The role of nitric oxide in the regulation of ion channels in airway epithelium: implications for diseases of the lung

The human respiratory tract is covered with airway surface liquid (ASL) that is essential for lung defense and normal airway function. The quantity and composition of ASL is regulated by active ion transport across the airway epithelium. Abnormal electrolyte transport produces changes in ASL volume and composition, inhibits mucociliary clearance and leads to chronic infection of airway surfaces, as is evident in cystic fibrosis. Agonists that induce intracellular increases in cAMP or Ca²⁺ are generally associated with electrolyte secretion. While these mechanisms have been studied in detail for many years, modulation of ion channels by nitric oxide (NO) has emerged only recently as a significant determinant of ion channel function. NO is a physiological regulator of transepithelial ion movement and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of ASL. This review presents the current understanding of regulation of airway epithelial ion channels by NO and attempts to highlight the importance of this regulation for lung defense.     

The role of nitric oxide in the regulation of ion channels in airway epithelium: Implications for diseases of the lung

The human respiratory tract is covered with airway surface liquid (ASL) that is essential for lung defense and normal airway function. The quantity and composition of ASL is regulated by active ion transport across the airway epithelium. Abnormal electrolyte transport produces changes in ASL volume and composition, inhibits mucociliary clearance and leads to chronic infection of airway surfaces, as is evident in cystic fibrosis. Agonists that induce intracellular increases in cAMP or Ca²⁺ are generally associated with electrolyte secretion. While these mechanisms have been studied in detail for many years, modulation of ion channels by nitric oxide (NO) has emerged only recently as a significant determinant of ion channel function. NO is a physiological regulator of transepithelial ion movement and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of ASL. This review presents the current understanding of regulation of airway epithelial ion channels by NO and attempts to highlight the importance of this regulation for lung defense.     

Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium

Nitric oxide (NO) plays an important role in airway function, and endothelial NO synthase (eNOS) is expressed in airway epithelium. To determine the basis of cell-specific eNOS expression in airway epithelium, studies were performed in NCI-H441 human bronchiolar epithelial cells transfected with the human eNOS promoter fused to luciferase. Transfection with 1624 base pairs of sequence 5' to the initiation ATG (position -1624) yielded a 19-fold increase in promoter activity versus vector alone. No activity was found in lung fibroblasts, which do not express eNOS. 5' deletions from -1624 to -279 had modest effects on promoter activity in H441 cells. Further deletion to -248 reduced activity by 65%, and activity was lost with deletion to -79. Point mutations revealed that the GATA binding motif at -254 is mandatory for promoter activity and that the positive regulatory element between -248 and -79 is the Sp1 binding motif at -125. Electrophoretic mobility shift assays yielded two complexes with the GATA site and three with the Sp1 site. Immunodepletion with antiserum to GATA-2 prevented formation of the slowest migrating GATA complex, and antiserum to Sp1 supershifted the slowest migrating Sp1 complex. An electrophoretic mobility shift assay with H441 versus fibroblast nuclei revealed that the slowest migrating GATA complex is unique to airway epithelium. Thus, cell-specific eNOS expression in airway epithelium is dependent on the interaction of GATA-2 with the core eNOS promoter, and the proximal Sp1 binding site is also an important positive regulatory element.     

Functional role of nitric oxide in guinea pig tracheal epithelium

Nitric oxide (NO) may play an important regulatory role in airway function. We have, thus, investigated in vitro whether epithelium derived NO may modulate cholinergic neurotrasmission, via release of NO in guinea pig trachea, by using L-arginine (L-ARG), a precursor of NO synthesis, and L-N^G-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase. Results show that L-ARG and L-NAME modify acetylcholine sensitivity in epithelium-intact smooth muscle preparations, suggesting a probable NO synthesis by tracheal guinea pig epithelium.     

Airway epithelium and apoptosis

Recent advances revealed that airway epithelium possesses versatile functions and plays a vital role in the mucosal defense and inflammatory responses. A maintenance of airway epithelium integrity is thus important and appears to be tightly regulated by a balanced cell proliferation and apoptosis. However, homeostasis of airway epithelium is likely affected by multiple environmental pathogens, irritants (reactive oxygen species, allergens, etc.), and toxins that may lead to various lung diseases. This review briefly summarizes airway epithelium apoptosis/proliferation in physiologic and pathological conditions along with various factors influencing airway epithelium homeostasis.     

Nitric oxide and reactive nitrogen species in airway epithelial signaling and inflammation

Nitric oxide (NO(.-)) is produced by many diverse cell types as a cellular or intracellular signaling molecule, by the activation of nitric oxide synthases (NOSs). All three known NOS isoforms are expressed within the respiratory tract and mediate various airway functional properties such as airway smooth muscle tone, ciliary function, epithelial electrolyte transport, and innate host defense. The respiratory epithelium is a major source of NO(.-), in which it regulates normal epithelial cell function and signaling as well as signaling pathways involved in airway inflammation. In addition to its normal physiological properties, increased airway NO(.-) production in inflammatory respiratory tract diseases such as asthma may activate additional signaling mechanisms to regulate inflammatory-immune pathways, and epithelial barrier (dys)function or repair. The biological actions of NO(.-) are controlled at various levels, including mechanisms that regulate NOS localization and activation, and variable oxidative metabolism of NO(.-), resulting in generation of bioactive reactive nitrogen species (RNS). Moreover, in addition to altered production of NO(.-) or RNS, the presence of various target enzymes and/or metabolic regulators of NO(.-)/RNS can be dramatically altered during airway inflammatory conditions, and contribute to alterations in NO(.-)-mediated signaling pathways in disease. This review summarizes current knowledge regarding NO(.-)-mediated epithelial signaling, as well as disease-related changes in airway NOS biology and target enzymes that affect NO(.-)/RNS signaling mechanisms. A detailed understanding of these various changes and their impact on NO(.-) signaling pathways are needed to fully appreciate the contributions of NO(.-)/RNS to airway inflammation and to develop suitable therapeutic approaches based on regulating NO(.-) function.     

