Showing papers on "Respiratory epithelium published in 1997"

Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.

Abstract: Eotaxin is a potent and specific eosinophil chemoattractant that is mobilized in the respiratory epithelium after allergic stimulation. Pulmonary levels of eotaxin mRNA are known to increase after allergen exposure in sensitized animals. In this study we demonstrate that TNF alpha and IL-1beta induce the accumulation of eotaxin mRNA in the pulmonary epithelial cell lines A549 and BEAS 2B in a dose-dependent manner. Cytokine-induced A549 cell mRNA accumulation was maximal at 4 h and was significantly enhanced when the cells were costimulated with IFNgamma. TNFalpha- and IL-1beta-induced increases in eotaxin mRNA were diminished in a dose-dependent manner by the glucocorticoid dexamethasone and were augmented by the protein synthesis inhibitor cycloheximide. Cytokine-induced increases in eotaxin mRNA expression correlated with increased eotaxin protein production and secretion, and dexamethasone inhibition of cytokine-induced eotaxin mRNA augmentation was associated with diminished eotaxin protein secretion. These findings, together with the known kinetics of TNF alpha and IL-1beta mobilization in asthmatic airways and the potent eosinophil chemotactic effects of eotaxin, define a mechanism linking inflammatory cytokine mobilization to eosinophil recruitment that may be relevant to the pathogenesis of asthma.

Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis.

Abstract: Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.

Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis.

Abstract: Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by alveolar inflammation, progressive proliferation of septal cells, increased production of septal matrix, and loss of lung architecture. The process of cellular injury in lung fibrosis is thought to be mediated by oxygen radicals produced by infiltrating inflammatory cells. Peroxynitrite is a potent oxidant produced by the rapid reaction of nitric oxide (NO) and superoxide. We investigated the production of nitrotyrosine, a byproduct of protein nitration by peroxynitrite, and the expression of the enzymes responsible for generating NO, in lungs of patients with IPF and compared them with lungs of normal control subjects. We used immunohistochemistry, histochemistry, and in situ hybridization to study the production of nitrotyrosine and the expression of inducible (iNOS) and constitutive endothelial (eNOS) nitric oxide synthases in 48 lungs of patients with different stages of IPF and 21 normal lungs. In lungs of control subjects, there was little expression of iNOS and nitrotyrosine in the airway epithelium and alveolar macrophages, and abundant expression of eNOS in the airway epithelium and vascular endothelium. By contrast, in lungs of patients with IPF, strong expression of nitrotyrosine and NOS was seen in macrophages, neutrophils, and alveolar epithelium. A significant increase in the expression of these molecules was only seen in lungs of patients with the early to intermediate stage of the disease. The active stage of IPF is associated with increased inflammatory and alveolar expression of nitrotyrosine and NOS. Increased production of NO and peroxynitrite may be responsible for the oxidative damage seen in this disease.

Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory Epithelium

Abstract: The respiratory epithelium is frequently injured by inhaled toxic agents or by micro-organisms. The epithelial wound repair represents a crucial process by which surface respiratory cells maintain the epithelial barrier integrity. The repair process involves both cell migration and proliferation, but as yet, the kinetic of these two mechanisms has not been extensively studied. Using an in vitro model of human respiratory epithelium wound repair, proliferative cell immunofluorescent staining and a computer-assisted technique allowing the tracking of living cells, we studied the cell proliferation and migration during the wound repair process. Respiratory epithelial cells were dissociated from human nasal polyps and cultured on a collagen I matrix. At confluency, a chemical wound was made on the culture. We observed that the cell mitotic activity peaked at 48 h after wounding (23% of the cells) and mainly concerned the cells located 160 to 400 microns from the wound edge. The migration speed was highest (35 to 45 microns/h) for the spreading cells at the wound edge and progressively decreased for the cells more and more distant from the wound edge. The temporal analysis of the cell migration speed during the wound repair showed that it was almost constant during the first 3 days of the repair mechanism and thereafter dropped down until the wound closure was completed (after 4 days). We also observed that over a 1-hour period, the intra-individual and interindividual variation of the cell migration speed was 43% and 37%, respectively. These results demonstrate that cell proliferation and cell migration during respiratory epithelial wound repair are differently expressed with regard to the cell location within the repairing area.

