Showing papers on "Respiratory epithelium published in 1999"

Epidermal growth factor system regulates mucin production in airways

Abstract: Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor α (TGFα), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor α (TNFα). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNFα. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFα induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue–periodic acid–Schiff staining (reflecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNFα, EGF, or TGFα alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFα-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.

The T Cell-Specific CXC Chemokines IP-10, Mig, and I-TAC Are Expressed by Activated Human Bronchial Epithelial Cells

Abstract: Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.

A protective role for protease-activated receptors in the airways

Abstract: The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2, and is a major source of PGE2, we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.

Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism

Abstract: Background Active sodium absorption is the dominant mechanism of ion transport in airway epithelium, but its role in pulmonary physiology and airway host defense is unknown. To address this question, we studied the function of airway epithelial cells and determined the frequency of pulmonary symptoms in patients with systemic pseudohypoaldosteronism, a salt-losing disorder caused by loss-of-function mutations in the genes for the epithelial sodium channel. Methods In nine patients 1.5 to 22 years of age who had systemic pseudohypoaldosteronism, we tested for mutations in the genes for the epithelial sodium channel, estimated the rate of sodium transport in the airway, determined the volume and ion composition of airway surface liquid, reviewed clinical features, collected laboratory data pertinent to pulmonary function, and, in three adults, measured mucociliary clearance. Results The patients with systemic pseudohypoaldosteronism had loss-of-function mutations in the genes for the epithelial sodium-chann...

Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development.

Abstract: In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693-1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.

Number and Proliferation of Clara Cells in Normal Human Airway Epithelium

Abstract: Experimental pathologic studies suggest that Clara cells are one of the types of airway stem cells but the proliferation of Clara cells in human lungs has not yet been examined. The purpose of this study was to assess in conducting airways of normal human lungs: (1) the number of Clara cells; and (2) the contribution of Clara cells to the proliferation compartment. Samples of histologic normal tissue were taken from seven lungs obtained by autopsy. A triple sequential (immuno)histochemical staining was performed, using MIB-1 as a proliferation marker and anti-CC10 for the identification of Clara cells; subsequently, a PAS stain was carried out as a marker for goblet cells, as these cells were reported to be CC10-immunoreactive in an unknown proportion. Clara cells were virtually absent in the proximal airway epithelium. The number of Clara cells in the terminal bronchioles was 11 ± 3% (mean ± SD) and in respiratory bronchioles 22 ± 5%. The overall proliferation compartment of the conducting airway epithel...

Respiratory Syncytical Virus-induced Chemokine Expression in the Lower Airways Eosinophil Recruitment and Degranulation

Abstract: Characterization of chemokine expression patterns in virus-infected epithelial cells provides important clues to the pathophysiology of such infections. The aim of this study was to determine the chemokine response pattern of respiratory epithelium when infected with respiratory syncytial virus (RSV). Macrophage inflammatory protein-1- α (MIP-1- α ), interleukin-8 (IL-8), and RANTES concentrations were measured from RSV-infected HEp-2, MRC-5, and WI-38 cell culture supernatants daily following infection. Additionally, MIP-1- α , IL-8, and RANTES concentrations were measured from lower respiratory secretions obtained from 10 intubated infants (0–24 mo) with RSV bronchiolitis, and from 10 control subjects. Our results indicate that respiratory epithelial cells respond to RSV infection by producing MIP-1- α , IL-8, and RANTES. Production of MIP-1- α required ongoing viral replication, whereas RANTES and IL-8 could be elicited by inactivated forms of the virus. MIP-1- α , RANTES, and IL-8 were also present in...

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus

Abstract: We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Airway Epithelial Cell Migration Dynamics: MMP-9 Role in Cell–Extracellular Matrix Remodeling

Abstract: Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell–ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell–collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

