LPS) and Aspergillus fumigatus (AFA) antigens.. Whole blood from adult patients with CF and from healthy volunteers was collected at the Rennes University Hospital (France). Blood was pretreated for 1 h with fluvastatin (0-300 µM) and incubated for 24 h with LPS (10 µg/mL) and/or AFA (diluted 1/200). IL-8 protein levels, quantified by ELISA, were increased in a concentration-dependent manner when cells were stimulated by LPS or AFA. Fluvastatin strongly decreased the levels of IL-8, in a concentration-dependent manner, in whole blood from CF patients. However, its inhibitory effect was decreased or absent in whole blood from healthy subjects. Furthermore, the inhibition induced by fluvastatin in CF whole blood was reversed in the presence of intermediates within the cholesterol biosynthesis pathway, mevalonate, farnesyl pyprophosphate or geranylgeranyl pyrophosphate that activate small GTPases by isoprenylation.. For the first time, the inhibitory effects of fluvastatin on CF systemic inflammation may reveal the important therapeutic potential of statins in pathological conditions associated with the over-production of pro-inflammatory cytokines and chemokines as observed during the manifestation of CF. The anti-inflammatory effect could be related to the modulation of the prenylation of signalling proteins.

Topics: Adolescent; Adult; Anti-Inflammatory Agents; Antigens, Fungal; Aspergillus fumigatus; Case-Control Studies; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fatty Acids, Monounsaturated; Female; Fluvastatin; Humans; Indoles; Inhibitory Concentration 50; Interleukin-8; Lipopolysaccharides; Male; Mevalonic Acid; Mutation; Pseudomonas aeruginosa; Terpenes; Young Adult

Nuclear Factor kappaB (NF-κB) plays a very important role in the control of gene expression and is deeply involved in several human pathologies. Accordingly, molecules targeting NF-κB dependent biological functions are considered of great interest. Virtual screening of furocoumarin libraries against NF-κB p50 allowed to rank compounds in respect to their expected ability to bind NF-κB and the identified compound might be considered for the development of analogs to be tested for biological activity on inhibition of NF-κB/DNA complex formation. The data reported in the present paper suggest that, following this approach, the best ranked compounds identified by virtual screening (a) strongly bind in silico to NF-κB and (b) efficiently inhibit the molecular interactions between (32)P-labeled NF-κB double stranded DNA and p50 or p50/p65 complex. These data allowed to develop a novel lead of great interest for inhibiting NF-κB dependent biological functions. This novel molecule (compound 2), bearing a methyl group in the 9 position of the psoralen nucleus, exhibits high efficiency in inhibiting NF-κB/DNA interactions. In addition, we found that compound 2 is a potent inhibitor of IL-8 gene expression in TNF-α treated IB3-1 cystic fibrosis cells. Taken together, our data indicate that compound 2 might find an important place in the set of molecules of interest for the development of pharmaceutical strategies against the inflammatory phenotype of cystic fibrosis.

Topics: Cell Line; Cystic Fibrosis; DNA; Drug Design; Furocoumarins; Gene Expression Regulation; Humans; Interleukin-8; Models, Molecular; NF-kappa B; Protein Binding; RNA, Messenger; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, causes retention of CFTR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca(2+) homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store-operated calcium entry (SOCE) through Orai1 is abnormal in CF. The significance of Orai1-mediated SOCE for increased interleukin-8 (IL-8) expression in CF was also investigated. CF and non-CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca(2+) imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orai1 function in CF vs. non-CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store-operated Ca(2+) entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ≈ 1.8- and ≈ 2.5-fold for total Ca(2+)(i) increase and Ca(2+) influx rate, respectively, and ≈ 2-fold increase in the I(CRAC) current) and is caused by increased exocytotic insertion (≈ 2-fold) of Orai1 channels into the plasma membrane, which is normalized by rescue of ΔF508-CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL-8 secretion (≈ 2-fold). CFTR normally down-regulates the Orai1/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells.

Topics: Base Sequence; Calcium Channels; Calcium Signaling; Cell Membrane; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Primers; Gene Knockdown Techniques; Humans; Interleukin-8; Membrane Potentials; Membrane Proteins; Mutant Proteins; Neoplasm Proteins; ORAI1 Protein; Respiratory Mucosa; RNA, Small Interfering; Signal Transduction; Stromal Interaction Molecule 1

Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells.. Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment.. Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

Topics: Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Antitoxins; Cathelicidins; Cell Line, Tumor; Cystic Fibrosis; DNA; Glycosaminoglycans; Humans; Interleukin-8; Lipopolysaccharides; Lung; Monocytes; Pseudomonas aeruginosa; Signal Transduction

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.

Topics: Blotting, Western; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Fluorescent Antibody Technique; Humans; Interleukin-8; Lung; NF-kappa B; Ozone; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases

The chemokine IL-8 is critically important in inflammatory processes in human tissues, and IL-8 interactions with sulfated glycosaminoglycans have been implicated in modification of inflammatory responses in bronchial epithelium. To determine the role of chondroitin-4-sulfate (C4S) in mediating effects of IL-8, we silenced the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B [ASB]) that removes the 4-sulfate group from C4S, in the IB3-1 and C38 bronchial epithelial cell lines and in normal primary bronchial epithelial cells. When ASB was silenced and IL-8 production stimulated by exposure to TNF-alpha, ASB activity declined by roughly 75%, cellular C4S content increased by over 7.5 microg/mg protein, cell-bound IL-8 increased by over 530 pg/mg protein, and secreted IL-8 declined by over 520 pg/mg protein in all cell lines (P < 0.001). When cell lysates were immunoprecipitated with C4S antibody after ASB silencing and TNF-alpha, the IL-8 content of the immunoprecipitate was approximately 500 pg/mg protein, indicating that most of the cell-bound IL-8 was associated with C4S. Cell fractionation demonstrated that the IL-8 content associated with the cell membranes was about twice that of the cytosolic fraction. Also, ASB appeared to localize in the cell membrane, as well as in lysosomes. Neutrophil attraction to the cell lysates increased after ASB silencing, consistent with increased attraction to the cell-bound IL-8. These findings provide evidence for the influential role of ASB and C4S in the regulation of IL-8 secretion, and suggest that changes in ASB activity and C4S content may have a significant impact on IL-8-mediated inflammatory responses.

Topics: Bronchi; Cell Line; Chondroitin Sulfates; Cystic Fibrosis; Epithelial Cells; Gene Silencing; Glycosaminoglycans; Humans; Immunohistochemistry; Inflammation; Interleukin-8; Lysosomes; Microscopy, Confocal; N-Acetylgalactosamine-4-Sulfatase; Subcellular Fractions; Tumor Necrosis Factor-alpha

Airway epithelial cells contribute to the inflammatory response of the lung, and their innate immune response is primarily mediated via Toll-like receptor (TLR) signaling. Cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. We investigated the TLR-4 expression and the inflammatory profile (IL-8 and IL-6 secretion) in CF bronchial epithelial cell line CFBE41o- and its CF transmembrane ion condcutance regulator (CFTR)-corrected counterpart grown under air-liquid interface conditions after stimulation with lipopolysaccharide (LPS) from gram-negative bacteria. In CFTR-corrected cells, IL-8 and IL-6 secretions were constitutively activated but significantly increased after LPS stimulation compared with CFBE41o-. Blocking TLR-4 by a specific antibody significantly inhibited IL-8 secretion only in CFTR-corrected cells. Transfection with specific siRNA directed against TLR-4 mRNA significantly reduced the response to LPS in both cell lines. Fluorescence-activated cell sorter analysis revealed significantly higher levels of TLR-4 surface expression in CFTR-corrected cells. In histologic lung sections of patients with CF, the TLR-4 expression in the bronchial epithelium was significantly reduced compared with healthy control subjects. In CF the loss of CFTR function appears to decrease innate immune responses, possibly by altering the expression of TLR-4 on airway epithelial cells. This may contribute to chronic bacterial infection of CF airways.

Topics: Adolescent; Adult; Antibodies; Bronchi; Cell Line; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Female; Gene Expression Regulation; Humans; Immunity, Innate; Interleukin-6; Interleukin-8; Lipopolysaccharides; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; RNA, Messenger; Signal Transduction; Toll-Like Receptor 4

Staphylococcus aureus is frequently isolated from lungs of patients with cystic fibrosis (CF). Upon lung infection with S. aureus, airway epithelial cells (AEC) produce high levels of chemokines that enhance T-cell chemotaxis. Although the number of lymphocytes is increased in the airways and bronchoalveolar lavage fluid of patients with CF, the mechanisms responsible for their accumulation and the role of S. aureus in this process are largely unknown. This study investigated early S. aureus impact on chemokine secretion by CF epithelial cells and chemotaxis of CF T cells. CF and non-CF AEC were grown in a cell culture model and apically stimulated with S. aureus. Supernatants were quantified for chemokine secretions and assayed for T-cell chemotaxis. CF AEC secreted constitutively larger amounts of IL-8, GROalpha, MIG, MIP-3beta, and MCP-1 than non-CF epithelial cells. S. aureus interaction with epithelial cells increased chemokine production by non-CF cells but had no effect on CF cells. Chemotaxis of T cells derived from patients with CF was greater than that of T cells from subjects without CF. Moreover, there were more CF T cells expressing CXCR1 as compared with non-CF T cells. Under our experimental conditions, inhibition of IL-8 or its receptor CXCR1 resulted in a considerable decrease in T-cell chemotaxis (up to 80%). These data suggest that IL-8 and its receptor CXCR1 are key players in the chemotaxis of CF T cells and could be used as targets to develop therapies for CF.

Topics: Adult; Antibodies, Monoclonal; Case-Control Studies; CD3 Complex; Cell Line; Chemotaxis, Leukocyte; Cystic Fibrosis; Electric Impedance; Female; Humans; Interleukin-8; Male; Receptors, Interleukin-8A; Recombinant Proteins; Respiratory Mucosa; Staphylococcus aureus; T-Lymphocytes; Time Factors; Young Adult

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular, lipid A is the endotoxic portion of LPS responsible for eliciting host innate immune response. In this work, the complete primary structure of the lipid A from B. multivorans C1576 has been defined and, further, its pro-inflammatory activity in a cystic fibrosis airways model is shown. The structure of B. multivorans lipid A was attained by chemical, mass spectrometry and nuclear magnetic resonance analyses whereas its biological activity was assessed on the intestinal epithelial cell line CACO-2 cells, on the airway epithelial IB3-1 cells, carrying the ΔF508/W1282X CFTR mutation and on an ex vivo model of culture explants of nasal polyps.

Topics: Bronchi; Burkholderia; Caco-2 Cells; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Host-Pathogen Interactions; Humans; Interleukin-8; Lipid A; Magnetic Resonance Spectroscopy; Microscopy, Confocal; Nasal Polyps; Spectrometry, Mass, Electrospray Ionization; Tumor Necrosis Factor-alpha

High-dose ibuprofen is clinically effective in cystic fibrosis (CF); however, its molecular mechanisms are poorly understood.. To test the hypothesis that clinically relevant concentrations of ibuprofen suppress activation of nuclear factor (NF)-kappaB and thus down-regulate stimulated interleukin (IL)-8 production in CF respiratory epithelial cells.. The majority of experiments were conducted in CFTE29o- cells (F508del-mutated CF transmembrane regulator, CFTR). Key experiments were confirmed in CFBE41o- cells (F508del-mutated CFTR) and 1HAEo- cells (wild-type CFTR). NF-kappaB and IL-8 were stimulated with tumour necrosis factor (TNF)-alpha or IL-1beta. NF-kappaB and IL-8 suppression by ibuprofen (480 microM) was compared to dexamethasone (5 nM).. Both TNF-alpha and IL-1beta activated NF-kappaB and stimulated IL-8 production. Both ibuprofen and dexamethasone demonstrated comparably modest suppression of NF-kappaB transcriptional activity. However, ibuprofen had no effect on stimulated IL-8 mRNA and protein. By contrast, dexamethasone significantly down-regulated stimulated IL-8 mRNA and protein.. The present data do not support the hypothesis that ibuprofen down-regulates IL-8 production in response to TNF-alpha and IL-1beta in CF respiratory epithelium. Suppression of NF-kappaB transcriptional activity does not discriminate between anti-inflammatory drugs with or without effects on IL-8 production. We speculate that NF-kappaB-independent mechanisms may be responsible for anti-IL-8 effects of dexamethasone.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cystic Fibrosis; Epithelial Cells; Humans; Ibuprofen; Interleukin-1beta; Interleukin-8; NF-kappa B; Respiratory Mucosa; Tumor Necrosis Factor-alpha

In Cystic Fibrosis (CF), the absence of functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is key to better therapies for CF. In this manuscript, we have found that the presence of IL-17 in the airways of CF patients not only exacerbates inflammation through the recruitment of neutrophils via secretion of CXCL8, but also by priming airway epithelial cells lacking functional CFTR to increase response to the bacterial sensor NOD1. IL-17 stimulation of airway epithelial cells (AECs) lacking functional CFTR increased the expression of NOD1, NOD2, TLR4 and its own receptors IL-17RA and IL-17RC. Moreover, prior stimulation of AECs expressing the CFTRDeltaF508 mutant with IL-17 showed much greater CXCL8 secretion in response to a NOD1 agonist and Pseudomonas aeruginosa diffusible material. Taken together our results show that IL-17 primes AECs expressing CFTRDeltaF508 to increase host defence response to bacteria through the up-regulation of PRRs, and in particular of NOD1, and identifies another mechanism of action through which the CFTRDeltaF508 mutation leads to increase inflammation in response to bacterial ligands. Therefore preventing IL-17 function in CF may prove an important strategy in decreasing lung inflammation due to both direct and indirect effects.

Topics: Adolescent; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-17; Interleukin-8; Lung; Neutrophils; Nod1 Signaling Adaptor Protein; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Interleukin-17; Respiratory Mucosa; Toll-Like Receptor 4

Achromobacter xylosoxidans infection may cause conspicuous chronic pulmonary inflammation in cystic fibrosis (CF) patients similar to Pseudomonas aeruginosa and the Burkholderia cepacia complex (Bcc). Evolution in lung function was compared in chronically infected patients. Cytokine concentrations in CF patients with and without chronic infection were compared to healthy controls.. Cytokines in serum and sputum were measured using multiplex bead based immunoassay.. Sixty CF patients, 11 with A. xylosoxidans, 11 with Bcc, 21 with P. aeruginosa and 17 non-infected CF patients were compared to 11 healthy controls. A. xylosoxidans patients were younger, but had a FEV(1) decline similar to P. aeruginosa patients. Bcc patients had the steepest decline in FEV(1). Serum levels of G-CSF, IL-6 and TNF-alpha were significantly higher in CF patients compared to healthy controls. Chronically infected CF patients had significantly higher serum levels of IFN-gamma and IL-6 compared to non-infected CF patients. Bcc patients had significantly lower serum G-CSF and A. xylosoxidans patients had significantly higher sputum TNF-alpha compared to the other groups of chronically infected patients.. A. xylosoxidans can cause a level of inflammation similar to P. aeruginosa in chronically infected CF patients. A. xylosoxidans is a clinically important pathogen in CF and should be treated accordingly.

Topics: Achromobacter denitrificans; Adolescent; Adult; Anti-Bacterial Agents; Biofilms; Breath Tests; Child; Cystic Fibrosis; Drug Resistance, Bacterial; Female; Forced Expiratory Volume; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-10; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Pneumonia, Bacterial; Retrospective Studies; Sputum; Tumor Necrosis Factor-alpha; Young Adult

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cystic Fibrosis; Humans; Ibuprofen; Interleukin-8; NF-kappa B; Respiratory Mucosa

Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines, including IL-8, which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability, activity, and interaction with GAGs, including chondroitin sulfate, hyaluronic acid, and heparan sulfate, present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28), non-CF controls (n = 14), and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production, as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was, however, a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8, which displaced IL-18 from these anionic-matrices, rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation, reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion, a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules, such as IL-18, within the inflammatory milieu of the CF lung.

Topics: Adolescent; Binding, Competitive; Bronchoalveolar Lavage Fluid; Cell Line, Transformed; Child; Child, Preschool; Cystic Fibrosis; Down-Regulation; Glycosaminoglycans; Humans; Inflammation Mediators; Interleukin-18; Interleukin-8; Jurkat Cells; Protein Binding; Protein Stability; Up-Regulation

An unexplained gender gap is observed in cystic fibrosis (CF). Females have poorer lung function, decreased survival, and earlier Pseudomonas colonization.. To evaluate the effect of 17beta-estradiol (E(2)) on CF bronchial epithelial cells in vitro and in vivo.. On exposure of CFBE41o- cultures to physiological concentrations of E(2), there was a significant dose-dependent inhibition of IL-8 release induced by toll-like receptor agonists, CF bronchoalveolar lavage fluid, or Pseudomonas-conditioned media. Estrogen receptor (ER)-alpha and -beta expression was quantified in cell lines and bronchial brushings from CF and non-CF patients.. Both receptors were expressed in vitro and in vivo, although ERbeta expression was significantly higher in CF. Using ER isoform-specific agonists and antagonists, we established that ERbeta mediates the inhibition of CF bronchoalveolar lavage fluid-induced IL-8 release. We also showed that secretory leucoprotease inhibitor gene expression and protein localization to the nucleus increased in response to E(2). Secretory leucoprotease inhibitor knockdown abrogated the inhibitory effects of E(2).. E(2) inhibits IL-8 release by ERbeta in CF bronchial epithelial cells through up-regulation of secretory leucoprotease inhibitor, inhibition of nuclear factor (NF)-kappaB, and IL-8 gene expression. These data implicate a novel anti-inflammatory mechanism for E(2) in females with CF, which predisposes to infection and colonization. This could, in part, account for the observed gender dichotomy in CF.

Topics: Adolescent; Adult; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Estradiol; Estrogen Receptor beta; Female; Humans; Interleukin-8; Male; NF-kappa B; Respiratory Mucosa; Secretory Leukocyte Peptidase Inhibitor; Sex Factors; Up-Regulation; Young Adult

Cystic Fibrosis (CF) is an inherited disorder characterised by chronic inflammation of the airways. The lung manifestations of CF include colonization with Pseudomonas aeruginosa and Staphylococcus aureus leading to neutrophil-dominated airway inflammation and tissue damage. Inflammation in the CF lung is initiated by microbial components which activate the innate immune response via Toll-like receptors (TLRs), increasing airway epithelial cell production of proinflammatory mediators such as the neutrophil chemokine interleukin-8 (IL-8). Thus modulation of TLR function represents a therapeutic approach for CF. Nicotine is a naturally occurring plant alkaloid. Although it is negatively associated with cigarette smoking and cardiovascular damage, nicotine also has anti-inflammatory properties. Here we investigate the inhibitory capacity of nicotine against TLR2- and TLR4-induced IL-8 production by CFTE29o- airway epithelial cells, determine the role of alpha7-nAChR (nicotinic acetylcholine receptor) in these events, and provide data to support the potential use of safe nicotine analogues as anti-inflammatories for CF.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Cell Line; Cell Proliferation; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; Laser Scanning Cytometry; Lipopolysaccharides; Nicotine; Peptidoglycan; Receptors, Nicotinic; Toll-Like Receptor 2; Toll-Like Receptor 4; Trachea; Zymosan

Probiotics reduce intestinal inflammation in, and Lactobacillus GG (LGG) reduces pulmonary exacerbation rate cystic fibrosis (CF) patients. We intended to determine the effect of a mixed probiotic preparation on pulmonary exacerbations and inflammatory characteristics of the sputum in CF patients.. A prospective pilot study of 10 CF patients with mild-moderate lung disease and Pseudomonas aeruginosa colonization, treated with probiotics for 6 months. Pulmonary function tests (PFT's), sputum cultures with semi-quantitative bacterial analysis, and sputum neutrophil count and interleukin-8 (IL-8) levels were compared to pre-treatment and post-treatment values. The rate of pulmonary exacerbations was compared to 2 years prior to the study.. The exacerbation rate was significantly reduced in comparison to the previous 2 years and to 6 months post-treatment (P = 0.002). PFT's have not changed at the end of treatment and during 6 months post-treatment. No change in sputum bacteria, neutrophil count, and IL-8 levels was observed.. Probiotics reduce pulmonary exacerbations rate in patients with CF. Probiotics may have a preventive potential for pulmonary deterioration in CF patients.

Topics: Adult; Cystic Fibrosis; Dietary Supplements; Disease Progression; Female; Humans; Interleukin-8; Male; Mutation; Neutrophils; Pilot Projects; Probiotics; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Sputum; Young Adult

Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D(2)S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D(2)S. D(2)S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at approximately 5 microM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D(2)S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D(2)S can improve transfection activity over common lipofection reagents, and D(2)S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation.

Topics: Animals; Anti-Infective Agents; Cations; Cattle; Cystic Fibrosis; Dexamethasone; DNA; Eukaryota; Gene Transfer Techniques; Genetic Therapy; Humans; Interleukin-8; Lipids; Phosphatidylethanolamines; Plasmids; Spermine; Transfection; Transgenes

Interleukin-8 (IL-8) and neutrophil elastase (NE) are commonly measured markers of inflammation in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis. Longitudinal analysis assumes uniform stability during storage, however the effect of extended low-temperature storage on these markers remains unclear.. BAL fluid from 104 children with cystic fibrosis was assayed for IL-8 and NE after storage at 4 ° C for 7 days and -80 ° C for up to 6 years and compared with the initial assays performed soon after collection.. IL-8 levels were stable after any measured length of time at -80 ° C or 4 ° C. NE levels were stable for 6 months at -80 ° C but decreased beyond that or after 7 days at 4 ° C.. Our data support the stability of IL-8 in BAL stored at -80 ° C for prolonged periods. NE in BAL decreases with storage and should be assayed as soon as practical after collection.

Topics: Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Cystic Fibrosis; Drug Stability; Drug Storage; Female; Humans; Infant; Interleukin-8; Leukocyte Elastase; Male; Temperature; Time Factors

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o(-) sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr(-/-) transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE(2), and PGF(2α) secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr(-/-) Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators.

Topics: Animals; Arachidonic Acid; Biomarkers; Bronchoalveolar Lavage Fluid; Cell Line; Cystic Fibrosis; Dietary Supplements; Disease Models, Animal; Eicosanoids; Fatty Acids; Humans; Inflammation; Interleukin-8; Linoleic Acid; MAP Kinase Signaling System; Mice; Mice, Inbred CFTR; Mice, Knockout; Mice, Transgenic; Pseudomonas aeruginosa; Respiratory Mucosa

alpha-Defensins, antimicrobial peptides produced mainly by neutrophils, have been reported to be associated with a wide variety of lung diseases, including idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), and diffuse panbronchiolitis (DPB). In each disease, alpha-defensins are located in different areas, such as around the alveolar septa in IPF and around the airways in CF and DPB, suggesting that alpha-defensins play different roles. Meanwhile, growth factors are known to contribute to IPF, CF, and DPB. alpha-Defensins are known to induce interleukin (IL)-8 in airway epithelial cells, but the effects of alpha-defensins on the release of growth factors from various components in the lung have not been sufficiently investigated. In the present study, the in vitro effects of human neutrophil peptide (HNP)-1 (a subtype of alpha-defensin) on the expressions of IL-8 and growth factors in lung fibroblasts, bronchial epithelial cells, and alveolar epithelial cells were examined. HNP-1 mainly enhanced the expression of IL-8 in epithelial cells, whereas it enhanced transforming growth factor-beta and vascular endothelial growth factor expressions in lung fibroblasts. These results suggest that alpha-defensins play different roles in the pathogenesis of IPF, CF, and DPB according to the location in the lung where the alpha-defensins are mainly produced.

Topics: alpha-Defensins; Bronchiolitis; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Fibroblasts; Haemophilus Infections; Humans; Idiopathic Pulmonary Fibrosis; Interleukin-8; Lung; Transforming Growth Factor beta; Vascular Endothelial Growth Factors

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Topics: Animals; Cell Death; Chemokine CXCL2; Cystic Fibrosis; Enzyme Inhibitors; Extracellular Space; Humans; Inflammation; Interleukin-8; Lung Diseases; Mice; NADPH Oxidases; Neutrophil Activation; Neutrophils; Onium Compounds; Receptors, Interleukin-8B

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells.

Topics: Alginates; Bronchi; Cell Survival; Cells, Cultured; Cystic Fibrosis; Drug Compounding; Epithelial Cells; Gene Expression; Glucuronic Acid; Hexuronic Acids; Humans; Interleukin-6; Interleukin-8; Microscopy; Microspheres; Particle Size; Proteome; Research Design; Tumor Necrosis Factor-alpha

In the present study, a structured-based virtual screening (VS) of differently substituted furocoumarins and analogues has been carried out against nuclear factor kappa B (NF-κB), with the objective of selecting molecules able to inhibit the binding of this transcription factor to the DNA. The focus library was developed starting from chemical structures obtained from the literature, as well as retrieving compounds from available commercial databases. A two dimensional substructure searching method based on four different chemical scaffolds was used for this purpose. Among the 10 highest-scored ligands selected from the docking studies, five commercially available molecules were investigated in biological assays. Four furocoumarin derivatives showed IC(50) values in the range of 40-100 μM in inhibiting NF-κB/DNA interactions studied by electrophoretic mobility shift assay (EMSA). Three compounds significantly inhibited NF-κB dependent biological functions (expression of IL-8) in cellular analysis based on Pseudomonas aeruginosa infection of cystic fibrosis IB3-1 cells. These findings validated the virtual screening approach here presented and reinforce the successful results of our previously computational studies aimed at the identification of molecules targeting NF-κB. The discovered novel compounds could be of relevance to identify more potent inhibitors of NF-κB dependent biological functions beneficial to control lung inflammation occurring in patients affected by cystic fibrosis.

Topics: Binding Sites; Cell Line; Computer Simulation; Cystic Fibrosis; Databases, Factual; Electrophoretic Mobility Shift Assay; Furocoumarins; Humans; Hydrogen Bonding; Interleukin-8; Ligands; NF-kappa B; Protein Structure, Tertiary

Dysregulated inflammation has been implicated in cystic fibrosis (CF) airway pathophysiology. The expression of inflammatory genes, like interleukin 8 (IL8), involves chromatin remodeling through histone acetylation. Inflammatory gene hyperacetylation could explain inflammatory mediator dysregulation seen in CF airways. CF airways are exposed to high levels of oxidative stress, and oxidative stress increases histone acetylation and inflammatory gene transcription. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) may even reduce protection against oxidative stress. Consequently, increasing oxidative stress would likely lead to an imbalance of histone acetyl-transferase (HAT) and deacetylase (HDAC) stoichiometry and contribute to the heightened inflammatory response seen in the CF airway. We hypothesize that oxidative stress in CF airways causes increased acetylation of inflammatory gene promoters, contributing to transcriptional activity of these loci. Messenger RNA levels of IL8, IL6, CXCL1, CXCL2, CXCL3, and IL1 are significantly elevated in CF epithelial cell models. Histone H4 acetylation is lower at the IL8 promoter of the non-CF cell lines than the CF models. The reducing agent N-acetyl-cysteine decreases IL8 message and promoter H4 acetylation to non-CF levels, suggesting that oxidative stress contributes to IL8 expression in these models. H(2)O(2) treatment causes increased IL-8 acetylation and mRNA in all cells, but less in the CF-model cells. Together these data suggest a model in which cells without functional CFTR are under increased oxidative stress. Our data suggest intrinsic alterations in the HAT/HDAC balance in CFTR-deficient cells, and that oxidative stress contributes to this alteration.

Topics: Acetylation; Acetylcysteine; Animals; Cell Line; Cystic Fibrosis; Cytokines; Epithelial Cells; Free Radical Scavengers; Humans; Hydrogen Peroxide; Interleukin-8; Oxidants; Oxidative Stress; Promoter Regions, Genetic; Respiratory Mucosa; Transcription, Genetic

To assess the effects of Pseudomonas aeruginosa and Staphylococcus aureus infection on lower airway inflammation and clinical status in young children with cystic fibrosis (CF).. We studied 111 children age < 6 years who had 2 P aeruginosa-positive oropharyngeal cultures within 12 months. We examined bronchoalveolar lavage fluid (BALF) inflammatory markers (ie, cell count, differential, interleukin [IL]-8, IL-6, neutrophil elastase), CF-related bacterial pathogens, exotoxin A serology, and clinical indicators of disease severity.. Young children with CF with both upper and lower airway P aeruginosa infection had higher neutrophil counts, higher IL-8 and free neutrophil elastase levels, increased likelihood of positive exotoxin A titers, and lower Shwachman scores compared with those with positive upper airway cultures only. S aureus was associated with increased lower airway inflammation, and the presence of both P aeruginosa and S aureus had an additive effect on concentrations of lower airway inflammatory markers. BALF markers of inflammation were increased with the number of different bacterial pathogens detected.. Young children with CF who have upper and lower airway P aeruginosa infection have increased endobronchial inflammation and poorer clinical status compared with those with only upper airway P aeruginosa infection. The independent and additive effects of S aureus on inflammation support the significance of polymicrobial infection in early CF lung disease.