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.

Abstract: The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.

Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators.

Abstract: Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known. We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA. IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk. Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC. IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not. Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation. In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

Telomerase Expression in Respiratory Epithelium during the Multistage Pathogenesis of Lung Carcinomas

Abstract: To investigate the role of telomerase in the multistage pathogenesis of lung cancer, we examined 205 fresh and archival tissue samples obtained from 40 patients, 34 of whom had invasive lung carcinoma, 5 with carcinoma in situ (CIS) without invasion, and 1 without lung carcinoma. We analyzed samples for telomerase enzyme activity using the semiquantitative PCR-based telomeric repeat amplification protocol assay (131 samples) or by a radioactive in situ hybridization method for expression of the RNA component of human telomerase (hTR; 74 samples). A subset of samples was assayed by both methods, and the correlation was excellent (30 of 36; 83%). With the exception of a carcinoid tumor and a necrotic squamous cell carcinoma, all tumor cells were moderate to strongly positive for both hTR and telomerase activity, except for foci of keratinization in squamous cell carcinomas. Telomerase positivity, with weak enzyme activity and/or low hTR expression, was present in basal epithelial cells of large bronchi, both histologically normal (26%) and hyperplastic (71%), and in 23% of peripheral lung samples (in epithelium of small bronchi and bronchioles or lymphoid aggregates). More advanced epithelial changes (metaplasia, dysplasia, and CIS) were associated with telomerase dysregulation. Dysregulation in preneoplasia was manifested in three ways: almost all such lesions expressed hTR, although enzyme activity levels were several-fold lower than in the corresponding invasive tumors; cells throughout these multilayered processes expressed hTR; and intense, focal up-regulation of hTR occurred in CIS foci in the vicinity of invasive cancers. Alveolar cells and areas of atypical adenomatous hyperplasia (possible precursor lesions for peripheral adenocarcinomas) were negative. Our studies demonstrate that dysregulation of telomerase occurs early in the multistage pathogenesis of bronchogenic lung carcinomas and that intense focal localized hTR expression in CIS may indicate imminent invasion.

Elevated expression of endothelin-1 and endothelin-converting enzyme-1 in idiopathic pulmonary fibrosis: possible involvement of proinflammatory cytokines.

Abstract: Endothelin-1 (ET-1) is a vasoconstrictor, bronchoconstrictor, and mitogenic peptide which is enzymatically converted from a biologically inactive big ET to mature ET (21 amino acid) by the ET-converting enzyme (ECE). Here, we investigate the expression of ECE-1, big ET-1, and ET-1 in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and compare it to those of normal subjects using immunohistochemistry and in situ hybridization. In normal lungs, focal moderate expression of all three molecules is localized to airway epithelium, pulmonary endothelium, and airway and vascular smooth muscle cells. Serous bronchial glands also expressed ET-1 and ECE-1. In IPF, strong diffuse expression of ECE-1 was seen in airway epithelium, proliferating type II pneumocytes, and in endothelial and inflammatory cells. ECE-1 immunostaining was colocalized to big ET-1 and ET-1 immunostaining, and correlated with disease activity (P < 0.05). To study regulatory mechanisms of ET-1 and ECE-1 expression, human normal bronchial epithelial (NBE) cells were treated with cytokines and analyzed by radioimmunoassay and Northern blot. Incubation of human NBE cells with IL-1alpha and -beta or tumor necrosis factor alpha (TNFalpha) resulted in a significant increase in ET-1 release and mRNA expression. TNFalpha resulted in a significant increase in ECE-1 mRNA expression. These findings demonstrated the colocalization of the precursor and active ET-1, and ECE-1 in the same cell, and that ECE-1 expression is elevated in IPF. In addition, increased expression of ET-1 and ECE-1 in IPF may be mediated by proinflammatory cytokines.

Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1β

Abstract: Exacerbations of asthma are often associated with respiratory infection caused by rhinoviruses. To study the effects of rhinovirus infection on respiratory epithelium, a primary target for respirat...

Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma.