Structure-Function Relationships

Abstract: Publisher Summary This chapter discusses the anatomy and physiology of respiratory surfaces, which constitute the interface between inhaled air and the organism. Throughout the airspaces, the respiratory epithelium is characterized by so-called tight junctions near the apical margin of adjoining cells. These structures, whose molecular anatomy is beginning to yield its secrets, impose a significant barrier to solute movement between the surface liquid and the intercellular space, which communicates with the submucosa across a relatively porous basement membrane. In addition, the tight junctional apparatus effectively separates the apical and basolateral membranes of each epithelial cell, thus allowing for selective insertion of various receptors and channels, which confer the polarity underlying vectorial transcellular transport, and selective secretion of epithelial cell products to one or the other face of the epithelium. The apical surfaces of the epithelial cells, which line the lumen of the airways, and air spaces are covered by a continuous layer of extracellular airways surface liquid (ASL) that contains about 93% water. In the cartilaginous airways, the ASL consists of a periciliary “sol” layer and a more lumenally located mucus “gel” layer. The thickness of the sol layer is appropriate to the height of the cilia so that during their power stroke, the cilia contacts the undersurface of the gel layer. Mucin is produced and stored in condensed form as membrane-enclosed granules in the apical portion of specialized secretory cells in bronchial epithelia and in the mucous glands.

Airway epithelial cells, cytokines, and pollutants.

Abstract: The airway epithelium is a complex physicochemical barrier that plays a pivotal role in host defense. Epithelial cells have been shown to be a rich source of several classes of modulatory compounds, of which the cytokines form the largest group and possibly play the most important role in the etiology of airway disease. Evidence suggests that there are differences in the airway epithelial cells of individuals with and without respiratory disease, both with regard to (1) their capacity to express and release different types and quantities of specific cytokines and (2) their reactivity to inhaled irritants. Consequently, it is tempting to speculate that differences in epithelial cell function are an important determinant of the predisposition to respiratory disease. However, whether the differences are a result of an intrinsic defect, an acquired property due to the disease process itself, or a combination of the two, remains to be determined. In view of advances that have been made in the understanding of the putative underlying mechanisms in airway diseases, it should be possible to formulate novel therapeutic agents in the form of specific monoclonal antibodies directed against specific proinflammatory cytokines. Mills PR, Davies RJ, Devalia JL. Airway epithelial cells, cytokines, and pollutants.

Cellular sources of enhanced brain-derived neurotrophic factor production in a mouse model of allergic inflammation.

Abstract: The aim of this study was to investigate production and cellular sources of brain-derived neurotrophic factor (BDNF) production in allergic asthma. For this purpose a mouse model of chronic and severe ovalbumin (OVA)-induced airway inflammation was developed. Allergen-exposed mice developed elevated immunoglobulin E titers; airway inflammation with influx of lymphocytes, monocytes, and eosinophils; and airway hyperresponsiveness. In addition to an influx of inflammatory cells, interleukin (IL)-4 and IL-5 production were enhanced, macrophages showed morphologic signs of activation, and airway epithelium was thickened and displayed a goblet-cell hyperplasia with a marked mucus production. BDNF was detected using in situ hybridization and enzyme-linked immunosorbent assay. Constitutive expression of BDNF messenger RNA (mRNA) was observed in the respiratory epithelium of sensitized and nonsensitized mouse lungs. In addition, BDNF mRNA was detected in airway inflammatory infiltrations and bronchoalveolar lavage fluid (BALF) cells of OVA-sensitized and aerosol-challenged mice. Highest BDNF protein levels were detected in BALF after long-term allergen aerosol exposure. Analysis of BDNF production by isolated lymphocyte subsets revealed T but not B cells as a cellular source of BDNF. In addition, activated alveolar macrophages were identified as BDNF-positive cells. These data indicate that in allergic airway inflammation BDNF production is upregulated and immune cells serve as a source of BDNF.

The Attenuated Fibroblast Sheath of the Respiratory Tract Epithelial–Mesenchymal Trophic Unit