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Exotoxins; Female; Humans; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Lung; Male; Neutrophils; Oropharynx; Pseudomonas aeruginosa; Pseudomonas Infections; Severity of Illness Index; Staphylococcal Infections; Staphylococcus aureus

Cystic fibrosis (CF) airway epithelia exhibit altered Cl(-) and Na(+) transport properties and increased IL-8 secretion. In the present study, we examined whether a small proportion of cells with a normal phenotype could normalize the ion transport and IL-8 secretion properties of a CF airway epithelial cell layer. We obtained three types of primary cultures of human bronchial epithelial cells: one composed of 100% non-CF cells, one of 100% CF cells, and one of 10% non-CF and 90% CF cells ("cocultures"). Measurement of the bioelectric properties in Ussing chambers revealed that the cocultures displayed Cl(-) and Na(+) transports similar to those observed in the 100% non-CF cultures and significantly different from CF cultures. IL-8 concentration in the coculture supernatant was not different from non-CF cultures, but was significantly lower than in CF cultures. This study provides evidence that 10% bronchial epithelial cells expressing a normal phenotype are sufficient to functionally correct a primary culture of CF bronchial epithelial cells in vitro. We postulate that 10% cells with a non-CF phenotype can be used as a goal for the design of gene therapy and cell therapy trials for CF lung disease.

Topics: Adult; Apoptosis; Bronchial Diseases; Cell Proliferation; Coculture Techniques; Cystic Fibrosis; Epithelial Cells; Genetic Diseases, Inborn; Genetic Therapy; Homozygote; Humans; Interleukin-8; Ions; Male; Phenotype

Anti-inflammatory properties of azithromycin (AZM) have been proposed as possible mechanisms of clinical beneficial effects in patients with cystic fibrosis (CF). Altered glutathione (GSH) transport in cystic fibrosis transmembrane regulator protein (CFTR)-deficient cells leads to the occurrence of oxidative stress that finally induces glutathione S-transferase (GST) activity. The present investigation was aimed to verify the effects of AZM on GST activity and expression in CF airway cells in vitro and in vivo. AZM exposure significantly decreased GSTT1 and GSTM1 mRNA and protein expression in IB3-1, restoring the levels to those observed in non-CF C38 cells, which also express lower levels of gamma-glutamyltransferase (GGT) activity than IB3-1. In another CF cell line, 2CFSMEo-, AZM produced 45% reduction in GSTT1 and GSTM1 mRNA levels. AZM reduced GST activity by approximately 25% and 40% in IB3-1 and 2CFSMEo- cells, respectively. GSTP1 was similarly expressed in all CF and non-CF cells and was unaffected by AZM. The anti-inflammatory cytokine IL-10 down-modulated GST activity at similar levels, supporting a link between GST inhibition and anti-inflammatory properties of AZM. In bronchoalveolar lavage fluid of CF mice homozygous for the F508 del mutation, GSTM1 protein levels were undetectable after AZM treatment. The association between increased GST expression and activity, together with its reversal by AZM treatment in vitro and in vivo, suggest novel antioxidant properties for this drug. The issue whether decreased GST activity may directly concur to anti-inflammatory properties of AZM or is rather a marker of the oxidative status of CF cells will require additional studies.

Topics: Animals; Anti-Inflammatory Agents; Azithromycin; Bronchoalveolar Lavage Fluid; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Female; Glutathione Transferase; Humans; Interleukin-8; Isoenzymes; Mice; Mice, Transgenic; Oxidation-Reduction; Respiratory Mucosa; Sulfhydryl Compounds; Tumor Necrosis Factor-alpha

Acid sphingomyelinase (ASMase) is a key enzyme in sphingolipid metabolism, which can be activated by various cellular stress mechanisms including bacterial pathogens. Activation of ASMase generates ceramide, which is important for innate immune response to eliminate infected pathogens. The current study reveals a defective ASMase pathway after Pseudomonas aeruginosa infection in both a cystic fibrosis (CF) bronchial epithelial cell line (IB3-1 cell) and in the lungs of CF transmembrane conductance regulator (CFTR) knockout (KO) mice as compared with S9 cells and wild-type C57BL/6 mice. ASMase activity and total ceramide levels significantly increased in S9 cells and C57BL/6 mice with P. aeruginosa infection, but not in IB3-1 cells and CFTR KO mice. The silencing of CFTR by CFTR RNAi in S9 cells significantly decreased ASMase activity after bacterial infection as compared with controls. This study also demonstrates that induction of ASMase is responsible for modulating the immune response to bacterial infection. Blocking ASMase activity with specific ASMase RNAi, an ASMase inhibitor, or an ASMase antibody in S9 cells significantly increased IL-8 levels with P. aeruginosa infection compared with controls. Reciprocally, adding exogenous bacterial sphingomyelinase to IB3-1 cells significantly decreased IL-8 levels compared with untreated cells. In addition, silencing of ASMase in S9 cells also significantly decreased bacterial internalization. Adding exogenous bacterial sphingomyelinase to IB3-1 cells reconstituted the cell death response to P. aeruginosa infection. This study demonstrates that the defective ASMase pathway in CF is a key contributor to the unabated IL-8 response with P. aeruginosa infection and to the compromised host response failing to eradicate bacteria.

Topics: Animals; Apoptosis; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Interleukin-8; Mice; Mice, Inbred C57BL; Mice, Knockout; Pseudomonas aeruginosa; Pseudomonas Infections; RNA Interference; Signal Transduction; Sphingolipids; Sphingomyelin Phosphodiesterase

P. aeruginosa chronically colonizes the lung in CF patients and elicits a proinflammatory response. Excessive secretion of IL-6 and IL-8 by CF airway cells in response to P. aeruginosa infection in the CF airway is though to contribute to lung injury. Accordingly, the goal of this study was to test the hypothesis that Corr4a and VRT325, investigational compounds that increase DeltaF508-CFTR mediated Cl(-) secretion in human CF airway cells, reduce the pro-inflammatory response to P. aeruginosa.. IL-6 and IL-8 secretion by polarized CF human airway epithelial cells (CFBE41o-) were measured by multiplex analysis, and DeltaF508-CFTR Cl- secretion was measured in Ussing chambers. Airway cells were exposed to P. aeruginosa (PAO1 or PA14) and Corr4a or VRT325.. Corr4a and VRT325 increased DeltaF508-CFTR Cl(-) secretion but did not reduce either constitutive IL-6 or IL-8 secretion, or IL-6 and IL-8 secretion stimulated by P. aeruginosa (PA14 or PAO1).. Corr4a and VRT325 do not reduce the inflammatory response to P. aeruginosa in human cystic fibrosis airway epithelial cells.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Piperazines; Pseudomonas Infections; Quinazolines

Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3'-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.

Topics: Adenine; Cell Line; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Humans; Interleukin-8; Phenotype; Respiratory Mucosa; RNA Stability; RNA, Messenger; Tristetraprolin

The relationship between lower airway markers of inflammation and infection with physiologic findings is poorly understood in young children with cystic fibrosis (CF). The goal of this study was to evaluate the association of bronchoalveolar lavage fluid (BALF) markers of infection and inflammation, including mediators linked to airway remodeling, to infant lung function values in young children with CF undergoing clinically indicated bronchoscopy.. Plethysmography and the raised volume rapid thoracoabdominal compression (RVRTC) technique were performed in 16 sedated infants and young children with CF prior to bronchoscopy. BALF was collected and analyzed for pathogen density, cell count, % neutrophils, transforming growth factor beta 1 (TGF-beta(1)), matrix metalloproteinases (MMP), and interleukin-8 (IL-8).. There was a significant direct correlation between functional residual capacity (FRC), the ratio of residual volume to total lung capacity (RV/TLC) and FRC/TLC with % neutrophils (P < 0.05). Forced expiratory flows were inversely correlated to % neutrophils (P < 0.01). Lung function parameters did not differentiate those with and without lower airway infection; however, pathogen density directly correlated with FRC and inversely correlated with flows (P < 0.05). In a subset of the population, MMP-2 directly correlated with RV/TLC and inversely correlated with flows (P < 0.05) and TGF-beta(1) directly correlated with FRC (P < 0.05).. Results from this study suggest that lower airway inflammation as well as mediators linked to airway remodeling play an active role in pulmonary deterioration in CF infants and young children undergoing clinically indicated bronchoscopy.

Topics: Bronchoalveolar Lavage Fluid; Child, Preschool; Cohort Studies; Colony Count, Microbial; Cystic Fibrosis; Female; Forced Expiratory Flow Rates; Functional Residual Capacity; Humans; Infant; Inflammation; Interleukin-8; Leukocyte Count; Male; Metalloproteases; Neutrophils; Plethysmography; Transforming Growth Factor beta1

Inflammatory cytokines, particularly the neutrophil chemoattractant IL-8, are elevated in the cystic fibrosis (CF) airway, even in the absence of detectable infection. The transcriptional regulation of many inflammatory genes, including IL8 (CXCL8), involves chromatin remodeling through histone acetylation. NF-kappaB is known to facilitate histone acetylation of IL8 and other proinflammatory gene promoters, but we find that increased NF-kappaB activation cannot explain the elevated IL8 expression and promoter acetylation seen in CFTR-deficient cells. Recognized components of the NF-kappaB-coactivator complex, acetyltransferase CBP, p300, and the histone deacetylase HDAC1, are unchanged by CFTR activity. However, we find that the histone acetyltransferase (HAT)/HDAC balance is sensitive to CFTR function, as cells with reduced or absent CFTR function have decreased HDAC2 protein, resulting in hyperacetylation of the IL8 promoter and increased IL8 transcription. Reduced HDAC2 and HDAC2 activity, but not HDAC2 mRNA, is observed in cells deficient in CFTR. Suppressing HDAC2 expression with HDAC2 short hairpin RNA (shRNA) results in increased IL8 expression and promoter acetylation comparable with CFTR-deficient cells. Treating CFTR-deficient cells with N-acetyl-cysteine (NAC) increases HDAC2 expression to near control levels. Our data suggest that there is an intrinsic alteration in the HAT/HDAC balance in cells lacking CFTR function in vitro and in native CF tissue and that oxidative stress is likely contributing to this alteration. This mechanism, found in other inflammatory airway diseases, provides an explanation for the apparent dysregulation of inflammatory mediators seen in the CF airway, as reduced histone deacetylation would potentially influence many genes.

Topics: Acetylation; Acetylcysteine; Cell Line; Cell Nucleus; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Knockdown Techniques; Histone Deacetylase 2; Histone Deacetylases; Humans; Interleukin-8; NF-kappa B; p300-CBP Transcription Factors; Promoter Regions, Genetic; Repressor Proteins; Respiratory System; RNA Interference; RNA, Messenger; RNA, Small Interfering

Cystic fibrosis (CF) lung disease is characterized by structural changes in the airways and parenchyma. No sputum biomarker exists to measure the degree of active structural destruction during pulmonary exacerbation in patients with CF. The noninvasive measurement of desmosine, a breakdown product of elastin, may reflect ongoing lung injury and serve as a biomarker of short-term damage. Our objectives were to measure desmosine in the sputum of patients with CF hospitalized for treatment of a pulmonary exacerbation and to explore the correlation between desmosine levels and other markers of clinical improvement, including lung function and inflammatory mediators, following hospitalization.. Sputum and blood samples collected and lung function measurements were made at multiple time points during hospitalization. We used a repeated measures model, adjusted for age and time between measurements, to compare log-transformed sputum desmosine levels across multiple time points and to correlate those levels with related variables.. Desmosine levels were measured by radioimmunoassay in 71 expectorated sputum samples from 19 patients with CF hospitalized for 26 pulmonary exacerbations (range of results, 0 to 200 pmol/L desmosine/mL). Sputum desmosine levels decreased significantly during the first week of hospitalization (p = 0.04). Desmosine levels were positively associated with plasma C-reactive protein (rho = 0.59; p = 0.03), sputum interleukin-8 (rho = 0.86; p < 0.01), and sputum neutrophil elastase (rho = 0.78; p < 0.01).. Sputum desmosine, a novel measure of acute structural lung injury, may serve as a marker of structural lung damage occurring during exacerbations of lung disease in CF.

Topics: Adolescent; Adult; Biomarkers; C-Reactive Protein; Child; Cohort Studies; Cystic Fibrosis; Desmosine; Female; Hospitalization; Humans; Interleukin-8; Leukocyte Elastase; Lung; Male; Prospective Studies; Severity of Illness Index; Sputum; Young Adult

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-kappaB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

Topics: Animals; Antioxidants; Bronchi; Bronchoalveolar Lavage; Cell Line; Cell Proliferation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Epithelial Cells; Flagellin; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Lipopolysaccharides; Mice; Neutrophils; NF-E2-Related Factor 2; NF-kappa B; Oleanolic Acid; Oxidation-Reduction; Proteomics; Trachea; Triterpenes

Persistent recruitment of neutrophils in the bronchi of cystic fibrosis patients contributes to airway tissue damage, suggesting the importance of intervening on the expression of the neutrophil chemokine IL-8. Extracts from plants have been investigated to select components able to reduce IL-8 expression in bronchial epithelial cells challenged with Pseudomonas aeruginosa. Extracts and purified components have been added to cells 24 h before pro-inflammatory challenge with P. aeruginosa and IL-8 transcription was quantified in the IB3-1 CF cells in vitro. P. aeruginosa-dependent IL-8 mRNA induction was increased by Argemone mexicana and Vernonia anthelmintica whereas no significant modification of transcription was observed with Aphanamixis polystachya, Lagerstroemia speciosa and Hemidesmus indicus. Finally, inhibition of IL-8 was observed with Polyalthia longifolia (IC50=200 microg/ml) and Aegle marmelos (IC50=20 microg/ml). Compounds from A. marmelos were isolated and identified by GC-MS. No significant effect was observed with butyl-p-tolyl sulphate, whereas the inhibition obtained with 6-methyl-4-chromanone concentration was accompanied by an anti-proliferative effect. On the contrary, 5-methoxypsoralen resulted in IL-8 inhibition at 10 microM concentration, without effects on cell proliferation. In synthesis, 5-methoxypsoralen can be taken into consideration to investigate mechanisms of neutrophil chemotactic signalling and for its potential application in modulating the excessive CF lung inflammation.

Topics: 5-Methoxypsoralen; Argemone; Bronchi; Cell Line; Cell Proliferation; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Humans; Interleukin-8; Methoxsalen; Neutrophils; Photosensitizing Agents; Plant Extracts; Pseudomonas aeruginosa; Vernonia

Cystic fibrosis pulmonary disease is characterized by excessive and prolonged inflammation. CF Pulmonary disease severity exhibits considerable variation that, to some extent, appears to be due to the presence of modifier genes. Several components of the inflammatory response are known to have altered regulation in the CF lung. Genetic variants in 52 inflammatory genes were tested for associations with lung disease indices in a CF patient population (n=737) homozygous for the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation. Variants in three inflammatory genes showed significant genotypic associations with CF lung disease severity, including IL8 and previously reported TGFbeta1 (P< or =0.05). When analyzed by gender, it was apparent that IL8 variant associations were predominantly due to males. The IL8 variants were tested in an additional CF population (n=385) and the association in males verified (P< or =0.01). The IL8 variants were in strong linkage disequilibrium with each other (R2> or =0.82), while variants in neighboring genes CXCL6, RASSF6 and PF4V1 did not associate (P> or =0.26) and were in weaker LD with each other and with the IL8 variants (0.01< or =R2< or =0.49). Studies revealed differential expression between the IL8 promoter variant alleles (P<0.001). These results suggest that IL8 variants modify CF lung disease severity and have functional consequences.

Topics: Cystic Fibrosis; Female; Humans; Interleukin-8; Male; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Sex Characteristics

Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

Topics: Animals; Antioxidants; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Hydrogen Peroxide; Interleukin-1beta; Interleukin-6; Interleukin-8; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Oxidants; Oxidation-Reduction; Tumor Necrosis Factor-alpha

The clinical course of cystic fibrosis (CF) varies considerably among patients carrying the same CF-causing gene mutation. Additional genetic modifiers may contribute to this variability. As airway inflammation is a key component of CF pathophysiology, we investigated whether major cytokine variants represent such modifiers in young CF patients. We tested 13 polymorphisms in 8 genes that play a key role in the inflammatory response: tumor necrosis factor, lymphotoxin alpha, interleukin (IL) 1B, IL1 receptor antagonist, IL6, IL8, IL10 and transforming growth factor beta 1 (TGFB1), for an association with lung disease progression and nutritional status in 329 CF patients. Variants in the TGFB1 gene at position +869T/C demonstrated a significant association with lung function decline. A less pronounced rate of decline in forced expiratory volume in 1 sec (FEV(1)) and forced vital capacity (FVC) were observed in patients heterozygous for TGFB1 +869 (+869CT), when compared to patients carrying either TGFB1 +869TT or +869CC genotypes. These findings support the concept that TGFB1 gene variants appear to be important genetic modifiers of lung disease progression in CF.

Topics: Adolescent; Child; Cystic Fibrosis; Disease Progression; Female; Genetic Variation; Humans; Inflammation Mediators; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Lymphotoxin-alpha; Male; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Many cystic fibrosis (CF) patients display airway hyperresponsiveness and have symptoms of asthma such as cough, wheezing and reversible airway obstruction. Chronic airway bacterial colonization, associated with neutrophilic inflammation and high levels of interleukin-8 (IL-8) is also a common occurrence in these patients. The aim of this work was to determine the responsiveness of airway smooth muscle to IL-8 in CF patients compared to non-CF individuals.. Experiments were conducted on cultured ASM cells harvested from subjects with and without CF (control subjects). Cells from the 2nd to 5th passage were studied. Expression of the IL-8 receptors CXCR1 and CXCR2 was assessed by flow cytometry. The cell response to IL-8 was determined by measuring intracellular calcium concentration ([Ca2+](i)), cell contraction, migration and proliferation.. The IL-8 receptors CXCR1 and CXCR2 were expressed in both non-CF and CF ASM cells to a comparable extent. IL-8 (100 nM) induced a peak Ca2+ release that was higher in control than in CF cells: 228 +/- 7 versus 198 +/- 10 nM (p < 0.05). IL-8 induced contraction was greater in CF cells compared to control. Furthermore, IL-8 exposure resulted in greater phosphorylation of myosin light chain (MLC20) in CF than in control cells. In addition, MLC20 expression was also increased in CF cells. Exposure to IL-8 induced migration and proliferation of both groups of ASM cells but was not different between CF and non-CF cells.. ASM cells of CF patients are more contractile to IL-8 than non-CF ASM cells. This enhanced contractility may be due to an increase in the amount of contractile protein MLC20. Higher expression of MLC20 by CF cells could contribute to airway hyperresponsiveness to IL-8 in CF patients.

Topics: Cells, Cultured; Cystic Fibrosis; Dose-Response Relationship, Drug; Humans; Interleukin-8; Lung; Muscle Contraction; Muscle, Smooth; Receptors, Interleukin-8A; Receptors, Interleukin-8B

Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis (CF). Infection is often associated with severe pulmonary inflammation, and some patients develop a fatal necrotizing pneumonia and sepsis ('cepacia syndrome'). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage, binds to tumour necrosis factor receptor 1 (TNFR1) and activates TNFR1-related signalling pathway similar to TNF-alpha, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain, do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared with non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patients who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia, ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism.

Topics: Bacterial Adhesion; Burkholderia cepacia complex; Burkholderia Infections; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; Receptors, Tumor Necrosis Factor, Type I; Respiratory Mucosa

Interleukin (IL)-8 is a potent neutrophil chemoattractant that drives the inflammatory response in cystic fibrosis (CF). Traditional approaches to the pathophysiology of this inflammation have focused on targeting NF-kappaB-dependent signaling and therapy with glucocorticoids. We test the hypothesis that an alternative pathway, independent of NF-kappaB, operates through prostaglandin E2 (PGE-2) receptor EP-2 and stimulates IL-8 chemokine secretion. Using CF bronchial epithelial cells (IB3-1) in vitro, exogenous PGE-2 induces IL-8 release in a dose-dependent manner. These events are associated with elevation in the EP-2 receptors. Inhibition of cyclooxygenase (Cox)-2 with NS-398 was associated with reductions in Cox-2 (2-fold) and IL-6 (1.3-fold) mRNA transcripts, and in IL-8 and PGE-2 chemokine secretion. The inhibition of Cox-2 signaling led to down-regulation of the downstream C/EBP homologous protein (CHOP) transcription factor, resulting in a decrease in IL-8 activation. We confirmed the regulation of IL-8 promoter by CHOP in CF cells using the IL-8 reporter assay. We conclude that PGE-2 stimulates IL-8 production through the CHOP transcription factor in CF cells.

Topics: Base Sequence; Blotting, Western; Bronchi; Cell Line; Chromatin Immunoprecipitation; Cyclic AMP; Cyclooxygenase 2; Cystic Fibrosis; Dinoprostone; DNA Primers; Epithelial Cells; Humans; Ibuprofen; Interleukin-8; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor CHOP

Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O(2)) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

Topics: Cell Line; Cystic Fibrosis; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Interleukin-8; Lung; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappaB-Inducing Kinase; Oxidative Stress; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Receptors, CXCR; Transcription Factor AP-1

Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.

Topics: Bronchi; Cystic Fibrosis; Epitopes; Fucosyltransferases; Glycosyltransferases; Humans; Interleukin-6; Interleukin-8; Lewis X Antigen; Mucous Membrane; Polymerase Chain Reaction; Sulfotransferases

Chronic inflammation and infection in patients with cystic fibrosis (CF) and other lung diseases begin early, making noninvasive diagnostic techniques vital. As induced sputum (IS) testing is useful in older patients, we investigated its adaptation to young nonexpectorating children.. Following the inhalation of a 4.5% saline solution, sputum was collected by nasopharyngeal or oropharyngeal suction for culture and testing for inflammatory markers, with paired preceding oropharyngeal cough swabs (OCSs) in a subgroup. Specimens from 48 IS procedures (46 successful) in 20 CF children (median age, 3 years) were compared with 8 specimens from 8 non-CF pulmonary patients (median age, 4.5 years).. The procedure was safe, with arterial oxygen saturation remaining at > or = 96%. Cultures from 14 of 46 CF patients (30%) grew Pseudomonas aeruginosa, whereas cultures from 19 of 46 CF patients (41%) had no growth. Cultures from seven of eight non-CF subjects grew bacteria, but none were P aeruginosa. Comparing 29 paired IS and OCS samples, 11 and 5 samples, respectively, cultured P aeruginosa (not significant), whereas 12 and 21 samples, respectively, had no growth (p = 0.02). A correlation was found between the independent inflammatory markers NE and both interleukin (IL)-8 (r = 0.85; p < 0.001) and the percentage of neutrophils (r = 0.35; p < 0.05), confirming the validity of IS samples in evaluating early airway disease. IL-8 levels also increased with age (r = 0.41; p < 0.05). Inflammation was similar in CF and non-CF subjects.. IS testing in the young is feasible, safe, and clinically useful, and could serve as an outcome measure for new therapies.

Topics: Child; Child, Preschool; Cystic Fibrosis; Early Diagnosis; Feasibility Studies; Female; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Male; Neutrophils; Respiratory Tract Infections; Sputum

This is the first report to describe a role for Lung Kruppel-like Factor (LKLF or KLF2) in inflammatory airways diseases. In the present study, we identify that LKLF is constitutively expressed in the small airways of normal lungs; however, its expression disappears in severe airway diseases, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease. LKLF from primary airway epithelial cells inhibits NF-kappaB-driven transcription induced by Pseudomonas aeruginosa 7-fold, but is down-regulated in the presence of TNF-alpha and activated human neutrophils. As a constitutively expressed protein, LKLF inhibits release of a key pro-inflammatory chemokine, IL-8, from airway epithelia. Its expression by lung epithelial cells is enhanced in the presence of TNF blockade. Thus, cytokine-mediated inhibition of LKLF by neutrophils may contribute to ongoing recruitment by promoting IL-8 release from airway epithelia. We conclude that, in neutrophil-dominated airway environments, such as that seen in CF, reduced LKLF activity releases a brake on pro-inflammatory cytokine production and thereby may contribute to the persistent inflammatory responses seen in CF airway disease.

Topics: Adult; Cell Line; Child; Cystic Fibrosis; Enzyme Activation; Enzyme Induction; Epithelial Cells; Humans; Interleukin-8; Kruppel-Like Transcription Factors; Lipopolysaccharides; Neutrophils; NF-kappa B; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Respiratory Mucosa; Transcription, Genetic; Tumor Necrosis Factor-alpha

Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-gamma, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1beta and IL-6. Since consensus sequences for the TF, NF-kappaB, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-kappaB consensus sequences. IL-8 NF-kappaB decoy ODN partially inhibited the P. aeruginosa-dependent transcription of IL-8, GRO-gamma, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-gamma, ICAM-1, IL-1beta, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-kappaB, is a promising strategy to obtain inhibition of IL-8 gene transcription.

Topics: Bronchi; Cell Nucleus; Cystic Fibrosis; HIV Long Terminal Repeat; Humans; Interleukin-8; NF-kappa B; Oligodeoxyribonucleotides; Pseudomonas aeruginosa; Transcription Factors; Transcription, Genetic

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased [Ca](i) but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca](i)) or Ca(2+)-free solution reduced increases in [Ca](i) due to ATP or thapsigargin and also reduced NF-kappaB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-kappaB-binding site was critical for activation by all agonists and for activation by [Ca](i). Thus increased [Ca](i) was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased [Ca](i) was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca](i) synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca](i) reduced these responses. Thus [Ca](i) plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in [Ca](i).

Topics: Adenosine Triphosphate; Base Sequence; Calcium Signaling; Cell Line; Cystic Fibrosis; DNA Primers; Egtazic Acid; Epithelial Cells; Flagellin; Humans; Interleukin-8; NF-kappa B; Promoter Regions, Genetic; Pseudomonas aeruginosa; Respiratory System; Thapsigargin

Topics: Animals; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Inflammation; Interleukin-8; Mice; Mutation; Respiratory System

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-alpha, IL-1beta, or LPS with or without 5-50 microg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-kappaB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-alpha and IL-1beta increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-alpha (P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-kappaB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-alpha increased, as did the p-p38, by 1.6- to 2-fold. MXF (5-50 microg/ml) and CIP (50 microg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-alpha-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-kappaB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-alpha and IL-1beta induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.

Topics: Anti-Infective Agents; Aza Compounds; Azithromycin; Bronchi; Cell Line; Ciprofloxacin; Cystic Fibrosis; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fluoroquinolones; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Moxifloxacin; NF-kappa B; Phosphorylation; Quinolines; Tumor Necrosis Factor-alpha

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr(-/-) mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr(-/-) mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Biological Transport; Cell Membrane; Cells, Cultured; Cholesterol; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Docosahexaenoic Acids; Enzyme Inhibitors; Epithelial Cells; Homeostasis; Humans; Interleukin-6; Interleukin-8; Lysosomal Storage Diseases; Mice; Microelectrodes; Nitric Oxide Synthase Type II; Smad3 Protein

Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasm; gamma-Glutamyltransferase; Glutathione; Humans; Hydrogen Peroxide; Interleukin-8; Leukocytes; Oxidative Stress; Respiratory Mucosa; Thiocyanates; Zinc

Cystic fibrosis (CF) at an advanced stage of the disease is characterized by airway epithelial injury and remodelling. Whether CF remodelling is related to infection and inflammation or due to an abnormal regenerative process is still undecided. We have recently established the expression and secretion profiles of interleukin (IL)-8, matrix metalloproteinase (MMP)-7, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 during non-CF airway epithelial regeneration in a humanized nude mouse xenograft model. To enhance our understanding of CF remodelling, we compared the regeneration process of non-infected human CF and non-CF nasal epithelia. In both CF and non-CF situations, epithelial regeneration was characterized by successive steps of cell adhesion and migration, proliferation, pseudostratification, and terminal differentiation. However, histological examination of the grafts showed a delay in differentiation of the CF airway epithelium. Cell proliferation was higher in the regenerating CF epithelium, and the differentiated CF epithelium exhibited a pronounced height increase and basal cell hyperplasia in comparison with non-CF epithelium. In addition, while the number of goblet cells expressing MUC5AC was similar in CF and non-CF regenerated epithelia, the number of MUC5B-immunopositive goblet cells was lower in CF grafts. The expression of human IL-8, MMP-7, MMP-9, and TIMP-1 was enhanced in CF epithelium, especially early in the regenerative process. Together, our data strongly suggest that the regeneration of human CF airway surface epithelium is characterized by remodelling, delayed differentiation, and altered pro-inflammatory and MMP responses.

