Chikako Kishioka

Regulation of secretion from mucous and serous cells in the excised ferret trachea.

Mucus hypersecretion is an important characteristic of many airway diseases. Mucin is the major component of mucus, and is secreted from surface goblet cells of the airway epithelium and mucous cells of submucosal glands. Lysozyme is an enzyme secreted by serous cells of airway submucosal glands. We hypothesized that secretagogues acting through different pathways would have different effects on tracheal mucin and lysozyme secretion. We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin-like glycoprotein secretion and a spectrophotometric method to measure lysozyme secretion from isolated ferret tracheal segments. We evaluated the secretory response to four secretagogues; prostaglandin F(2alpha) (PGF(2alpha)), adenosine triphosphate (ATP), methacholine (MCh), and human neutrophil elastase (HNE). Each agent stimulated mucin and lysozyme secretion. The relative potency was PGF(2alpha)< or =ATP<MCh<HNE for mucin and ATP< or =PGF(2alpha)<MCh<HNE for lysozyme secretion. We showed that there is an anatomic gradient for constitutive and stimulated mucin and lysozyme secretion with the distal tracheal segments secreting more mucin and lysozyme per gram of tissue than the proximal segments. This robust model system can be used to evaluate the regulation of airway mucous and serous cell secretion and to assess the effect of agents that might alter the secretory response. We confirm that on an equimolar basis, HNE is one of the most potent mucus secretagogues.

Pranlukast Inhibits NF-κB Activation and MUC2 Gene Expression in Cultured Human Epithelial Cells

Pranlukast is a selective cysteinyl leukotriene1 (cysLT1) receptor antagonist, and is now widely used in the treatment of asthma. The anti-asthmatic effect of pranlukast may be rendered not only by antileukotriene activity, but also by other pharmacological activity. This study was designed to investigate whether pranlukast had inhibitory effects on nuclear factor-ĸB (NF-ĸB) activation and mucin gene expression in cultured human epithelial cells. Luciferase assay was mainly used for analysis. Cultured epithelial cells were transfected with NF-ĸB luciferase vector, MUC2 or MUC5AC luciferase vectors. Lipopolysaccharide (LPS) significantly increased NF-ĸB activation in NCI-H292 cells, which was inhibited by the pretreatment by pranlukast in a dose-dependent manner. Either LTD4 or pranlukast alone did not increase NF-ĸB activation in NCI-H292 cells. Pranlukast also inhibited NF-ĸB activation induced by phorbol 12-myristate 13-acetate (PMA). Pranlukast also significantly inhibited LPS-induced MUC2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis in NCI-H292 cells. Pranlukast also inhibited LPS-induced MUC2 gene expression in HM3-MUC2 cells. However, pranlukast did not inhibit MUC5AC gene transcription activity induced by lipoteichoic acid (LTA) in NCI-H292 cells. These results suggest that pranlukast may inhibit NF-ĸB activation and MUC2 gene transcription through pathways distinct from cysLT1 receptor antagonism in cultured human epithelial cells.

Physical and Transport Properties of Sputum From Children With Idiopathic Bronchiectasis

BACKGROUND Childhood idiopathic bronchiectasis (IB) unrelated to cystic fibrosis (CF) or known immunodeficiency remains a common problem among indigenous populations in developed and developing countries. The physical and transport properties of sputum among children with IB have not been described, and these properties may suggest therapies that would be particularly effective for this group of children. METHODS Sputum from children in stable condition with IB and chronic daily productive cough was collected to measure viscosity, elasticity, cohesivity, adhesivity, and mucociliary and cough transportability in vitro. The results were compared to banked data from the sputa of children with CF and adults with chronic bronchitis (CB) measured by the same methods. RESULTS Sputa from children with CF and adults with CB had similar values for viscosity, elasticity, frictional adhesion, cough transportability, and mucociliary transportability. The elasticity of sputum from children with IB was 12 to 20%, respectively, of the value of CB and CF sputum (p < 0.01). The viscosity of sputum from children with IB was 23 to 32%, respectively, of the value of CB and CF sputum (p < 0.02). The surface frictional adhesion for sputum from children with IB was 55% of the values from both CF and CB sputa (p < 0.0001). Cough transportability for sputum from children with IB was 43 to 54% greater, respectively, than that for sputum from CB and CF patients (p < 0.0001). Mucociliary transportability was similar for all three groups (p > 0.05). CONCLUSIONS The physical and transport properties of sputum from children with IB who are stable in the outpatient setting are substantially different and lead to improved cough transportability compared to sputum from children with CF or adults with CB. Therapies that focus on cough may be sufficient to improve airway mucus clearance in children with IB. Sputum properties may explain in part the different clinical course of children with IB compared to children with CF.

Mucin gene expression in the effusions of otitis media with effusion.