Abstract: Interactions between epithelial, mesenchymal, and neural tissue and also the extracellular matrix are necessary to initiate numerous cellular functions of the lung (1). The most common of these functions include differentiation during lung growth, repair of damaged tissue, and regulation of the inflammatory response. Each of these processes requires a localized response to a specific stimulus. Fibroblasts, especially those in close proximity to the airway epithelium, are likely regulators of local responses. In a recent commentary, Smith and colleagues discussed the possibility that resident fibroblasts may act as sentinel cells for these responses (2). In addition to their role as connective tissue cells, fibroblasts also produce cytokines and chemokines in response to various stimuli. Their fixed position in the tissue suggests that they can respond in a local manner to bacterial products, tissue injury, or other environmental factors. The relationship between cytokines and inflammatory cells in asthmatic airways also indicates a similar role for fibroblasts. In the asthmatic lung, the fibroblast plays a key role as a resident mesenchymal cell beneath the epithelium, receiving and sending information to epithelial and inflammatory cells (3, 4). Additionally, these fibroblasts are thought to be responsible for the subepithelial fibrosis associated with asthma. The significance of resident fibroblasts in the airway during inflamation has been described; however, the concept of an anatomically distinct group of fibroblasts associated with airway epithelium has not been explored. In 1990, Brewster and associates described a layer of subepithelial fibroblasts in the bronchi of normal and asthmatic human subjects that were positioned to allow close interaction with the epithelium, neural tissues, and extracellular matrix (5). The population of cells was shown to comprise fibroblasts and myofibroblasts, and individual cells were reported to be as large as 100 m m in diameter. A detailed description of the subepithelial layer of resident fibroblasts in the rat trachea was reported by Evans and coworkers (6). In tissue sections, the cells appear as a layer of attenuated cell processes closely opposed to the lamina reticularis of the basement membrane zone, about 1.9 m m beneath the epithelial basal lamina. The cells are intermeshed with each other, about 40 m m in diameter with the attenuated portions about 0.55 m m in thickness. The cell is thicker near the nucleus and contains abundant rough endoplasmic reticulum near the nucleus. There were no apparent bundles of microfilaments in the thin or thick portions of the cell as there are in myofibroblasts. The cells were determined to be stellate in shape. They exist as a layer of large, flat cells covering about 70% of the interstitial surface of the lamina reticularis, and make numerous contacts with the lamina densa of the basement membrane zone (approximately 7,000 times per mm 2 ). This layer of thin mesenchymal cells was named the attenuated fibroblast sheath (6), with properties of a layer of similar cells in the gut identified as the pericryptal fibroblast sheath (7). On the basis of the data from this previous paper (6), we constructed a three-dimensional model of the attenuated fibroblast sheath (Figure 1). The total attenuated fibroblast sheath, with its large surface area and close proximity to the epithelial/environmental interface, defines an anatomic unit of resident fibroblasts that could respond in a local manner to various stimuli. In this role, the attenuated fibroblast sheath represents the mesenchymal component of interactions with the epithelium, extracellular matrix, neural tissues, and migratory cells of the inflammatory response. The anatomic and functional relationship between the attenuated fibroblast sheath, epithelial and neural tissue, and also the extracellular matrix appears to serve as an epithelial–mesenchymal trophic unit. The epithelial–mesenchymal trophic unit would allow local exchange of information between the different tissue elements in response to various stimuli. This concept is similar to that of other investigators, with the exception that it recognizes that the subepithelial fibroblasts exist as a specific layer of resident fibroblasts beneath the epithelium instead of being randomly distributed in the lamina propria (2–4). Of the tissues in the epithelial–mesenchymal trophic unit, the least is known about the attenuated fibroblast sheath. Examples of the attenuated fibroblast sheath can be seen in most published electron microscope studies of the upper respiratory tract as a thin layer of attenuated cell processes immediately beneath the epithelium. Present in all animal species examined to date, the attenuated fibroblast sheath extends from the proximal to the distal regions of the conducting airways (5, 6, 8). Conceptually, it continues into the gas exchange region as interstitial fibroblasts in ( Received in original form May 14, 1999 and in revised form July 9, 1999 )

Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.

Abstract: Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable H. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

Nitric oxide synthase isoform expression in the developing lung epithelium

Abstract: Nitric oxide (NO), generated by NO synthase (NOS), is an important mediator of physiological processes in the airway and lung parenchyma, and there is evidence that the pulmonary expression of the ...

The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.

Abstract: Gene transfer to the respiratory epithelium is currently suboptimal and may be helped by the identification of limiting biological barriers. We have, therefore, developed an ex vivo model which retains many of the characteristics of in vivo native airways including mucociliary clearance, mucus coverage and an intact cellular structure. Using this model we have demonstrated several barriers to gene transfer. Liposome-mediated gene transfer was inhibited by normal mucus, with removal of this layer increasing expression approximately 25-fold. In addition both liposome and adenovirus were inhibited by CF sputum. The apical membrane represented a significant barrier to both agents. Adenovirus-mediated expression could be significantly augmented by increasing contact time or by pretreatment of tissues with a nominally calcium-free medium. The presence of these extracellular and plasma membrane barriers appeared to be the key parameters responsible for the approximately three log difference in gene expression found in vitro compared with our ex vivo model. Cytoskeletal elements and the cell cycle also influenced in vitro gene transfer, and represent further barriers which need to be overcome.

Airway epithelium expresses interleukin-18.