Topics: Adolescent; Adult; Animals; Cell Differentiation; Cells, Cultured; Chi-Square Distribution; Cystic Fibrosis; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression; Humans; Immunohistochemistry; Interleukin-8; Male; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Nude; Rats; Rats, Wistar; Regeneration; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Tissue Inhibitor of Metalloproteinase-1; Trachea; Transplantation, Heterologous

Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the DeltaF508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073-4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl- efflux was confirmed in MPB-07-treated IB3-1 and CuFi-1 cells. In conclusion, the DeltaF508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.

Topics: Anti-Inflammatory Agents; Bronchi; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Genistein; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; NF-kappa B; Protein Binding; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolizines; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic

Stenotrophomonas maltophilia is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections and especially from patients with cystic fibrosis (CF). While clinicians feel compelled to treat infections involving this organism, its potential for virulence is not well established. We evaluated the immunostimulatory properties and overall virulence of clinical isolates of S. maltophilia using the well-characterized opportunistic pathogen Pseudomonas aeruginosa PAO1 as a control. The properties of CF isolates were examined specifically to see if they have a common phenotype. The immunostimulatory properties of S. maltophilia were studied in vitro by stimulating airway epithelial and macrophage cell lines. A neonatal mouse model of pneumonia was used to determine the rates of pneumonia, bacteremia, and mortality, as well as the inflammatory response elicited by S. maltophilia infection. Respiratory and nonrespiratory S. maltophilia isolates were highly immunostimulatory and elicited significant interleukin-8 expression by airway epithelial cells, as well as tumor necrosis factor alpha (TNF-alpha) expression by macrophages. TNF-alpha signaling appears to be important in the pathogenesis of S. maltophilia infection as less than 20% of TNFR1 null mice (compared with 100% of wild-type mice) developed pneumonia and bacteremia following intranasal inoculation. The S. maltophilia isolates were weakly invasive, and low-level bacteremia with no mortality was observed. Despite the lack of invasiveness of S. maltophilia, the immunostimulatory properties of this organism and its induction of TNF-alpha expression specifically indicate that it is likely to contribute significantly to airway inflammation.

Topics: Animals; Bacteremia; Cell Line; Cystic Fibrosis; Epithelial Cells; Gram-Negative Bacterial Infections; Humans; Interleukin-8; Lipid A; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phagocytosis; Pneumonia, Bacterial; Pseudomonas aeruginosa; Receptors, Tumor Necrosis Factor, Type I; Respiratory Mucosa; Respiratory Tract Infections; Stenotrophomonas maltophilia; Tumor Necrosis Factor-alpha

Sputum biomarkers of infection and inflammation are noninvasive measures that enable quantification of the complex pathophysiology of cystic fibrosis (CF) lung disease. Validation of these biomarkers as correlates of disease severity is a key step for their application.. We constructed a large database from four multicenter studies to quantify the strength of association between expectorated sputum biomarkers and FEV(1.). FEV(1) (range, 25-120% predicted) and quantitative data on expectorated sputum biomarkers including free neutrophil elastase, IL-8, neutrophils, Pseudomonas aeruginosa, and Staphylococcus aureus were obtained from 269 participants (ages, 9-54 years) from 33 centers. Cross-sectional and longitudinal statistical analyses were performed to estimate associations between the markers and FEV(1), including the use of multivariable analyses.. Elastase was negatively correlated with FEV(1) (correlation [r] = -0.35; 95% confidence interval [CI]: -0.46, -0.22). On average, patients with CF who differed in their elastase measurements by 0.5 log differed in their FEV(1) values by -7.3% (95% CI: -9.7, -4.6). Neutrophil counts and IL-8 were also each negatively correlated. In a multivariable regression, elastase and neutrophil counts were able to explain the majority of variation in FEV(1). Elastase was further shown to have a significant longitudinal association with FEV(1), specifically a -2.9% decline in FEV(1) (95% CI: -5.0, -0.9) per 1-log increase in elastase. Although correlated with FEV(1), bacterial densities were unable to explain clinically meaningful differences in FEV(1) within and across patients.. These data support the role of sputum biomarkers as correlates of disease severity in a diverse CF population.

Topics: Adolescent; Adult; Biomarkers; Child; Cohort Studies; Cross-Sectional Studies; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Male; Middle Aged; Neutrophils; Retrospective Studies; Sputum

There is little information on the reproducibility of measurement of cytokine levels in sputum obtained from cystic fibrosis (CF) patients. Our aim was to investigate whether assay of cytokine levels in CF sputum is reproducible or is hampered by proteolytic degradation.. Interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) levels were measured in native and spiked samples (fresh or after freezing for 15 and 23 days at -70 degrees C) obtained from nine CF patients using an immunometric assay with chemiluminescent substrate run on a IMMULITE analyzer.. For both cytokines, linearity was >0.98 for dilutions up to 1:32. After storage, cytokine concentrations in native samples varied between -2.9% and -5.6% for IL-8 and between 0.4% and 3.0% for TNF-alpha. In spiked samples, concentrations increased by 5.8%-12.6% for TNF-alpha and decreased by 3.8%-14.3% for IL-8 after 15 and 23 days of storage. In samples spiked with cytokines, the mean recovery rates for IL-8 and TNF-alpha were 109.4% and 106.3%, respectively.. Measurement of IL-8 and TNF-alpha in CF sputum is reproducible and is not hampered by freezing and thawing of samples.

Topics: Adolescent; Adult; Automation; Biomarkers; Child; Cystic Fibrosis; Female; Humans; Immunoassay; Interleukin-8; Male; Sputum; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-kappaB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-kappaB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-kappaB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1beta and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-kappaB activation, IkappaBalpha degradation, and IkappaB Kinase complex activity. CFTR-KO and wild-type mice were pretreated with parthenolide or vehicle alone then challenged intratracheally with LPS. Bronchoalveolar lavage was performed 3, 6, and 8 h later. Parthenolide pretreatment inhibited PMN influx as well as cytokine and chemokine production. This was also associated with inhibition of IkappaBalpha degradation and NF-kappaB activation. We thus conclude that parthenolide inhibits IkappaB kinase, resulting in stabilization of cytoplasmic IkappaBalpha, which in turn leads to inhibition of NF-kappaB translocation and attenuation of subsequent inflammatory responses. IkappaB kinase may be a good target, and parthenolide and/or feverfew might be promising treatments for the excessive inflammation in CF.

Topics: Active Transport, Cell Nucleus; Animals; Bronchoalveolar Lavage Fluid; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; I-kappa B Kinase; Inflammation; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Lung; Male; Mice; Mice, Knockout; NF-kappa B; Sesquiterpenes; Tumor Necrosis Factor-alpha

Transcription nuclear factor-kappaB (NF-kappaB) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-kappaB occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1beta stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1beta-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-kappaB. Within 30min, exposure to IL-1beta caused high activation of NF-kappaB, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1beta-stimulated CF cells with a series of chemical inhibitors of NF-kappaB, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1beta-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-kappaB and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.

Topics: Cell Line; Cystic Fibrosis; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Interleukin-1beta; Interleukin-8; Lung; Mitogen-Activated Protein Kinases; NF-kappa B p50 Subunit; Respiratory Mucosa; Signal Transduction

The pathophysiologic mechanisms causing inflammation in cystic fibrosis (CF) remain obscure. The effects of proapoptotic agents on pancreatic and tracheal cell lines expressing wild-type CFTR (PANC-1 and NT-1, respectively) or the homozygous CFTRDeltaF508 mutation (CFPAC-1 and CFT-2, respectively) were assessed. An increased susceptibility to apoptosis was observed in CFPAC-1 and CFT-2 cells. Apoptosis was reduced by treatment with a pan-caspase inhibitor and by incubation at 27 degrees C, allowing recruitment of CFTR deltaF508 at the plasma membrane. Inhibition of CFTR function in wild-type cells induced an increase of apoptosis. Apoptosis in CFPAC-1, but not in CFT-2 cells, was associated with overexpression of the proinflammatory mediators interleukin-6 and interleukin-8. In CF cells, apoptosis was linked to NF-kappaB pathway activation. Conditioned medium from actinomycin D-treated CFPAC-1 cells produced an increase in apoptosis of wild-type cells, suggesting that proinflammatory mediators secreted by mutant cells promote apoptosis. This was confirmed through the induction of apoptosis in wild-type cells by exogenous interleukin-6 and interleukin-8. These results suggest that CFTR deltaF508 mutation, apoptosis, and activation of the NF-kappaB pathway contribute to the self-perpetuating inflammatory cycle, at least in pancreatic cells, and provide evidence that excessive apoptosis may account for the exaggerated proinflammatory response observed in CF patients.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Caspases; Cell Membrane; Cells, Cultured; Culture Media, Conditioned; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorescent Antibody Technique; Humans; I-kappa B Proteins; Interleukin-6; Interleukin-8; Mutation; Necrosis; NF-kappa B; Pancreatic Neoplasms; Trachea

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality and may precede bacterial colonization. The aim of the present study was to investigate the molecular mechanisms underlying intrinsic inflammation in cystic fibrosis airways. Using different cystic fibrosis cell models, we first demonstrated that, beside a high constitutive nuclear factor of kappaB (NF-kappaB) activity, CF cells showed a higher activator protein-1 (AP-1) activity as compared to their respective control cells. Gene expression profiles, confirmed by RT-PCR and ELISA, showed over-expression of numerous NF-kappaB and AP-1-dependent pro-inflammatory genes in CF cells in comparison with control cells. Activation of NF-kappaB was correlated with higher inhibitor of kappaB kinase (IKK) activity. In addition, Bio-plex phosphoprotein assays revealed higher extracellular signal-regulated kinase (ERK) phosphorylation in CFT-2 cells. Inhibition of this kinase strongly decreased expression of pro-inflammatory genes coding for growth-regulated proteins (Gro-alpha, Gro-beta and Gro-gamma) and interleukins (IL-1beta, IL-6 and IL-8). Moreover, inhibition of secreted interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) with neutralizing antibodies reduced pro-inflammatory gene expression. Our data thus demonstrated for the first time that the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) at the plasma membrane leads to an intrinsic AP-1, in addition to NF-kappaB, activity and consequently to a pro-inflammatory state sustained through autocrine factors such as IL-1beta and bFGF.

Topics: Cell Line, Transformed; Chemokine CXCL1; Chemokine CXCL2; Chemokines, CXC; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Gene Expression; Genes, Reporter; HeLa Cells; Homozygote; Humans; I-kappa B Kinase; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Luciferases; Models, Biological; Mutation; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Trachea; Transcription Factor AP-1

Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury. Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF.. Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out.. There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus 41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1).. These data suggest a modifiable systemic response to i.v. antibiotics but a local sustained inflammatory response in the lung.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Biomarkers; CD11b Antigen; Cell Survival; Cystic Fibrosis; Fas Ligand Protein; fas Receptor; Histocytochemistry; Humans; Injections, Intravenous; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocyte Count; Lung Diseases; Neutrophils; Receptors, Tumor Necrosis Factor, Type I; Sputum

Airway inflammation in cystic fibrosis (CF) is exaggerated and characterized by neutrophil-mediated tissue destruction, but its genesis and mechanisms remain poorly understood. To further define the pulmonary inflammatory response, we conducted a proteome-based screen of bronchoalveolar lavage fluid (BALF) collected from young children with and without CF experiencing endobronchial infection.. We collected BALF samples from 45 children younger than 5 years and grouped them according to the presence of respiratory pathogens: > or = 1 x 10(5) colony-forming units (CFU)/mL BALF (18 and 12 samples with and without CF, respectively) and <1 x 10(5) CFU/mL (23 and 15 samples). BALF proteins were analyzed with SELDI-TOF mass spectrometry (MS) and H4 ProteinChips. Proteins were identified and characterized using trypsin digestion, tandem MS, Fourier transform ion cyclotron resonance MS, immunoblotting, and ELISA.. The SELDI-TOF MS BALF profiles contained 53 unique, reliably detected proteins. Peak intensities of 24 proteins differed significantly between the CF and non-CF samples. They included the neutrophil proteins, alpha-defensin 1 and 2, S100A8, S100A9, and S100A12, as well as novel forms of S100A8 and S100A12 with equivalent C-terminal deletions. Peak intensities of these neutrophil proteins and immunoreactive concentrations of selected examples were significantly higher in CF than non-CF samples.. Small neutrophil-derived BALF proteins, including novel C-terminal truncated forms of S100A proteins, are easily detected with SELDI-TOF MS. Concentrations of these molecules are abnormally high in early CF lung disease. The data provide new insights into CF lung disease and identify novel proteins strongly associated with CF airway inflammation.

Topics: Bronchitis; Bronchoalveolar Lavage Fluid; Child, Preschool; Colony Count, Microbial; Cystic Fibrosis; Female; Humans; Infant; Interleukin-8; Leukocyte Count; Male; Neutrophils; Proteome; S100 Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.

Topics: Bronchi; Calcium; Calcium Signaling; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Flagellin; Humans; Inflammation; Interleukin-8; Microscopy, Confocal; Mutation; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Topics: alpha 1-Antitrypsin; Animals; Cystic Fibrosis; Glycosylation; Humans; Interleukin-8; Lung; Mice; Models, Biological; Neutrophils; Receptors, Interleukin-8A; Toll-Like Receptor 2

Topics: Animals; Cystic Fibrosis; Humans; Immune System; Inflammation; Interleukin-8; Lung; Models, Biological; Neutrophils; Phagocytes; Phagocytosis; Pseudomonas aeruginosa

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies.

Topics: Biological Transport; Cell Culture Techniques; Cell Differentiation; Cell Polarity; Cell Shape; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Epithelial Cells; Fibroblasts; Humans; Interleukin-8; Mucins; Respiratory Mucosa; Stem Cells

Antimicrobial human neutrophil peptides (HNPs) play a pivotal role in innate host defense against a broad spectrum of prokaryotic pathogens. In addition, HNPs modulate cellular immune responses by producing the chemokine interleukin-8 (IL-8) in myeloid and epithelial cells and by exerting chemotaxis to T cells, immature dendritic cells, and monocytes. However, the mechanisms by which HNPs modulate the immune responses in the eukaryotic cells remain unclear. We demonstrated that, as with adenosine triphosphate (ATP) and uridine diphosphate (UDP), HNP stimulation of human lung epithelial cells selectively induced IL-8 production in 10 pro- and anti-inflammatory cytokines examined. HNP-induced IL-8 release was inhibited by treatment with the nucleotide receptor antagonists suramin and reactive blue. Transfection of lung epithelial cells with antisense oligonucleotides targeting specific purinergic P2Y receptors revealed that the P2Y6 (ligand of UDP) signaling pathway plays a predominant role in mediating HNP-induced IL-8 production.

Topics: Base Sequence; Cell Survival; Chemotaxis, Leukocyte; Cystic Fibrosis; Humans; Interleukin-8; L-Lactate Dehydrogenase; Neutrophils; Oligonucleotides, Antisense; Peptide Fragments; Receptors, Purinergic P2; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

It has been assumed that cystic fibrosis (CF) lung disease is due in part to abnormal airway mucus. Primary ciliary dyskinesia (PCD) is a form of bronchiectasis that is similar to CF in many ways but is caused by congenital defects in mucociliary clearance. Our objective was to compare the biophysical and transport properties of CF and PCD sputa in subjects matched for age and degree of lung function impairment.. PCD patients (n = 19; mean age, 9.5 +/- 3.0 years [+/- SD]; FEV1, 65.0 +/- 7.8 L) were recruited from the clinic at the Royal Brompton Hospital. Patients with CF (n = 30, mean age, 10.8 +/- 2.6 years; FEV1, 61.8 +/- 22.8 L) were identified from the Wake Forest University School of Medicine CF Center. Pulmonary function testing and sputum collection were performed as part of routine, scheduled clinic visits.. Pulmonary function was measured by spirometry, and sputum was collected during the pulmonary function test maneuver. Some patients were longitudinally assessed at visits during the course of 3 years. Sputum properties measured were dynamic viscoelasticity, wettability, cohesivity, interfacial (surface) tension, solids composition, DNA and interleukin (IL)-8 concentration, in vitro mucociliary transportability, and cough transportability.. Inflammation as measured by IL-8 concentration was three times greater in the PCD sputa (p < 0.0001). There were no significant differences in the sputum biophysical or transport properties comparing CF with PCD sputum.. It is unlikely that established CF lung disease is principally due to abnormal sputum properties, and it is more likely that the biophysical and transport properties reflect disease severity regardless of whether bronchiectasis is due to CF or PCD.

Topics: Biomarkers; Child; Cystic Fibrosis; Elasticity; Humans; Interleukin-8; Kartagener Syndrome; Mucus; Respiratory Function Tests; Severity of Illness Index; Spirometry; Sputum; Viscosity

It is not clear whether cystic fibrosis (CF) airway inflammation is a consequence of bacterial infection or is intrinsically dysregulated. The aim of this study was to investigate IL-8 secretion and NF-kappaB activity in primary respiratory epithelial cells cultured from nasal polyps obtained from CF and non-CF subjects.. NF-kappaB activity was studied by electrophoretic mobility-shift and quantitative colorimetric assays in nuclear extracts. Immunoreactive IL-8 levels were assessed by ELISA in cell culture supernatants. Both parameters were studied at baseline and following challenge with Pseudomonas aeruginosa or stimulation with pro-inflammatory cytokines.. Under basal conditions, CF cells presented a significant higher activity of NF-kappaB than non-CF cells (P=0.0004). P. aeruginosa challenge and IL-1beta/H2O2 co-stimulation caused four and two fold induction of NF-kappaB activity in non-CF and CF cells, respectively. IL-8 levels in unstimulated CF cells were significantly higher than in non-CF cells (P=0.0025). Upon incubation with P. aeruginosa and IL-1beta/H2O2, non-CF cells produced 6.3 times more IL-8 than unstimulated cells, whereas IL-8 secretion increased only of 1.4 times in CF cells.. CF respiratory epithelial cells exhibit a basal dysregulated production of IL-8 that partially correlates to enhanced NF-kappaB activity. Our data corroborate the hypothesis of a basal exaggerated inflammatory response in the CF respiratory epithelium.

Topics: Cells, Cultured; Cystic Fibrosis; Humans; Hydrogen Peroxide; Interleukin-1; Interleukin-8; Nasal Mucosa; NF-kappa B; Pseudomonas aeruginosa

Cystic fibrosis (CF) is inherited as an autosomal recessive disorder. It is caused by mutations in the protein-coding gene of chromosome 7, resulting in chronic pulmonary disease and pancreatic insufficiency. The disease affects all secretory epithelia, including the eye. The pathogenesis of ocular changes in CF is still unknown, but the involvement of immunologic processes in patients with CF has been studied in recent years. We measured interleukin-8 (IL-8) and interferon-gamma (IFN-gamma) levels in tears in a group of patients and a group of normal controls to determine if the levels of these cytokines are elevated in CF. The levels of these cytokines in tears and the clinical severity of CF and eye disease were compared. Tear samples were collected from 24 patients with CF at the department of pediatric diseases, Medical University of Bialystok, Poland. Cytokine levels were determined by ELISA. Ophthalmic examinations, including tests for keratoconjunctivitis sicca (dry eye), were used to study the ocular surface. The tear levels of IL-8 and IFN-gamma in the CF patients were significantly higher than those in controls. The clinical severity of CF correlated significantly with the IL-8 and IFN-gamma levels. We found positive correlation between the tear levels of IFN-gamma and dry eye findings in CF patients. Our results suggest that the inflammatory cytokines IL-8 and IFN-gamma may play key roles in the regulation of ocular surface inflammation and the immunologic reaction in patients with CF. The tear levels of IL-8 and IFN-gamma may be candidate markers for evaluation of the clinical status of CF and eye disease. These findings help to provide a new insight into the pathogenesis of dry eye in patients with CF and provide potential targets for therapy.

Topics: Adolescent; Adult; Biomarkers; Cystic Fibrosis; Eye Diseases; Female; Humans; Inflammation Mediators; Interferon-gamma; Interleukin-8; Male; Severity of Illness Index; Surface Properties; Tears

Chronic Pseudomonas aeruginosa lung infection is the major reason for premature death in patients with cystic fibrosis (CF). Infected patients experience a progressive deterioration of the lung tissue caused by a persistent accumulation of PMNs. We investigated if the pulmonary accumulation of PMNs is reflected as a migration of PMNs through the blood in chronically infected CF patients.. Blood and sputum samples from 37 stable, chronically (CF+P) and 6 non-infected (CF-P) CF patients without exacerbations were compared using FACS, leukocyte counting, and ELISA. Within the CF+P patients, the blood parameters were compared to the lung function (FEV1 and FVC) and to the sputum. Similar measurements were performed on 15 chronically infected CF patients before and after elective antibiotic treatment.. In the CF+P patients the concentration of G-CSF in the sera and PMNs in the blood was increased and correlated to poor lung function. However, only the concentration of G-CSF in the sera was correlated to the concentration of TNF-alpha in the sputum. After the antibiotic treatment, the lung function was improved and the concentration of PMNs in the blood and G-CSF in the sera was reduced.. G-CSF in the sera may contribute to the pulmonary inflammation in CF patients with chronic P. aeruginosa lung infection by regulating the number of PMNs available for migration and may be considered as an indicator of clinical status.

Topics: Adolescent; Adult; Blood Cell Count; Cell Movement; Cystic Fibrosis; Female; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Humans; Inflammation; Interleukin-8; Male; Middle Aged; Neutrophils; Pneumonia; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with DeltaF508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between DeltaF508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between DeltaF508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype.

Topics: Adolescent; Adult; Cystic Fibrosis; Dual Oxidases; Flavoproteins; Gene Expression; Gene Expression Profiling; Humans; Interleukin-8; NADPH Oxidases; Nasal Mucosa; Salivary Proteins and Peptides

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, DeltaF508-cystic fibrosis transmembrane regulator (CFTR), is destroyed by ERAD to cause cystic fibrosis (CF). p97/valosin-containing protein (VCP) physically interacts with gp78/autocrine motility factor receptor to couple ubiquitination, retrotranslocation, and proteasome degradation of misfolded proteins. We show here that p97/VCP and gp78 form complexes with CFTR during translocation from the ER for degradation by the cytosolic proteasome. Interference in the VCP-CFTR complex promoted accumulation of immature CFTR in the ER and partial rescue of functional chloride channels to the cell surface. Moreover, under these conditions, interleukin-8 (IL8), the expression of which is regulated by the proteasome, was reduced. Inhibition of the proteasome with bortezomib (PS-341/Velcade) also rescued CFTR, but with less efficiency, and suppressed NFkappaB-mediated IL8 activation. The inhibition of the major stress-inducible transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 together with bortezomib was most effective in repressing NFkappaB-mediated IL8 activation compared with interference of VCP, MLN-273 (proteasome inhibitor), or 4-phenylbutyrate (histone deacetylase inhibitor). Immunoprecipitation of DeltaF508-CFTR from primary CF bronchial epithelial cells confirmed the interaction with VCP and associated chaperones in CF. We conclude that VCP is an integral component of ERAD and cellular stress pathways induced by the unfolded protein response and may be central to the efficacy of CF drugs that target the ubiquitin-proteasome pathway.

Topics: Adenosine Triphosphatases; Adolescent; Cell Cycle Proteins; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Interleukin-8; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Valosin Containing Protein

Pseudomonas aeruginosa is a critical colonizer of the respiratory tract in cystic fibrosis. The chronic infections with this microorganism contribute to excessive inflammation and progressive lung damage in cystic fibrosis patients. The full repertoire of Pseudomonas products that promote inflammation in the cystic fibrosis lung is not known. Here we show that P. aeruginosa DNA released from the bacterium, but not human DNA from epithelial cells or Escherichia coli DNA, displays proinflammatory properties and induces human respiratory epithelial cells to secrete interleukin-8 (IL-8), a key chemokine causing excessive neutrophil infiltration in the cystic fibrosis lung. IL-8 secretion was not due to an increase in NF-kappaB- or activator protein-1-dependent IL-8 promoter transcription, but instead depended on p38 and Erk mitogen-activated protein kinases. No secretion of IL-8 was observed using conventional Toll-like receptor 9 ligands (CpG oligonucleotides), although it could be demonstrated that parts of the Toll-like receptor 9-signaling pathway were functional, since class B and C CpG oligonucleotide ligands stimulated production of RANTES chemokine. The IL-8 secretion in response to P. aeruginosa DNA was decreased by treatments that inhibit acidification of intracellular organelles, using chloroquine, a pH-neutralizing compound, or bafilomycin A1, an inhibitor of vacuolar H+-ATPase. These data indicate that DNA released from P. aeruginosa during chronic infections may significantly contribute to the proinflammatory processes in cystic fibrosis. Our findings also show that treatments with drugs diminishing organellar acidification may reduce the inflammatory response in cystic fibrosis.

Topics: Cells, Cultured; Chemokines; Cystic Fibrosis; DNA, Bacterial; Epithelial Cells; Humans; Hydrogen-Ion Concentration; Interleukin-8; MAP Kinase Signaling System; NF-kappa B; Oligodeoxyribonucleotides; Protein Biosynthesis; Pseudomonas aeruginosa; Signal Transduction

Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF), especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF.. This study investigated ability of NTHi to form biofilms and its impact on airway epithelia using in vivo and in vitro analyses.. We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we developed a novel in vitro coculture model of NTHi biofilm formation on polarized human airway epithelial cells grown at the air-liquid interface.. In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF, we found morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms on plastic surfaces. NTHi formed biofilms on the apical surface of cultured airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial cells remained viable throughout 4 d of coculture, and responded to NTHi with nuclear factor-kappaB signaling, and increased chemokine and cytokine secretion.. NTHi formed adherent biofilms on the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory cells exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data presented here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H. influenzae.

Topics: Biofilms; Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Coculture Techniques; Cystic Fibrosis; Gentamicins; Haemophilus influenzae; Humans; Infant; Interleukin-8; Microbial Sensitivity Tests; NF-kappa B; Respiratory Mucosa

Chronic lung rejection is characterized by obliterative bronchiolitis (OB) diagnosed based on spirometric criteria reflecting an already advanced process. Biologic markers such as bronchoalveolar lavage (BAL) neutrophilia or increased levels of chemokines (interleukin-8, RANTES [regulated on activation: normal T cell expressed and secreted]) have been proposed as early diagnosis tools. However, BAL is too invasive to be used as a routine strategy. Induced sputum (IS), however, is a non-invasive method of recovering bronchial cells.. The aim of this study was to compare BAL and IS differential cellular counts as well as IL-8 and RANTES levels between patients with bronchiolitis obliterans syndrome (BOS), recipients with good outcome and well-preserved lung function (non-BOS) and non-transplanted controls. We compared 34 BAL and IS findings obtained consecutively from 34 lung transplant recipients (LTRs), including 22 non-BOS and 12 BOS patients.. IS results were compared with 19 samples from non-transplanted controls. IS was well tolerated. There was no correlation between BAL and sputum cell populations. BAL neutrophils and IL-8 levels were increased in BOS, and these parameters were positively correlated. Moreover, BAL neutrophils and IL-8 levels were both negatively correlated with respiratory function. Sputum evaluation allows discrimination of BOS from non-BOS by the presence of higher neutrophil and eosinophil counts. Moreover, IS neutrophils and eosinophils were both correlated with lung function parameters. In contrast to BAL, IL-8 level in sputum was not a useful predictive marker of BOS development. IS RANTES levels were higher in BOS than in healthy recipients and correlated significantly with IS eosinophils.. IS and BAL provide different but complementary data. In this study, IS appeared to be a useful, non-invasive tool for LTR monitoring. Furthermore, IS provides new insights into BOS pathogenesis, especially with regard to implication of eosinophils and its chemokine, RANTES, at the bronchial level.

