Showing papers on "Respiratory epithelium published in 2001"

Collaboration of epithelial cells with organized mucosal lymphoid tissues

Abstract: Immune surveillance of mucosal surfaces requires the delivery of intact macromolecules and microorganisms across epithelial barriers to organized mucosal lymphoid tissues. Transport, processing and presentation of foreign antigens, as well as local induction and clonal expansion of antigen-specific effector lymphocytes, involves a close collaboration between organized lymphoid tissues and the specialized follicle-associated epithelium. M cells in the follicle-associated epithelium transport foreign macromolecules and microorganisms to antigen-presenting cells within and under the epithelial barrier. Determination of the earliest cellular interactions that occur in and under the follicle-associated epithelium could greatly facilitate the design of effective mucosal vaccines in the future.

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression.

Abstract: Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

Increased expression of transforming growth factor-beta1 in small airway epithelium from tobacco smokers and patients with chronic obstructive pulmonary disease (COPD).

Abstract: Tobacco smoke is believed to cause small airway disease and then chronic obstructive pulmonary disease (COPD), but the molecular mechanisms by which small airway obstruction occurs remain unknown. To study the gene expression levels of transforming growth factor (TGF)-beta1, a potent fibrogenic factor, in small airway epithelium from smokers and patients with COPD, we harvested highly pure samples of epithelial cells from small airways under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm with a biopsy channel of 0.8 mm in diameter). The expression levels of TGF-beta1 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of TGF-beta1 corrected by beta-actin transcripts were significantly higher in the smoking group and patients with COPD than those in nonsmokers (p < 0.01). Furthermore, among smokers and patients with COPD, TGF-beta1 mRNA levels correlated positively with the extent of smoking history (pack-years) and the degree of small airway obstruction as assessed by measurements of flow-volume curves. Immunocytochemistry of the cells demonstrated more intense stainings for TGF-beta1 in samples from smokers and patients with COPD than from nonsmokers. Spontaneously released immunoreactive TGF-beta1 levels from cultured epithelial cells were more elevated in subjects with a history of smoking and patients with COPD than in nonsmokers. Our study showed a close link between smoking and expression of TGF-beta1 in small airways. Our results also suggested that small airway epithelial cells might be involved in obstructive changes found in smokers and patients with COPD.

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Abstract: Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.

Interaction of Mite Allergens Der P3 and Der P9 with Protease-Activated Receptor-2 Expressed by Lung Epithelial Cells

Abstract: The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca2+ mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