Abstract: Interleukin (IL)-18 is an interferon (IFN)-gamma-inducing cytokine suggested to be important in regulating inflammatory responses. This study investigated the pulmonary expression of IL-18 under conditions characterized by T-helper (Th)1 (lipopolysaccharide (LPS) treatment/sarcoidosis) and Th2 (ovalbumin (OVA) challenge/asthma) cytokine production. In situ hybridization and immunocytochemistry were used to determine the number of cells expressing IL-18, IFN-gamma, IL-5 and major basic protein (MBP) within lung tissue from Balb/c mice stimulated with LPS, OVA and in normal control mice. Bronchial biopsies from patients with sarcoidosis, asthma and control individuals were also examined. IL-18 was localized primarily to airway epithelium and mononuclear cells. Constitutive expression was observed within the lungs of control mice. Animals challenged with LPS exhibited more IL-18 messenger ribonucleic acid (mRNA)-positive and IFN-gamma immunoreactive cells, compared to control mice (p<0.01). OVA-challenged mice had fewer IL-18 mRNA positive and more IL-5 and MBP immunoreactive cells, compared to control mice (p<0.01). Similarly, constitutive expression of IL-18 protein was observed within the airway epithelium of control individuals, with more positive cells found within sarcoidosis tissue (p<0.01) and fewer within asthmatic tissue (p<0.01), compared to controls. These results demonstrate the expression of interleukin-18 within airway epithelium and the regulation of this cytokine under conditions of both T-helper1 and T-helper2 cytokine production.

Localization of TFF3, a new mucus-associated peptide of the human respiratory tract.

Abstract: Trefoil factor family (TFF)-domain peptides (formerly P-domain peptides, trefoil factors) represent major mucin-associated peptides of the gastrointestinal tract. Here, the first localization studies on TFF3 in the lower respiratory tract of human material are presented. Immunohistochemistry revealed significant accumulation of TFF3 to mucous cells in the acini of submucosal glands and varying amounts in goblet cells at the ductular portions and the surface epithelium. TFF3 appears also as a component of the mucus, for example from patients with chronic bronchitis. Expression of TFF3 was also shown by use of the polymerase chain reaction. In contrast, TFF1 and TFF2 transcripts were hardly detectable in the human respiratory tract. Thus, a structural function of TFF3 for the airway mucus is discussed, possibly together with the mucins MUC5B and MUC5AC.

Superoxide mediates cigarette smoke-induced infiltration of neutrophils into the airways through nuclear factor-kappaB activation and IL-8 mRNA expression in guinea pigs in vivo.

Abstract: We examined the hypothesis that superoxide mediates infiltration of neutrophils to the airways through nuclear factor (NF)-kappaB and interleukin-8 (IL-8) after acute exposure to cigarette smoke (CS) in vivo. Male Hartley strain guinea pigs were exposed to air or 20 puffs of CS and killed 5 h after the exposure. The differential cell count of bronchoalveolar lavage fluid and specific myeloperoxidase enzyme assay demonstrated that acute exposure to CS caused neutrophil accumulation to the airways and parenchyma, respectively. Acute exposure to CS increased DNA-binding activity of NF-kappaB in the lung. Acute exposure to CS also increased IL-8 messenger RNA (mRNA) expression in the lung. Pretreatment of guinea pigs with recombinant human superoxide dismutase (rhSOD) aerosols reduced the CS-induced neutrophil accumulation to the airways. Both activation of NF-kappaB and increased IL-8 mRNA expression were also inhibited by the pretreatment of rhSOD aerosols. Strong immunoreactivities for p65 and p50 were detected in the nuclei of alveolar macrophages after acute exposure to CS. The signal for IL-8 mRNA expression was demonstrated in the alveolar space after acute exposure to CS. Neither significant immunoreactivities for p65 and p50 nor IL-8 mRNA signals were observed in airway epithelium. These observations suggest that acute exposure to CS initiates superoxide-dependent mechanism that, through NF-kappaB activation and IL-8 mRNA expression, produces infiltration of neutrophils to the airways in vivo. It was also suggested that the alveolar macrophage is one potential source of NF-kappaB activation and IL-8 mRNA expression after acute exposure to CS.

Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

Abstract: Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to t...