Abstract: BACKGROUND: There are two isoforms of cyclo-oxygenase (COX), namely COX- 1 and COX-2. COX-1 is constitutively expressed in most tissues and in blood platelets. The metabolites derived from COX-1 are probably involved in cellular housekeeping functions. COX-2 is expressed only following cellular activation by inflammatory stimuli and is thought to be involved in inflammation. METHODS: The expression of COX-1 and COX-2 isoenzymes has been studied in the bronchial mucosa of 10 normal and 18 asthmatic subjects, 11 of whom had aspirin-sensitive asthma (ASA) and seven had non-aspirin-sensitive asthma (NASA) RESULTS: There was a significant fourfold and 14-fold increase, respectively, in the epithelial and submucosal cellular expression of COX-2, but not of COX- 1, in asthmatic patients. There was no significant difference in the total number of cells staining for either COX-1 or COX-2 between subjects with ASA and NASA, but the number and percentage of mast cells that expressed COX-2 was significantly increased sixfold and twofold, respectively, in individuals with ASA. There was a mean fourfold increase in the percentage of COX-2 expressing cells that were mast cells in subjects with ASA and the number of eosinophils expressing COX- 2 was increased 2.5-fold in these subjects. CONCLUSION: COX-2-derived metabolites may play an essential part in the inflammatory processes present in asthmatic airways and development of drugs targeted at this isoenzyme may have therapeutic potential in the treatment of asthma. Mast cells and eosinophils may also have a central role in the pathology of aspirin-sensitive asthma.

Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators

Abstract: Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).

Developmental Expression of Mucin Genes in the Human Respiratory Tract

Abstract: Mucin glycoproteins play a key role in the normal function of the airway epithelium. We examined the expression of mucin genes, MUC3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues to establish the localization and age of onset of expression of each mucin gene during human development. We detected expression of MUC4, 5AC, 5B, and 7 in the mid-trimester airway epithelium but did not detect expression of MUC3, 6, or 8. MUC4 was expressed in the trachea and large airways in the majority of cells in the airway epithelium. Expression of MUC5AC was only seen in individual goblet cells in the trachea, while MUC5B was expressed in the surface epithelium of the trachea at 13 wk but was largely restricted to submucosal glands by 23 wk of gestation.

Decreased Cu,Zn-SOD activity in asthmatic airway epithelium: correction by inhaled corticosteroid in vivo

Abstract: To investigate the antioxidant response of respiratory epithelium to the chronic airway inflammation in asthma, the major intracellular antioxidants [copper and zinc-containing superoxide dismutase (Cu,Zn-SOD) and manganese-containing SOD (Mn-SOD), catalase, and glutathione peroxidase] were quantitated in bronchial epithelial cells of healthy control and asthmatic individuals. Although catalase and glutathione peroxidase in bronchial epithelium of asthmatics were similar to control SOD activity in asthmatics not on inhaled corticosteroid (-CS) was lower than asthmatics on inhaled corticosteroid (+CS) and controls. Investigation of Mn-SOD and Cu,Zn-SOD activities revealed that the lower SOD activity in asthmatics -CS was because of decreased Cu,Zn-SOD activity. However, Mn-SOD and Cu,Zn-SOD mRNA and protein levels were similar among asthmatics -CS, asthmatics +CS, and controls. Importantly, Cu,Zn-SOD specific activity in asthmatics -CS was decreased in comparison with control and asthmatics +CS. Furthermore, in paired comparisons of asthmatics -CS and +CS, inhaled corticosteroids resulted in normalization of bronchial epithelial Cu,Zn-SOD specific activity. These findings suggest loss of Cu,Zn-SOD activity in asthma is related to inflammation, perhaps through oxidant inactivation of Cu,Zn-SOD protein.