Topics: Adult; Bronchi; Bronchiolitis Obliterans; Bronchoalveolar Lavage Fluid; Cell Count; Chemokine CCL5; Cystic Fibrosis; Female; Graft Rejection; Heart-Lung Transplantation; Humans; Immunosuppressive Agents; Interleukin-8; Lung Transplantation; Male; Middle Aged; Neutrophils; Respiratory Function Tests; Sputum

Polymorphonuclear neutrophil (PMN)-dominated inflammation is prominent in the airways of subjects with cystic fibrosis (CF) and chronic bronchitis (CB). Interleukin (IL)-8, myeloperoxidase (MPO), and DNA are markers of neutrophilic inflammation. We hypothesized that sputum MPO, DNA, and IL-8 concentrations would negatively correlate with pulmonary function and sputum transportability.. We measured pulmonary function and analyzed sputum IL-8, MPO, and DNA concentrations, as well as the transport properties of sputum samples obtained from 16 subjects with CF and 15 subjects with CB. We also evaluated changes in these measurements in paired sputum samples from these subjects obtained 2 to 12 months apart.. IL-8 and MPO concentrations in the sputum of CF subjects was inversely correlated with FEV(1) percent predicted (IL-8: r = -0.40; p = 0.003; MPO: r = -0.38; p = 0.003) and FVC percent predicted (IL-8: r = -0.4; p = 0.02; MPO: r = -0.4; p = 0.02). IL-8 and DNA concentrations were inversely correlated with sputum cough transportability (CTR) [IL-8: r = -0.4; p = 0.02; DNA: r = -0.36; p = 0.048]. Changes in DNA concentration in sputum samples from CF subjects over time were inversely correlated with changes in FEV(1) percent predicted (r = -0.58; p = 0.02), FVC percent predicted (r = -0.74; p = 0.002), and CTR (r = -0.59; p = 0.02). There was no correlation among pulmonary function, sputum properties, and inflammatory markers in the sputum from subjects with CB.. The sputum concentrations of IL-8, MPO, and DNA appear to be closely associated with pulmonary function in subjects with CF but not in subjects with CB.

Topics: Adolescent; Adult; Aged; Biomarkers; Bronchitis, Chronic; Child; Cough; Cystic Fibrosis; DNA; Enzyme-Linked Immunosorbent Assay; Female; Forced Expiratory Volume; Humans; Interleukin-8; Male; Middle Aged; Peroxidase; Respiratory Function Tests; Severity of Illness Index; Sputum

Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation.

Topics: Bacterial Outer Membrane Proteins; Cystic Fibrosis; Electrophoresis, Gel, Two-Dimensional; Epithelial Cells; Humans; Interleukin-8; Lung; Mass Spectrometry; Pseudomonas aeruginosa; Pseudomonas Infections; Secretory Vesicles

Leptin plays an important role in the energy balance and may be affected by hormonal and metabolic derangement associated with chronic disease. The aim of this study was to assess the correlation between leptin, proinflammatory cytokines and nutritional status with regard to clinical status in homozygous delta F 508 cystic fibrosis patients.. Patients with mild (Shwachman score 71-100 points, group A) disease were compared with those with moderate disease (Shwachman score 41-55 points, group B) and age-matched controls (group C, n = 22). Leptin was assessed by enzyme-linked immunosorbent assay and cytokines (interleukin-8, tumor necrosis factor alpha) before and after stimulation with 5 ng lipopolysaccharide by a chemiluminescent immunometric assay.. Twenty-two patients were recruited for each group (median A/B/C forced expiratory volume in 1 second 80%/59%/-; median age 12/13.5/12.5 years). Leptin (median 3.25/2.65/3.3 pg/mL; P = 0.083) and body mass index were lower (group A/B/C 18.55/16.75/20.5 kg/m(2); P = 0.023), but dietary intake was significantly higher (group A/B/C 50.5/68/43 kcal/kg body weight; P = 0.026) in moderate disease. Cytokines before stimulation with lipopolysaccharide were highest in moderate disease, but there was no significant difference after stimulation (interleukin-8 median A/B/C before--15/25.1/8.0 pg/mL, P < 0.005; after--570.5/573.5/415.5 pg/mL, not significant; tumor necrosis factor alpha median A/B/C 43/56/30 pg/mL, P < 0.0001; 580/427/720.5 pg/mL, not significant.).. There is a physiological regulation of leptin even in more advanced states of disease with significantly lower body mass index than controls. However, our data do not support the idea of elevated cytokine levels inducing anorexia in homozygous delta F 508 cystic fibrosis patients.

Topics: Adolescent; Adult; Anorexia; Body Mass Index; Case-Control Studies; Child; Child, Preschool; Cystic Fibrosis; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Interleukin-8; Leptin; Lipopolysaccharides; Luminescence; Male; Nutritional Status; Severity of Illness Index; Tumor Necrosis Factor-alpha

Neutrophil elastase in the cystic fibrosis airways inhibits opsonophagocytosis and induces the expression of interleukin-8, a neutrophil chemoattractant. Prolastin is a therapeutic preparation of alpha-1 proteinase inhibitor (alpha1,-PI), a neutrophil elastase inhibitor. The objective of this study was to determine the effects of Prolastin aerosol therapy on airway inflammation in cystic fibrosis.. The primary endpoint of this study was sputum taurine, an amino-acid present in high concentrations in neutrophils. Sputum taurine correlates with respiratory exacerbations of cystic fibrosis. Seventeen patients with cystic fibrosis were each assigned to three sequential 10-day periods including first, aerosol therapy of 5 ml saline solution bid; second, aerosol therapy of 250 mg Prolastin bid; third, no aerosol therapy. On days 8, 9 and 10 of each period, early morning sputum was collected for the quantification of alpha1-PI, neutrophil elastase activity, IL-8 and taurine.. During Prolastin therapy, a 3-fold increase in sputum alpha1-PI was observed (P = 0.002). Baseline values of sputum alpha1-PI correlated with the values obtained after Prolastin aerosol (R = 0.77, P < 0.01). Sputum neutrophil elastase activity remained unchanged but taurine decreased after Prolastin therapy (during therapy P = 0.052, after therapy P = 0.026). Prolastin aerosol therapy had no adverse effect on pulmonary function.. Aerosol therapy with Prolastin in patients with cystic fibrosis leads to a progressive decrease in sputum taurine. This suggests that even in the absence of sustained elastase inhibition, Prolastin aerosol therapy may have a beneficial effect on airway inflammation in patients with cystic fibrosis.

Topics: Administration, Inhalation; Adolescent; Adult; alpha 1-Antitrypsin; Cystic Fibrosis; Female; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Male; Sputum; Taurine

To evaluate IL-8 concentration in tears fluid in cystic fibrosis patients.. Tears samples were collected from 18 CF Patients and 18 normal controls. Cytokine level was determined by ELISA.. A significant increase of IL-8 concentration in tears fluid was found in CF patients compared with controls.. Increase IL-8 concentration in tears fluid in CF patients suggests a role of immunologic processes in the pathogenesis of the ocular changes, and may be a marker of the inflammatory status in ocular surface in cystic fibrosis.

Topics: Adolescent; Adult; Biomarkers; Child; Cystic Fibrosis; Eye; Female; Humans; Interleukin-8; Male; Reference Values; Tears

We aimed at identifying molecular mechanisms for anti-inflammatory effects of azithromycin (AZM) suggested by clinical evidences. IL-8 expression and DNA binding activity of two key pro-inflammatory transcription factors (TF), NF-kappaB and AP-1, were investigated in cystic fibrosis (CF) and isogenic non-CF airway epithelial cell lines. AZM reduced about 40% of IL-8 mRNA and protein expression (n=9, p=0.02, and n=4, p=0.00011) in CF cells reaching the levels of non-CF cells. In the presence of AZM we found about 50% and 70% reduction of NF-kappaB and AP-1 DNA binding, respectively (n=3, p=0.01, and n=3, p=0.0017), leading to levels of non-CF cells. The relevance of NF-kappaB and AP-1 in regulating IL-8 promoter transcriptional activity was demonstrated by gene reporter assays (n=4, p=8.54x10(-7), and n=4, p=6.45x10(-6)). Our data support the anti-inflammatory effects of AZM in CF cells, indicating inhibition of transcription of pro-inflammatory genes as possible mechanism, thus providing a rationale for the possible use of specific TF inhibitors for therapy.

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents; Azithromycin; Cell Line; Cystic Fibrosis; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Interleukin-8; NF-kappa B; Transcription Factor AP-1

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-kappaB (NF-kappaB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-kappaB in cells isolated from five CF (DeltaF508/DeltaF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-kappaB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-kappaB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-kappaB inhibitor IkappaBalpha were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-kappaB to nuclei of primary CF epithelial cell cultures, but intranuclear IkappaBalpha may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

Topics: Biological Transport; Cell Extracts; Cell Nucleus; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Gene Expression; Humans; I-kappa B Proteins; Interleukin-8; Lung; NF-kappa B; Protein Isoforms; Pseudomonas aeruginosa; RNA, Messenger; Transcription Factor RelA

Cytokines and polymorphonuclear leukocytes play a key role in immune mediated inflammation in progressive pulmonary damage due to cystic fibrosis. The aim of this study is to establish a simple measure of the host's propensity to secrete inflammatory cytokines and to correlate this with clinical status. Patients (n=44, median age 16 years) with the DeltaF 508 mutation (homozygous) were grouped according to their Shwachman score: Patients with mild disease (Shwachman score 71-100 points, group A, n=22, median FEV(1) 79%) were compared with those with more severe disease (Shwachman score 41-55 points, group B, n=22, median FEV(1) 55%) and age-matched controls (group C, n=22, median FEV(1) 102%). Whole blood was stimulated with 5 ng of lipopolysaccharide (LPS). Interleukin-8 (IL-8) was measured by chemiluminescent immunometric assay (DPC, Bad Nauheim, Germany). Though there was a significant difference at baseline for IL-8 (median group A/B/C 6.1/30.5/5.8 pg/ml; p<0.001), there was no significant difference after stimulation. Moreover, in Pseudomonas aeruginosa positive (Psa+) patients (n=26) there was a significant negative correlation (r=-0.539; p<0.004) between baseline IL-8 and FEV(1) (%). Clinical course and lung function (in Psa+) correlate with IL-8 levels.

Topics: Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Cytokines; Female; Homozygote; Humans; Immunoassay; Infant; Inflammation; Interleukin-8; Lipopolysaccharides; Lung; Male; Middle Aged; Mutation; Neutrophils; Pseudomonas aeruginosa; Spirometry

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o(-), which is homozygous for the DeltaF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o(-). Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o(-) cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-kappaB-linked reporter gene and increased IL-8 protein production in CFTE29o(-) cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.

Topics: Adaptor Proteins, Signal Transducing; Antigens, Differentiation; Bacterial Proteins; Bronchoalveolar Lavage Fluid; Cell Line; CpG Islands; Cystic Fibrosis; Humans; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipopeptides; Lipopolysaccharides; Lipoproteins; Membrane Glycoproteins; Myeloid Differentiation Factor 88; NF-kappa B; Oligodeoxyribonucleotides; Oligopeptides; Pseudomonas Infections; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Interleukin-1; Respiratory Mucosa; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 9; Toll-Like Receptors; U937 Cells; Up-Regulation

Lung disease in cystic fibrosis (CF) is established in early childhood with recurrent bacterial infections and inflammation. Using spirometry, the effect of this early lung damage cannot be measured until a child is 6 years of age when some irreversible lung damage may already have occurred. Techniques for measurement of lung function in infants and young children include raised volume rapid thoracic compression (RVRTC) and low frequency forced oscillation (LFFOT). The aim of this study was to investigate the role of inflammation and infection on a population of infants and young children with CF and to determine whether lung function in this population (measured by LFFOT) is affected by early lung disease.. Lung function was measured by LFFOT in 24 children undergoing bronchoalveolar lavage (BAL) on 27 occasions as part of an annual programme while still under general anaesthesia. Following lung function testing, three aliquots of saline were instilled into the right middle or lower lobe. The first aliquot retrieved was processed for the detection of microbes, and the remaining aliquots were pooled to assess inflammatory markers (cytology, IL-8, NE, LTB(4)).. Inflammation (percentage and number of neutrophils) was significantly higher in children with infections (p<0.001, p = 0.04, respectively), but not in those with symptoms. Several markers of inflammation significantly correlated with LFFOT parameters (R, G, and eta).. Infections and inflammation are established before symptoms are apparent. Inflammation is correlated with measures of parenchymal changes in lung function measured by LFFOT.

Topics: Airway Resistance; Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Female; Humans; Infant; Interleukin-8; Male; Oscillometry; Pneumonia; Respiratory Function Tests; Respiratory Tract Infections

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.

Topics: Adolescent; Adult; Calcium; Cells, Cultured; Cystic Fibrosis; Female; Humans; Inflammation; Interleukin-8; Intracellular Fluid; Male; Middle Aged; Respiratory Mucosa

The aim of this study was to determine whether nasal inflammation reflects pulmonary inflammation in young children with cystic fibrosis (CF), as assessed by inflammatory markers in nasal wash (NW) and bronchoalveolar lavage (BAL), respectively.. CF patients younger than 6 years of age who were to undergo bronchoscopy for routine BAL from May 2000 to October 2001 were recruited for this study. NW was collected immediately after the patient was sedated for bronchoscopy. Total cell counts (TCC), differential cell counts and interleukin (IL)-8 levels (enzyme-linked immunosorbent assay) were assessed in NW and BAL.. In total, 19 children with CF (mean age, 1.9 years; SD, 1.7 years) were included in the study. There was a significant relationship between IL-8 and the percentages of neutrophils in NW (r (2) = 0.76; P < 0.001) and in BAL fluid (r 2 = 0.62; P = 0.006). Similarly, IL-8 concentrations in the NW correlated with those in the BAL (r 2 = 0.48; P = 0.036) and neutrophil percentages in NW correlated significantly with those in BAL (r 2 = 0.7; P = 0.004).. When measured under 'ideal' conditions, nasal IL-8 reflects lower airway levels and may reflect the inflammatory stimulus that results in neutrophilic inflammation. These data encourage further assessment of nasal wash under clinically appropriate conditions to determine its utility for assessing inflammation in young children with CF.

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Cell Count; Chemokines, CXC; Child, Preschool; Cystic Fibrosis; Early Diagnosis; Female; Humans; Infant; Interleukin-8; Male; Nasal Lavage Fluid; Nasal Provocation Tests; Neutrophils; Pneumonia

The dysfunction of the mucosal interface of the upper respiratory tract in cystic fibrosis (CF) patients is clinically visible by the development of nasal polyps (NP) at a young age. Innate defence markers and inflammatory mediators in NP from patients with CF were compared with non-cystic fibrosis nasal polyps (non-CF-NP) to determine a possible different immunological background in macroscopically similar tissue.. Surgical samples were obtained from patients with non-CF-NP, cystic fibrosis patients with nasal polyps (CF-NP) and control patients (CO). With real time PCR, the mRNA expression of human beta defensins (HBD) 2 and 3, toll-like receptors (TLR) 2 and 4 and the macrophage mannose receptor (MMR) were measured. On homogenates of the surgical samples eotaxin, myeloperoxidase (MPO), IL-5 and IL-8 protein content was measured using commercial ELISA kits; IgE and eosinophilic cationic protein (ECP) were measured by the Unicap system.. In CF-NP we found a statistically significant higher mRNA expression of HBD 2 compared with non-CF-NP and CO and of TLR 2 compared with non-CF-NP. In the non-CF-NP group, MMR mRNA expression was significantly elevated compared with CO and CF-NP. For TLR 4 mRNA expression no statistically significant differences were found between groups. IL-5 was below detection level in all CO and CF-NP, but was measurable in 80% of the non-CF-NP. MPO and IL-8 concentrations were significantly higher in CF-NP compared with CO and non-CF-NP, whereas ECP, eotaxin and IgE were significantly higher in the non-CF-NP group.. We here demonstrate that CF-NP and non-CF-NP not only differ in terms of inflammatory mediator profile, but also in terms of innate markers.

Topics: Anti-Infective Agents; beta-Defensins; Biomarkers; Cystic Fibrosis; Humans; Inflammation Mediators; Interleukin-5; Interleukin-8; Lectins, C-Type; Macrophages; Mannose Receptor; Mannose-Binding Lectins; Membrane Glycoproteins; Nasal Polyps; Peroxidase; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

The aim of this study was to evaluate the anti-inflammatory properties of intermittent inhaled tobramycin.. To establish this, we initiated a prospective study to measure the concentration of the three pro-inflammatory cytokines IL-8, IL-6 and TNF-alpha in the sputum from 20 cystic fibrosis (CF) patients (15 teenagers and 5 young adults) during cycles and off cycles.. A significant decrease in IL-8 (P = 0.001) and a more moderate decrease in IL-6 (P = 0.046) and TNF-alpha (P = 0.052) levels were observed during cycles, even if no significant decrease in the number of leucocytes was observed.. These results associated with a decrease in the Pseudomonas aeruginosa population can contribute in part to the beneficial effect of intermittent inhaled tobramycin on pulmonary function.

Topics: Administration, Inhalation; Adolescent; Adult; Cystic Fibrosis; Cytokines; Humans; Interleukin-6; Interleukin-8; Prospective Studies; Pseudomonas aeruginosa; Sputum; Tobramycin; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is a common, lethal genetic disease, which is due to mutations in the CFTR gene. The CF lung expresses a profoundly proinflammatory phenotype, due to constitutive hypersecretion of IL-8 from epithelial cells lining the airways. In a systematic search for candidate drugs that might be used therapeutically to suppress IL-8 secretion from these cells, we have identified a potent and efficacious series of amphiphilic pyridinium salts. The most potent of these salts is MRS2481, an (R)-1-phenylpropionic acid ester, with an IC50 of ca. 1microM. We have synthesized 21 analogues of MRS2481, which have proven sufficient to develop a preliminary structure-activity relationship (SAR). For optimal activity, we have found that the ester must be connected to the pyridinium derivative by an eight-carbon chain. An optical isomer of the lead compound, containing an (S)-1-phenylpropionic acid ester, has been found to be a much less active. The mechanism of action of MRS2481 appears to involve inhibition of signaling of the NF(kappa)B and AP-1 transcription factors to the IL-8 promoter. MRS2481 is a potent inhibitor of TNFalpha-induced phosphorylation and proteosomal destruction of I(kappa)B(alpha). Inasmuch as I(kappa)B(alpha) is the principal inhibitor of the NF(kappa)B signaling pathway, preservation of intact I(kappa)B(alpha) would serve to keep the IL-8 promoter silent. We also find that MRS2481 blocks TNF(alpha)-activated phosphorylation of JNK, the c-JUN kinase. The IL-8 promoter is also activated by an AP-1 site, which requires a phospho-c-JUN/c-FOS dimer for activity. We therefore interpret these data to suggest that the mechanism of MRS2481 action is to inhibit both NF(kappa)B and AP-1 signaling on the IL-8 promoter. Given the medicinally promising properties of water-solubility, potency in the low muM concentration range, and high efficacy, we anticipate that MRS2481, or a further optimized derivative, may find an important place in the armamentarium of pharmaceutical strategies yet to be arrayed against the inflammatory phenotype of the CF lung.

Topics: Cystic Fibrosis; Dose-Response Relationship, Drug; HeLa Cells; Humans; Interleukin-8; NF-kappa B; Pyridinium Compounds; Respiratory Mucosa; Salts; Signal Transduction; Surface-Active Agents; Tumor Necrosis Factor-alpha

Pulmonary disease in cystic fibrosis (CF) is characterized by an exaggerated interleukin (IL)-8-driven, neutrophilic, inflammatory response to infection. Binding of IL-8 to heparan sulfate (HS)-containing proteoglycans (HSPG) facilitates binding of the chemokine to its specific receptor, stabilizes and prolongs IL-8 activity, and protects it from proteolysis. We hypothesized that increased expression of HSPG contributes to the sustained inflammatory response in CF bronchial tissue.. Our objectives were to analyze the distribution and abundance of IL-8 and HS, in intact and cleaved forms, in bronchial tissue from adult patients with CF or chronic obstructive pulmonary disease (COPD) and a control group without inflammatory airway disease.. Immunostaining and quantitative image analysis were applied to ethanol-fixed and paraffin-embedded tissue obtained at transplant in patients with CF or COPD, or postmortem in the control group.. Quantitative immunohistochemical analysis demonstrated significant disease-related differences. Intact HS was significantly more abundant in epithelial and endothelial basement membranes in CF than in COPD or the control group. Conversely, cleaved HS was significantly more abundant in COPD than the other groups. More IL-8-positive blood vessels were observed in CF and COPD compared with the control group, whereas more extensive IL-8 expression in the epithelium was observed in CF compared with COPD.. Sustained neutrophil recruitment in the CF airway may therefore be related not only to increased IL-8 expression but also to the increased stability and prolonged activity and retention of IL-8 when it is bound to HSPG in bronchial tissue.

Topics: Adult; Cystic Fibrosis; Endothelium, Vascular; Female; Heparan Sulfate Proteoglycans; Humans; Immunohistochemistry; Interleukin-8; Male; Middle Aged; Neutrophil Infiltration; Pulmonary Disease, Chronic Obstructive

Cystic fibrosis (CF) airways are characterised by chronic inflammation, increased interleukin (IL)-8 secretion, and neutrophil activation which are considered the principal factors of morbidity and mortality in CF patients. Optimising management of this chronic inflammatory response is therefore a key issue of basic and clinical CF research. Several reports have addressed ways to manage CF airways inflammation, and an attractive therapeutic strategy may be the inhibition of the p38-mitogen activated protein kinase (p38-MAP-k) pathway.. A new ex vivo model was used to study the mucosal inflammatory response to environmental airways stimuli. Nasal biopsy tissues from CF patients and controls were cultured ex vivo for 20 minutes, 4 hours, and 24 hours in the presence of lipopolysaccharide (LPS) from Pseudomonas aeruginosa (PA) with and without the p38-MAP-k inhibitor SB203580. Quantitative mRNA assessment, immunohistochemistry, and Western blots were used to detect the expression and modulation of inflammatory markers.. PA-LPS challenge induced a time dependent mucosal inflammation indicated by rapid epithelial activation, IL-8 release, COX-2 upregulation, and neutrophil migration to the upper mucosal layers. Some of these LPS induced changes (IL-8 release and neutrophil migration) were specific to CF tissues. SB203580 significantly controlled all LPS induced mucosal changes in CF tissues.. These findings provide a rationale and proof of principle for the potential use of p38-MAP-k inhibitors to control inflammation in patients with CF.

Topics: Adolescent; Adult; Blotting, Western; Bronchitis; Cells, Cultured; Cyclooxygenase 2; Cystic Fibrosis; Female; Humans; Interleukin-8; Lipopolysaccharides; Male; Membrane Proteins; p38 Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Pseudomonas aeruginosa; Respiratory Mucosa; RNA, Messenger

Recurrent pulmonary exacerbations are associated with progressive lung disease in cystic fibrosis (CF). Current definitions of an exacerbation, although not precisely defined, include new/worsening symptoms, declining lung function, and/or changing radiologic appearance. Early diagnosis of exacerbations by rapid noninvasive means should expedite therapeutic intervention, thereby minimizing lung damage.. To identify biomarkers of lung exacerbation for point-of-care monitoring of CF lung disease progression.. Saline-induced sputum was collected from adults with CF with an exacerbation and requiring hospitalization (FEV(1) < 60%), a subset of these adults at hospital discharge, children with stable CF and preserved lung function (FEV(1) > 70%), and control subjects (FEV(1) > 80%). Sputum was arrayed by two-dimensional electrophoresis and differentially expressed proteins were identified by proteomic analysis.. Sputum profiles from adults with CF with an exacerbation were characterized by extensive proteolytic degradation and influx of inflammation-related proteins, with some adults with CF approaching a "healthy" protein profile after hospitalization. Two children with CF showed profiles and biomarker expression resembling those of adults with an exacerbation. Levels of differentially expressed myeloperoxidase, cleaved alpha(1)-antitrypsin, IgG degradation, interleukin-8, and total protein concentration, together with their correlation to FEV(1), were statistically significant. Statistical correlation analyses indicated that changes in myeloperoxidase expression and IgG degradation were the strongest predictors of FEV(1).. We identified extensive protein degradation and differentially expressed proteins as biomarkers of inflammation relating to pulmonary exacerbations. Prediction of exacerbation onset and more precise evaluation of the extent of resolution with treatment could be achieved by including biomarkers in standard assessment.

Topics: Adolescent; Adult; alpha 1-Antitrypsin; Biomarkers; Blotting, Western; Child; Cystic Fibrosis; Disease Progression; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Forced Expiratory Volume; Humans; Immunoglobulin G; Interleukin-8; Male; Mass Spectrometry; Peroxidase; Prognosis; Proteomics; Severity of Illness Index; Sputum

A role of autoreactive T cells for type 1 diabetes pathogenesis is considered crucial. In our pilot study we addressed if autoreactive mononuclear cells are present also in peripheral blood of patients with other specific forms of diabetes as cystic fibrosis related diabetes (CFRD).. Cellular immune responses to a known beta-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-gamma) and by protein microarray analysis in four patients suffering from CFRD, in four cystic fibrosis (CF) patients without diabetes, in eight type 1 diabetes patients (without CF) and in four healthy controls.. Response to the autoantigen GAD65 (protein and peptides) was observed in 7/8 patients suffering from CF and in all type 1 diabetes patients. Post-stimulation production of Th1 cytokines (IFN-gamma, TNF-beta) was observed in 2/4 CFRD, 1/4 CF patients and in 7/8 type 1 diabetes patients. All these patients carry prodiabetogenic HLA-DQ genotype. Th2- and Th3 type of cytokine pattern was observed in 2/4 CF patients. Production of IL-8 was observed in the third CFRD as well as in the third CF patient and in 1/8 type 1 diabetes patient and borderline production of this chemokine was also observed in 2/4 healthy controls. No reaction was observed in the other 2/4 healthy controls and in the fourth CFRD patient who carried a strongly protective genotype and did not produce autoantibodies. The most potent peptide of GAD65 was amino acids 509-528.. We consider our observations as a sign of a reaction directed against the self-antigen GAD65 that are closely connected to type 1 diabetes. In CF patients who do not develop diabetes autoreactive mechanisms are very probably efficiently suppressed by immune self-tolerance mechanisms. CFRD patients are a heterogenic group. To disclose those who may display features of autoimmune diabetes could have an impact for their therapy and prognosis.

Topics: Adolescent; Adult; Autoantibodies; Child; Cystic Fibrosis; Diabetes Mellitus; Female; Glutamate Decarboxylase; HLA-DQ Antigens; Humans; Interferon-gamma; Interleukin-8; Isoenzymes; Leukocytes, Mononuclear; Lymphotoxin-alpha; Male; Pilot Projects; Protein Array Analysis

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

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

Topics: Adult; Asthma; Biomarkers; Child; Cystic Fibrosis; Female; Glycosylation; Humans; Interleukin-8; Male; Phenotype; Probability; Prognosis; Reference Values; Respiratory Mucosa; Secretory Component; Sensitivity and Specificity; Severity of Illness Index; Sputum

It is controversial whether mutations in cystic fibrosis transmembrane conductance regulator intrinsically dysregulate inflammation. We characterized passage 2 human tracheobronchial epithelial cell cultures morphologically and physiologically and determined whether cytokine production or nuclear factor-kappaB activation was systematically altered in cystic fibrosis (CF) cells. Non-CF and CF cells originating from a total of 33 and 25 lungs, respectively, were available for culture on plastic or at an air-liquid interface until well differentiated. Forskolin-stimulated short-circuit currents were present in representative polarized non-CF cultures and were absent in CF cultures, whereas uridine 5'-triphosphate-stimulated currents were present in both. Constitutive or interleukin (IL)-1beta-induced IL-8 or IL-6 secretion or nuclear factor-kappaB activity was not significantly different between non-CF and CF cells. The cytokines regulated upon activation, normal T cell expressed and secreted (RANTES) and IL-10 were not detectable. Stimulation with tumor necrosis factor-alpha or a synthetic toll-like receptor 2 agonist or variable doses and times of Staphylococcus aureus culture filtrate revealed a single dose- and time-dependent difference in IL-8 production by CF cells. Interestingly, although IL-8 secretion after stimulation with Pseudomonas aeruginosa filtrates was not greater in CF cells in the absence of human serum, it was variably greater in its presence. Thus, although exaggerated responses may develop under certain conditions, our results do not support an overall intrinsically hyperinflammatory phenotype in CF cells.