Abstract: Transfer of therapeutic genes to the lung may provide a cure for diseases such as cystic fibrosis (CF), which affects 1 in 3,000 Caucasian births Inactivating mutations in the CF transmembrane regulator (CFTR), a chloride ion channel, result in gradual lung destruction, which is the major cause of morbidity Although the normal CFTR protein has been localized to both the apical surface of the airway epithelium (38) and the submucosal glands beneath the epithelium (10), the airway is the site of microbial obstruction associated with mortality This clinical manifestation provides the rationale for targeting the airway epithelium for CF gene therapy Among the many gene transfer systems being investigated are viral vectors such as those based on adeno-associated virus (AAV), a single-stranded DNA parvovirus AAV can integrate and promote persistent gene expression in cultured cells and in dividing and nondividing cells in multiple somatic tissues of animals (19, 20, 22–24, 26, and 32) The ability to transduce nondividing cells is an important feature of AAV vectors for gene transfer to the airway epithelium, which has a low rate of proliferation (3, 21) Additionally, the fact that wild-type (wt) AAV has been isolated from human airways (4, 5, 27) is consistent with the idea that AAV has tropism for the lung epithelium However, although AAV type 2 (AAV2) vectors can transduce multiple cell types in the lung, animal data thus far have shown low to modest rates of transduction by AAV2 vectors in the lung (11, 16, 17) Indeed, even introduction of a high dose of an AAV2 vector (1012 genome-containing particles) resulted in an overall transduction efficiency of 2% in the mouse airway epithelium (2) Animal studies show that the efficiency of AAV transduction is affected by several factors and can be enhanced by various treatments AAV transduction in the developing neonatal rabbit lung was more efficient than that in adult lung and was observed in a variety of airway and alveolar cell types (40) AAV vector-transduced cells in adult mouse lungs were rare, but their numbers could be increased by addition of adenovirus to provide helper functions (11), DNA-damaging reagents (1, 23, 30), or tissue injury (13, 16) Additionally, administration of much higher doses of AAV vector could also increase transduction in the lung epithelium (2) Tissue culture models have shown that proliferation rates (29) and polarity of epithelia influence the efficiency of AAV vectors (9) Indeed, reagents that help AAV vectors to bypass the natural resistance of the apical surface to infection can augment AAV transduction 10- to 100-fold (9, 35) These results show that dosage, proliferation rates, and target cell access are factors involved in efficient transduction by AAV vectors in lung epithelia Although AAV vector expression can persist for months to years, there may still be a need for readministration of vector to increase or replenish the population of modified cells In readministration studies, few to no new transduction events have been detected in the rabbit or mouse lung or in skeletal muscle (12, 16, 17, 37), and these results were associated with the detection of neutralizing antibodies (16, 17) Several approaches have been used to achieve effective readministration These include immune suppression in the lung (17) and muscle (25) and the use of other AAV types in the muscle and liver (36) and the lung (18) We showed that AAV vectors utilizing an AAV6 capsid (AAV6 pseudotype vector) have properties that could be useful for gene therapy, including low immunogenicity and the lack of cross-reactive antibodies generated against AAV2 (18) Quantitation of the number of vector-expressing cells in our previous study indicated that the AAV6 pseudotype vector was as efficient as, if not more efficient than, AAV2 vectors in the mouse lung and that the transduction rates in various cell types were different between AAV2 and AAV6 (18) In this study, we evaluated the transduction of lung cells by vectors based on the AAV6 serotype more thoroughly to determine the combination of vector components that mediated the most efficient transduction of airway epithelia To achieve this goal, we made separate expression plasmids for the three components of the AAV6 vector: rep, cap, and the packaged genome containing terminal repeats (TR) from AAV6 and expressing human placental alkaline phosphatase (AP) In conjunction with similar constructs for AAV2 (2), complementation of AAV2 and AAV6 components to generate infectious virions was assessed and then transduction was evaluated in a mouse model of lung gene transfer Our data show that AAV2 and AAV6 pseudotype vectors could be generated from all combinations of rep, cap, and genome from the two viruses and that transduction efficiencies of these vectors in tissue-cultured cells were primarily determined by the vector pseudotype, defined as the source of the capsid While both AAV2 and AAV6 pseudotype vectors bind heparin, only AAV2 is inhibited by heparin in cell transduction assays, indicating that AAV2 and AAV6 interact with different receptors In mouse lung delivery, the AAV2 vector gave high transduction rates in alveolar cells and much lower rates in airway epithelia, similar to the results obtained in previous studies In contrast, an AAV6 pseudotype vector showed preferential transduction of epithelial cells in large and small airways at rates up to 80% Transduction of the mouse airway epithelium by AAV6 pseudotype vectors was 15 to 74 times more efficient than transduction by an AAV2 vector These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2, indicate that AAV6 vectors may provide significant advantages for gene therapy for CF

Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma.

Abstract: Background: Protease-activated receptors (PARs), which are G protein–coupled receptors that are activated after proteolytic cleavage of the amino terminus of the receptor, are likely to play a major role in airway inflammation. PARs are activated by endogenous proteases, including thrombin (PAR-1, -3, and -4) and tryptase (PAR-2 and -4), both of which are present in inflamed airways. Objective: The purpose of this study was to compare the expression and distribution of PARs in biopsy specimens obtained from asthmatic and normal subjects and to examine the effect of inhaled corticosteroids on PAR expression. Methods: Biopsy specimens were obtained from 10 normal and 20 asthmatic patients, and sections were stained for PAR-1, -2, -3, and -4 through use of specific antibodies. Staining was scored semiquantitatively for both intensity and distribution. Results: Staining for all PARs was seen on the epithelium and smooth muscle in biopsy specimens from both normal and asthmatic subjects. In the epithelium, PAR-1 and -3 staining appeared to be apically concentrated, whereas PAR-2 and -4 staining was more diffuse. In normal subjects, epithelial staining intensity of PAR-1 and –3 was significantly greater than for PAR-4 ( P Conclusion: Asthma per se is associated with increased PAR-2 expression in bronchial epithelium. Importantly, staining was not influenced by inhaled corticosteroids. These results suggest that PAR-2 might be involved in airway inflammation. (J Allergy Clin Immunol 2001;108:797-803.)