Airborne particles evoke an inflammatory response in human airway epithelium. Activation of transcription factors

Abstract: PM10, the commonly used indicator of respirable environmental suspended particulate matter with a mean aerodynamic diameter of less than 10 microm, is composed of organic or elemental carbon aggregates containing various metals, acid salts, organic pollutants (polyaromatic hydrocarbons, quinones, nitroaromatic hydrocarbons, etc.), and biological contaminants. In urban and industrial areas, fossil fuel combustion products (e.g., diesel exhaust particles and residual oil fly ash) are the main contributors to PM10. Epidemiological data show that air pollution particulates cause adverse pulmonary health effects, especially in individuals with preexisting lung diseases. A critical cell type that encounters particles after inhalation and that is affected in a number of respiratory diseases is the epithelial cell of the airway and alveoli. In vitro studies have shown that PM10 is responsible for the production and the release of inflammatory cytokines by the respiratory tract epithelium as well as for the activation of the transcription factor NFkappaB. As many of the adsorbed materials on the particle surface are direct oxidants (metals, quinones) and indirectly produce reactive oxygen species, it is hypothesized that oxidative stress may be a component of the mechanisms by which particles activate cytokine production and NFkappaB in epithelial cells.

Fibronectin and alpha5beta1 integrin mediate binding of Pseudomonas aeruginosa to repairing airway epithelium

Abstract: Initial infection of the airway by Pseudomonas aeruginosa may occur through a variety of bacterial strategies including binding to epithelial receptors present at the surface of the respiratory epithelium. In order to characterize the adherence sites for P. aeruginosa in damaged and repairing bronchial tissue, an ex vivo model of airway epithelial injury and repair was developed using primary cell cultures of nasal cells from 14 subjects with polyposis. P. aeruginosa strongly adhered to flattened dedifferentiated (FD) bronchial and nasal cytokeratin 13-positive epithelial cells in the process of migration for repair. In in vitro experiments, competitive binding inhibition assays demonstrated that alpha5beta1 integrins and cellular fibronectin, in particular the RGD sequence, are receptors involved in P. aeruginosa adherence to FD nasal epithelial cells. Fluorescent cell sorting analysis and immunofluorescence techniques revealed that the alpha5beta1 integrins are overexpressed and apically exposed in FD nasal epithelial cells. One 50 kDa outer membrane protein was identified in piliated and nonpiliated strains of P. aeruginosa that was involved in binding to cellular fibronectin and alpha5beta1 epithelial integrins. These results demonstrate that Pseudomonas aeruginosa adherence is related to the dedifferentiation of airway epithelium during the repair process which unmasks and upregulates the alpha5beta1 integrin expression and induces active synthesis of cellular fibronectin. These epithelial receptors are then used by a Pseudomonas aeruginosa 50 kDa outer membrane protein as sites of bacterial adherence.

Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling

Abstract: Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

Could the airway epithelium play an important role in mucosal immunoglobulin A production

Abstract: Immunoglobulin (Ig) A is the major immunoglobulin of the healthy respiratory tract and is thought to be the most important immunoglobulin for lung defence. The basis for the preferential generation of IgA-secreting cells in the airway mucosa remains unclear. Given the half-life of 5 days for the majority of IgA plasma cells, many IgA plasma cells must develop daily from B cells to guarantee a continuous supply of IgA antibodies in the airway mucosa. For this, the surrounding cells must provide a constant supply of cytokines necessary for B-cell isotype switch, growth and differentiation into IgA-secreting plasma cells. Studies with CD4+ T-cell knockout mice, T-cell receptor knockout mice and mice made transgenic for CTLA4-Ig demonstrate normal mucosal IgA isotype switch, differentiation and IgA production, thereby suggesting that T cells are not critical for mucosal IgA production, and that other cell sources may be more important. Also, the bronchus-associated lymphoid tissue (BALT), which is believed to be the major site where IgA isotype switch and differentiation of B cells into plasma cells occur with the help of cytokines released by T cells, is not a constitutive feature of the normal human lung. This indicates that other parts of the respiratory tract must carry out the BALT function. We have recently demonstrated that healthy human airway epithelial cells constitutively produce IL-5, a major cytokine implicated in the growth and differentiation of post-switch mIgA+ B cells to IgA-producing plasma cells. Several studies have recently reported that the human airway epithelium also constitutively produces IL-2, TGFbeta, IL-6 and IL-10, factors which are essential for B-cell clonal proliferation, IgA isotype switch and differentiation into IgA-producing plasma cells. The close proximity of B cells to the airway epithelium probably ensures a constant supply of growth and differentiation factors necessary for mucosal IgA production. In addition, the epithelial cells produce a glycoprotein, called the secretory component, which not only confers increased stability to S-IgA, but is also quantitatively the most important receptor of the mucosal immune system, since it is responsible for the external transport of locally produced polymeric IgA and IgM. Recent studies also suggest a possible role for epithelial cells in antigen presentation. Dendritic cells situated within the airway epithelium could directly present antigens to B cells and direct their isotype switch towards IgA1 and IgA2 with the help of cytokines produced by epithelial cells. Airway epithelial cells could therefore play a major role in the production of mucosal IgA antibodies which are essential for airway mucosal defence.