Phenotyping and Cytokine Regulation of the BEAS-2B Human Bronchial Epithelial Cell: Demonstration of Inducible Expression of the Adhesion Molecules VCAM-1 and ICAM-1

Abstract: Airway epithelium may actively participate in inflammatory responses, such as occur in asthma. The presence and regulation of surface molecules on the airway epithelium, however, is incompletely understood. We have determined the phenotype of the human bronchial epithelial cell line BEAS-2B by flow cytometry. We confirmed previous observations that human bronchial epithelial cells constitutively express CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1. BEAS-2B cells were also found to constitutively express CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50, CD71, and CD102 (ICAM-2). Culture of BEAS-2B cells with tumor necrosis factor (TNF)-α or interleukin (IL)-1β (1 ng/ml) was found to enhance intercellular adhesion molecule-1 (ICAM-1) expression (severalfold) and induce de novo CD106 [vascular cell adhesion molecule-1 (VCAM-1)] expression. TNF-α or IL-1β did not change the expression of CD9, CD13, CD16, CD23, CD29, CD31, CD32, CD35, ...

RSV infection of human airway epithelial cells causes production of the beta-chemokine RANTES.

Abstract: Infection of airway epithelial cells with respiratory syncytial virus (RSV) results in the production of a restricted number of cytokines, which may modulate the inflammatory response to infection. To get a better understanding of epithelial cell-mediated inflammatory processes in RSV disease, the aim of the present study was to identify the production of mononuclear cell/eosinophil/mast cell inflammatory chemokines [monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein-1beta, and RANTES] during productive RSV infection in airway epithelial cells. Normal human primary bronchial epithelial cell cultures, nasal epithelial cell explants, and the BEAS-2B airway epithelial cell line were inoculated with RSV, and chemokine induction was assessed during the phase of logarithmic increase in infectious virus production. Only RANTES was found to increase in epithelial cell cultures in an infection-dependent manner. Furthermore, RANTES was released only by RSV-producing cells. To determine whether RANTES was induced by RSV infection in vivo, RANTES was measured in nasal lavage fluids (NLF) from children with RSV-positive and RSV-negative upper respiratory infection and children when they were well. RANTES was increased significantly during RSV infection (128 +/- 38 pg/ml NFL) compared with non-RSV infection (42 +/- 12 pg/ml NFL) and with asymptomatic baseline (13 +/- 4 ng/ml NFL) in the same children. Because RANTES is an effective eosinophil and memory T cell chemoattractant and activator and because eosinophil-dominated inflammation is a hallmark of asthmatic airways, RANTES may play a role in the pathogenesis of RSV-induced exacerbations of airway reactivity and wheezing.

Expression and activity of nitric oxide synthases in human airway epithelium

Abstract: Nitric oxide (NO) synthesized by airway epithelium may be important in the regulation of airway inflammation and reactivity. As such, the expression and localization of nitric oxide synthase (NOS) isoforms was assessed in human airway tissue obtained following thoracotomy, and in cultured human airway epithelial (BEAS-2B) cells. NOS expression was determined by reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot analysis, and localized by in situ hybridization and immunohistochemistry. No synthesis by cell cultures was detected by nitrite assay. Endothelial and neuronal constitutive NOS mRNAs were not detected in airways or cell cultures. Inducible NOS (iNOS) mRNA was detected in 5 of 6 airway specimens, and in situ hybridization demonstrated iNOS mRNA expression in columnar epithelial cells. This was confirmed by immunohistochemistry using an iNOS specific antibody. BEAS-2B cell cultures were stimulated with (I) combinations of tumor necrosis factor alpha (TNF alpha) (50-2,000 U/ml)/in...

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

Abstract: 1 In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2 The eAect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNg) 100 u ml 71 , interleukin-1b (IL-1b )1 um l 7 1 and lipopolysaccharide (LPS) 10 m gm l 7 1 induced nitrite formation which could be inhibited by the competitive NOS inhibitor N G -nitro-L-arginine-methylester (L-NAME). IL-1b alone (1‐50 u ml 71 ) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNg 100 u ml 71 , IL-1b 1um l 7 1 and LPS 10 m gm l 7 1 to induce both the iNOS and COX-2 pathways, and IL-1b 3um l 7 1 to induce COX-2 without iNOS activity. 3 Cells treated with IFNg 100 u ml 71 , IL-1b 1um l 7 1 and LPS 10 m gm l 7 1 for 48 h either alone, or with the addition of L-NAME (0 to 10 72 M), demonstrated inhibition by L-NAME of PGE2 (3.61+0.55 to 0.51+0.04 pg/10 4 cells; P50.001) and nitrite (34.33+8.07 to 0 pmol/10 4 cells; P50.001) production. Restoration of the PGE2 response (0.187+0.053 to 15.46+2.59 pg/10 4 cells; P50.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10 76 M, but with the addition of the NOS substrate L-arginine (0 to 10 75 M). 4 Cells incubated with IL-1b 3um l 7 1 for 6 h, either alone or with addition of the NO donor Snitroso-acetyl-penicillamine (SNAP) (0 to 10 74 M), demonstrated increased PGE2 formation (1.23+0.03 to 2.92+0.19 pg/10 4 cells; P5 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 mM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1b 3um l 7 1 for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10 73 M) also demonstrated an increased PGE2 response (2.56+0.21 to 4.53+0.64 pg/10 4 cells; P50.05). 5 These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this eAect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells

Abstract: Ozone, one of the most reactive oxidant gases to which humans are routinely exposed, induces inflammation in the lower airways. The airway epithelium is one of the first targets that inhaled ozone will encounter, but its role in airway inflammation is not well understood. Expression of inducible genes involved in the inflammatory response, such as interleukin (IL)-8, is controlled by transcription factors. Expression of the IL-8 gene is regulated by the transcription factors nuclear factor (NF)-kappaB, NF-IL-6, and possibly activator protein-1 (AP-1). Type II-like epithelial cells (A549) were grown on a collagen-coated membrane and exposed in vitro to 0.1 ppm ozone or air. Exposure to ozone induced DNA-binding activity of NF-kappaB, NF-IL-6, and AP-1. IL-8 mRNA and IL-8 protein levels were also increased after ozone exposure. These results link ozone-induced DNA-binding activity of transcription factors and the production of IL-8 by epithelial cells thus demonstrating a potential cellular cascade resulting in the recruitment of inflammatory cells into the airway lumen.

Active replication of HIV-1 at the lymphoepithelial surface of the tonsil.

Abstract: Cells that are infected with HIV-1 were visualized at the mucosal surface of the nasopharyngeal and palatine tonsils in 14 specimens from patients with CD4+ T-cell counts of 200 to 900/microliter and 2- to 10-year histories of HIV-1 infection. Most of the cells with intracellular HIV-1 protein were small but multinucleated. The majority of these syncytia could be double labeled for HIV-1 RNA and a dendritic cell marker S100. In the palatine tonsil, the infected cells were not found in the stratified squamous epithelium that is adjacent to the pharynx. Instead, the S100+ infected syncytia were localized to the surface of tonsil invaginations or crypts. This mucosa, termed lymphoepithelium, contains antigen-transporting M cells that lie above regions where S100+ dendritic cells are juxtaposed with CD4+ lymphocytes. Likewise, infected cells were found in lymphoepithelium and not respiratory epithelium of nasopharyngeal tonsils or adenoids. We propose that lymphoepithelia, the histological term that describes the specialized regions where antigens access mucosa-associated lymphoid tissue, are sites where HIV-1 replication can be enhanced in syncytia derived from dendritic cells.

Airway goblet cell mucin: its structure and regulation of secretion

Abstract: Mucociliary clearance is a major function of the airway epithelium. This important function depends both on the physicochemical properties of the airway mucus and on the activity of the cilia. The former, in turn, is dependent mainly on the quality and quantity of mucous glycoproteins or mucins, which are produced by two different cell types, namely, goblet cells of the epithelium and mucous cells of the submucosal gland. Neither the structural nor the functional differences of mucins produced by these two cell types are yet known. The availability of primary airway epithelial cell culture systems, however, has made it possible to study the structure and regulation of airway goblet cells to some extent. The epithelial mucins are extremely hydrophobic and are associated with various macromolecules, the quality and quantity of which may also affect the physicochemical properties of the mucus. Secretion of epithelial mucins is stimulated by various factors, including a number of inflammatory agents. The recent progress in mucin molecular biological research will allow us to identify different mucin core proteins produced by those different cell types, and, hopefully, the differential functions of these mucins in health and disease.