Phenotypic characteristics of a distinctive multilayered epithelium suggests that it is a precursor in the development of Barrett's esophagus.

Abstract: A distinctive type of multilayered epithelium (ME) has been described at the neo-squamocolumnar junction and within columnar mucosa in patients with Barrett's esophagus (BE). This epithelium has morphologic and ultrastructural features of both squamous and columnar epithelium. Multilayered epithelium may represent an early or intermediate stage of columnar metaplasia; therefore, we performed this study to determine the morphologic and biologic characteristics of this epithelium and to gain insight into its derivation. Esophageal mucosal biopsies containing ME from 17 patients with BE were evaluated morphologically, stained with a variety of mucin histochemical stains; and also immunostained with antibodies against cytokeratins (CK) 13 (squamous epithelium marker); 14 (basal squamous epithelium marker) 7, 8/18, 19, and 20 (columnar epithelium markers), MIB-1 (proliferation marker); villin (intestinal brush border protein); and TGFalpha, EGFR, pS2, and hSP (enteric proliferation/differentiation regulatory peptides). The results were compared with normal esophageal squamous epithelium, normal gastric cardia epithelium, specialized-type intestinal epithelium (BE), and esophageal mucosal and submucosal gland duct epithelium. Multilayered epithelium expressed a pattern of mucin production (neutral mucin, sialomucin, and sulfomucin in 88%, 100%, and 71% of cases, respectively) and cytokeratin expression (CK 13 and 19 in the basal "squamoid" cells, CK 7, 8/18, 19, and 20 in the superficial "columnar" cells) similar to that of columnar epithelium in BE, and showed a high capacity for cellular proliferation (Ki-67-positive in 88% of cases) and differentiation (TGFalpha, EGFR, pS2 and villin-positive in 100%, 100%, 93%, and 66% of cases, respectively). The mucosal gland duct epithelium showed a similar phenotypic pattern and, in one case, was seen to give rise to ME at the surface of the mucosa. These data provide evidence in support of the hypothesis that ME represents an early or intermediate stage in the development of esophageal columnar metaplasia (BE). The mucosal gland duct epithelium may contain progenitor cells that can give rise to ME.

Relationship of Epidermal Growth Factor Receptors to Goblet Cell Production in Human Bronchi

Abstract: To determine the relationship of epidermal growth factor receptor (EGFR) expression to mucin synthesis in human airways, we examined EGFR and MUC5AC expression at both gene and protein levels using in situ hybridization and immunohistochemical analysis in human bronchi. Bronchial mucosal biopsy specimens were obtained from 12 asthmatic subjects and 11 healthy subjects. In asthmatic airways, EGFR mRNA was expressed in the airway epithelium. EGFR immunoreactivity staining patterns varied among the asthmatic airways: staining was positive mainly in goblet cells, in basal cells, or in both. In contrast, healthy airways showed little expression of EGFR mRNA; EGFR immunoreactivity was observed mainly in goblet cells. In parallel to EGFR expression, MUC5AC mRNA expression was greater in asthmatic airways; mucous glycoconjugates that stained positively with Alcian blue/PAS were also increased in asthmatic airways. Ciliated cells were negative for EGFR and MUC5AC both in asthmatic and in healthy subjects at both mRNA and protein levels. There was a significant positive correlation between EGFR immunoreactivity and the area of MUC5AC-positive staining in both asthmatics and healthy subjects. These findings suggest a sequence of events by which EGFR activation is involved in mucin expression in asthmatic airway epithelium.

Regulation of Peroxisome Proliferator-activated Receptor γ Expression in Human Asthmatic Airways: Relationship with Proliferation, Apoptosis, and Airway Remodeling