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.     

Toll-Like Receptor Expression and Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/β) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.     

Airway Epithelial Expression of TLR5 Is Downregulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10(-10)) and smokers with COPD (1.6-fold, p < 10(-11)). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.     

Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Nasal nitric oxide levels in cystic fibrosis patients are associated with a neuronal NO synthase (NOS1) gene polymorphism.

Nitric oxide (NO) plays an important role in a number of physiological processes in the airways, including host defense. Although the exact cellular and molecular source of the NO formation in airways is unknown, there is recent evidence that neuronal NO synthase (NOS1) contributes significantly to NO in the lower airways of cystic fibrosis (CF) patients. NOS1 protein has been shown to be expressed in nasal epithelium, suggesting an involvement of NOS1-derived NO in upper airway biology. We here hypothesized that nasal NO concentrations in CF patients are related to genotype variants in the NOS1 gene. Measurements of nasal NO concentration and pulmonary function were performed in 40 clinically stable CF patients. Genomic DNA from all patients was screened for an intronic AAT-repeat polymorphism in the NOS1 gene using polymerase chain reaction and simple sequence length polymorphism (SSLP) analysis. The allele size at that locus was significantly (P = 0.001) associated with upper airway NO. Mean (+/- SD) nasal NO concentrations were 40.5 +/- 5.2 ppb in CF patients (n = 12) with high repeat numbers (i.e., both alleles > or =12 repeats) and 72.6 +/- 7.4 ppb in patients (n = 28) with low repeat numbers (i.e., at least one allele <12 repeats). Furthermore, in the group of CF patients harboring NOS1 genotypes associated with low nasal NO, colonization of airways with P. aeruginosa was significantly more frequent than in patients with NOS1 genotypes associated high nasal NO concentrations (P = 0.0022). We conclude that (1) the variability in CF nasal NO levels are related to naturally occurring variants in the NOS1 gene, and (2) that nasal NOS1-derived NO affects the susceptibility of CF airways to infection with P. aeruginosa.     

Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1,25(OH)₂ vitamin D₃

The airway epithelium plays a role in host defense through the binding of innate immune receptors, which leads to the activation of inflammatory mediators, including antimicrobial peptides. The active form of vitamin D, 1,25(OH)(2)D(3), induces the expression of the antimicrobial peptide LL-37 in both myeloid cells and airway epithelial cells (AEC). Here, we demonstrate that mRNA encoding triggering receptor expressed on myeloid cells (TREM)-1 was induced up to 12-fold by 1,25(OH)(2)D(3) in normal human bronchial epithelial (NHBE) cells and in well-differentiated cultures of six airway epithelial cell lines from patients with cystic fibrosis and healthy individuals. TREM-2 and DAP12 were also expressed in airway cultures, but not induced by vitamin D. Induction occurs through a vitamin D response element identified in its proximal promoter region, and was regulated by PU.1 expressed in the AEC. Activation of TREM-1 by a cross-linking antibody led to an induction of both human β-defensin-2 and TNF-α mRNA, demonstrating its functionality in these cells. Our results expand on the role played by the airway epithelium in innate immunity and suggest that vitamin D can modulate the innate immune defense of the airway epithelium, and could potentially be developed as an adjunctive therapy for airway infections.     

Airway nitric oxide release is reduced after PBS inhalation in asthma.

Exhaled nitric oxide (NO) is elevated in asthma, but the underlying mechanisms remain poorly understood. Recent results in subjects with asthma have reported a decrease in exhaled breath pH and ammonia, as well as altered expression and activity of glutaminase in both alveolar and airway epithelial cells. This suggests that pH-dependent nitrite conversion to NO may be a source of exhaled NO in the asthmatic airway epithelium. However, the anatomic location (i.e., airway or alveolar region) of this pH-dependent NO release has not been investigated and could impact potential therapeutic strategies. We quantified airway (proximal) and alveolar (peripheral) contributions to exhaled NO at baseline and then after PBS inhalation in stable (mild-intermittent to severe) asthmatic subjects (20-44 yr old; n = 9) and healthy controls (22-41 yr old; n = 6). The mean (SD) maximum airway wall flux (pl/s) and alveolar concentration (ppb) at baseline in asthma subjects and healthy controls was 2,530 (2,572) and 5.42 (7.31) and 1,703 (1,567) and 1.88 (1.29), respectively. Compared with baseline, there is a significant decrease in the airway wall flux of NO in asthma as early as 15 min and continuing for up to 60 min (maximum -28% at 45 min) after PBS inhalation without alteration of alveolar concentration. Healthy control subjects did not display any changes in exhaled NO. We conclude that elevated airway NO at baseline in asthma is reduced by inhaled PBS. Thus airway NO may be, in part, due to nitrite conversion to NO and is consistent with airway pH dysregulation in asthma.     

Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl^- channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung’s innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca²⁺ in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca²⁺ largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca²⁺ elevation.     

Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.