Acute safety and effects on mucociliary clearance of aerosolized uridine 5′-triphosphate ± amiloride in normal human adults

Abstract: Impaired mucociliary clearance contributes to the pathophysiology of several airways diseases including cystic fibrosis, asthma, and chronic bronchitis. Extracellular triphosphate nucleotides (adenosine 5'-triphosphate [ATP], uridine 5'-triphosphate [UTP]) activate several components of the mucociliary escalator, suggesting they may have potential as therapeutic agents for airways diseases. We conducted initial (Phase I) studies of acute safety and efficacy of aerosolized UTP alone and in combination with aerosolized amiloride, the sodium channel blocker, in normal human volunteers. Safety was assessed by measurement of pulmonary function. Neither UTP alone nor in combination with amiloride caused any clinically significant adverse effects on airway mechanics, (subdivisions of) lung volumes, or gas exchange. Acute efficacy of UTP and amiloride alone and in combination, was assessed by measuring changes in the clearance of inhaled radiolabeled particles. A 2.5-fold increase in mucociliary clearance was seen in response to UTP alone and in combination with amiloride. We conclude that aerosolized UTP +/- amiloride clearly enhances mucociliary clearance without acute adverse effects in normal adults, and may have therapeutic potential to enhance airways clearance in diseases characterized by retained airways secretions.

Ontogeny of the digestive tract during larval development of yellowtail flounder: a light microscopic and mucous histochemical study

Abstract: The histological development and mucous histochemistry of the alimentary tract in larval yellowtail flounder were studied using light microscopy. Samples were taken when the larvae were first offered food at 3 days post-hatch, then at 7, 10, 29, 36, and 46 days post-hatch, at which time they were metamorphosing. Regional partitioning of the digestive tract into the buccal cavity, pharynx, oesophagus, post-oesophageal swelling (PES), intestine, and rectum was complete by day 10. Goblet cells were present only in the buccal cavity, pharynx and intestine by day 7, but increased in number and distribution as development continued. By day 29, the posterior zone of the oesophagus had a marked increase in goblet cell density and mucosal folding. At the transition from oesophagus to PES/stomach stratified epithelium with goblet cells changed abruptly to a columnar epithelium with no goblet cells. Multicellular glands in the PES of 36-day larvae allowed it to be defined as a stomach. The distinct brush border of columnar epithelium and the presence of goblet cells characterize the intestine and rectum. All goblet cells throughout the digestive tract were strongly positive for acid mucins as was the luminal layer of the stratified epithelia lining the buccal cavity, pharynx and oesophagus. The PES/stomach epithelium stained weakly for neutral mucins. No mucin staining was associated with the gastric glandular epithelium. The brush borders of the intestine and rectum were strongly positive for combinations of neutral and acid mucins.

Noncoordinated expression of alpha-, beta-, and gamma-subunit mRNAs of epithelial Na+ channel along rat respiratory tract.

Abstract: Na+ reabsorption from the epithelial surface of the respiratory tract plays a fundamental role in respiratory physiology. As in the epithelia of the renal collecting tubule and distal colon, Na+ en...

Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo

Abstract: Airway epithelium is subject to injury during inflammation and exposure to a variety of inhaled and infectious agents. Little is known about the expression of integrins during human airway epithelial regeneration and differentiation after injury. We therefore characterized integrin subunit expression after mechanical injury in an in vivo xenograft model of human bronchial epithelium. On the migrating cells at the edges of surface epithelial wounds, there was increased expression of the alpha v-, beta 5-, beta 6-, and alpha 5-integrin subunits. During the later phase of repair, the increased expression of alpha v-, beta 5-, and beta 6-subunits persisted, but the expression of the beta 8-subunits was restricted to basal cells. In addition, there was a redistribution of the alpha 2- and alpha 6-collagen/laminin-binding integrins to suprabasal epithelial layers. There was no expression of the beta 3- or alpha 4-integrin subunit on reparative epithelium. A similar upregulation of alpha v-, beta 5-, and beta 6-integrin receptor subunits was observed in areas of undifferentiated airway from cystic fibrosis patients. Injured epithelium was found to be significantly more susceptible to gene transfer with a recombinant adenovirus, suggesting that the increased integrin expression has implications for the acquisition of adenovirus infections and for lung-directed gene therapy.