Abstract: Airway inflammation and alterations in cellular turnover are histopathologic features of asthma. We show that the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma), a nuclear hormone receptor involved in cell activation, differentiation, proliferation, and/or apoptosis, is augmented in the bronchial submucosa, the airway epithelium, and the smooth muscle of steroid-untreated asthmatics, as compared with control subjects. This is associated with enhanced proliferation and apoptosis of airway epithelial and submucosal cells, as assessed by the immunodetection of the nuclear antigen Ki67, and of the cleaved form of caspase-3, respectively, and with signs of airway remodeling, including thickness of the subepithelial membrane (SBM) and collagen deposition. PPAR gamma expression in the epithelium correlates positively with SBM thickening and collagen deposition, whereas PPAR gamma expressing cells in the submucosa relate both to SBM thickening and to the number of proliferating cells. The intensity of PPAR gamma expression in the bronchial submucosa, the airway epithelium, and the smooth muscle is negatively related to FEV(1) values. Inhaled steroids alone, or associated with oral steroids, downregulate PPAR gamma expression in all the compartments, cell proliferation, SBM thickness, and collagen deposition, whereas they increase apoptotic cell numbers in the epithelium and the submucosa. Our findings have demonstrated that PPAR gamma (1) is a new indicator of airway inflammation and remodeling in asthma; (2) may be involved in extracellular matrix remodeling and submucosal cell proliferation; (3) is a target for steroid therapy.

Remodeling in response to infection and injury. Airway inflammation and hypersecretion of mucus in smoking subjects with chronic obstructive pulmonary disease.

Abstract: Airway epithelium represents the first line of defense against toxic inhalants. In some subjects, cigarette smoking causes airway inflammation, hypersecretion of mucus, and poorly reversible airflow limitation through mechanisms that are still largely unknown. Likewise, it is unclear why only some smokers develop chronic obstructive pulmonary disease (COPD). Two cell types consistently result in relation to chronic airflow limitation in COPD: neutrophils and CD8(+) cells. Neutrophils are compartmentalized in the mucosal surface of the airways and air spaces, that is, the epithelium and lumen, whereas CD8(+) cells exhibit a more extensive distribution along the subepithelial zone of the airways and lung parenchyma, including alveolar walls and arteries. This pattern of inflammatory cell distribution is observed in mild or moderate COPD, and in patients who have developed COPD, it is not modified by smoking cessation. The number of neutrophils further increases in the submucosa of patients with severe COPD, suggesting a role for these cells in the progression of the disease. Hypersecretion of mucus is a major manifestation in COPD. Mucus is produced by bronchial glands and goblet cells lining the airway epithelium. Unlike mucous gland enlargement, greater mucosal inflammation is associated with sputum production. Whereas neutrophil infiltration of submucosal glands occurs only in smokers with COPD, goblet cell hyperplasia in peripheral airways occurs both in smokers with or without COPD, suggesting that the major determinant of goblet cell hyperplasia is cigarette smoke itself.

Remodeling of the Airway Epithelium in Asthma

Abstract: Several pathologic changes occur in the airway epithelium in asthma, but the relationship between these changes and the initiation and progression of asthma remains poorly understood. One possibility is that changes in the structure and function of the epithelium induced by environmental exposure in genetically susceptible subjects represent primary pivotal events that occur early in the pathogenesis of asthma. Alternatively, these epithelial changes may occur simply as a consequence of pivotal early events in other systems, such as immune deviation in childhood to a helper T cell type 2 (Th2) subtype of CD4(+) cells. Epithelial desquamation in asthma represents a pathologic change that is frequently cited as important for the mechanisms of airway remodeling and airway hyperresponsiveness. Desquamation of the epithelium may not represent true pathology, however, but may instead be an artifact of tissue sampling and handling. Evidence is more firm for other pathologic changes in the epithelium. For example, goblet cell numbers are increased in asthma, leading to increases in stored mucins in the epithelium and in secreted mucins in sputum. The functional consequences of these changes include sputum production and airway narrowing, which lead to asthma exacerbations. Currently available data suggest that an important mechanism for goblet cell hyperplasia in asthma is the action of Th2 cytokines. Improved understanding of epithelial goblet cell abnormalities in asthma will hopefully lead to novel therapies for mucin hypersecretion, which is an important cause of morbidity and mortality.

Lung mucosal immunity: immunoglobulin‐A revisited

Abstract: Mucosal defence mechanisms are critical in preventing colonization of the respiratory tract by pathogens and penetration of antigens through the epithelial barrier. Recent research has now illustrated the active contribution of the respiratory epithelium to the exclusion of microbes and particles, but also to the control of the inflammatory and immune responses in the airways and in the alveoli. Epithelial cells also mediate the active transport of polymeric immunoglobulin-A from the lamina propria to the airway lumen through the polymeric immunoglobulin receptor. The role of IgA in the defence of mucosal surfaces has now expanded from a limited role of scavenger of exogenous material to a broader protective function with potential applications in immunotherapy. In addition, the recent identification of receptors for IgA on the surface of blood leukocytes and alveolar macrophages provides an additional mechanism of interaction between the cellular and humoral immune systems at the level of the respiratory tract.