Topics: Adolescent; Adult; Aged; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Chemokine CCL5; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Female; Genotype; Humans; Inflammation; Interleukin-10; Interleukin-8; Male; Middle Aged; Mutation; NF-kappa B; Phenotype; Respiratory Mucosa; Tumor Necrosis Factor-alpha

It is well documented that patients with cystic fibrosis (CF) are unable to clear persistent airway infections in spite of strong local inflammation, suggesting a dysregulation of immunity in CF. We and others have reported previously that T lymphocytes may play a prominent role in this immune imbalance. In the present work, we compared the reactivity of CD3+ T cells obtained from young CF patients in stable clinical conditions (n = 10, aged 9-16.5 years) to age-matched healthy subjects (n = 6, aged 9-13.5 years). Intracellular levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-8 and IL-10 were determined by flow cytometry after whole blood culture. The data identified T lymphocyte subsets producing either low levels (M1) or high levels (M2) of cytokine under steady-state conditions. We found that the production of IFN-gamma and IL-10 by T lymphocytes was similar between young CF patients and healthy subjects. In contrast, after 4 h of activation with PMA and ionomycin, the percentage of T cells producing high levels of IL-2 (M2) was greater in CF patients (P = 0.02). Moreover, T cells from CF patients produced lower levels of IL-8, before and after activation (P = 0.007). We conclude that a systemic immune imbalance is present in young CF patients, even when clinically stable. This disorder is characterized by the capability of circulating T lymphocytes to produce low levels of IL-8 and by the emergence of more numerous T cells producing high levels of IL-2. This imbalance may contribute to immune dysregulation in CF.

Topics: Adolescent; CD3 Complex; Child; Cystic Fibrosis; Cytokines; Cytoplasm; Female; Flow Cytometry; Humans; Interleukin-2; Interleukin-8; Lymphocyte Activation; Male; T-Lymphocyte Subsets

Controversy exists concerning abnormalities of the nitric oxide (NO) pathway in cystic fibrosis (CF) lung disease. Although some studies suggested that NO activity is impaired in CF, changes in NO production in young children have not been studied. We hypothesized that nitric oxide synthase (NOS II) expression is decreased in young children with CF, leading to decreased production of lower airway NO, and that decreased NOS II expression is related to airway inflammation. Accordingly, we measured lower airway exhaled NO, nitrate, and NOS II expression in airway epithelium and macrophages by bronchoscopy, bronchoalveolar lavage (BAL), and bronchial brushing in 13 children with CF, 4 adolescent patients with CF, and 14 disease control children. Lower airway NO and nitrate were not different between CF and disease controls. Immunostaining studies of NOS II expression in airway epithelial cells and macrophages were similar in CF and control patients. Within the CF group, however, expression of NOS II was inversely related to BAL neutrophil counts and IL-8, two markers of airway inflammation. We conclude that lower airway NO, nitrate levels, and NOS II expression are not different in young children with CF and disease control patients, but that NOS II expression decreases in CF as airway inflammation increases.

Topics: Adolescent; Adult; Bronchoalveolar Lavage Fluid; Case-Control Studies; Child; Child, Preschool; Cystic Fibrosis; Female; Humans; Immunohistochemistry; Infant; Interleukin-8; Male; Neutrophils; Nitric Oxide Synthase

Bronchoalveolar lavage fluid (BALF) in cystic fibrosis (CF) shows increased inflammation, which could be due to abnormal cytokine regulation. Bronchial epithelial cells and migratory inflammatory cells produce these cytokines, but few quantitative in vivo data are available comparing young CF patients with controls. We hypothesized that IL-8 mRNA abundance was higher in young CF vs. non-CF disease control patients in lung epithelium and inflammatory cells. Bronchial epithelial cells (BEC) were obtained by brush biopsy, and airway inflammatory cells (BALFC) by bronchoalveolar lavage, in 17 CF and 21 non-CF patients <5 years old undergoing clinically indicated bronchoscopy. Cellular mRNA expression was quantified by real-time PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Abundance of IL-8/GAPDH in BEC was significantly higher in CF (14.8 +/- 3.3) than non-CF (4.2 +/- 0.6) samples, and this difference was also significant when patients were stratified according to infection. In BALFC, the difference in IL-8 expression did not reach statistical significance: CF (17.1 +/- 6.5) vs. non-CF (6.8 +/- 1.9), but BALF cell number/ml was significantly higher in CF. IL-10 mRNA was very low in all samples, without showing a decrease in CF vs. non-CF patients. We conclude that early in the disease, IL-8 mRNA expression in BEC is increased in CF in vivo. Although IL-8 mRNA in migratory cells was not significantly higher in CF, these cells may still contribute to elevated IL-8 in airway secretions, secondary to increased cell density in BALF.

Topics: Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Humans; Infant; Interleukin-10; Interleukin-8; Lung Diseases; RNA, Messenger

Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.

Topics: Apoptosis; Bacterial Adhesion; Cell Line; Cell Polarity; Colony Count, Microbial; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasm; Epithelial Cells; Gene Deletion; Humans; Interleukin-8; Microscopy, Confocal; Microscopy, Electron; Mutation; Pseudomonas aeruginosa; Respiratory Mucosa

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-kappa B (I kappa B alpha). I kappa B alpha phosphorylation is a required step in the activation of the NF-kappa B signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF.

Topics: Cardiac Glycosides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Digitoxin; Enzyme Inhibitors; Epithelium; Genetic Therapy; I-kappa B Proteins; Interleukin-8; Lung

Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (10(4) to 10(5) CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients.

Topics: Burkholderia; Burkholderia Infections; Cystic Fibrosis; Cytokines; Epithelial Cells; Fluorescent Antibody Technique; Humans; Interleukin-8; Microscopy, Electron; Mucins; Mucus; Permeability; Respiratory System

Studies on mucociliary clearance (MCC) in cystic fibrosis (CF) have produced conflicting results. This study aimed to differentiate primary (ion transport-related) from secondary (inflammatory) causes of delayed MCC in CF. Nasal MCC was measured in 50 children (CF, primary ciliary dyskinesia (PCD) and no respiratory disease). Nasal lavage fluid was analysed for interleukin (IL)-8 and tumour necrosis factor-alpha. Similar measurements were obtained in adult CF patients with and without chronic sinusitis (CS). Children with CF had neither delayed MCC nor increased levels of cytokines. Conversely, children with PCD had prolonged MCC times (all >30 min) and significantly raised levels of IL-8. CS-positive CF adults had significantly slower MCC than CS-negative subjects, but IL-8 levels were low and similar in both groups. Decreased airway surface liquid and delayed mucociliary clearance are the postulated primary mechanisms in cystic fibrosis. However, the current study reports that cystic fibrosis children have normal nasal mucociliary clearance. Abnormalities appeared in cystic fibrosis adults with symptoms of chronic sinus disease, suggesting a secondary rather than primary phenomenon. Studies to explore this mechanism in the distal, more sparsely-ciliated airways could aid an understanding of pathogenesis and the development of new treatments.

Topics: Adolescent; Adult; Age Factors; Case-Control Studies; Child; Child, Preschool; Ciliary Motility Disorders; Cystic Fibrosis; Female; Follow-Up Studies; Humans; Incidence; Interleukin-8; Male; Middle Aged; Mucociliary Clearance; Nasal Lavage Fluid; Nasal Mucosa; Probability; Reference Values; Risk Assessment; Severity of Illness Index; Sex Factors; Statistics, Nonparametric; Tumor Necrosis Factor-alpha

Morbidity and mortality in cystic fibrosis patients is mainly attributed to pulmonary infection and inflammation. Chemokines play a pivotal role in the inflammatory process. Although genotype-phenotype correlation in cystic fibrosis patients has been defined, a clear relationship between the defect in the cystic fibrosis transmembrane regulator (CFTR) gene and pulmonary inflammation has not been established. The aim of this study was to assess whether serum chemokines levels in cystic fibrosis patients correlate with genotype and pulmonary function tests, as well as with other clinical characteristics. Serum levels of interleukin-8, RANTES, and monocyte chemoattractant protein-1 were measured in 36 cystic fibrosis patients grouped according to their genotype. Group A included 25 patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (deltaF508, W1282X, G542X, N1303K, S549R). Group B included 11 compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat test and pancreatic sufficiency (3849+10kb C to T, 5T). Associations between chemokine levels, genotype, pulmonary function, Pseudomonas aeruginosa colonization, age, sweat chloride level, and pancreatic and nutritional status were examined. Mean interleukin-8 and monocyte chemoattractant protein-1 levels were significantly higher in group A than group B (11.4 +/- 2.1 pg/ml vs. 5 +/- 0.9 pg/ml and 157 +/- 16 pg/ml vs. 88.8 +/- 16.4 pg/ml, respectively) (P < 0.01). No difference in RANTES levels were found between groups. interleukin-8 levels were inversely related to forced expiratory volume in 1 s (r = -0.37, P < 0.02), while there was no association between the latter and RANTES and monocyte chemoattractant protein-1 levels. The Pseudomonas colonization rate was higher among group A patients than group B (88% vs. 40%, P < 0.01). No relationship was found between measured chemokines and age, sweat chloride levels, and pancreatic and nutritional status. Our study demonstrates an association between interleukin-8, forced expiratory volume, and cystic fibrosis genotype. Hence, elevated interleukin-8 serum levels could serve as an indicator of an early inflammatory process and encourage the initiation of anti-inflammatory treatment.

Topics: Adolescent; Adult; Chemokine CCL2; Chemokine CCL5; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Forced Expiratory Volume; Genotype; Humans; Infant; Inflammation Mediators; Interleukin-8; Pseudomonas aeruginosa

Ligation of the asialoGM1 Pseudomonas aeruginosa pilin receptor has been demonstrated to induce IL-8 expression in airway epithelial cells via an NF-kappaB-dependent pathway. We examined the signaling pathways required for asialoGM1-mediated NF-kappaB activation in IB3 cells, a human bronchial epithelial cell line derived from a cystic fibrosis (CF) patient, and C-38 cells, the rescued cell line that expresses a functional CF transmembrane regulator. Ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells. AsialoGM1-mediated activation of NF-kappaB, IkappaB kinase (IKK), and ERK was also greater in IB3 cells. With the use of genetic inhibitors, we found that IKK-beta and NF-kappaB-inducing kinase are required for maximal NF-kappaB transactivation and transcription from the IL-8 promoter. Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no effect on IKK or NF-kappaB activation, suggesting that ERK regulates IL-8 expression in an NF-kappaB-independent manner.

Topics: Antibodies; Bronchi; Cell Line; Cystic Fibrosis; Enzyme Activation; Epithelial Cells; G(M1) Ganglioside; Humans; I-kappa B Kinase; Interleukin-8; Mitogen-Activated Protein Kinases; NF-kappa B; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Tumor Necrosis Factor-alpha

Accumulating evidence suggests that in cystic fibrosis (CF) patients, airway fluids are characterized by decreased antibacterial activity, elevated NaCl concentration, and high levels of chemokines, resulting in exaggerated activation of the transcriptional nuclear factor (NF)-kappaB in airway epithelial cells. The present study was undertaken to evaluate the effects of anti-inflammatory cytokine interleukin-10 (IL-10) on NaCl-induced chemokine IL-8 and regulated on activation normal T cell expressed and secreted (RANTES) expression through the NF-kappaB signaling in primary deltaF508 CF and non-CF (control) human bronchial epithelial cells. Exposure of CF and non-CF bronchial epithelial cells to hypertonic (170 mmol/L NaCl) milieu compared to isotonic (115 mmol/L NaCl) and hypotonic (85 mmol/L NaCl) milieu caused a significant, NaCl-dependent increase in IL-8 and RANTES gene expression and protein production. Compared to non-CF cells, CF bronchial epithelial cells were characterized by a higher susceptibility to produce elevated IL-8 and RANTES production in an hypertonic NaCl milieu in response to IL-1beta activation. Treatment with IL-10 suppressed IL-8 and RANTES gene expression in both non-CF and CF bronchial epithelial cells was associated with a reduced expression of I(k)B (IKK) alpha/beta kinases, particularly for IKKalpha which is greater expressed in CF bronchial epithelial cells, and resulting in reduced NF-kappaB activation. These findings suggest that IL-10 might have anti-inflammatory benefits in airways of CF patients.

Topics: Bronchi; Cells, Cultured; Chemokine CCL5; Chemokines; Cystic Fibrosis; Epithelial Cells; Gene Expression; Humans; I-kappa B Kinase; I-kappa B Proteins; Interleukin-1; Interleukin-10; Interleukin-8; Macromolecular Substances; NF-kappa B; Protein Serine-Threonine Kinases; Respiratory Mucosa; Sodium Chloride; Tumor Necrosis Factor-alpha

Inflammation plays a critical role in lung disease progression in cystic fibrosis (CF). This inflammatory process is dominated by a neutrophil influx in the airways. To determine whether the accumulation of neutrophils in the airways of CF patients is associated with an altered function, we analyzed the capacity of neutrophils isolated from the lung compartment and the blood to release the major neutrophil pro- and anti-inflammatory cytokines IL-8 and IL-1-receptor antagonist (ra) spontaneously and in the presence of LPS. Comparison of cytokine production by blood neutrophils from CF patients and from control subjects showed significantly increased IL-8 and decreased IL-1ra release by CF neutrophils. Comparison of cytokine production by airway and blood neutrophils from CF patients also documented distinct profiles: the spontaneous release of IL-8 and IL-1ra by airway neutrophils was significantly higher than that from blood neutrophils. Culture in the presence of LPS failed to further enhance cytokine production. Analysis of the effect of dexamethasone confirmed the difference in the responsiveness of lung and blood neutrophils in CF. Used at a concentration effective in reducing IL-8 production by blood neutrophils, dexamethasone (10(-6) M) was unable to repress secretion of IL-8 by airway neutrophils. In addition, comparison of cytokine production by airway neutrophils from children with CF and children with dyskinetic cilia syndrome also documented distinct profiles of secretion. These results are consistent with a dysregulated cytokine production by lung and blood neutrophils in CF. They provide support to the hypothesis that not only the CF genotype but also the local environment may modify the functional properties of the neutrophils.

Topics: Adolescent; Cells, Cultured; Child; Cystic Fibrosis; Dexamethasone; Female; Glucocorticoids; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-8; Lung; Male; Neutrophils; Pneumonia; Sialoglycoproteins

Sputum induction (SI) is a noninvasive tool for sampling inflamed airways. The purpose of this study was to determine the optimal duration of collection in patients with cystic fibrosis (CF). The hypothesis was that the duration of SI collection would quantitatively and qualitatively alter the content of the induced sputum.. In 10 clinically stable patients with CF (mean +/- SD age, 28 +/- 7 years; mean FEV(1), 2.6 +/- 0.7 L), SI was performed with 3% hypertonic saline solution at five time points over 20 min.. SI was well tolerated, with an average maximum fall in FEV(1) of 7 +/- 7%. The sample volumes, urea concentrations, interleukin-8 concentrations, total cell counts, and nonsquamous cell counts remained constant (p > 0.05). The percentage of neutrophils decreased from 89 +/- 5% to 86 +/- 4% (p = 0.03), and the percentage of alveolar macrophages increased 5 +/- 2% to 8 +/- 4% (p < 0.01). The mean quantitative microbiological counts of nonmucoid Pseudomonas aeruginosa and Staphylococcus aureus decreased over the 20-min time period each by half a log (p = 0.05 and p < 0.01, respectively). Surfactant protein-A concentration increased from 1.6 +/- 0.3 to 2.4 +/- 0.4 ng/mL (log(10); p < 0.001).. We conclude that aliquots of induced sputum are similar in clinically stable patients with CF during 4-min intervals, although there is more alveolar sampling after 20 min. When induced-sputum samples are fractionated for research monitoring of inflammatory or microbiologic indexes, power calculations accounting for these variations over time are required.

Topics: Adult; Biomarkers; Cell Count; Colony Count, Microbial; Cystic Fibrosis; Female; Humans; Interleukin-8; Male; Pilot Projects; Pulmonary Surfactant-Associated Proteins; Regression Analysis; Saline Solution, Hypertonic; Specimen Handling; Sputum; Time Factors; Urea

Inhaled fluticasone propionate (FP) is widely used to reduce pulmonary inflammation in chronic obstructive pulmonary disease, but the potential effects of FP on airway epithelial cells from patients with cystic fibrosis (CF) are unknown. In CF disease, a nonregulated inflammatory lung response occurs through exaggerated nuclear factor (NF)-kappaB activation and elevated pro-inflammatory cytokines production by airway epithelial cells. To determine whether FP reduces cytokine production in bronchial epithelial cells via NF-kappaB, the authors investigated the nonstimulated and the Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulated production of NF-kappaB-dependent interleukin (IL)-6, IL-8 and RANTES (regulated on activation, T-cell expressed and secreted) along with the activation of NF-kappaB in non-CF and CF human bronchial gland epithelial cells. It was demonstrated that a relevant concentration of FP (10(-8) M) inhibited constitutive and P. aeruginosa LPS-induced IL-6 and IL-8 production of non-CF and CF bronchial epithelial cells. Interestingly, the expression of two IkappaB kinases (IKK)-alpha/beta, the degradation of cytosolic IkappaB-beta inhibitor and the NF-kappaB deoxyribonucleic acid binding activity were markedly reduced after FP treatment in both CF and non-CF bronchial epithelial cells. It was shown by the authors that fluticasone propionate exerts an anti-inflammatory effect by blocking a signal transduction leading to a reduced level of IkappaB-alpha/beta kinases in bronchial epithelial cells. In particular the strong effect on the IkappaB-beta kinase, which is known to be elevated in bronchial epithelial cells in cystic fibrosis patients, was observed.

Topics: Analysis of Variance; Androstadienes; Blotting, Western; Bronchi; Bronchodilator Agents; Case-Control Studies; Cells, Cultured; Chemokine CCL5; Chemokines; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Fluticasone; Humans; I-kappa B Kinase; I-kappa B Proteins; Interleukin-6; Interleukin-8; Protein Serine-Threonine Kinases; Respiratory Mucosa; Tumor Necrosis Factor-alpha

Intravenous (IV) antibiotics are a mainstay of therapy in children with cystic fibrosis. It is unclear, however, over what period associated improvements in pulmonary function are maintained, and to what extent the underlying inflammatory process is impeded in children admitted for a course of IV antibiotics. This was a prospective, interventional study of 14 children (median age, 14 years; interquartile range, 10-14) with cystic fibrosis who were regular sputum producers and who required admission for a 2-week course of IV antibiotics. Children performed spirometry and provided a sputum sample prior to starting IV antibiotics and then weekly for 6 weeks, the first 2 weeks of which IV antibiotics were given. Sputum IL-8, TNF-alpha, IL-6, IL-10, MIP1-alpha, and elastase were measured. Seven children were asked to repeat the protocol in a subsequent exacerbation to assess similarities in response to therapy. Significant improvements were seen in forced expired volume in 1 sec (FEV(1)) in association with IV antibiotics (27% relative improvement in predicted from baseline to end of week 1, median FEV(1) 41.3% increasing to 52.2%), but this continued only 1 week following cessation of antibiotics. Although IL-8 demonstrated a trend for reduction in association with antibiotics, no significant profile was demonstrated for any of the cytokines assessed. IL-10 was detectable in 64% of samples (all <100 pg/ml). In children with two episodes assessed, although there was a close correlation of FEV(1) and FVC between exacerbations (before antibiotics), no significant correlation was seen for IL-8, TNF-alpha, or IL-10 measured in both sets of samples at any sample point (indeed, a discordant response was seen between sample points in the two exacerbations). Although FEV(1) temporarily improves in response to admission for IV antibiotics, no such response is seen in sputum cytokine values. In addition, assessment of cytokines in subsequent exacerbations does not show a similar pattern of response to treatment.

Topics: Adolescent; Age Factors; Anti-Bacterial Agents; Bronchial Provocation Tests; Child; Cohort Studies; Cystic Fibrosis; Cytokines; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Follow-Up Studies; Humans; Infusions, Intravenous; Interleukin-8; Male; Probability; Risk Assessment; Sampling Studies; Severity of Illness Index; Sex Factors; Spirometry; Sputum; Statistics, Nonparametric; Treatment Outcome

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin-8 (IL-8) expression via an IL-1 receptor-associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll-like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK-TLR4) express TLR4 mRNA and protein and induce IL-8 promoter activity in response to NE. Treatment of both HEK-TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE-induced IL-8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL-8 expression in bronchial epithelium.

Topics: Blotting, Western; Cell Line; Cell Membrane; Cells, Cultured; Cystic Fibrosis; DNA, Complementary; Genes, Reporter; Humans; Interleukin-8; Lasers; Leukocyte Elastase; Membrane Glycoproteins; Protein Binding; Receptors, Cell Surface; RNA; RNA, Messenger; Toll-Like Receptor 4; Toll-Like Receptors; Transfection; Up-Regulation

Chronic Pseudomonas aeruginosa infection in cystic fibrosis (CF) leads to a damaging host inflammatory response. There are an increasing number of reports of P. aeruginosa cross-infection at CF centres. The clinical significance of acquisition of a transmissible strain for patients who already harbour P. aeruginosa is unclear. In this study, levels of inflammatory markers in clinically stable adult CF patients who harbour transmissible and sporadic strains of P. aeruginosa have been compared. Patients with CF and chronic P. aeruginosa infection were grouped into those who harbour a transmissible P. aeruginosa and those who harbour their own sporadic strains. Total white cell and differential counts, sputum neutrophil elastase (NE), interleukin (IL)-8, tumour necrosis factor (TNF)-alpha, plasma IL-6 and NE/alpha1-antitrypsin complexes, serum C-reactive protein, and urine TNF receptor 1 were all measured in clinically stable patients 4-6 weeks following completion of intravenous antibiotic therapy. The two groups (both n=20) were well matched for per cent predicted forced expiratory volume in one second, per cent predicted forced vital capacity and body mass index. There were no significant differences in levels of white cell counts or inflammatory markers between the two groups. At times of clinical stability, cystic fibrosis patients infected with transmissible Pseudomonas aeruginosa do not have a heightened inflammatory response above that of those harbouring sporadic strains.

Topics: Adult; alpha 1-Antitrypsin; Biomarkers; C-Reactive Protein; Case-Control Studies; Cell Count; Cystic Fibrosis; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Leukocyte Elastase; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Tumor Necrosis Factor; Sputum; Tumor Necrosis Factor-alpha

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.

Topics: Burkholderia cepacia; Cell Line; Cell Line, Tumor; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Dinoprostone; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-1; Interleukin-6; Interleukin-8; Mutation; NF-kappa B; Prostaglandin-Endoperoxide Synthases; Respiratory Mucosa; Transcriptional Activation

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E(2) both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.

Topics: Arachidonic Acid; Bradykinin; Cell Line; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cystic Fibrosis; Dinoprostone; Epithelial Cells; Humans; Indomethacin; Interleukin-8; Isoenzymes; Membrane Proteins; Nitrobenzenes; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Sulfonamides; Trachea

Airway inflammation, one of the major factors leading to lung damage in cystic fibrosis (CF) patients, is associated with an abnormal increase in proinflammatory cytokines. In this work, we demonstrate the increased release of the proinflammatory cytokines after lipopolysaccharide (LPS) stimulation: human interleukin (hIL)-8 in CF and non-CF airway xenografts, and hIL-6 and human growth-related oncogene-alpha (hGRO-alpha), which could be only analyzed in non-CF xenografts. Under basal conditions, we observed that hIL-8 was higher in CF xenografts compared with non-CF. We also report the anti-inflammatory effect of a glucocorticoid, fluticasone propionate (FP), on CF airway epithelium using a humanized model of airway inflammation developed in nude mice. In CF and non-CF tracheal xenografts, airway inflammation was induced by inoculating Pseudomonas aeruginosa LPS (4 h; 1 microg/ml) in the lumen of the xenografts. FP pretreatment (2 h; 10(-8) M) followed by P. aeruginosa LPS stimulation induced a significant reduction of LPS-induced hIL-8 release in airway liquid collected from CF and non-CF tracheal xenografts (85 and 80%, respectively). In non-CF tracheal xenografts, FP treatment before LPS stimulation induced a significant decrease in hIL-6 and hGRO-alpha. From these data, we suggest that FP exerts anti-inflammatory properties that may be appropriate to CF therapy, at an early stage of the disease. In addition, these results demonstrate that the humanized airway model of inflammation provides a relevant tool for analyzing the effects of anti-inflammatory drugs in different diseases in which airway inflammation is implicated.

Topics: Androstadienes; Animals; Anti-Inflammatory Agents; Body Fluids; Child; Cystic Fibrosis; Cytokines; Female; Fluticasone; Humans; Immunoenzyme Techniques; Inflammation; Interleukin-8; Lipopolysaccharides; Lung; Male; Mice; Mice, Nude; Pseudomonas aeruginosa; Trachea; Transplantation, Heterologous

To validate a sputum induction technique in cystic fibrosis (CF), we examined the relation between airway inflammation and pulmonary function in children with CF by correlating inflammatory indexes in induced sputum with FEV(1).. We measured baseline spirometry and oxygen saturations and then performed sputum inductions with 3% hypertonic saline in 20 clinically stable children with CF (11 girls). We examined the relation of airway inflammation and lung function in the 19 individuals (95%) who expectorated an adequate sputum sample. Measures of airway inflammation in induced sputum included total cell counts, neutrophil (PMN) counts, interleukin-8 levels, and free neutrophil elastase activity.. There were significant inverse relations between FEV(1) and total cell counts and PMN counts (r = -0.57, P <.01 for both), interleukin-8 (r = -0.72, P =.002), and elastase (r = -0.75, P =.001). Airway infection, as assessed by bacterial density in induced sputum, did not correlate with lung function or indexes of inflammation.. We conclude that measures of inflammation in induced sputum correlate with FEV(1) in clinically stable children with CF with normal to mildly abnormal lung function and that they may be useful as surrogate outcome measures in clinical trials.

Topics: Adolescent; Cell Count; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Leukocyte Elastase; Lung; Male; Spirometry; Sputum

Nasal polyposis (NP) frequently complicates the course of cystic fibrosis (CF). The aim of this study was to determine the pattern of inflammatory cells and mediators in nasal secretions from patients with or without NP compared to patients with idiopathic NP and healthy controls.. Eighteen CF patients with NP (NP+ group: 6 untreated, 12 treated with nasal steroids), and 15 without NP (NP- group) were included in this prospective study and compared to 9 patients with idiopathic NP and 12 healthy controls. Differential cell count eosinophil cationic protein (ECP), interleukin-5 (IL-5) and IL-8 were determined in nasal lavage fluids.. The total cell count, the number and the percentage of neutrophils and eosinophils, the levels of IL-8, IL-5 and ECP were significantly higher in nasal secretions from both NP+ and NP- as compared with controls. No difference was found between untreated and treated CF patients with NP. No difference was found between NP+ and NP- groups. Compared to idiopathic NP group, both NP+ and NP- groups had higher percentage of neutrophils and lower percentage of eosinophils. There were no differences according to the use of topical steroids, systemic antibiotherapy, or the type of mutation. CF patients with positive nasal culture had a higher percentage of neutrophils than those with negative culture. CF patients with atopy had a higher percentage of eosinophils than non-atopic patients.. Our results demonstrate that nasal inflammation is a prominent feature in patients with CF and does not differ according to the presence of NP. IL-8 and IL-5 may play crucial roles in recruitment and activation of neutrophils and eosinophils in upper airways of CF patients.