Interleukin-5 Production by Human Airway Epithelial Cells

Abstract: Interleukin (IL)-5 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoieitic lineages. IL-5 enhances mediator release from human basophils and plays a pivotal role in the chemoattraction, proliferation, differentiation, survival, and activation of eosinophils. Th2- and Tc2-like T cells, mast cells, basophils, and eosinophils are the known cellular sources of this cytokine. Using a sensitive and novel reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay system, we found that IL-5 messenger RNA (mRNA) was constitutively expressed in bronchial biopsies obtained from healthy individuals, and that the levels of IL-5 mRNA expression decreased 1. 5 h after exposure to 0.12 ppm ozone for 2 h. Because the oxidative effects of ozone are confined to the epithelial cell surface and it is known that ozone induces epithelial damage and shedding, we hypothesized that epithelial cells might be a source of IL-5 mRNA. We demonstrate here that both transformed human bronchial epithelial cell lines (A549 and 16HBE14o-) and primary human bronchial and nasal epithelial cells grown in culture constitutively express IL-5 mRNA, which is upregulated on stimulation with tumor necrosis factor (TNF)-alpha. Culture supernatants derived from A549 cells exposed to TNF-alpha and interferon-gamma demonstrated detectable levels of IL-5 protein, and immunostaining of bronchial biopsies obtained from healthy human airways revealed the presence of IL-5 protein localized to the bronchial epithelium. To our knowledge, this is the first report demonstrating IL-5 production by human airway epithelial cells. This observation provides further evidence for the role of airway epithelium in regulating airway immune responses, in particular enhancing chemotaxis, activation, and survival of eosinophils, which could play an important role in the pathogenesis of bronchial asthma.

HNF-3/forkhead homologue-4 (HFH-4) is expressed in ciliated epithelial cells in the developing mouse lung.

Abstract: HNF-3/forkhead homologue-4 (HFH-4), a transcription factor of the winged-helix/forkhead family, was detected by immunohistochemistry in tissue of the developing mouse. HFH-4 protein was present in epithelial cells of the lung, trachea, oviduct, and embryonic esophagus, and in ependymal cells lining the spinal column and ventricles of the brain. In lung, trachea, and nose, HFH-4 was expressed in a distinct subset of epithelial cells that also expressed beta-tubulin IV, a ciliated cell marker. Cellular sites of HFH-4 and beta-tubulin IV expression were distinct from that of Clara cell secretory protein (CCSP), which was detected in nonciliated epithelial cells in the conducting airway of the lung. HFH-4 and beta-tubulin IV, but not CCSP, were detected in the respiratory epithelium of thyroid transcription factor-1 (TTF-1) gene-targeted mice. The presence of HFH-4 and beta-tubulin IV in TTF-1 gene-targeted mice demonstrates that differentiation of ciliated epithelium does not require TTF-1. Co-localization of HFH-4 and beta-tubulin IV staining in various tissues during mouse development supports a role for HFH-4 in the differentiation of ciliated cell lineages.

Expression of P2Y receptors in cell lines derived from the human lung.

Abstract: 1. Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo- and 16HBE14o- epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU-3 serous cells, the 6CFSMEo- submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC-1. These experiments revealed that P2Y2 and P2Y6 mRNA are co-expressed in the IHAEo-, 16HBE14o- and A549 epithelial cell lines. The CFPAC-1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. 2. We have then performed RT-PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo-, 16HBE14o-, 6CFSMEo-, HASMSC1 and CFPAC-1. P2Y1 mRNA was only detected in the CALU-3 cell line. 3. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo- and the 16HBE14o- airway epithelial cell lines which co-express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo- submucosal cells expressed a UTP-specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-α-induced oxidant stress

Abstract: We have shown previously that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific chemok...