Interleukin-13 upregulates eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism.

Abstract: Interleukin (IL)-13 is a T helper 2‐derived cytokine that has recently been implicated in allergic airway responses. We hypothesized that IL-13 may regulate expression of eotaxin in airway epithelium. We found that IL-13 upregulated eotaxin messenger RNA and protein synthesis in the airway epithelial cell line BEAS-2B; this effect showed synergy with tumor necrosis factor (TNF)- � and also was inhibited by the glucocorticoid budesonide. To establish the mechanisms of eotaxin upregulation by IL-13, cells were transfected with an eotaxin promoter‐luciferase reporter plasmid and transcription was activated by IL-13 (1.7-fold) and TNF- � (2.8-fold). The combination of IL-13 and TNF- � additively activated the promoter constructs (4.1-fold). Activation of signal transducer and activator of transcription (STAT) 6 by IL-13 was confirmed by nuclear protein binding to a DNA probe derived from the eotaxin promoter. Activation of eotaxin transcription by IL-13 and the additive effect with TNF- � were lost in plasmids mutated at a putative STAT6 binding site. Cotransfection with a wild-type STAT6 expression vector significantly enhanced activation of the eotaxin promoter after IL-13 stimulation (6-fold induction). A significant increase of eotaxin protein secretion in the supernatant of STAT6 wild-type‐transfected cells was observed after IL-13 stimulation. Cotransfection with a dominant negative STAT6 mutant expression vector inhibited activation of the eotaxin promoter by IL-13. These results indicate that IL-13 stimulates eotaxin expression in airway epithelial cells and that STAT6 plays a pivotal role in this response. Asthma is a disease characterized by the infiltration of eosinophils and lymphocytes into airway epithelium and subsequent epithelial damage and tissue remodeling (1‐4). T helper (Th) 2 cells and their cytokine products play a crucial role in this process. Interleukin (IL)-13 is one of the important Th2-type cytokines which have been implicated and are upregulated in asthma (5‐7). Recent studies indicate that IL-13 can induce pathologic changes reminiscent of asthma in animals, including infiltration of eosinophils and mononuclear cells, epithelial damage, hyperplasia of goblet cells, and subepithelial fibrosis (8‐10). IL-13 probably plays important roles as a mucus-stimulating cytokine (8, 11) as well as in the recruitment of eosinophils. Endothelial cell expression of vascular cell adhesion molecule (VCAM)-1, an adhesion molecule involved in eosinophil recruitment, has been shown to be induced by IL-13 (12, 13).

Reduced Epithelial Expression of Secretory Component in Small Airways Correlates with Airflow Obstruction in Chronic Obstructive Pulmonary Disease

Abstract: The epithelial polymeric immunoglobulin receptor/transmembrane secretory component (pIgR/SC) transports into secretions polymeric immunoglobulin A (pIgA), which is considered the first line of defense of the respiratory tract. The present study, done with quantitative immunohistochemistry, evaluated epithelial expression of secretory component (SC) and Clara cell protein (CC16) and neutrophil infiltration into the airways of eight patients with severe chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation, as compared with these processes in six nonsmoking patients with pulmonary hypertension who were used as controls and in lung specimens from five smokers without chronic bronchitis. Staining for SC was significantly decreased in the COPD patients as compared with the controls, both in large (mean optical density [MOD]: 23.4 [range: 21.1 to 27.8] versus 42.2 [range: 28.2 to 49.3], p = 0.003) and in small airways (MOD: 30.8 [range: 20.3 to 39.4] versus 41.5 [range: 39.2 to 46.2], p = 0.003). SC expression in small airways correlated strongly with functional parameters such as FEV1 (Kendall's tau (K) = 0.76, p = 0.008), FVC (K = 0.64, p = 0.03), and midexpiratory flow at 50% of VC (MEF50) (K = 0.74, p = 0.01). The reduced expression of SC in large airways correlated with neutrophil infiltration in submucosal glands (K = -0.47, p = 0.03). Expression of CC16 in the bronchial epithelium of COPD patients was also significantly decreased as compared with that of controls, especially in small airways (MOD: 28.3 [range: 26.8 to 32.4] versus 45.8 [range: 40.7 to 56.0], p = 0.002), but no correlation was observed with lung function tests. In conclusion, this study shows that reduced expression of SC in airway epithelium is associated with airflow obstruction and neutrophil infiltration in severe COPD.