Topics: Adolescent; Biomarkers; Blood Proteins; Cell Count; Cystic Fibrosis; Cytokines; Eosinophil Granule Proteins; Epithelial Cells; Female; Humans; Interleukin-5; Interleukin-8; Leukocytes; Male; Nasal Lavage Fluid; Nasal Mucosa; Prospective Studies; Ribonucleases

Nasal polyps frequently appear in patients with cystic fibrosis (CF). The aims of this study were to focus on what problems (symptoms, endoscopic findings, and laboratory correlates) nasal polyps cause the CF patient, and how these correlate to the total health situation of this patient group.. The clinical histories, endoscopic investigations of the nasal cavity, and analyses of nasal lavage fluid of 44 patients with CF complicated with nasal polyposis have been compared with those of 67 CF control subjects. The patients were examined at annual control examinations (with pulmonary tests, working capacity, liver tests, and bacterial and blood tests) from 1995 to 1996 at Stockholm Cystic Fibrosis Center, Huddinge University Hospital. All patients were > 2 years of age. The endoscopic findings were related to the actual pulmonary function, inflammatory blood parameters, colonizing pathogens, antibodies (Staphylococcus aureus and Pseudomonas aeruginosa), and genotype.. The patients with nasal polyps differed with respect to chronic colonization of P aeruginosa in sputum samples and had a higher occurrence of serum antibodies against the same species. The two groups did not differ in pulmonary functions, inflammatory parameters, or genotype. The polyps found were mainly small (within the meatus media) and gave no significant increase in ongoing clinical symptoms such as rhinorrhea, nasal obstruction, or hyposmia. Neither was any significantly marked finding concerning the nose (mucosal swellings, secretion, etc.) made in the polyp patients. The patients with CF scored slightly lower in a smell identification test in comparison with the healthy control group. The nasal lavage fluid was analyzed (in 93 of the 111 patients) for the occurrence of P aeruginosa (by polymerase-chain reaction [PCR]), interleukin [IL]-5, IL-8, and lysozyme. The lysozyme and IL-8 content was equal in the two CF groups but increased in comparison with the healthy control group. P aeruginosa was not detected with PCR in any nasal lavage fluid. No measurable levels of IL-5 in the nasal lavage were found.. There was a higher frequency of chronic colonization of P aeruginosa in the lower respiratory tract in patients with nasal polyps. Otherwise, nonsevere nasal polyposis was not an indicator of lower respiratory tract morbidity in CF patients.

Topics: Adolescent; Adult; Antibodies, Bacterial; Child; Child, Preschool; Cystic Fibrosis; Endoscopy; Female; Humans; Interleukin-5; Interleukin-8; Male; Muramidase; Nasal Lavage Fluid; Nasal Polyps; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Risk Factors; Staphylococcal Infections

The conversion of Pseudomonas aeruginosa to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF). A major pathway of conversion to mucoidy in clinical strains of P. aeruginosa is dependent upon activation of the alternative sigma factor AlgU (P. aeruginosa sigma(E)). Here we initiated studies of AlgU-dependent global expression patterns in P. aeruginosa in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy. Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (sigma(E)) promoter sites on the P. aeruginosa chromosome. Each candidate promoter was individually tested by reverse transcription and mRNA 5'-end mapping using RNA isolated from algU(+) and algU::Tc(r) mutant cells. A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped. Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes. Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes, lptA and lptB, had proinflammatory activity. In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung. Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given sigma factor (collectively termed here sigmulon) using conventional molecular tools. In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity. This link may be of significance for infections and inflammatory processes in CF.

Topics: Amino Acid Sequence; Bacterial Proteins; Cells, Cultured; Chromosome Mapping; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genome, Bacterial; Humans; Interleukin-8; Lipoproteins; Macrophages; Molecular Sequence Data; Porins; Promoter Regions, Genetic; Pseudomonas aeruginosa; Sigma Factor; Transcription Factors; Transcription, Genetic

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

Topics: Bacterial Adhesion; Cell Adhesion; Chemokines; Cystic Fibrosis; Cytokines; Enzyme-Linked Immunosorbent Assay; Epithelium; Flow Cytometry; Gentamicins; Green Fluorescent Proteins; Humans; Interleukin-8; Luminescent Proteins; Microscopy, Confocal; Microscopy, Fluorescence; NF-kappa B; Protein Binding; Recombinant Fusion Proteins

Our aim was to study the effect of lower airway infection on clinical parameters, pulmonary function tests, and inflammation in clinically stable infants and young children with cystic fibrosis (CF). To accomplish this goal, a prospective cohort of screened CF patients under 4 years of age were studied, using elective anesthesia and intubation for: passive respiratory mechanics (single breath occlusion passive deflation) and lung volumes (nitrogen washout), under neuromuscular blockade; and bronchoalveolar lavage (BAL) of 3 main bronchi for cytology, cytokine interleukin (IL)-8, and quantitative microbiology. There were 22 children studied, with a mean age of 23.2 months (6.7-44 months). A greater relative risk of lower airway pathogens was associated with prior respiratory admission (3.60, 95% confidence interval [CI] 2.87-4.51), history of asthma (1.75, 95% CI 1.52-2.03), and chronic symptoms (1.50, 95% CI 1.23-1.83), especially wheeze (1.88, 95% CI 1.61-2.19). Lower respiratory pathogens (> or = 10 cfu/ml BAL) were found in 14 out of 22, and greater than 10(5) cfu/ml in 8 out of 22 subjects. The level of pathogens in BAL (log10 cfu/ml) explained 78% of the variability in percent neutrophils and 34% of the variability in IL-8 levels. Pathogen level also correlated with pulmonary function tests of specific respiratory system compliance (r -0.49, p = 0.02) and functional residual capacity over total lung capacity (r 0.49, p = 0.03). We conclude that the presence of pathogens in the lower airways correlated with levels of inflammation, respiratory system compliance, and degree of air trapping.

Topics: Bronchoalveolar Lavage Fluid; Cell Count; Child, Preschool; Cohort Studies; Cystic Fibrosis; Female; Humans; Infant; Inflammation; Inflammation Mediators; Interleukin-10; Interleukin-8; Lung; Male; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mechanics; Respiratory Tract Infections

Chronic endobronchial infection frequently caused by gram-negative organisms and an increased, neutrophil-dominated inflammation are characteristics of cystic fibrosis (CF). The present study examines endotoxin levels in bronchoalveolar lavage fluids of CF versus non-CF (N) control children, and correlates these with the inflammatory markers interleukin-8 and neutrophils. Fifty-five patients with CF and 56 patients without CF between the ages of 0.04 to 13.25 years were included. Infection, defined as a bacterial count above 50,000 cfu/ml, was present in 27 CF and 25 N patients. Endotoxin levels were not different between patients with and without CF (infected: 74.9 +/- 12.1 EU/ml versus 51.4 +/- 12.5 EU/ml, p = 0.16; noninfected: 5.9 +/- 4.8 EU/ml versus 11.1 +/- 4.3 EU/ml, p = 0.28). Endotoxin activity correlated to the number of gram-negative organisms in CF and N patients, and endotoxin activity per bacterial colony forming unit did not differ with various gram-negative species. Both interleukin-8 and neutrophils were positively correlated with endotoxin, but this slope was shifted toward higher levels of inflammation in CF patients. We conclude that it is unlikely that higher levels of endotoxin in the absence of viable bacteria explain the increased inflammatory response in CF.

Topics: Adolescent; Bacteria; Bacterial Infections; Bronchoalveolar Lavage Fluid; Cell Count; Child; Child, Preschool; Cystic Fibrosis; Endotoxins; Humans; Infant; Interleukin-8; Neutrophils; Respiratory Tract Infections

High-resolution computed tomography (HRCT) is a sensitive technique for early visualisation and location of cystic fibrosis (CF) bronchopathology, and has been shown to detect acute reversible and chronic changes. It would be expected to correlate with markers of the underlying pathological processes, such as sputum cytokines and cytology, as well as with pulmonary function tests (PFTs). Our aim was to study the relationship between PFTs, sputum cytology, and sputum cytokine interleukin-8 (IL-8) and HRCT in CF patients. Prospective standardized collection of sputum samples was performed at the time of routine annual high-resolution CT scans. Forced expired volume in 1 sec (FEV(1)) and forced vital capacity (FVC) were recorded. Sputum processing was selective, with dispersal by the three-enzyme technique. IL-8 measurements were by kit assay. HRCT scans were scored by a pediatric radiologist, blinded to clinical condition, using a modified Bhalla score.Forty-three CT scans were performed on 34 children with CF between March 1998 and April 2000. Mean age was 12.3 years (range, 6-21 years), FEV(1) (% predicted) was 67% (range, 23-120%), and mean modified Bhalla score was 11.2 (range, 0-22). Sputum IL-8 concentration (mean, 86; range, 4-150 ng/mL) and total cell count (mean, 31.9 x 10(6)/mL; range, 21.8-42.0 x 10(6)/mL) were high. FEV(1) and FVC correlated with modified Bhalla score (r = -0.66, P < 0.0001 for both), and most individual components of the score, especially mosaic perfusion (r = -0.64, r = -0.61 respectively, P < 0.0001) and extent of bronchiectasis (r = -0.61, P < 0.0001 for both). The combination of these two predicted 58% of the variability in FEV(1) on analysis of variance (P < 0.0001). Sputum total cell count correlated weakly with modified Bhalla score (r = 0.38, P < 0.05) and with FEV(1) and FVC (r = -0.36, P < 0.05; and r = -0.46, P < 0.01). Differential cell counts, cell viability, and IL-8 did not correlate with modified Bhalla scores, or with reversible components such as mucus plugging, centrilobular nodules, or peribronchial thickening. In conclusion, pathological changes on HRCT correlated with lung function but not with sputum markers of inflammation.

Topics: Adolescent; Adult; Biomarkers; Cell Count; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Male; Sputum; Tomography, X-Ray Computed; Ultrasonography; Vital Capacity

Much of the pulmonary disease in cystic fibrosis is associated with polymorphonuclear leukocyte-dominated airway inflammation caused by bacterial infection. Respiratory epithelial cells express the polymorphonuclear chemokine interleukin-8 (IL-8) in response to ligation of asialylated glycolipid receptors, which are increased on damaged or regenerating cells and those with cystic fibrosis transmembrane conductance regulator mutations. Because both Pseudomonas aeruginosa and Staphylococcus aureus, the most common pathogens in cystic fibrosis, bind asialylated glycolipid receptors such as asialoGM1, we postulated that diverse bacteria can activate a common epithelial signaling pathway to elicit IL-8 expression. P. aeruginosa PAO1 but not pil mutants and S. aureus RN6390 but not the agr mutant RN6911 stimulated increases in [Ca(2+)](i) in 1HAEo- airway epithelial cells. This response stimulated p38 and ERK1/2 mitogen-activated protein kinase (MAPK) signaling cascades resulting in NF-kappaB activation and IL-8 expression. Ligation of the asialoGM1 receptor or thapsigargin-elicited Ca(2+) release activated this pathway, whereas P. aeruginosa lipopolysaccharide did not. The rapid kinetics of epithelial activation precluded bacterial invasion of the epithelium. Recognition of asialylated glycolipid receptors on airway epithelial cells provides a common pathway for Gram-positive and Gram-negative organisms to initiate an epithelial inflammatory response.

Topics: Adhesins, Bacterial; Blotting, Western; Calcium; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Egtazic Acid; Enzyme Activation; Epithelial Cells; G(M1) Ganglioside; Genes, Reporter; Humans; Inflammation; Interleukin-8; Kinetics; Lipopolysaccharides; Luciferases; Lung; MAP Kinase Signaling System; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; NF-kappa B; Pseudomonas aeruginosa; Receptors, Cell Surface; Signal Transduction; Spectrophotometry; Staphylococcus aureus; Thapsigargin; Time Factors; Trachea

Topics: Biomarkers; Cystic Fibrosis; Enterocolitis; Humans; Interleukin-8; Intestines; Reproducibility of Results; Sputum; Therapeutic Irrigation

It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.

Topics: Adolescent; Adult; Animals; Bacterial Adhesion; Bacterial Infections; beta-Galactosidase; Bronchi; Cell Line; Cells, Cultured; COS Cells; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Genotype; Humans; Inflammation; Interleukin-8; Lung; Male; Mice; Mice, Inbred CFTR; Middle Aged; Mutation; Nasal Mucosa; NF-kappa B; Pseudomonas aeruginosa; Respiratory Mucosa; Trachea; Transcriptional Activation; Transfection

Bronchoalveolar lavage (BAL) performed in specialist centres has improved the understanding of infant cystic fibrosis (CF) lung disease. As most researchers sample from a single lobe, it was determined whether BAL results could be generalized to other lung segments. Thirty-three CF children, aged 1.5-57 months, underwent in random order sequential BAL of their right middle and lingula lobes. Specimens from each lobe had separate quantitative bacteriology, cytology and cytokine analysis. Bacterial counts > or = 1 x 10(5) colony forming units (cfu) x mL(-1) were observed in nine (27%) subjects, including six involving only the right middle lobe. These six children had similar inflammatory indices in their right middle and lingula lobes, and interleukin (IL)-8 concentrations in the latter were significantly higher than that observed within the lingula lobes of the 24 CF children with bacterial counts < 1 x 10(5) cfu x mL(-1). Lingula neutrophil and IL-8 levels correlated best with right middle lobe bacteria numbers. This observational study in cystic fibrosis children suggests that while inflammation is detected in both lungs, bacterial distribution may be more inhomogeneous. Bronchoalveolar lavage microbiological findings from a single lobe may therefore, not be generalized to other lung segments. When performing bronchoalveolar lavage in cystic fibrosis children, it is important to sample from multiple sites.

Topics: Bronchoalveolar Lavage Fluid; Cell Count; Child; Child, Preschool; Colony Count, Microbial; Cystic Fibrosis; Female; Humans; Infant; Interleukin-8; Lung; Lymphocytes; Macrophages; Male; Neutrophils

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) alpha and IL-1beta stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNFalpha stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNFalpha stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1beta. Finally, when CF cells were grown at 27 degrees C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNFalpha-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.

Topics: Bronchi; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Epithelial Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-1; Interleukin-6; Interleukin-8; Tumor Necrosis Factor-alpha

To determine whether macrolide antibiotics improve pulmonary function and decrease airway inflammation in cystic fibrosis (CF), we treated 10 patients (females; aged 19-26 years, all colonized with P. aeruginosa, none with atypical Mycobacteria) with 3 weeks of placebo, followed by 6 weeks of clarithromycin (500 mg BID) in a single-blind prospective study. We also determined the safety of sputum induction and the reproducibility of assessing inflammatory markers in induced sputum. Subjects performed spirometry and underwent sputum induction (12-min inhalation of 3% saline) at 3-week intervals. We found that sputum induction was well-tolerated. We also found that the reproducibility was high for neutrophil (PMN) number (R = 0.87, P = 0.009), interleukin (IL)-8 (R = 0.73, P < 0.05, free neutrophil elastase (NE) (R = 0.82, P < 0.05), and myeloperoxidase (MPO) levels (R = 0.86, P < 0.05), but was less so for tumor necrosis factor (TNF)-alpha (R = -0.15, P = 0.7). We found no significant difference in pulmonary function after 6 weeks of treatment with clarithromycin (FEV(1) (% predicted) (mean +/- SEM), 2.2 +/- 0.9 (60 +/- 24%) vs. 2.3 +/- 1 (61 +/- 29%)), and no significant differences in any of the inflammatory indices measured. The median (and range) values before and after treatment for indices of airway inflammation in the induced sputum samples were: for PMNs, 8 (1-326) and 21 (0.2 -175) x 10(6) cells/mL sputum; for IL-8, 156 (24-656) and 202 (16-680) ng/mL; for free NE, 260 (31-1,264) and 237 (49-1,048) microg/mL; for TNF-alpha, 20 (7-128) and 35 (17-87) pg/mL; and for MPO, 169 (13-960) and 195 (14-816) microg/mL. We conclude that clarithromycin is not uniformly effective in improving airway obstruction or in decreasing airway inflammation in patients with CF.

Topics: Adult; Airway Obstruction; Anti-Bacterial Agents; Biomarkers; Clarithromycin; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Male; Neutrophils; Peroxidase; Predictive Value of Tests; Prospective Studies; Reproducibility of Results; Respiratory Function Tests; Single-Blind Method; Sputum; Treatment Outcome; Tumor Necrosis Factor-alpha

Neutrophil-dominated inflammation is prominent in the cystic fibrosis (CF) and chronic bronchitis (CB) airways. We assessed the degree of airway inflammation by measuring the sputum concentrations of interleukin (IL)-8, myeloperoxidase (MPO), and deoxyribonucleic acid (DNA). We determined the relationship among the concentrations of these mediators and investigated methodological problems that may be responsible for reported variability in measurements. Sputa obtained from 31 patients were solubilized with phosphate-buffered saline, dithiothreitol (DTT) (0.1% or 1%), or dornase alfa (0.2 mg/mL). The sputum concentration of IL-8 and MPO was measured by enzyme-linked immunosorbent assay (ELISA), and DNA was measured using microfluorimetry. There was a significant relationship among sputum IL-8, MPO, and DNA. For MPO (means +/- SD), CF was 1,392 +/- 771 vs. CB at 75 +/- 65 mcg/mL; P < 0.0001. For IL-8: CF was 239 +/- 154 vs. CB at 121 +/- 108 ng/mL; P = 0.0002. For DNA, CF was 1.707 +/- 1.25 vs. CB at 0.184 +/- 0.272 mg/mL; P < 0.0001. The MPO concentration in CF sputum was approximately double after in vitro treatment with dornase alfa (P < 0.0001). There is a greater concentration of IL-8, MPO, and DNA in CF than in CB sputa. There is a significant relationship among these inflammatory markers in sputum. DNA polymers bind myeloperoxidase in the sputum, and we speculate that treatment with dornase alfa may remove a source of MPO inhibition.

Topics: Adolescent; Adult; Biomarkers; Bronchitis; Child; Cystic Fibrosis; Deoxyribonuclease I; DNA; Expectorants; Female; Humans; Inflammation; Interleukin-8; Male; Peroxidase; Polymers; Recombinant Proteins; Specimen Handling; Sputum

Inhaled corticosteroids are commonly used in cystic fibrosis (CF), but there are few studies evaluating their safety in young children. We, therefore, prospectively administered beclomethasone diproprionate (BDP) to 12 clinically stable young children with CF to examine the safety of this therapy with respect to adrenal suppression and airway infection. To determine potential mechanisms of corticosteroid action in CF, we also examined airway markers of inflammation before and after inhaled steroid treatment. BDP 210 microg twice a day was given via spacer for 2 months. Twelve-hour serum and urine cortisols and response to low-dose synthetic ACTH cortisol stimulation were assessed. Bronchoalveolar lavage fluid (BALF) was examined pre- and posttreatment with BDP by quantitative bacteriology and indices of airway inflammation, including levels of total neutrophils, neutrophil elastase-alpha-1 antiprotease complexes (NEAP), CA 19-9 mucin-associated antigen, interleukin-8 (IL-8), and macrophage IL-8 mRNA. Following 2 months of treatment, serum and urine cortisol levels were unchanged. Response to low-dose ACTH cortisol stimulation was not significantly decreased at 30 min. Posttreatment BALF bacterial density was not statistically different from pretreatment; however, one patient who was initially culture negative became culture-positive with Hemophilus influenzae. BALF total neutrophil counts, corrected for epithelial lining fluid dilution, were decreased to approximately one third of pretreatment values (P = 0.03). NEAP and CA 19-9 mucin-associated antigen demonstrated similar decreases. BALF IL-8 levels and macrophage IL-8 mRNA levels were not statistically changed. These findings suggest that treatment with BDP 420 microg per day for 2 months in young children with CF does not affect urine and blood cortisol, causes no decrease in adrenal reserve, and does not result in a clinically significant increase in airway infection. In addition, the fall in bronchoalveolar lavage fluid inflammatory markers following BDP suggests possible modulation of neutrophil influx into the CF airway and provides justification for further studies of inhaled corticosteroids in CF.

Topics: Administration, Inhalation; Adolescent; Adrenal Glands; Airway Resistance; Analysis of Variance; Anti-Inflammatory Agents; Beclomethasone; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child; Child, Preschool; Cystic Fibrosis; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Follow-Up Studies; Humans; Inflammation Mediators; Interleukin-8; Male; Nebulizers and Vaporizers; Pilot Projects; Probability; Prospective Studies; RNA, Messenger; Statistics, Nonparametric; Treatment Outcome

Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe. Extensive work is being performed to develop both gene and drug therapies. The principal mutation causing CF is in the CFTR gene ([Delta F508]CFTR). This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity. CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit. CPX also activates mutant CFTR chloride channel activity. CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells. IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway. In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint.. To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells. The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX. CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients. cDNA microarrays were used to assess global gene expression under the different conditions. A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8.. We report here that IB3 CF cells secrete massive levels of IL-8. However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion. Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P. aeruginosa. Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells. Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy. Additionally, the same result obtains in the presence of P. aeruginosa. Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion. The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells. Certain other genes, previously known to be positively associated with CF, also fall into this category. Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion.. Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.

Topics: Algorithms; Cell Line; Child; Cluster Analysis; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Genetic Therapy; Humans; Interleukin-8; Lung; Models, Biological; NF-kappa B; Oligonucleotide Array Sequence Analysis; Pseudomonas aeruginosa; Receptors, Tumor Necrosis Factor; Respiratory Mucosa; Time Factors; Xanthines

A noninvasive method to characterize inflammation and infection in the airways of nonexpectorating children with cystic fibrosis (CF) is needed for clinical and research purposes. Accordingly, we performed sputum inductions by administering 3% saline to 11 healthy control children and 20 children with CF, composed of 7 sputum producers (capable of spontaneously expectorating sputum) and 13 nonproducers. Induced sputum weights were comparable in each group, whereas the amount of induced sputum collected from the CF producers was over 10-fold higher than the spontaneously expectorated samples. We found a significant increase in indices of airway inflammation, including total cell counts, absolute neutrophil counts, interleukin-8 (IL-8) levels, and neutrophil elastase activity in the CF subjects compared with the healthy control subjects. These same indices in the induced sputum specimens from CF producers were significantly correlated with levels in the matched expectorated sputum specimens. Sputum total protein concentration was elevated in the CF groups, whereas urea and albumin levels were not significantly different. Salivary analysis, performed separately, revealed higher levels of IL-8 and total protein in the CF groups. Airway infection, as assessed by quantitative counts of CF-related bacterial pathogens, was also higher in the CF subjects. The same bacterial pathogens, in similar colony counts, were isolated from both the induced and expectorated sputum samples from the CF producers. We conclude that airway inflammation and infection, assessed through sputum induction, are significantly increased in children with CF as compared with healthy children. Furthermore, induced sputum samples are similar to spontaneously expectorated samples in describing both inflammation and infection in the CF airway.

Topics: Child; Cough; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Prospective Studies; Retrospective Studies; Saliva; Sputum

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.

Topics: Animals; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA-Binding Proteins; Enzyme Induction; Epithelial Cells; Female; Gene Expression Regulation; Genes, Reporter; Humans; Inflammation; Interleukin-8; Liver; Luciferases; Lung; Male; Mice; Mice, Knockout; Nasal Mucosa; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organ Specificity; Promoter Regions, Genetic; Recombinant Fusion Proteins; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta

Several recent reports have suggested that airway inflammation may precede infection and relate to an endogenous dysregulation of pro-inflammatory cytokines in cystic fibrosis (CF) airways. Evidence suggests that activation of the nuclear factor kappa B (NFkappaB), which regulates the inflammatory gene transcription, depends on the degradation of the inhibitory factor IkappaBalpha. We show that, in in situ human DeltaF508 CF bronchial tissues, inhibitor factor IkappaBalpha is not present in gland cells, although endogenous levels of chemokine IL-8 are high. These data are confirmed by studying cultured CF human bronchial gland cells, in which a lack of cytosolic IkappaBalpha and high levels of activated NFkappaB, concomitant with IL-8 overproduction (a 13-fold increase) are found when compared to non-CF bronchial gland cells. Interestingly, treatment of CF gland cells with the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, results in a significant decrease ( P < 0.001) in IL-8 production down to levels released by non-CF gland cells. The addition of genistein also reverses the effects of lipopolysaccharide (LPS) Pseudomonas-aeruginosa-induced nuclear translocation of NFkappaB by increasing IkappaBalpha protein level (65%) in CF gland cells. Our data indicate that the induction of IkappaBalpha protein in CF airway glandular epithelial cells may be a novel mechanism by which IL-8-mediated lung inflammatory events are markedly reduced in CF patients, at least at the airway glandular level.

Topics: Bronchi; Cells, Cultured; Cystic Fibrosis; Cytosol; DNA-Binding Proteins; Enzyme Inhibitors; Genistein; Humans; I-kappa B Proteins; Interleukin-8; NF-kappa B; NF-KappaB Inhibitor alpha; Protein-Tyrosine Kinases; Pseudomonas Infections; Respiratory Mucosa

Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.

Topics: Cell Line; Cystic Fibrosis; Cytokines; Endotoxins; Enzyme-Linked Immunosorbent Assay; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Macrophages; Respiratory Tract Infections; Tumor Necrosis Factor-alpha

Interleukin-1beta (IL-1beta) and neutrophil elastase (NE) are present in the epithelial lining fluid (ELF) of patients with cystic fibrosis (CF). Both factors activate surrounding cells including lung epithelial cells, causing release of IL-8, a potent chemoattractant for neutrophils. Previous studies showed up-regulation of IL-8 release by lung epithelial cells as a function of NE in CF; however, few studies addressed the relationship between IL-1beta and activation of lung epithelial cells in CF lungs. Confluent layers of A549 cells, a type II-like human lung epithelial cell line, were incubated overnight with IL-1beta (0-5 ng/ml) or NE (100 nM), and supernatants were analyzed for IL-8 by enzyme-linked immunosorbent assay (ELISA). Both IL-1beta and NE led to a significant increase in IL-8: 12.8 +/- 2.8 ng/ml and 0.8 +/- 0.3 ng/ml, respectively. Next, bronchoalveolar lavage (BAL) samples were obtained from one healthy adult volunteer and six patients with CF and measured for IL-8 and IL-1beta concentrations by ELISA. Both IL-8 (range 169.00 +/- 56.57 to 1742.04 +/- 338.98 pg/ml) and IL-1beta (range 0-24.26 +/- 0.52 pg/ml) were detected in CF specimens, whereas neither was detected in the volunteer's specimen. Normal and CF BALs then were incubated overnight at a 1:10 dilution with confluent A549 cells. Analysis by ELISA of cell-free supernatants revealed increased IL-8 production from cells stimulated with CF BALs only. Similar experiments were performed with BAL supernatants that had been incubated with soluble IL-1 type II receptor, soluble IL-1 receptor antagonist, or a peptide inhibitor of NE. Addition of IL-1 inhibitors had a marginal effect on the amount of IL-8 release after incubation with CF BAL samples, whereas inhibition of NE had no effect. Our results indicate that other factors present in ELF in CF account for IL-8 release from lung epithelial cells.

Topics: Adolescent; Adult; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cell Line; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Epithelium; Female; Humans; Interleukin-1; Interleukin-8; Leukocyte Elastase; Lung; Male

Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung. We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects. Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli. To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis. These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE. This may help explain the increased NE burden seen in this condition.

Topics: Antigens, CD; Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Cystic Fibrosis; Humans; Interleukin-8; Leukocyte Elastase; Macrophage-1 Antigen; Neutrophils; Norepinephrine; Precipitin Tests; Protein Isoforms; Receptors, Fc; Receptors, Interleukin; Receptors, Interleukin-8A; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is characterised by chronic airway inflammation. Pro-inflammatory mediators in the lung are regulated by the transcription factor nuclear factor kappa B (NFkappaB). We have assessed the effect of adenovirus and liposome-mediated overexpression of the NFkappaB inhibitor IkappaBalpha, as well as liposome-mediated transfection with oligonucleotides resembling NFkappaB consensus binding sites (decoys) in a cystic fibrosis airway epithelial cell line (CFTE). Electrophoretic mobility shift assays (EMSA) were used to assess NFkappaB activity and secretion of the pro-inflammatory cytokine interleukin-8 (IL-8) was measured by ELISA. At a MOI of 30, Ad-IkappaBalpha significantly decreased IL-8 secretion to 60% and 43% of control unstimulated and TNF-alpha stimulated cells, respectively. At this MOI, approximately 70% of cells are transduced. EMSA showed an approximately 50% decrease in NFkappaB activation. Liposome-mediated transfection of IkappaBalpha did not reduce IL-8 secretion, probably due to low transfection efficiency (approximately 5% of cells). Liposome-mediated transfection of CFTE cells with rhodamine-labeled decoy oligonucleotides indicated a transfection efficiency close to 100%. TNF-alpha stimulated IL-8 secretion was reduced by approximately 40% using this approach. EMSA confirmed a significant decrease of NFkappaB activation. Decoy oligonucleotides may be a promising approach for reduction of NFkappaB-mediated pulmonary inflammation. Gene Therapy (2000) 7, 306-313.

