Mammalian Airway Research Articles

Transforming Growth Factor-β-Smad Signaling Pathway Cooperates with NF-κB to Mediate NontypeableHaemophilus influenzae-induced MUC2 Mucin Transcription

Transforming growth factor-beta (TGF-beta) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF-beta-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKKbeta/gamma-IkappaBalpha pathway to mediate NF-kappaB-dependent MUC2 mucin transcription. The NTHi-induced TGF-beta receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF-beta neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-kappaB p65/p50 with Smad3/4 appears to positively mediate NF-kappaB-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF-beta signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

Mucus clearance as a primary innate defense mechanism for mammalian airways.

The conducting airways branch 20–25 times between the trachea and the alveoli as inhaled air passes from the relatively constricted nasal/tracheal passages to the large surface area of alveoli (70 m2), where gas exchange occurs. This branching anatomy leads to a surface area that expands greatly from proximal airways (e.g., third generation; ∼50 cm2) to distal airways (20th to 25th generation; ∼2 m2). The regional differences in airway surface area (or airway perimeters; ref. 1), which is often depicted by showing the airways as an inverted funnel (Figure ​(Figure1),1), pose interesting challenges for lung defense. Because many of the particles that settle on airway surfaces are infectious, airways have evolved innate defense mechanisms that constantly protect airways against bacterial and other types of infection. Figure 1 Pulmonary defense mechanisms preventing chronic bacterial infection. The lung is depicted as an inverted funnel, reflecting the relative surface area of distal versus proximal airways. The mechanical-clearance-of-mucus hypothesis is shown on the left. ... There is still little agreement on the nature of these innate airway defense mechanisms (2, 3) (Figure ​(Figure1).1). In the more traditional view, mechanical clearance of mucus is considered the primary innate airway defense mechanism (4–6). In this view, the role of the epithelia lining airway surfaces is to provide the integrated activities required for mucus transport, including ciliary activity and regulation of the proper quantity of salt and water on airway surfaces via transepithelial ion transport. More recently, a second view of innate airway defense has emerged as a result of studies of the pathogenesis of cystic fibrosis (CF) (7). This view emphasizes a role for a “chemical shield” in protecting the lung against inhaled bacteria (8). In this hypothesis, the two important functions for epithelia are the production of salt-sensitive defensins that are secreted into airway lumens, and the production of a low-salt (<50 mM NaCl) liquid on airway surfaces that renders defensins active (9). The predictions of each of these models and the relevant data have been extensively reviewed (2, 3, 10, 11). Here, we will focus on the role of mucus clearance in the lung as the more important innate defense mechanism in health and disease, including CF. We will attempt to fill in the gaps in our knowledge regarding important aspects of the mucus clearance system, and, where relevant, point out differences between the two views of innate airway defense.

Mucus clearance as a primary innate defense mechanism for mammalian airways

Mucus clearance as a primary innate defense mechanism for mammalian airways

Mucus clearance as a primary innate defense mechanism for mammalian airways

Mucus clearance as a primary innate defense mechanism for mammalian airways

Pseudomonas aeruginosa and Tumor Necrosis Factor- α Attenuate Clara Cell Secretory Protein Promoter Function

The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-alpha, respectively. A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

Mucus clearance as a primary innate defense mechanism for mammalian airways

Mucus clearance as a primary innate defense mechanism for mammalian airways

Aquaporin-5 dependent fluid secretion in airway submucosal glands.

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to play an important role in airway defense and surface liquid homeostasis and in the pathogenesis of cystic fibrosis. Immunocytochemistry revealed strong expression of aquaporin water channel AQP5 at the luminal membrane of serous epithelial cells in submucosal glands throughout the mouse nasopharynx and upper airways and AQP4 at the contralateral basolateral membrane in some glands. Novel methods were applied to measure secretion rates and composition of gland fluid in wild type mice and knockout mice lacking AQP4 or AQP5. In mice breathing through a tracheotomy, total gland fluid output was measured from the dilution of a volume marker present in the fluid-filled nasopharynx and upper trachea. Pilocarpine-stimulated fluid secretion was 4.3 +/- 0.4 microl/min in wild type mice, 4.9 +/- 0.9 microl/min in AQP4 null mice, and 1.9 +/- 0.3 microl/min in AQP5 null mice (p < 0.001). Similar results were obtained when secreted fluid was collected in the oil-filled nasopharyngeal cavity. Real-time video imaging of fluid droplets secreted from individual submucosal glands near the larynx in living mice showed a 57 +/- 4% reduced fluid secretion rate in AQP5 null mice. Analysis of secreted fluid showed a 2.3 +/- 0.2-fold increase in total protein in AQP5 null mice and a smaller increase in [Cl(-)], suggesting intact protein and salt secretion across a relatively water impermeable epithelial barrier. Submucosal gland morphology and density did not differ significantly in wild type versus AQP5 null mice. These results indicate that AQP5 facilitates fluid secretion in submucosal glands and that the luminal membrane of gland epithelial cells is the rate-limiting barrier to water movement. Modulation of gland AQP5 expression or function might provide a novel approach to treat hyperviscous gland secretions in cystic fibrosis and excessive fluid secretions in infectious or allergic bronchitis/rhinitis.

Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na(+)] and pH but elevated viscosity.

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na(+)], [Cl(-)], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1--0.5 microl of fluid accumulated in spherical droplets at gland orifices in approximately 3--5 min. In gland fluid from normal human airways, [Na(+)] was 94 +/- 8 mM, [Cl(-)] was 92 +/- 12 mM, and pH was 6.97 +/- 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 +/- 0.3-fold greater than that of saline. Neither [Na(+)] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by approximately 2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na(+)], [Cl(-)], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.

Genomic Organization of the Mouse plunc Gene and Expression in the Developing Airways and Thymus

Few genes have been isolated which display specific expression in the proximal airways. A recently identified mouse cDNA, plunc, appears to be confined to the upper airways and nasopharyngeal epithelium, and may prove a useful marker for these regions. We now report the genomic cloning and characterization of the mouse plunc gene as well as its developmental expression in the nasal and airway epithelium. We also report the novel finding that plunc is also expressed in the medullary compartment of the murine thymus. The mouse gene contains nine exons and the intron–exon boundaries are conserved with those in the human homologue. At e14.5 plunc is expressed in the nasal epithelium and several days later is seen in the thymic lobes, but not in the lining of the tracheobronchial tree. Expression in the trachea and main-stem bronchi first appears at 1–2 days after birth. Tracheobronchial expression persists at high levels throughout adulthood, as do regional areas of nasal and thymic expression. Finally, we show that the human homologue is expressed in bronchial epithelium, suggesting a transcript that is evolutionarily conserved in the mammalian airway.

Role of aquaporin water channels in airway fluid transport, humidification, and surface liquid hydration.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 +/- 5 microm and [Na(+)] was 115 +/- 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by approximately 40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.

Expression and Distribution of Vasoactive Intestinal Polypeptide Receptor VPAC2 mRNA in Human Airways

Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter of the inhibitory non-adrenergic non-cholinergic nervous system and influences many aspects of mammalian airway function. VIP binds to two G-protein-coupled VPAC receptors that are highly homologous structurally but distinguished by their different affinities for peptide analogues of VIP. As VIP binding sites in the respiratory tract have only been examined by ligand binding and cytochemical techniques, we studied the distribution of the mRNA that encodes the inducible receptor subtype VPAC2 in the human respiratory tract. Northern blots demonstrated the expression of VPAC2 mRNA in human airways and other tissues. A human-specific VPAC2 cRNA probe was used to detect VPAC2 mRNA expression in human lung by nonradioactive in situ hybridization. In larger airways, positive VPAC2 mRNA signals were localized to tracheal and bronchial ciliated epithelial cells. There was also marked staining of mucous and serous cells of submucosal glands. No signals were obtained in airway and vascular smooth muscle myocytes and endothelial cells. In peripheral lung tissues, VPAC2 mRNA expression was localized to epithelial cells of the bronchioles. Specific staining was detected in immune cells and alveolar macrophages. In summary, VPAC2 is localized in airway epithelial, glandular, and immune cells of the lung but not in airway and vascular smooth muscle. The absence of VPAC2 mRNA in vascular and airway smooth muscle myocytes may indicate that the effects of VIP on vasodilation and bronchodilation are mediated by VPAC1 or undefined receptors. However, a paracrine modulation of the two most prominent effects of VIP in the respiratory tract by VPAC2 cannot be excluded.

Ciliogenesis and Axonemal Dynein Expression in Human Airway Epithelium Cultured in an Air/Liquid Interface Environment

Abstract Axonemal dynein is a high molecular weight Mg2+-ATPase which occurs across the eukaryotic phylogenetic spectrum and interacts with the microtubular doublets of ciliary axonemes to effect ciliary beating. Unlike cytoplasmic dyneins which serve intracellular functions and are relatively ubiquitous in all cells, axonemal dynein is expressed exclusively in mature and nascent ciliated cells. Ciliated cells figure prominently in mucociliary clearance, a primary defense mechanism of the mammalian respiratory airways. Acting in concert with mucus secreting cells, the mucociliary escalator maintains virtual sterility in the airways and lung. Although ciliated cells are the predominant cell type in the airways and serve a critical function in respiratory health, they also are particularly vulnerable to injury. The strategic functional importance of the ciliated cell as well as its susceptibility to injury by infectious agents and irritants has led to development of epithelial cell culture systems which facilitate epithelium-specific studies of injury.