The effects of coronavirus on human nasal ciliated respiratory epithelium

Abstract: Human coronavirus (HCoV) accounts for 15-30% of common colds, but only one case report has described the effect of a coronavirus infection, that was asymptomatic, on human respiratory epithelium. The authors examined the effects of infection with HCoV on ciliary structure and function in healthy volunteers infected by intranasal inoculation with HCoV 229E. A further four volunteers were sham infected with ultraviolet-inactivated virus. Immediately before inoculation (day 0) and 3 days later (day 3), ciliated epithelium was obtained by brushing the inferior nasal turbinate. Ciliary beat frequency was determined and beat pattern analysed for evidence of dyskinesia (0=normal, 3=severely dyskinetic) using digital high-speed video photography. Ciliary ultrastructure was examined by transmission electron microscopy. Symptom diaries were kept for the duration of the study. All subjects inoculated with HCoV, including the three who did not develop symptoms of an upper respiratory tract infection, had disruption of their respiratory epithelium on day 3. Although there was no difference in the mean ciliary beat frequency between day 0 (11.3 Hz (95% confidence interval (CI): 8.6-14.0) and day 3 (9.4 Hz (95% CI 7.2-11.6)), there was a significant increase (p<0.05) in the ciliary dyskinesia score between day 0 (0.2 (95% CI 0-0.5)) and day 3 (1.1 (95% CI 0.5-1.7). In sham-infected subjects, no differences in epithelial integrity, or ciliary structure and function were found between day 0 and day 3. Inoculation of healthy volunteers with human coronavirus caused disruption of the ciliated epithelium and ciliary dyskinesia. This is likely to impair mucociliary clearance. Damage to the respiratory epithelium, due to human coronavirus infection, may occur without overt clinical symptoms.

Cellular and molecular characteristics of basal cells in airway epithelium.

Abstract: Basal cells exist as a separate layer of cells covering most of the airway basal lamina. In this central position, they can interact with columnar epithelium, neurons, basement membrane, and the underlying mesenchymal cells. In addition, they interact with inflammatory cells, lymphocytes and dendritic cells. These interactions take place in the lateral intercellular space between basal cells. In this central position basal cells become a very important part of the epithelial-mesenchymal trophic unit of larger airways. In this review it is shown that basal cells may function as progenitor cells of airway epithelium and have a role in attachment of columnar epithelium with the basement membrane. They also have the potential to function in regulation of neurogenic inflammation, the inflammatory response, transepithelial water movement, oxidant defense of the tissue and formation of the lateral intercellular space. Other characteristics of basal cells were not clearly associated with a particular function. The functions for basal cells listed attempt to explain the presence of recently identified molecules in basal cells of airway epithelium. It should be pointed out that specific studies have not been carried out which test the relationship between the molecular functions we describe in this review and the basal cell in airway epithelium.

Role of Latent Viral Infections in Chronic Obstructive Pulmonary Disease and Asthma

Abstract: Acute viral respiratory tract infections are well known to precipitate asthma attacks and acute exacerbations of chronic obstructive pulmonary disease, but their role in the pathogenesis of chronic disease is poorly defined. Double-stranded DNA viruses have the ability to persist in airway epithelial cells long after the acute infection has cleared. During these latent infections, viral genes are expressed at the protein level without replication of a complete virus. The expression of the adenoviral trans-activating protein has been demonstrated in the airway epithelium of both human and animal lungs and is associated with an amplification of the cigarette smoke-induced inflammatory response. Studies of cultured human airway epithelial cells have also shown that transfection with this viral gene upregulates the expression of intercellular adhesion molecule 1 and interleukin 8 by these cells when they are challenged with endotoxin. In guinea pigs, cigarette smoke-induced emphysema is amplified by latent adenoviral infection. Furthermore, this infection independently increased the number of CD-8 cells, whereas the cigarette smoke independently increased the number of CD-4 cells in the inflammatory infiltrate. On the other hand, allergen-induced lung inflammation was uninfluenced by latent adenoviral infection in the guinea pig, but the latent infection caused the eosinophilic component of this response to become steroid resistant. These studies suggest that latent adenoviral infections may have a role in the pathogenesis of obstructive airway disease by amplifying the response to cigarette smoke and inducing steroid resistance.