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Abstract: Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates.

Abstract: Epidemiologic and occupational studies indicate adverse health effects due to inhalation of particulate air pollutants, but precise biologic mechanisms responsible have yet to be fully established. The tracheobronchial epithelium forms the body's first physiologic barrier to such airborne pollutants, where ciliary movement functions to remove the offending substances caught in the overlying mucus layer. Resident and infiltrating phagocytic cells also function in this removal process. In this paper, we examine the role of reactive oxygen and nitrogen species (ROS/RNS) in the response of airway epithelium to particulates. Some particulates themselves can generate ROS, as can the epithelial cells, in response to appropriate stimulation. In addition, resident macrophages in the airways and the alveolar spaces can release ROS/RNS after phagocytosis of inhaled particles. These macrophages also release large amounts of tumor necrosis factor alpha (TNF-alpha), a cytokine that can generate responses within the airway epithelium dependent upon intracellular generation of ROS/RNS. As a result, signal transduction pathways are set in motion that may contribute to inflammation and other pathobiology in the airway. Such effects include increased expression of intercellular adhesion molecule 1, interleukin-6, cytosolic and inducible nitric oxide synthase, manganese superoxide dismutase, cytosolic phospholipase A2, and hypersecretion of mucus. Ultimately, ROS/RNS may play a role in the global response of the airway epithelium to particulate pollutants via activation of kinases and transcription factors common to many response genes. Thus, defense mechanisms involved in responding to offending particulates may result in a complex cascade of events that can contribute to airway pathology.

Fas expression in pulmonary alveolar type II cells

Abstract: Fas, a type I membrane receptor protein, transduces a signal culminating in apoptosis after binding to the Fas ligand. Information regarding the expression of Fas in nonlymphoid tissues, although limited, suggests a role for Fas in epithelial progenitor cell populations. In this paper, we provide several lines of evidence indicating that the progenitor cell of the alveolus, the type II cell, displays restricted expression of Fas. We found 1) Fas gene expression in RNA derived from fresh isolates of primary rat type II cells; 2) restriction of Fas expression to a subset of alveolar type II cells by in situ hybridization and immunohistochemistry of the normal mouse lung; 3) induction of apoptosis in a mouse lung type II epithelial cell line (MLE) after activation of Fas; and 4) induction of apoptosis in a subpopulation of type II cells after the intratracheal instillation of an activating anti-Fas antibody in mice. These findings suggest that Fas-dependent apoptosis is involved in regulating turnover of the alveolar epithelium.

Expression of CD44 isoforms is increased in the airway epithelium of asthmatic subjects.

Abstract: Since shedding of columnar, but not basal, epithelial cells is common in asthma, cell adhesion molecules such as CD44, which are differentially expressed on these cell types, are likely to be important in this disease. In bronchial epithelium of asthmatic and nonasthmatic subjects, CD44 isoforms have been localized by light- and electron-microscopic immunocytochemistry. Immunoreactivity for total CD44 (mAb Hermes-3/mAb 25.32) and for isoforms containing CD44v9 (mAb 11.24), CD44v6 (mAb 11.9), and CD44v4 (mAb 11.10) have been compared. In nonasthmatic samples, CD44s and CD44v9 were seen on basal but not columnar epithelial cells. Weak CD44v6 immunoreactivity was found infrequently in the bronchus, whereas CD44v4 immunoreactivity was absent. This indicates the presence of a distinct population of basal cells that express CD44. No CD44 was detected in areas of close cell-cell or cell-matrix contact, thus precluding the involvement of CD44 in stable adhesion in these areas. CD44 immunoreactivity was locally increased in areas showing morphologic damage to the epithelium. In epithelium from asthmatic subjects, the mean level of CD44 immunoreactivity on basal-cell membranes was doubled (4.3 versus 2.0 gold particles/microns membrane) as compared with nonasthmatic subjects. Increased expression of CD44 in asthmatic subjects, suggests that it has a significant role in the pathobiology of this disease, whereas the restricted distribution of this increase supports an association with repair rather than with inflammatory processes.

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.

Abstract: Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.