OBJECTIVES The purpose of the study is to know if mucin gene expression can be detected in the middle ear effusion and if so, which mucin genes are expressed in the effusions. METHODS Mucin gene expression in the middle ear effusions obtained from five patients with otitis media with effusion were analyzed by reverse transcription-polymerase chain reaction. Ribonucleic acids (RNAs) were extracted from the effusion and the expression of 12 mucin genes was analyzed by reverse transcription-polymerase chain reaction. RESULTS Mucin gene expression examined by reverse transcription-polymerase chain reaction indicated the expression of MUC1, MUC4, MUC5AC, MUC6, MUC7, MUC8, MUC9, MUC11 and MUC12 mRNA in the effusion. This mucin gene expression was similar to that in BEAS-2B cell, a bronchial epithelial cell line. CONCLUSION Middle ear effusion can give us valuable information on mucin gene expression in the middle ear. There is similarity between mucin gene expression in the middle ear effusion and that in the bronchial epithelia.

Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells : Use of a monoclonal antibody to study human nasal mucin

We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyps were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and secretory cell and ciliated cell differentiation appeared after 7 days in an air-liquid interface. After 21 days, about half of the epithelial cells were stained with Alcian blue—periodic acid—Schiff stain or monoclonal antibody HCS18, which was directed against human nasal mucin specific for epithelial secretory (goblet) cells. The quantitative examination using the antibody HCS 18 revealed that the antibody-reactive nasal mucin was secreted only on the apical side of the cultures, and interleukin-1 β and tumor necrosis factor α stimulated these mucus secretions. The culture system with an antimucin monoclonal antibody developed in this study should be useful for studying polarized mucus secretion from human nasal epithelial cells.

Roxithromycin Suppresses Mucin Gene Expression in Epithelial Cells

Macrolide antibiotics are believed to inhibit mucus secretion but the mechanism of action is unclear. This study was designed to investigate an effect of roxithromycin on MUC2 gene expression in cultured intestinal epithelial cells (HM3-MUC2 cells). A reporter gene assay was used for analysis. Roxithromycin suppressed MUC2 gene transcriptional activity in a dose-dependent manner in HM3-MUC2 cells. Phorbol 12-myristate 13-acetate (PMA), lipoteichoic acid (LTA), lipopolysaccharide (LPS) and leukotriene D4 (LTD4) significantly increased MUC2 luciferase activities in the following order: PMA > LTA > LTD4 > LPS. Roxithromycin also decreased MUC2 gene transcriptional activity induced by PMA in a dose-dependent manner. NF-ĸB activation, but not AP-1 activation, was significantly suppressed by roxithromycin in HM3-MUC2 cells. A suppression of NF-ĸB activation was also observed in NCI-H292 cells. These results suggest that roxithromycin suppresses MUC2 gene expression in epithelial cells and that this suppression is probably via inhibition of NF-ĸB activation.

Quantitative Analysis of Mucin and Lectin in Maxillary Sinus Fluids in Patients With Acute and Chronic Sinusitis

Objectives Sinusitis is characterized by quantitative and qualitative changes in mucus biosynthesis that contribute to sinus disease. In general, patients with acute sinusitis complain of purulent rhinorrhea, whereas those with chronic sinusitis complain of mucoid or mucopurulent rhinorrhea. Locally produced mucin largely contributes to the high viscoelasticity of mucus in sinusitis. In this study, the authors attempt to quantify the concentrations of mucin and lectin in the maxillary sinus fluids from these patients.

Pseudomonas aeruginosa alginate is a potent secretagogue in the isolated ferret trachea

Airway mucus hypersecretion is in part a response to infection and inflammation. Pseudomonas aeruginosa is nearly universal in advanced cystic fibrosis (CF) lung disease. Mucoid strains of P. aeruginosa produce an exopolysaccharide product called alginate. The purpose of this study was to determine whether P. aeruginosa alginate stimulates secretion from mucous or serous cells in the ferret trachea exposed to alginate at concentrations reported to be present in the CF airway. We used a sandwich enzyme‐linked lectin assay (ELLA) to measure mucin secretion and spectrophotometry to measure lysozyme secretion from isolated ferret tracheal segments.

Effects of dexamethasone on mucin gene expression in cultured human nasal epithelial cells

Objective To clarify 1) which mucin gene expression is influenced by glucocorticoid and 2) whether glucocorticoid influences steady‐state mucin expression or mucin gene expression induced by lipopolysaccharide.

Regulation of Mucin Secretion in the Ferret Trachea

Mucin is the major component of mucus and can be used as a marker for mucus secretion. The purpose of this study was to develop an in vitro method to evaluate the regulation of mucin secretion. To do this, we used a sandwiched enzyme-linked lectin assay to measure mucin secretion from isolated ferret tracheal segments. This assay entailed coating microtiter plate wells with dolichos biflorus agglutinin and detecting the bound mucin that was secreted into a buffer solution by the tracheal segments. We used this method to evaluate the secretory response to four secretagogues: prostaglandin F2alpha (PGF2alpha), adenosine triphosphate (ATP), methacholine, and human neutrophil elastase (HNE). Each agent stimulated mucin secretion above baseline secretion (ATP (p = 0.022), PGF2alpha (p = 0.009), and HNE (p < 0.05)), and the relative potency of these secretagogues was PGF2alpha < or = ATP < MCh < HNE. We also demonstrated that there is an anatomic gradient for both constitutive and stimulated mucin secretion, with the distal tracheal segments secreting more mucin per gram of weight than the proximal segments. This fairly simple and reproducible technique can be used to evaluate the regulation of mucin secretion in the airway and to assess the efficacy of agents that might alter the secretory response.