Airway mucus obstruction: mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia

Abstract: The mucus layer that coats the airway epithelium provides a protective barrier against pathogenic and noxious agents and participates in the mucosal response to inflammation and infection. Airway mucus is composed of water, ions, lung secretions, serum protein transudates, and mucin glycoproteins (mucins). Mucins are the major components of mucus and the macromolecules that impart rheologic properties to airway mucus (1, 2). Airway mucus is overproduced in the upper and/or lower respiratory tracts during acute challenges and in chronic conditions (asthma, cystic fibrosis, bronchitis, and sinusitis), thereby contributing to mucus obstruction of the airways (3). Mucus obstruction is the culmination of several complex processes including mucin ( MUC ) gene regulation, mucin secretion and goblet cell hyperplasia (GCH) (Figure 1). Insight into each of these processes is limited; more detailed information about fundamental cellular mechanisms will be required to better understand their interrelationships. For example, pathogenic agents and inflammatory mediators initiate secretion of mucins (4–7) and sustain mucin production by increasing expression of mucin genes (8, 9). Nevertheless, each process has markedly different kinetics and their response to mediators likely involves different cellular signaling and pathways. The increasing availability of molecular probes for specific mucin genes and gene products is beginning to provide some insights into the pathogenesis of mucus overproduction. However, detailed information about the expression and regulation of respiratory tract mucins in specific disease entities is still incomplete. The report by Chen and colleagues (10) provides new information on altered expression of a specific mucin gene, MUC5B.

FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo.

Abstract: Transgenic mice in which fibroblast growth factor (FGF)-10 was expressed in the lungs of fetal and postnatal mice were generated with a doxycycline-inducible system controlled by surfactant protein (SP) C or Clara cell secretory protein (CCSP) promoter elements. Expression of FGF-10 mRNA in the fetal lung caused adenomatous malformations, perturbed branching morphogenesis, and caused respiratory failure at birth. When expressed after birth, FGF-10 caused multifocal pulmonary tumors. FGF-10-induced tumors were highly differentiated papillary and lepidic pulmonary adenomas. Epithelial cells lining the tumors stained intensely for thyroid transcription factor (TTF)-1 and SP-C but not CCSP, indicating that FGF-10 enhanced differentiation of cells to a peripheral alveolar type II cell phenotype. Withdrawal from doxycycline caused rapid regression of the tumors associated with rapid loss of the differentiation markers TTF-1, SP-B, and proSP-C. FGF-10 disrupted lung morphogenesis and induced multifocal pulmonary tumors in vivo and caused reversible type II cell differentiation of the respiratory epithelium.

Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment

Abstract: BACKGROUND—Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment. METHODS—Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with β2 agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry. RESULTS—Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups. CONCLUSIONS—These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease.

New insights into the role of zinc in the respiratory epithelium

Abstract: Over the past 30 years, many researchers have demonstrated the critical role of zinc (Zn), a group IIb metal, in diverse physiological processes, such as growth and development, maintenance and priming of the immune system, and tissue repair. This review will discuss aspects of Zn physiology and its possible beneficial role in the respiratory epithelium. Here we have detailed the mechanisms by which Zn diversely acts as: (i) an anti-oxidant; (ii) an organelle stabilizer; (iii) an anti-apopototic agent; (iv) an important cofactor for DNA synthesis; (v) a vital component for wound healing; and (vi) an anti-inflammatory agent. This paper will also review studies from the authors' laboratory concerning the first attempts to map Zn in the respiratory epithelium and to elucidate its role in regulating caspase-3 activated apoptosis. We propose that Zn, being a major dietary anti-oxidant has a protective role for the airway epithelium against oxyradicals and other noxious agents. Zn may therefore have important implications for asthma and other inflammatory diseases where the physical barrier is vulnerable and compromised.