Topics: Adenoviridae; Cells, Cultured; Cystic Fibrosis; Electrophoresis; Genetic Therapy; Genetic Vectors; Humans; Interleukin-8; Liposomes; NF-kappa B; Oligonucleotides; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Increasing evidence suggests that in airways from cystic fibrosis (CF) patients, inflammation may precede bacterial infection and be related to an endogenous dysregulation of proinflammatory cytokines in airway epithelial cells. Several investigators have reported that, in CF airway fluids, elevated NaCl concentrations may also contribute to the diseased state by inhibiting the bactericidal properties of airway fluid. Because many proinflammatory cytokines are transcriptionally regulated by the NF-kappa B, we investigated whether an elevated extracellular NaCl content in airway fluids significantly impaired the regulation of the NF-kappa B/I kappa B alpha complex and the chemokine IL-8 production in primary non-CF and CF human bronchial gland epithelial cells. Exposure of non-CF gland cells to hypotonic (85 mM) NaCl solution, compared with isotonic (115 mM) NaCl and hypertonic (170 mM) NaCl solutions, resulted in a significant decrease in IL-8 production that was paralleled by a strong inhibition of activated NF-kappa B associated with an increased cytosolic expression of I kappa B alpha and a decrease in the I kappa B kinase alpha protein level. In CF gland cells, we demonstrated that, compared with the high IL-8 in an hypertonic solution, the release of IL-8 was significantly reduced 2-fold in an isotonic solution and 5-fold in a hypotonic solution. Strikingly, exposure of CF bronchial gland cells to either hypotonic or isotonic milieu did not result in a marked inhibition of the activated NF-kappa B/I kappa B alpha system. This is the first demonstration that primary human CF bronchial gland cells exhibit abnormally high IL-8 production through constitutively activated NF-kappa B and high I kappa B kinase alpha level, whatever the hypo-, iso-, and hypertonic NaCl milieu.

Topics: Adolescent; Adult; Bronchi; Cells, Cultured; Child; Cystic Fibrosis; Extracellular Space; Female; Humans; I-kappa B Kinase; Interleukin-8; Male; Middle Aged; NF-kappa B; Protein Serine-Threonine Kinases; Respiratory Mucosa; Sodium Chloride

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

Proinflammatory cytokines in sputum are useful markers of the activity of lung disease in cystic fibrosis (CF). Tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) concentrations in sputum of 10 CF patients were determined during exacerbation and IL-8 in sputum of 48 patients at a yearly follow-up when patients were in optimal clinical condition. In 9 patients of the former group, TNF-alpha levels were increased during exacerbation. In 4 patients, the peak occurred within 2 d (median value > 1500 ng/l), whereas the remaining 5 had peak values on days 3-6 (median value 720 ng/l). IL-8 levels were > 800 microg/l in all 10 patients, and in 9 cases there was a positive correlation between IL-8 and TNF-alpha. Baseline IL-8 levels of 48 patients showed considerable variation (median 207 microg/l, range 1.5-392). There was a significant correlation between IL-8 concentrations and current colonization with either Pseudomonas aeruginosa or Staphylococcus aureus in the lower airways (p = 0.002), immunoglobulin G levels (p = 0.02) and the severity of the pathological findings shown by chest X-ray (p = 0.008). High IL-8 and TNF-alpha values correlated with symptoms of deterioration. IL-8 levels seemed to be markers of both current bacterial colonization and the degree of lung damage.

Topics: Acute Disease; Adolescent; Adult; Anti-Bacterial Agents; Antibodies, Bacterial; Antibody Specificity; Bacterial Toxins; Biomarkers; Child; Cystic Fibrosis; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Interleukin-8; Male; Pseudomonas aeruginosa; Radiography; Respiratory Tract Infections; Sputum; Staphylococcus aureus; Statistics, Nonparametric; Tumor Necrosis Factor-alpha

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.

Topics: Adenoviridae; Bacterial Adhesion; Cell Movement; Cell Polarity; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; Interleukin-8; Mutation; Nasal Mucosa; Neutrophils; Pseudomonas aeruginosa; Reference Values; Transgenes

There is controversy about whether the inflammatory response observed in the cystic fibrosis (CF) lung occurs secondary to bacterial infection or is caused by a dysregulation of the inflammatory response associated with the basic cellular defect of CF.. To study the inflammatory response in the gastrointestinal tract of children with CF; and to investigate whether there is increased inflammation in the gastrointestinal tract of CF children with fibrosing colonopathy.. Whole gut lavage was performed on 21 pancreatic insufficient children with CF, who were clinically well, five children with CF and fibrosing colonopathy, and 12 controls. Intestinal outputs of plasma derived proteins (albumin, alpha(1) antitrypsin, IgG), secretory immunoglobulins (IgA and IgM), cellular constituents (eosinophil cationic protein and neutrophil elastase), and cytokines (interleukin 8 and interleukin 1beta) were measured.. Compared to controls, the 21 CF patients, with no intestinal complications, had increased intestinal outputs of albumin, IgG, IgM, eosinophil cationic protein, neutrophil elastase, interleukin 1beta, and interleukin 8. Similar values were obtained for the CF patients with fibrosing colonopathy.. These data suggest that there is immune activation in the gastrointestinal mucosa of children with cystic fibrosis, which may result from the basic cellular defect. Fibrosing colonopathy does not appear to be associated with increased inflammation.

Topics: Adolescent; Albumins; Case-Control Studies; Child; Child, Preschool; Cystic Fibrosis; Enterocolitis; Eosinophils; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Infant; Interleukin-1; Interleukin-8; Intestinal Mucosa; Leukocyte Elastase; Trypsin Inhibitors

Identifying noninvasive markers of pulmonary inflammation would be useful in assessing new therapies in children. Breath condensate is a simple and potentially acceptable sample medium even in small children. The technique has previously been used in adults, but not children with cystic fibrosis. The technique was assessed in 36 children with cystic fibrosis (mean age 10.4 yrs) and 17 control subjects, analysing samples for nitrite, interleukin(IL)-8 and salivary and nasal contamination. Correlations were made between levels of the inflammatory markers and forced expiratory volume in one second/forced vital capacity, chest radiograph score and use of inhaled steroids. On samples without significant contamination (<10 u x L(-1) amylase) nitrite was detected in 93% of samples at a median concentration of 3.0 microM compared with 50% of control samples at a median of 0.5 microM. Condensate amylase levels did not correlate with the nitrite value obtained (r=0.31). IL-8 was detected in 33% of CF samples. Breath condensate is an acceptable method of sample collection in children. Nitrite was raised in breath condensate from patients with cystic fibrosis when compared with control subjects.

Topics: Breath Tests; Child; Cystic Fibrosis; Humans; Interleukin-8; Nitrites; Saliva

Cystic fibrosis (CF) is a common, serious, and frequently fatal autosomal recessive genetic disorder associated with the poor function of chloride channels. Chronic endobronchial inflammation and bacterial infection are main causes of morbidity and mortality due to CF. The study dealt with a relationship between progression and inflammation markers. Twenty one CF children with acute pulmonary exacerbation were examined. The signs of peripheral blood inflammation (responses of lymphocytes to PHA and their sensitivity to the antiproliferative effect of glucocorticoids) and in situ inflammation markers (sputum elastase activity, IL-8 and TNF-alpha, and protein concentrations in the same sputum specimens). These laboratory findings were used to calculate a "laboratory index" (LI). The clinical status of each patient was evaluated with a "clinical index" (CI), a parameter that includes respiratory secretion cultures, pulmonary function test results, nutritional status, and the presence of disease-related complications. There was a positive linear correlation between LI and CI. The presence of P. aeruginosa was strongly associated with the changes of inflammatory markers. CF patients with prolonged P. aeruginosa infection demonstrated extremely enhanced elastase activity and elevated amounts of sputum IL-8 and TNF-alpha as compared to uninfected subjects. The lung elastase activities, sputum protein contents, and TNF-alpha levels in individuals with short-term colonization were at or below those without P. aeruginosa infection. In patients with or without short-term colonization, the normalization of laboratory parameters was strongly related to evident clinical improvement. At the same time, antibiotic treatment failed to suppress an excessive inflammatory response in the lungs of patients with prolonged P. aeruginosa infection. The importance of individual inflammation markers is discussed in the paper.

Topics: Adolescent; Biomarkers; Child; Child, Preschool; Cystic Fibrosis; Disease Progression; Humans; Immunologic Techniques; Inflammation; Interleukin-8; Pancreatic Elastase; T-Lymphocytes; Tumor Necrosis Factor-alpha

Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease. Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial. We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice. This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways. Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001). CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria. In non-CF grafts, these three sets of events were not observed before 6 h. Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state. After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Fetal Tissue Transplantation; Fetus; Humans; Inflammation; Interleukin-8; Leukocytes; Mice; Mice, SCID; Microscopy, Electron; Pseudomonas Infections; Trachea; Transplantation, Heterologous

In cystic fibrosis (CF), inflammatory mediator production by airway epithelial cells is a critical determinant of chronic airway inflammation. To determine whether altered signal transduction through the nuclear factor (NF)-kappaB pathway occurs in CF epithelial cells and results in excessive generation of inflammatory cytokines, we evaluated tumor necrosis factor (TNF)-alpha-induced production of the NF-kappaB-dependent cytokine interleukin (IL)-8 and activation of NF-kappaB in three different human bronchial epithelial cell lines: (1) BEAS cells that express wild-type CF transmembrane conductance regulator (CFTR), (2) IB3 cells with mutant CFTR, and (3) C38 cells, which are "corrected" IB3 cells complemented with wild-type CFTR. Treatment of cells with TNF-alpha (30 ng/ml) resulted in markedly elevated NF-kappaB activation and production of IL-8 by IB3 cells compared with BEAS and C38 cells. Despite the differences in NF- kappaB activation, no differences in basal levels of IkappaB-alpha or TNF-alpha- induced IkappaB-alpha processing and degradation were detected among the cell lines. In contrast, the basal level of IkappaB-beta was increased in the IB3 cells. Treatment with TNF-alpha resulted in increased formation of hypophosphorylated IkappaB-beta and increased nuclear localization of IkappaB-beta in IB3 cells compared with the other cell types. These findings provide additional evidence of a dysregulated inflammatory response in CF.

Topics: Bronchi; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; I-kappa B Proteins; Immunoblotting; Interleukin-8; Ligases; Mutagenesis; NF-kappa B; Phosphorylation; Respiratory Mucosa; Tumor Necrosis Factor-alpha

Infection of the cystic fibrosis (CF) airways elicits an exaggerated, interleukin-8 (IL-8) mediated, neutrophil inflammatory response. Necrosing neutrophils release DNA and actin into the airways, increasing the viscoelasticity of airway secretions. Mucolytics aim to improve airway clearance by reducing this viscoelasticity. DNase I reduces the viscoelasticity of CF sputum, and a human recombinant form of this enzyme is widely administered to patients with CF. Gelsolin, which cleaves actin polymers, is also known to reduce CF sputum viscosity in vitro, and it has been proposed as a future mucolytic agent. We have shown that the anionic polymers DNA and actin bind and mask immunologic recognition of the basic peptide IL-8 and prevent this chemokine from binding to neutrophil receptors. Reduction of CF sputum viscosity by DNase I or gelsolin in vitro was demonstrated to increase the proportion of free IL-8 and the IL-8-dependent neutrophil chemotactic activity of sputum supernatants. We hypothesize that an electrostatic interaction between polymer and chemokine may limit the inflammatory potential of the latter, but that this interaction may be weakened by polymer cleavage. The potential risk of increased inflammation via this mechanism suggests a caveat should be attendant on treatment of patients with CF with these mucolytic agents.

Topics: Actins; Chemotaxis, Leukocyte; Cystic Fibrosis; Deoxyribonuclease I; DNA; Expectorants; Gelsolin; Humans; In Vitro Techniques; Interleukin-8; Neutrophils; Sputum; Viscosity

Various treatment regimens and difficulties with research design are encountered with cystic fibrosis (CF) because no standard diagnostic criteria exist for defining acute respiratory exacerbations. This study evaluated the role of serial monitoring of concentrations of selected cytokines and inflammatory mediators in serum and sputum as predictors of respiratory exacerbation, as useful outcome measures for CF, and to guide therapy. Interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), neutrophil elastase-alpha-1-protease inhibitor complex (NE complex), protein, and alpha-1-protease inhibitor (alpha-1-PI) were measured in serum and sputum collected from CF patients during respiratory exacerbations and periods of well-being. Levels of NE complex, protein, and alpha-1-PI in sputum rose during respiratory exacerbations and fell after institution of antibiotic therapy (P = 0.078, 0.001, and 0.002, respectively). Mean (+/- standard error of the mean) levels of IL-8 and TNF-alpha were extremely high in sputum (13,780 +/- 916 and 249.4 +/- 23.5 ng/liter, respectively) but did not change significantly with clinical deterioration of the patient (P > 0.23). IL-8 and TNF-alpha were generally undetectable in serum, and therefore these measures were unhelpful. Drop in forced expiratory volume in 1 s was the only clinical or laboratory parameter that was close to being a determinant of respiratory exacerbation (P = 0.055). This study provides evidence of intense immunological activity occurring continually within the lungs of adult CF patients. Measurement of cytokines and inflammatory mediators in CF sputum is not helpful for identifying acute respiratory exacerbations.

Topics: Acute Disease; Adolescent; Adult; alpha 1-Antitrypsin; Cystic Fibrosis; Disease Progression; Female; Humans; Inflammation Mediators; Interleukin-8; Leukocyte Elastase; Male; Pneumonia, Bacterial; Predictive Value of Tests; Pseudomonas Infections; Reproducibility of Results; Respiratory Function Tests; Sputum; Tumor Necrosis Factor-alpha

To delineate the mechanisms that facilitate leukocyte migration into the cystic fibrosis (CF) lung, expression of chemokines, including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES, was compared between CF and non-CF airway epithelia. The findings presented herein demonstrate that, under either basal conditions or tumor necrosis factor-alpha (TNF-alpha)- and/or interferon-gamma (IFN-gamma)-stimulated conditions, a consistent pattern of differences in the secretion of IL-8 and MCP-1 between CF and non-CF epithelial cells was not observed. In contrast, CF epithelial cells expressed no detectable RANTES protein or mRNA under basal conditions or when stimulated with TNF-alpha and/or IFN-gamma (P

Topics: Bronchi; Chemokine CCL2; Chemokine CCL5; Chemokines; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Combinations; Epithelial Cells; Humans; Interferon-gamma; Interleukin-8; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The acquisition of Pseudomonas aeruginosa in the airways of patients with cystic fibrosis (CF) is the initial event leading to bronchiectasis and lung disease. Although the host factors that permit initial airway colonization are largely unknown, recent studies suggest that secretion of interleukin (IL)-8 by airway epithelia and local recruitment of neutrophils is the final pathway in a pulmonary cytokine network. To determine whether differences in cytokine production exist between normal and CF airway epithelia, secretion of immunoreactive IL-8 and IL-10 as well as specific messenger RNA (mRNA) abundance were compared in airway epithelia expressing normal and mutant CF transmembrane regulator. After induction with IL-1beta, a CF airway cell line engineered to express the wild-type CF gene (CFT1-LCFSN) secreted significantly more immunoreactive IL-8 than did its isogenic parent that expressed the mutant CF gene (CFT1) or an isogenic vector control line (CFT1-LC3). Further studies with the three related cell lines demonstrated that expression of CFT1-LCFSN was associated with a significant increase in uninduced secretion of immunoreactive IL-8 as well as a 10- to 20-fold increase in IL-8 mRNA abundance when compared with the isogenic lines expressing the mutant gene. IL-1beta induction and intracellular accumulation of IL-8 appeared to be unaffected by CF genotype. These studies suggest that IL-8 secretion by CF airway epithelial cells is defective and may contribute to Pseudomonas persistence in the CF airway. Further studies are needed to confirm this difference in other cell lines and determine the linkage between IL-8 production and CF gene expression.

Topics: Bronchi; Cell Line, Transformed; Cystic Fibrosis; Epithelial Cells; Homozygote; Humans; Interleukin-10; Interleukin-8; Lipopolysaccharides; Pseudomonas aeruginosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF). The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems. Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications. Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P. aeruginosa. The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen. Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF. We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P. aeruginosa). These data may provide further rationale for anti-inflammatory therapy early in CF.

Topics: Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Colony Count, Microbial; Cystic Fibrosis; Female; Humans; Infant; Infant, Newborn; Inflammation Mediators; Interleukin-8; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Reference Values; Systemic Inflammatory Response Syndrome

Reports that lung inflammation in patients with cystic fibrosis (CF) might precede infection raise the possibility that the excessive inflammatory response in lungs of patients with CF might be directly related to defects in epithelial cell cystic fibrosis transmembrane regulator.. We sought to determine the relationship of epithelial cell cytokine production to CF lung disease.. Immunofluorescence and cultures of freshly obtained bronchial epithelial cells and ELISA for IL-10, IL-8, and IL-6 were used to study alterations in epithelial cell cytokine production.. Fresh bronchial epithelial cells from healthy control subjects (HCs) secreted 98 +/- 20 pg/mL of the anti-inflammatory cytokine IL-10 when placed in primary culture in vitro but little or no IL-8 or IL-6. In contrast, fresh epithelial cells from patients with CF did not secrete detectable IL-10 but produced 38 +/- 17 pg/mL IL-8 and 40 +/- 17 pg/mL IL-6. These data correlated very well with the immunofluorescence data. The correlation between the immunofluorescent staining of fresh bronchial epithelial cells from both the HCs and patients with CF and the concentrations of cytokines in epithelial lining fluid suggests a reciprocal relationship between anti-inflammatory (IL-10) and proinflammatory (IL-6 and IL-8) cytokine production by the epithelial cells in HCs versus patients with CF.. Alterations in epithelial cell cytokine production in the lungs of patients with CF may contribute to the excessive local inflammation.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Bronchi; Cells, Cultured; Cystic Fibrosis; Cytokines; Epithelial Cells; Female; Humans; Infant; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Male; Tumor Necrosis Factor-alpha

The inflammatory pathogenesis in airways of patients with cystic fibrosis (CF) is still unresolved. We demonstrate here that in in situ human DeltaF508 homozygous CF bronchial tissues, submucosal gland cells exhibit an absence of inhibitor factor kappaBalpha (IkappaBalpha) and high levels of chemokine interleukin-8 (IL-8) expression. These results were confirmed by cultured human CF bronchial gland cells in which a lack of cytosolic IkappaBalpha and high levels of constitutively activated nuclear factor kappaB (NFkappaB) associated with an up-regulation of IL-8 production (13-fold increase) were found when compared to non-CF (control) disease bronchial gland cells. We also demonstrated that the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, significantly reduces the endogenous and Pseudomonas aeruginosa lipopolysaccharide-induced IL-8 production in cultured CF bronchial gland cells by increasing cytosolic IkappaBalpha protein levels. Overall, results show that genistein is a potent inhibitor of the activated NFkappaB identified in CF gland cells. This strong inhibition of constitutively activated NFkappaB and the resulting down-regulation of IL-8 production by genistein in the CF gland cells highlights the key role played by cytosolic IkappaBalpha in the regulation of inflammatory processes in CF human airway cells.

Topics: Adolescent; Adult; Aged; Bronchi; Cell Nucleus; Cells, Cultured; Child; Cystic Fibrosis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Genistein; Humans; Immunohistochemistry; Interleukin-8; Lipopolysaccharides; Lung Transplantation; Male; Middle Aged; NF-kappa B; Pseudomonas aeruginosa; Time Factors

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Topics: Acylation; Antimicrobial Cationic Peptides; Arabinose; Bacterial Proteins; Cells, Cultured; Cystic Fibrosis; Drug Resistance, Microbial; Humans; Infant; Interleukin-8; Lipid A; Lipopolysaccharides; Magnesium; Mutation; Palmitates; Peptides; Polymyxins; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory System; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Virulence

Chronic endobronchial inflammation and bacterial infection are the main causes of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive genetic disorder associated with improper function of chloride channels. Inflammation in CF lung is greatly amplified after Pseudomonas aeruginosa infection. In this study the relationship between P. aeruginosa status and inflammatory markers has been investigated. Seventeen CF children in acute lung exacerbation were examined. CF patients without P. aeruginosa infection were characterized by elevated activity of sputum elastase, reduced response of peripheral blood lymphocytes to PHA and significant resistance to the antiproliferative action of glucocorticoids. These parameters were normalized after antibiotic treatment. The patients with prolonged P. aeruginosa infection demonstrated extremely high levels of elastase activity and elevated amounts of sputum IL-8 and TNF-alpha. Although antibiotic treatment resulted in clinical improvement, it failed to suppress excessive immune response in the lung. The data indicate that CF patients with prolonged P. aeruginosa need the modified treatment, which should include immunomodulating drugs and protease inhibitors as well as antibacterial therapy.

Topics: Anti-Bacterial Agents; Cells, Cultured; Child; Cystic Fibrosis; Dexamethasone; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Lung Diseases; Lymphocyte Activation; Lymphocytes; Phytohemagglutinins; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum; Tumor Necrosis Factor-alpha; Vital Capacity

The neutrophil-dominated inflammation of the lung in cystic fibrosis (CF) has traditionally been seen as a physiological response to continuous opportunistic infection. Recent studies suggest, however, that regulation of the inflammatory response itself may be altered in CF. Neutrophil migration from the bloodstream involves alterations in surface expression of the adhesion molecules L-selectin and Mac-1 (CD11b/CD18). The aim of this study was to assess neutrophil adhesion molecule expression and responsiveness in CF. Neutrophils from chronic (n = 16) and acutely infected (n = 13) CF patients and 15 normal control subjects were directly assessed by Fluorescence-activated cell sorter (FACS) analysis for surface expression of L-selectin and CD11b before and after stimulation with interleukin 8 (IL-8) or f-Met-Leu-Phe (fMLP). Neutrophils from stable (n = 5) and acutely infected (n = 5) non-CF bronchiectasis patients were also assessed. Surface upregulation of CD11b was similar in all groups. Basal levels of L-selectin were also comparable among all groups, however, when stimulated, neutrophils from both stable and acutely infected CF patients shed significantly less L-selectin than those from control subjects (p < 0.05 and p < 0.01, respectively). This decreased responsiveness was not observed in either stable or acutely infected non-CF bronchiectasis patients. These results add to the accumulating evidence suggestive of a defective inflammatory response in CF.

Topics: Acute Disease; Adolescent; Adult; Aged; Bronchiectasis; CD11 Antigens; CD18 Antigens; Cell Movement; Cell Separation; Chemotaxis, Leukocyte; Chronic Disease; Cystic Fibrosis; Female; Flow Cytometry; Gene Expression Regulation; Humans; Interleukin-8; L-Selectin; Macrophage-1 Antigen; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Up-Regulation

PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease. Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8. Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P. aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells. Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1. IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC). Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P. aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum.

Topics: Bacterial Adhesion; Bacterial Outer Membrane Proteins; Bronchi; Cell Nucleus; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Fimbriae Proteins; Humans; Interleukin-8; NF-kappa B; Protease Inhibitors; Pseudomonas aeruginosa; Trachea

High levels of neutrophils and the neutrophil-attracting chemokine interleukin (IL)-8 have been observed in the airways of patients with cystic fibrosis (CF). We hypothesized that CF respiratory epithelium produces excessive amounts of IL-8 either at baseline or after stimulation. To test this hypothesis we compared immunoreactive IL-8 release by primary nasal epithelial cell (NEC) cultures established from young children with or without CF, at several time points after stimulation of cultures with tumor necrosis factor-alpha (TNF-alpha) or infection with respiratory syncytial virus (RSV). Both stimuli induced significantly increased IL-8 release by both CF and control cultures. However, there was no difference between CF and control cells in either the magnitude or duration of the IL-8 response. The effect of transduction of CF cells with Ad5-CBCFTR, an adenovirus vector mediating expression of cystic fibrosis transmembrane regulator (CFTR), on IL-8 production was also determined. TNF-alpha stimulated IL-8 production was not different in Ad5-CBCFTR-transduced, -untransduced, or Ad5-CMVLacZ-transduced control cells. Lastly, immortalized CF tracheal epithelial cell lines, both uncorrected and retrovirally corrected with CFTR, were compared. Again, TNF-alpha-stimulated IL-8 production did not differ significantly between cell lines with and without functioning CFTR. Our data suggest that isolated CF NECs cultured under these conditions do not produce more IL-8 than do non-CF control cultures, either at baseline or after incubation with the nonspecific stimuli TNF-alpha and RSV. We conclude that the absence of functioning CFTR alone is not sufficient to cause excessive production of IL-8.

Topics: Adenoviridae; Adolescent; Adult; Cell Line, Transformed; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Gene Transfer Techniques; Humans; Interleukin-8; Male; Middle Aged; Nasal Mucosa; Respiratory Syncytial Viruses; Tumor Necrosis Factor-alpha

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Topics: Adolescent; Adult; Bronchi; Cell Count; Cells, Cultured; Child; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dexamethasone; DNA Probes; Epithelial Cells; Exocrine Glands; Female; Fluorescent Antibody Technique, Indirect; Humans; Interleukin-8; Male; Middle Aged; Up-Regulation

Recently, some investigators have observed elevated concentrations of chloride in the airway surface fluid (ASF) overlying respiratory epithelia from cystic fibrosis (CF) patients compared with ASF overlying non-CF epithelia. Others have shown that this elevated ASF salt concentration can inactivate human beta-defensin-1, an antimicrobial peptide secreted by respiratory epithelia. This could impair the primary epithelial defense against bacteria in the CF airway, thereby forcing a greater reliance on polymorphonuclear leukocyte (PMN)-mediated defenses. Pseudomonas aeruginosa (Psa) flourishes in the CF airway despite the presence of abundant PMN. We therefore investigated whether elevated ASF chloride concentration in CF might also compromise PMN function. We employed a cell-culture model in which halide concentrations and osmolarity were varied independently. We examined the effects of chloride concentration on three aspects of PMN function: recruitment of PMN to the airway (production of interleukin-8 [IL-8]), PMN antimicrobial activity (killing of Psa), and PMN clearance from the airways (apoptosis and lysis). We found that exposure to elevated chloride concentration increased PMN synthesis of IL-8, decreased PMN killing of Psa, and accelerated PMN apoptosis and lysis. In CF airways, elevated chloride therefore could contribute to the increased number of PMN recruited into the airways, the increased survival of Psa, and the increased quantity of toxic mediators released by PMN into the airways. These effects of elevated chloride on PMN function may provide another causal link between loss of cystic fibrosis transmembrane conductance regulator function and CF lung disease.

Topics: Adult; Apoptosis; Cells, Cultured; Chlorides; Cystic Fibrosis; Dose-Response Relationship, Drug; Humans; Interleukin-8; Lung; Neutrophil Activation; Neutrophils; Phagocytosis; Pneumonia; Pseudomonas aeruginosa

Cytokine levels in nasal and lower airways in young cystic fibrosis (CF) patients were compared with those in controls. Nasal (NLF) and bronchoalveolar (BALF) lavage fluids were obtained from children with or without CF who were undergoing bronchoscopy for clinical indications. In NLF, neither inflammatory cells nor cytokine concentrations differed between patients and controls. However, interleukin (IL)-8 levels in infected BALF from children with CF were markedly elevated compared with levels in infected and uninfected controls, even after standardization of IL-8 concentrations to bacterial counts. BALF IL-6 was modestly elevated in infected CF patients compared with uninfected but not infected controls; IL-10 did not differ among the groups. NLF and BALF IL-8 levels were not significantly correlated. Excessive airway inflammation in early CF thus appears to be confined to the lower respiratory tract, and IL-8 levels are markedly increased in children with CF compared with control children with a bacterial infection of the lower airways.

Topics: Bacterial Infections; Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Cystic Fibrosis; Cytokines; Humans; Infant; Interleukin-10; Interleukin-6; Interleukin-8; Leukocyte Count; Nasal Lavage Fluid; Nasal Mucosa; Neutrophils; Time Factors

Airway inflammation is an important component of cystic fibrosis (CF) lung disease. To determine whether this begins early in the illness, before the onset of infection, we examined bronchoalveolar lavage (BAL) fluid from 46 newly diagnosed infants with CF under the age of 6 mo identified by a neonatal screening program. These infants were divided into three groups: 10 had not experienced respiratory symptoms or received antibiotics and pathogens were absent in their BAL fluid; 18 had clear evidence of lower respiratory viral or bacterial (> or = 10(5) CFU/ml) infection; and the remaining 18 had either respiratory symptoms, taken antibiotics, or had < 10(5) CFU/ml of respiratory pathogens. Their BAL cytology, interleukin-8, and elastolytic activity were compared with those from 13 control subjects. In a longitudinal study to assess if inflammation develops or persists in the absence of infection, the results of 56 paired annual BAL specimens from 44 CF infants were grouped according to whether they showed absence, development, clearance, or persistence of infection. In newly diagnosed infants with CF, those without infection had BAL profiles comparable with control subjects while those with a lower respiratory infection had evidence of airway inflammation. In older children, the development and persistence of infection was accompanied by increased inflammatory markers, whereas these were decreased in the absence, or with the clearance, of infection. We conclude that airway inflammation follows respiratory infection and, in young children, improves when pathogens are eradicated from the airways.