Early cytotoxic effects of mechlorethamine, a nitrogen mustard, on mammalian airway epithelium

The aim of this in vitro study was to characterize the early effects of short-term exposure to low concentrations of mechlorethamine (HN2), a nitrogen mustard, on rabbit tracheal primary cultures. Marked inhibition of cell growth was observed without recovery until 14 days after the treatment with sublethal doses of HN2. Cell detachment associated with rearrangement of the cytoskeletal proteins was also noted as early as 1 hr after treatment. Moreover, cells treated with HN2 at the sublethal dose of 50 μm (LC 10) for 1 hr showed early lipid peroxidation and cellular membrane damage. This was correlated with a significant increase in the activities of antioxidant enzymes associated with an increase in glutathione content. These early events appeared several hours before arrest of the cell cycle progression. On the other hand, long-term treatment with HN2 at a sublethal dose led to modification of cytokeratin expression and appeared to induce squamous metaplasia.

Variability in distribution and populations of gap junctions in ferret trachea during postnatal development.

Immunocytochemical probes have been used to characterize gap junction distribution in the postnatal ferret trachea by epifluorescence and by laser scanning confocal and electron microscopy. A battery of antibodies directed against fragments of different connexins localized beta 1- and beta 2-gap junction antigens (connexins 32 and 26, respectively) at the intercellular borders of the superficial epithelium while alpha 1-gap junction antigen (connexin 43) was localized to the loose connective tissues. Gap junction labeling in the superficial epithelium declined in the first weeks of life but persisted in the developing submucosal glands to the weanling stage. Localization of the alpha- and beta-antigens was specific for connective tissues and epithelial layers, respectively. These observations suggest that communication competence is an important component of early development in the mammalian airways.

Gene expressions of transforming growth factor-ß1 and extracellular matrix components during gastric ulcer healing in rats

This chapter describes ganglia within the mammalian gut, heart, urinary bladder, and airways. Autonomic ganglia are present in almost all organs of the body and exhibit considerable differences in their structures and properties. The most well-studied intrinsic autonomic ganglia, both in culture and in situ, are those of the enteric nervous system (ENS). The ganglia of the ENS are grouped into two major plexuses: (1) the myenteric (or Auerbach's), which lies between the outer longitudinal and circular smooth-muscle layers and (2) the submucous (or Meissner's), which lies in the connective tissue of the submucosa. The two plexuses are connected by nerves that pass through the circular muscle layer. The enteric neurons are a highly diverse population functionally, morphologically, and with respect to their neurotransmitter phenotypes. The unique advantages of culture preparations, including the increased accessibility of the cultured cells, the ability to observe cells over extended periods of time, and the ability to control their physical and chemical environment, are pertinent in the study of the regulation of phenotypic differentiation.

Morphometric approaches for evaluating pulmonary toxicity in mammals: implications for risk assessment.

Recent advances in quantitative morphology provide all the tools necessary to obtain structural information in the lung that can be quantified and interpreted in the three-dimensional world of toxicology. Structural hierarchies of conducting airways and parenchyma of the lung provide: (1) numbers of cells per airway, lobe, or lung; (2) surface areas of cells, airways, and alveoli; (3) length of airways and vessels; and (4) volumes of cells, alveoli, airways, vessels, and individual lobes or the entire lung. Unbiased sampling of these subcompartments of the lung requires fractionation of lobes or individual airways. Individual airways of proximal and distal generations are obtained by airway microdissection along one axial pathway and comparisons made between airway generations. Vertical sections of selected airways are used to sample epithelium and interstitium. Using this unbiased approach of quantitative morphology, we have shown that inhalation of low ambient concentrations of ozone ([O3]0.15 ppm) near or at the United States National Ambient Air Quality Standard (NAAQS) (0.12 ppm O3) induces significant alterations in bronchiolar epithelium and interstitium in nonhuman primates but not rats. The alterations do not appear to be concentration- or time-dependent, thereby bringing into question the current NAAQS that may be at or above the threshold for distal airway injury in primates. Unbiased morphometric methods are critical in a quantitative evaluation of toxicological injury of mammalian tracheobronchial airways.

Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl

Chloride secretion in mammalian airway epithelia is stimulated by beta-adrenergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism and by apical triphosphate nucleotides (ATP, UTP) via a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cotransport must be upregulated in parallel with apical Cl efflux. To examine the regulation of basolateral cotransport activity and its relationship to apical Cl efflux, we examined Cl transport in confluent primary cultures of dog tracheal epithelial cells treated with nystatin, an antibiotic that increases the permeability of plasma membranes to small monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transport across the basolateral membrane, which reflects primarily the activity of the cotransporter. In cultures of tracheal cells not treated with nystatin, transepithelial (basolateral-to-apical) 36Cl flux was increased two- to threefold by exposure to isoproterenol (5 microM, basolateral) or apical UTP (10 microM). Apical application of nystatin (400 units/ml) increased the basal level of transepithelial 36Cl flux approximately 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [3H]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular Cl concentration ([Cl]i) by incubating cultures with apical media containing nystatin and reduced [Cl] (NO3 replacement) increased both basolateral-to-apical 36Cl flux and [3H]bumetanide binding in the absence of secretagogues or cell shrinkage. The results support our previous suggestion, based entirely on [3H]bumetanide binding [M. Haas, D. G. McBrayer, and J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200, 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in airway epithelial cells is entirely secondary to, and requires, an increase in apical Cl efflux, and further suggest that a decrease in [Cl]i may be a signal for cotransport activation in response to UTP. In addition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.

Endotoxin Upregulates Expression of an Antimicrobial Peptide Gene in Mammalian Airway Epithelial Cells

Tracheal antimicrobial peptide (TAP) is a 38-amino acid peptide isolated from the bovine tracheal mucosa, which has a broad spectrum of antimicrobial activity. It is structurally similar to antimicrobial peptides present in circulating phagocytes and has comparable microbicidal activity.1 We now show by Northern blot analysis that TAP is expressed along the entire length of the airway, from nasal to bronchiolar tissue, and in situ hybridization of airway sections further localizes the message to the columnar epithelial cells lining the trachea.

Origin of galanin in nerves of cat airways and colocalization with vasoactive intestinal peptide

Galanin is a 29 amino acid residue neuropeptide. In mammalian airways, galanin is found in nerve fibers associated with airway smooth muscle, bronchial glands, and blood vessels, and in nerve cell bodies of airway ganglia. The present study was conducted to determine if galanin-containing fibers in the walls of feline airways originate from the nerve cell bodies of airway ganglia. The colocalization of galanin with vasoactive intestinal peptide was also investigated. Organotypic cultures of cat airways were held in culture for 0 (nonculture control), 3, 5, and 7 days. After each culture period, the distribution of galanin and the colocalization of galanin with vasoactive intestinal peptide were determined by immunocytochemistry. Galanin-containing fibers were found in bronchial smooth muscle, around bronchial glands and in the walls of bronchial arteries and arterioles throughout the culture period. Nerve fibers and cell bodies containing both galanin and vasoactive intestinal peptide were observed after all culture periods. Nerve fibers and cells bodies that contained galanin frequently contained vasoactive intestinal peptide as well, but nerve fibers with only galanin or vasoactive intestinal peptide were also observed. Galanin- and vasoactive intestinal peptide-containing nerve fibers and cell bodies were both well maintained throughout the culture period. The findings show that galanin-containing nerve fibers associated with bronchial smooth muscle, bronchial glands, and bronchial arteries, originate from nerve cell bodies of intrinsic airway ganglia, and that galanin and vasoactive intestinal peptide are frequently colocalized in these neurons.

Effects of 5-hydroxytryptamine and 5-hydroxytryptamine receptor agonists on ion transport across mammalian airway epithelia.

1. 5-Hydroxytryptamine has been suggested as a candidate for an endogenous inhibitor of airway sodium transport. Amiloride, an inhibitor of epithelial sodium channels, has therapeutic potential in disorders of airway ion transport such as cystic fibrosis, but its duration of action in vivo is short. 5-Hydroxytryptamine and related compounds have been studied to investigate whether any might be a useful alternative to amiloride for clinical use, and to further assess the possible physiological role of 5-hydroxytryptamine in the regulation of airway ion transport. 2. Sheep tracheal epithelium was mounted in Ussing chambers under short-circuit conditions. Mucosal application of 5-hydroxytryptamine resulted in an immediate, reversible, concentration-related decrease in the short-circuit current, maximal with 38% inhibition of the short-circuit current at 25 mmol/l. This response was completely inhibited by pretreatment of tissues with mucosal amiloride (100 mumol/l). These features are consistent with a direct effect of 5-hydroxytryptamine on amiloride-sensitive sodium channels. Similar results were obtained in a limited number of studies using human bronchial epithelium. 3. The effects of mucosal addition of a range of 5-hydroxytryptamine agonists were studied to determine if any was a more potent blocker of amiloride-sensitive sodium transport than 5-hydroxytryptamine. The 5-HT3 agonist 2-methyl-5-hydroxytryptamine had no effect on the short-circuit current at concentrations of up to 5 mmol/l. The 5-HT1D agonist sumatriptan had no effect at concentrations below 5 mmol/l and at 5 mmol/l had only a transient effect.(ABSTRACT TRUNCATED AT 250 WORDS)