Apoptosis of airway epithelial cells induced by corticosteroids

Abstract: Damage to the airway epithelium is one prominent feature of chronic asthma. Corticosteroids induce apoptosis in inflammatory cells, which in part explains their ability to suppress airway inflammation. However, corticosteroid therapy does not necessarily reverse epithelial damage. We hypothesized that corticosteroids may induce airway epithelial cell apoptosis as one potential explanation for persistent damage. We tested this hypothesis in cultured primary central airway epithelial cells and in the cell line 1HAEo−. Treatment with dexamethasone, beclomethasone, budesonide, or triamcinolone each elicited a time-dependent and concentration-dependent cell death. This cell death was associated with cleavage of nuclear chromatin, mitochondrial depolarization, cytochrome c extrusion, activation of caspase-9, and expression of phosphatidylserine on the outer cell membrane. Inhibitors of caspase activity blocked apoptotic cell death, as did overexpression of the apoptosis regulators Bcl-2 or Bcl-xL. We demonstrat...

Transfer of heme oxygenase 1 cDNA by a replication-deficient adenovirus enhances interleukin 10 production from alveolar macrophages that attenuates lipopolysaccharide-induced acute lung injury in mice.

Abstract: By using a direct, intratracheal inoculation of an adenovirus encoding heme oxygenase 1 (Ad.HO-1), model gene therapy for acute lung injury induced by inhaled pathogen was performed. Data demonstrated that Ad.HO-1 administration is as effective as the pharmacologic upregulation of the endogenous HO-1 gene expression by hemin to attenuate neutrophilic inflammations of the lung after aerosolized lipopolysaccharide (LPS) exposure. Interestingly, immunohistochemical analysis revealed that the HO-1 gene was transferred not only to the airway epithelium, but to the alveolar macrophages (AMs). Moreover, overexpression of exogenous HO-1 in the macrophages provided a high level of endogenous interleukin 10 (IL-10) production from the macrophages, and additional experiments using IL-10 knockout mice demonstrated that the increase in IL-10 in the macrophages was critical for the resolution of neutrophilic migration in the lung after LPS exposure. These results suggest that AMs not only are barriers for efficient gene transfer to the respiratory epithelium, but also represent logical targets for Ad-mediated, direct, in vivo gene therapy strategies for inflammatory disorders in humans.

Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: Use of alternative chemokine receptors as a function of inducing stimulus

Abstract: Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50–60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.

Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.

Abstract: BACKGROUND: Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium MATERIAL AND METHODS: To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals RESULTS: Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p < 00005) Western analysis of BAL fluid proteins showed the major VEGF isoform in respiratory epithelial lining fluid is VEGF165 CONCLUSIONS: With the knowledge that proteins of molecular mass like VEGF (34 to 46 kDa) slowly diffuse across the alveolar epithelium, it is likely that this high level "reservoir" of VEGF protein on the respiratory epithelial surface plays a role in normal lung endothelial biology However, this compartmentalized VEGF reservoir may also be a "Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached

Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice.

Abstract: The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. The possible cause of this protein excess in gastric and other lesions and its possible functions are discussed.

Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5'-flanking region.

Abstract: Human mucin (MUC) 5B gene expression in human airway epithelium was studied in both tissue sections and cultures of tracheobronchial epithelial (TBE) cells. In situ hybridization demonstrated that MUC5B message was expressed mainly in the mucous cells of submucosal glands of normal human airway tissues. Nevertheless, an elevated MUC5B message level could be seen in surface goblet cells from patients with airway diseases and inflammation. Regardless of the airway tissue sources, MUC5B message was regulated by all-trans-retinoic acid (RA) and culture conditions in both primary and passage-1 cultures of TBE cells. MUC5B message, to a lesser extent, was also found in the immortalized epithelial cell line HBE1, but not in BEAS-2B cells. To elucidate the molecular mechanism of MUC5B gene expression, a genomic clone was obtained and sequenced for the amino terminal and the 5'-flanking region of MUC5B gene. A luciferase reporter construct containing 4,169 base pairs of the 5'-flanking region of MUC5B gene demonstrated a cell type-specific basal promoter activity in transfection studies. Both RA and the air-liquid interface culture condition further enhanced this promoter activity. These results suggest that the 5'-flanking region of MUC5B gene contains cis-elements that are potentially involved in the regulation of MUC5B gene expression.