Topics: Anti-Bacterial Agents; Bacterial Infections; Biomarkers; Bronchoalveolar Lavage Fluid; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Drug Therapy, Combination; Female; Humans; Infant; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Longitudinal Studies; Male; Neutrophils; Pneumonia, Bacterial; Pneumonia, Viral; Respiratory Tract Infections; Virus Diseases

As collections of lower respiratory tract specimens from young children with cystic fibrosis (CF) are difficult, we determined whether oropharyngeal cultures predicted lower airway pathogens. During 1992-1994, 75 of 90 (83%) infants with CF diagnosed by neonatal screening had 150 simultaneous bronchoalveolar lavage (BAL) and oropharyngeal specimens collected for quantitative bacterial culture at a mean age of 17 months (range, 1-52). Ten children undergoing bronchoscopy for stridor served as controls. Total and differential cell counts and interleukin-8 concentrations were measured in BAL fluid. A subset of bacterial pathogens were typed by pulsed field gel electrophoresis. A non-linear relationship with inflammatory markers supported a diagnosis of lower airway infection when > or = 10(5) colony-forming units/ml were detected. This criterion was met in 47 (31%) BAL cultures from 37 (49%) children. Staphylococcus aureus (19%), Pseudomonas aeruginosa (11%), and Hemophilus influenzae (8%) were the major lower airway pathogens. In oropharyngeal cultures, S. aureus (47%), Escherichia coli (23%), H. influenzae (15%), and P. aeruginosa (13%) predominated. The sensitivity, specificity, and positive and negative predictive values of oropharyngeal cultures for pathogens causing lower respiratory infections were 82%, 83%, 41%, and 97%, respectively. When there was agreement between paired oropharyngeal and BAL cultures, genetic fingerprinting showed some strains of the same organism were unrelated. We conclude that oropharyngeal cultures do not reliably predict the presence of bacterial pathogens in the lower airways of young CF children.

Topics: Bacteriological Techniques; Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Escherichia coli Infections; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Interleukin-8; Male; Oropharynx; Pneumonia, Bacterial; Predictive Value of Tests; Pseudomonas Infections; Respiratory Tract Infections; Staphylococcal Infections

In cystic fibrosis (CF), neutrophil-dominated airway inflammation results in high levels of neutrophil elastase (NE). Some of these proteases are sequestered by the large amounts of deoxyribonucleic acid (DNA) present in purulent sputum. Recombinant human deoxyribonuclease (rhDNase), a new treatment in CF, depolymerizes DNA. Our concerns were that this might release proteases bound to DNA, which could be potentially harmful. The in vitro and in vivo effects of rhDNase on NE and interleukin-8 (IL-8) were evaluated. The acute effects of rhDNase were evaluated in CF patients during the first 6 days of treatment. Medium-term effects were evaluated in stable CF patients observed in rhDNase over 6 months. Sputum samples were collected at regular intervals and NE activity was measured by a fluorimetric assay and IL-8 with a radioimmunoassay. In vitro addition of rhDNase resulted in a twofold increase in protease activity and this was reflected in an acute transient rise on initiation of treatment with rhDNase. Medium-term treatment was associated with a decline in NE activity and IL-8. These in vivo results are encouraging, since the increase in protease activity was transient and the trend over 6 months was a reduction in both inflammatory markers.

Topics: Cystic Fibrosis; Deoxyribonuclease I; Expectorants; Fluorometry; Humans; Interleukin-8; Leukocyte Elastase; Pancreatic Elastase; Radioimmunoassay; Recombinant Proteins; Sputum; Treatment Outcome

The cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and intercellular adhesion molecule-1 (ICAM-1) have important roles in regulating neutrophil migration and the inflammatory response. To determine whether the concentration of these cytokines and soluble ICAM-1 (sICAM-1) in sputum was increased in patients with cystic fibrosis during acute exacerbations, we conducted (1) a cross-sectional study of 40 patients, 22 who were clinically well and 18 with acute pulmonary exacerbations; and (2) an 11 months longitudinal study of 16 patients. Significant differences in clinical scores, pulmonary function, and sputum neutrophil density were found between the acutely ill and the well group. There was a strong linear relationship (P < 0.0005) between TNF-alpha and IL-8 concentrations in sputum, but no association between clinical status and cytokine concentrations. The concentration of sICAM-1 was lower in acutely ill compared with well patients in the cross-sectional study. Recovery of exogenous IL-8 added to sputum was complete, while recovery of TNF-alpha averaged 70%. Recovery of exogenous sICAM-1 was only 43%, and the recoveries were lower in sputum samples from acutely ill patients than those from stable patients (P = 0.018). These data indicate that in cystic fibrosis patients, sputum concentrations of TNF-alpha and IL-8 are not increased during acute exacerbations of pulmonary inflammation.

Topics: Adult; Cell Movement; Cross-Sectional Studies; Cystic Fibrosis; Cytokines; Disease Progression; Female; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Longitudinal Studies; Male; Neutrophils; Sputum; Tumor Necrosis Factor-alpha

Neutrophil-activating peptide ENA-78 is a novel chemotactic cytokine isolated from a human type II pulmonary epithelial cell line. It is a member of the chemokine family of proinflammatory polypeptides and exhibits structural homology to interleukin-8 (IL-8) and GROalpha. The immunohistochemical identification of ENA-78 in pulmonary alveolar leukocytes of bovine pneumonic lungs supports a role for ENA-78 in the pathogenesis of pulmonary inflammation. Although ENA-78 is able to stimulate polymorphonuclear neutrophils (PMN), neither its binding specificities nor its expression in human pulmonary disease states have been determined. 125I-labeled ENA-78 binds with high affinity to human PMN. Its actions on PMN appear to be mediated by the IL-8 type B receptor, to which it binds with a K(d) of 2.2 nM. Human IL-8, GROalpha, and murine KC compete with high affinity for 125I-ENA-78 binding to the human IL-8 type B receptor. In contrast, 125I-ENA-78 does not bind to the IL-8 type A receptor nor does it compete significantly for 125I-IL-8 binding to this same receptor. ENA-78 is a potent upregulator of Mac-1 cell surface expression. In addition, ENA-78 mRNA is detected in cystic fibrosis lung but is not detected in normal donor lung. Thus, ENA-78 mRNA levels appear to be increased in human pulmonary inflammation and its stimulatory activities on PMN appear to be a function mediated primarily by the IL-8 type B receptor.

Topics: Animals; Base Sequence; Binding, Competitive; Cell Line; Chemokine CXCL1; Chemokine CXCL5; Chemokines; Chemokines, CXC; Chemotactic Factors; Chlorocebus aethiops; Cloning, Molecular; Cystic Fibrosis; Cytokines; Gene Expression; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lung; Macrophage-1 Antigen; Mice; Molecular Sequence Data; Neutrophils; Receptors, Interleukin; Receptors, Interleukin-8B; RNA, Messenger; Up-Regulation

In cystic fibrosis (CF), large amounts of free leucocyte proteases are present in bronchial secretions, contributing to progressive lung damage. Recombinant, human deoxyribonuclease (rhDNase) is a new therapeutic agent that decreases sputum viscosity. However, deoxyribonuclease has been shown, in vitro, to release cationic enzymes from complexes with deoxyribonucleic acid (DNA). The present study was conducted to assess this effect in vivo. Free human leucocyte elastase (HLE), human leucocyte cathepsin G (HCG), total chemotactic activity, and interleukin-8 (IL-8) were determined in sputum from eight patients before, during and after rhDNase treatment. After 15 days of treatment, HLE activity increased by 81+/-44% (NS), and HCG by 189+/-70% (p<0.05). One week after stopping a 4-6 months treatment, HLE activity decreased by 35+/-18% (p<0.05), and HCG by 43+/-11% (p<0.05). Sputum bacterial density, chemotactic activity, and IL-8 concentration did not change. Thus, treatment with rhDNase can indeed increase the activity of HLE and HCG in the bronchial secretions of CF patients, and this effect is still detectable after several months of treatment. If this can be shown to be clinically relevant, combination therapy of recombinant human deoxyribonuclease with protease inhibitors should be considered as an approach to the problem.

Topics: Adult; Aerosols; Cathepsin G; Cathepsins; Chemotaxis, Leukocyte; Cystic Fibrosis; Deoxyribonucleases; Female; Humans; Interleukin-8; Leukocyte Elastase; Male; Pseudomonas aeruginosa; Recombinant Proteins; Serine Endopeptidases; Sputum; Staphylococcus aureus

Topics: Bronchitis; Cystic Fibrosis; Haemophilus influenzae; Humans; Interleukin-8; Mucociliary Clearance; Mucous Membrane; Respiratory Tract Infections; Streptococcus pneumoniae

We used a whole-gut perfusion technique to study subclinical gut inflammation in children with cystic fibrosis (18 elective tests, three lavages to treat distal intestinal obstruction syndrome); and in 12 control children with constipation or pre-colonoscopy. We assayed for haemoglobin, IgG, albumin, alpha-1-antitrypsin, granulocyte elastase, interleukin-1 beta (IL-1 beta) and IL-8 concentrations in whole-gut lavage fluid. Results for two children with distal intestinal obstruction syndrome, the only children in the series taking Nutrizym 22, were strikingly abnormal. This new test has revealed subclinical gut mucosal inflammation in a minority of CF children, for which distal intestinal obstruction syndrome, Nutrizym 22 treatment, or both, may be risk factors.

Topics: Adolescent; Albumins; alpha 1-Antitrypsin; Amylases; Bromelains; Case-Control Studies; Child; Child, Preschool; Colitis; Colonoscopy; Constipation; Cystic Fibrosis; Drug Combinations; Female; Gastrointestinal Agents; Hemoglobins; Humans; Immunoglobulin G; Interleukin-1; Interleukin-8; Intestinal Obstruction; Leukocyte Elastase; Lipase; Male; Pancreatic Elastase; Pancreatic Extracts; Pancreatin; Pancrelipase; Risk Factors; Syndrome; Therapeutic Irrigation; Trypsin

Bronchopulmonary disease in patients with cystic fibrosis (CF) is a paradigm of neutrophil-dominated airway inflammation. We hypothesized that proinflammatory cytokines contribute to a localized neutrophil-dominated inflammatory state as present in CF airways. In a cross-sectional study, we analyzed 63 sputum samples from 33 CF patients for concentrations of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), and granulocyte-colony stimulating factor (G-CSF) by means of enzyme-linked immunosorbent assay. Furthermore, the activity of neutrophil elastase (NE) in the sputum samples was determined using a specific chromogenic substrate. Compared to sputum samples from 10 healthy controls, there were significantly increased concentrations of IL-1 beta, IL-8 and TNF-alpha in the CF sputum samples. The concentration of IL-8 correlated significantly with NE activity in the CF sputum samples. In CF patients with airways chronically colonized with Pseudomonas aeruginosa, IL-8 concentrations in sputum were significantly enhanced. In glucocorticoid-treated patients, IL-1 alpha and G-CSF sputum concentrations were significantly lower when compared to levels in the other patients. These results show that there are high concentrations of proinflammatory cytokines in CF airways which may contribute to the localized neutrophil-dominated inflammatory state found clinically.

Topics: Adolescent; Adult; Child; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Female; Glucocorticoids; Granulocyte Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Interleukins; Leukocyte Elastase; Lung; Male; Neutrophils; Pancreatic Elastase; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum; Tumor Necrosis Factor-alpha

Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) concentrations were measured in faecal samples from nine patients with cystic fibrosis and nine healthy age matched controls. The patients were assessed with Shwachman score, apparent energy absorption, pancreatic enzyme dosage, simple spirometry, and presence of pseudomonal colonisation. Median (range) wet stool IL-8 and TNF-alpha concentrations in patients were 32,113 pg/g (21,656-178,128) and 3187 pg/g (368-17,611) respectively, compared with < 43.5 pg (IL-8)/g (< 22-4079) and 99 pg (TNF-alpha)/g (< 0.26-231) in controls. IL-8 concentration was negatively correlated with Shwachman score (r = -0.79) and pancreatic enzyme dosage (r = -0.77), but not with energy absorption. Seven patients were mature enough to cooperate with spirometry. Their IL-8 concentrations correlated with percentage predicted forced expiratory volume in one second (r = -0.78). IL-8 concentration was greater in four patients with, than five without, established pseudomonal colonisation: median difference 134,583 pg/g. TNF-alpha concentration was not correlated with measures of disease severity. Faecal IL-8 concentration might reflect the severity of pulmonary inflammation in cystic fibrosis and could provide an easily obtainable marker of disease activity.

Topics: Adolescent; Adult; Biomarkers; Child; Cystic Fibrosis; Feces; Humans; Interleukin-8; Lipase; Lung; Pancreatic Extracts; Pancrelipase; Pseudomonas Infections; Spirometry; Tumor Necrosis Factor-alpha

The mechanisms underlying the initiation of lung disease and early respiratory morbidity in cystic fibrosis (CF) are poorly understood. By identifying infants with CF through a statewide neonatal screening program, we investigated whether airway inflammation was present in these infants, with the goal of furthering our understanding of the early events in this lung disease. Bronchoalveolar lavage fluid (BALF) from 16 infants with CF (mean age, 6 mo) and 11 disease control infants (mean age, 12 mo) was examined for the following inflammatory parameters: (1) neutrophil count; (2) activity of free neutrophil elastase; (3) elastase/alpha 1-antiprotease inhibitor complexes; and (4) the level of interleukin-8 (IL-8). We also quantified the spontaneous level of expression of IL-8 mRNA transcripts by airway macrophages. Each index of airway inflammation was increased in the BALF of infants with CF as compared with control infants. In addition, both the number of neutrophils and IL-8 levels were increased in infants with CF who had negative cultures (n = 7) for common bacterial CF-related pathogens, as well as for common respiratory viruses and fungi at the time of bronchoalveolar lavage (BAL). These findings suggest that airway inflammation is already present in infants with CF who are as young as 4 wks. Furthermore, although many different cell types (e.g., epithelial cells) may express IL-8, airway macrophages appear to be a source of this chemokine, and may thus play a prominent role in early neutrophil influx into the lung.

Topics: alpha 1-Antitrypsin; Base Sequence; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Humans; Infant; Infant, Newborn; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Lung Diseases; Macrophages, Alveolar; Molecular Sequence Data; Neutrophils; Pancreatic Elastase; RNA, Messenger

We examined the production of reactive nitrogen intermediates in the tracheo-bronchial tree of patients with cystic fibrosis (CF). Examination of the soluble phase of sputa from 17 CF patients revealed the presence of high levels of NO2-/NO3- assayed by the Greiss reaction. We also examined the presence of the chemotactic cytokine interleukin-8 (IL-8) in these samples so as to assess another important inflammatory marker; high levels of IL-8 were present in the sputa of cystic fibrosis subjects. The elevated nitrite was not produced by the presence of Pseudomonas bacteria in the sputa, inasmuch as bacteria in culture released undetectable amounts of nitrite in culture media. Neutrophils from the sputa of CF patients with disease exacerbation released higher amounts of nitrite and IL-8. Neutrophils from the sputa were also shown to spontaneously release substantial amounts of nitrite in the supernatants, and this release was partly blocked by the antagonist NG-mono-methyl-L-arginine (L-NMMA). Blood neutrophils were shown to release nitrite only in response to challenge with CF-associated strains of Pseudomonas, and not exposure to cytokines. There was no significant differences in nitrite release between normal and CF blood polymorphonuclear leucocytes (PMNs). A study of upper airway epithelial cell lines showed that these cells released low amounts of nitrite after infection with CF-associated strains of Pseudomonas but not after cytokine exposure. Epithelial cell lines with CF or normal phenotypes were shown to release similar quantities of nitrite, upon stimulation with Pseudomonas. These data demonstrate that elevated levels of reactive nitrogen intermediates and IL-8 are produced in the tracheo-bronchial tree of subjects with CF. Levels of IL-8 and nitrite were higher in the secretions of CF subjects with disease exacerbation. The involvement of nitric oxide and other reactive nitrogen intermediates produced by neutrophils and other cells in the tissue damaging processes in CF deserves further investigation.

Topics: Adolescent; Adult; Bronchi; Child; Cystic Fibrosis; Humans; Inflammation Mediators; Interleukin-8; Nasal Mucosa; Neutrophils; Nitric Oxide; Nitrites; Pseudomonas aeruginosa; Sputum; Trachea

Inflammation associated with neutrophil infiltration is a commonly observed feature of children with cystic fibrosis. Production of the major neutrophil chemotactic cytokine interleukin 8 (IL-8) is potentially of great importance in the pathology of cystic fibrosis. Concentrations of IL-8 in both sputum and bronchoalveolar lavage fluid have been found to be higher in children with cystic fibrosis than in controls. The IL-8 induced chemotactic response and numbers of IL-8 receptors on peripheral neutrophils obtained from children with cystic fibrosis have been compared with a control group of children.. Cells were isolated from 18 patients with cystic fibrosis (aged 4-20 years) and 13 controls (aged 5-12 years) by dextran centrifugation followed by separation on Lymphoprep. Chemotaxis was assayed using multiwell microchemotaxis chambers and 5 microns polycarbonate filters. Filters were fixed and stained with Haema-Gurr for counting. Results were expressed as numbers of neutrophils per high power field (HPF).. At the optimum concentration (1 x 10(-8) mol/l) the number of cells migrating were similar for controls (150 (12)/HPF) and for the cystic fibrosis group (140 (14)/HPF)). At lower concentrations the numbers of neutrophils migrating were lower for the cystic fibrosis group. Scatchard analysis of 125I-labelled IL-8 binding revealed lower numbers of receptors on neutrophils from patients with cystic fibrosis (22,000 per cell) than from controls (75,000 per cell).. Reduced responsiveness to IL-8 of neutrophils from patients with cystic fibrosis is associated with receptor desensitisation as a result of exposure to high systemic levels of IL-8.

Topics: Adolescent; Adult; Chemotaxis, Leukocyte; Child; Child, Preschool; Cystic Fibrosis; Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Interleukin-8; Leukocyte Count; Male; Neutrophils; Receptors, Interleukin; Receptors, Interleukin-8A

Polymorphonuclear leukocytes (PMNL) in bronchoalveolar lavage (BAL) fluid of cystic fibrosis patients express increased levels of receptor for the Fc portion of IgA (Fc alpha R), similar to those found on PMNL stimulated with FMLP in vitro. Since tumor necrosis factor-alpha (TNF alpha) is an activator of PMNL and is found at inflammatory sites, its effects on Fc alpha R expression and IgA-mediated PMNL functions were investigated. Exposure of PMNL to TNF alpha increased surface expression of Fc alpha R 2- to 3-fold, increased superoxide production in response to aggregated IgA 5-fold, and increased phagocytosis of IgA aggregates 3- to 4-fold. Interleukin-8 did not increase Fc alpha R expression and did not enhance IgA-mediated superoxide generation. IgA-dependent PMNL killing of Pseudomonas aeruginosa was enhanced when PMNL were pretreated with TNF alpha. Thus, interactions between phagocytic host defense mechanisms and mucosal IgA may be enhanced by TNF alpha in the inflammatory milieu of the cystic fibrosis lung.

Topics: Adolescent; Adult; Cystic Fibrosis; Humans; Immunoglobulin A; Immunoglobulin G; Interleukin-8; Neutrophils; Phagocytosis; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Fc; Superoxides; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is characterized by a dramatic neutrophil recruitment and repeated Pseudomonas infections in the lungs. To evaluate cytokine releasibility by airway epithelial cells in the context of CF, we studied primary nasal epithelial cells isolated from the upper airways and continuous epithelial cell lines from normal and CF subjects. Relatively low levels of interleukin (IL)-8, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were produced spontaneously by primary epithelial cells (< 50 pg/10(6) cells) and higher levels of colony-stimulating factor-1 (CSF-1) (1 to 2 ng/10(6) cells). Cells were stimulated with substances that are likely to be present in the inflamed lungs of CF patients-namely, the proinflammatory monokines IL-1 and tumor necrosis factor-alpha (TNF alpha) as well as neutrophil elastase and bacterial products from Pseudomonas (mucoid exopolysaccharide [MEP] and rhamnolipids). Both IL-1 and TNF alpha induced a dose-dependent release of IL-6 (5 to 10 ng/10(6) cells) and GM-CSF (2 to 3 ng/10(6) cells) by primary epithelial cells from eight normal volunteers. The TNF alpha/IL-1-stimulated GM-CSF release was blocked by the addition of 1 microM dexamethasone, whereas basal CSF-1 release was unaffected. Neutrophil elastase was a potent inducer of IL-8 and GM-CSF both in primary epithelial cells and in cell lines. Dexamethasone (1 microM) did not inhibit elastase-induced IL-8 release in either normal or CF epithelial cells. Rhamnolipids and MEP were found to stimulate the copious release of IL-8, GM-CSF, and IL-6 from epithelial cells, in a steroid-sensitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Cells, Cultured; Colony-Stimulating Factors; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dexamethasone; Epithelial Cells; Epithelium; Humans; Interleukin-6; Interleukin-8; Membrane Proteins; Nasal Mucosa; Phenotype

Chronic airway inflammation is an important feature of cystic fibrosis (CF), markedly influencing morbidity and mortality. We wanted to assess the contribution of the respiratory epithelium in the mediation of local inflammatory events, and, more particularly, its regulating role through cytokine secretion. We have studied the regulation of interleukin-6 and 8 (IL-6 and IL-8) production by the SV40 transformed airway epithelial cell line JME/CF15 (homozygous for the deletion of Phe 508). We show that unstimulated JME/CF15 cells secrete IL-6 and IL-8. Neutrophil chemotactic activity (NCA) is detected in supernatants. The secretion of IL-6 and IL-8 is increased following stimulation of the JME/CF15 cells by IL-1 beta and neutrophil elastase. Lipopolysaccharide and granulocyte macrophage colony stimulating factor (GM-CSF) have no effect on secretion of IL-6 or IL-8. Neutrophil elastase inactivates recombinant human IL-6 at 37 degrees C in vitro, but has no effect at 4 degrees C, suggesting a proteolytic effect of elastase on IL-6. IL-8 activity remains preserved, even after prolonged exposure to elastase. Our data suggest that the airway epithelium may play an active role in the mediation of neutrophil chemotaxis. Local production of IL-8 in response to elastase and IL-1 beta, together with the inactivation of the anti-inflammatory protein IL-6, may result in a significant upregulation of airway inflammation in cystic fibrosis.

Topics: Cells, Cultured; Chemotaxis, Leukocyte; Cystic Fibrosis; Cytokines; Epithelial Cells; Epithelium; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Neutrophils; Pancreatic Elastase; Respiratory System

Concurrent pulmonary inflammation and neutrophil infiltration are characteristic of children with cystic fibrosis (CF). The production of the major neutrophil chemotactic cytokine IL-8 by alveolar macrophages or other cells could be of great importance in the pathology of acute lung disease, but its role in the persistent lung inflammation characteristic of CF has not been evaluated. In this study, we have measured, by ELISA, the concentration of IL-8 in sputum, bronchoalveolar lavage, and sera specimens obtained from children with CF. For comparison, IL-8 in bronchoalveolar lavage obtained from asthmatic patients and from non-CF children with or without lung infection and in sera from age-matched controls was measured. High levels of IL-8 were measured in sputum (mean = 2952 pM) and in bronchoalveolar lavage (mean = 6624 pM) from CF patients. In both cases, there was a significant correlation between clinical status (Schwachman score) and IL-8 levels. This was not true for IL-8 levels measured in sera, which nevertheless were significantly higher in CF patients (p = 0.0001) than in normal controls in the over-10-y age group.

Topics: Adolescent; Biomarkers; Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Cystic Fibrosis; Humans; Inflammation; Interleukin-8; Pneumonia; Sputum

Sputum from patients with cystic fibrosis, bronchiectasis, and chronic bronchitis contains neutrophils and neutrophil proteases, which have been implicated in the pathophysiology of mucus hypersecretion in airways. We asked whether interleukin-8 (IL-8), a potent neutrophil chemoattractant, might be involved in recruiting neutrophils into airways of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis. We found significant neutrophil chemotactic activity in sputum obtained from these patients. The IL-8 concentrations that we measured in sputum of patients with cystic fibrosis (7.1 +/- 1.0 x 10(-9) M, mean +/- SE), bronchiectasis (9.6 +/- 2.9 x 10(-9) M), and chronic bronchitis (2.8 +/- 1.0 x 10(-9) M) have been reported to cause significant chemotaxis in vitro and in airways in vivo, whereas concentrations measured in induced sputum from healthy subjects (1.1 +/- 0.3 x 10(-10) M) do not. A monoclonal antibody to IL-8 significantly inhibited the chemotactic activity in patients' sputum by 75-98%, but not in induced sputum from healthy subjects (9%). We conclude that IL-8 is an important chemotactic factor in sputum of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis, and we suggest that IL-8 accounts, at least in part, for neutrophil recruitment into airways of patients with these diseases.

Topics: Adult; Biomarkers; Bronchiectasis; Bronchitis; Chemotaxis, Leukocyte; Chronic Disease; Cystic Fibrosis; Female; Humans; Inpatients; Interleukin-8; Male; Middle Aged; Neutrophils; Outpatients; Reference Values; Sputum

Based on the knowledge that neutrophil elastase (NE) in cystic fibrosis (CF) epithelial lining fluid (ELF) can induce human bronchial epithelial cells to express the gene for interleukin 8 (IL-8), an 8.5-kD neutrophil chemoattractant, we have evaluated CF ELF for the presence of IL-8, and investigated the ability of aerosolized recombinant secretory leukoprotease inhibitor (rSLPI) to suppress NE, and hence IL-8, levels on the respiratory epithelial surface in CF. Enzyme-linked immunoassay revealed 21.9 +/- 4.8 nM IL-8 in CF ELF compared with none in normals. Active NE was detectable in ELF of all individuals with CF and was significantly decreased (P < 0.03) after aerosolization of rSLPI. Human bronchial epithelial cells exposed to CF ELF recovered before rSLPI therapy expressed IL-8 mRNA transcripts, but ELF recovered after rSLPI therapy induced far less bronchial epithelial cell IL-8 gene expression. Consistent with this, rSLPI aerosol therapy caused a marked reduction in CF ELF IL-8 levels (P < 0.05) and neutrophil number (P < 0.02). There was also a clear association between CF ELF active NE and IL-8 levels (r = 0.94). These data suggest that rSLPI therapy not only suppresses respiratory epithelial NE levels, but also breaks a cycle of inflammation on the CF epithelial surface.

Topics: Adult; Aerosols; Cystic Fibrosis; Dimercaprol; Epithelium; Female; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Male; Pancreatic Elastase; Proteinase Inhibitory Proteins, Secretory; Proteins; Recombinant Proteins; Respiratory System; Serine Proteinase Inhibitors

The respiratory manifestations of cystic fibrosis (CF) are characterized by neutrophil-dominated airway inflammation. Since a variety of inflammatory stimuli are capable of inducing bronchial epithelial cells to express the gene for IL-8, a cytokine that attracts and activates neutrophils, mediators in respiratory epithelial lining fluid (ELF) of CF individuals might induce IL-8 production by epithelial cells, thus recruiting neutrophils to the airways. BET-1A human bronchial epithelial cells at rest or incubated with normal ELF showed little IL-8 gene expression, but after incubation with CF ELF, a marked increase in IL-8 transcript levels was observed. CF ELF contained high levels of neutrophil elastase (NE) and various serine protease inhibitors prevented CF ELF from inducing IL-8 gene expression in BET-1A cells, suggesting that NE was the dominant inducer for IL-8 production in CF ELF. The addition of purified NE caused BET-1A cells to increase IL-8 gene transcription with accumulation of mRNA transcripts and to release IL-8-like neutrophil chemotactic activity. These observations suggest a self-perpetuating inflammatory process on the CF bronchial surface where NE released by neutrophils induced the bronchial epithelium to secrete IL-8, which in turn recruits additional neutrophils to the bronchial surface.

Topics: Base Sequence; Bronchi; Bronchoalveolar Lavage Fluid; Cell Line; Chemotaxis, Leukocyte; Cystic Fibrosis; Epithelium; Gene Expression; Humans; Interleukin-8; Molecular Sequence Data; Neutrophils; Oligodeoxyribonucleotides; Pancreatic Elastase; Polymerase Chain Reaction; RNA, Messenger