Cultures Of Human Bronchial Epithelial Cells Research Articles

Acute Regulation of the Epithelial Sodium Channel in Airway Epithelia by Proteases and Trafficking

Effective clearance of inhaled pathogens is the primary innate defense mechanism in the lung, and requires the maintenance of a proper airway surface liquid (ASL) volume to facilitate ciliary beat and optimize mucociliary clearance. Na(+) absorption via the epithelial sodium channel (ENaC) is tightly regulated and, together with chloride movement, provides the optimal osmotic gradients to absorb excessive fluid in the airway lumen while preventing excessive ASL dehydration, which would compromise mucus clearance from the lung. To absorb excessive fluid from the luminal surface, a local mechanism of ENaC activation allows for an increase in Na(+) absorption at times when the ASL volume is expanded. To help define these regulatory mechanisms, we examined the effects of ASL volume expansion on ENaC activity in primary human bronchial epithelial (HBE) cell cultures. We found that ENaC activity increases dramatically after rapid dilution of endogenous ASL. Approximately 35% of the increase in Na(+) absorption was attributable to activation of ENaC by proteases. The remainder of the increase in Na(+) current was prevented when membrane trafficking was disrupted with brefeldin A, nocodazole, or myosin light chain kinase inhibitors, demonstrating that trafficking is involved with ENaC regulation in the airway. These findings demonstrate that Na(+) absorption in the airway is acutely modulated by the coordinated trafficking of channels to the luminal surface and by the proteolytic activation of ENaC in response to ASL volume expansion.

Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

BackgroundCigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity.Methodology/Principal FindingsTo address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture).ConclusionsOur results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure.

Regulation of Neuregulin 1β1–Induced MUC5AC and MUC5B Expression in Human Airway Epithelium

Excessive mucus production has been linked to many of the pathologic features of respiratory diseases, including obstruction of the airways, decline in lung function, increased rates of mortality, and increased infections. The mucins, MUC5AC and MUC5B, contribute to the viscoelastic properties of mucus, and are found at elevated levels in the airways of individuals with chronic respiratory diseases. The T helper type 2 cell cytokine, IL-13, is known to regulate MUC5AC expression in goblet cells of the airways, although much less is known about the regulation of MUC5B expression. In a study to further understand the mediators of MUC5AC and MUC5B expression, neuregulin (NRG) 1beta1 was identified as novel regulator of goblet cell formation in primary cultures of human bronchial epithelial cells (HBECs). NRG1beta1 increased expression of MUCAC and MUC5B proteins in a time- and dose-dependent fashion in HBEC cultures. NRG1beta1-induced expression of MU5AC and MUC5B was shown to involve v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) and ErbB3 receptors, but not ErbB4 receptors. Treatment of HBECs with inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, and phosphatidylinositol 3-kinase indicated that these kinases were involved in NRG1beta1-induced MUC5AC and MUC5B expression. Additionally, NRG1beta1 was shown to induce the phosphorylation of the ErbB2 receptor, AKT, and extracellular signal-regulated kinase 1/2. NRG1beta1 protein was found increased in the airways of antigen-challenged mice, together with increases in MUC5AC and MUC5B message. Together, these data indicate that NRG1beta1 is a novel mediator of MUC5AC and MUC5B expression in HBECs, and may represent a novel therapeutic target for mucus hypersecretion in respiratory diseases.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects

Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.

Airway inflammation in employees involved in cultivating Japanese mushrooms (bunashimeji)

Chronic inhalation of spores may cause respiratory symptoms such as productive cough and sputum. The purpose of this study was to determine the clinical pathophysiology of airway inflammation caused by bunashimeji spores and to investigate whether the spores have direct toxic inflammatory effects. Sensitized employees with respiratory symptoms and a stimulation index (SI) > 200%, and non-sensitized employees with a SI < 200% were enrolled. They underwent sputum induction and chest high-resolution computed tomography (HRCT). The in vitro effect of bunashimeji spore solutions on normal human bronchial epithelial (NHBE) cell cultures was investigated using the air-liquid interface method. Bunashimeji spore solution was added at 10(4) or 10(6) spores per 20 microL/well. The interleukin (IL)-8 and epithelial neutrophil-activating peptide-78 (ENA-78) concentrations in the medium and IL-8 mRNA expression of NHBE cells were assessed after each stimulation. Sensitized employees were divided into 14 with normal HRCT and 9 with abnormal HRCT. Fifteen of the sensitized group and five of the non-sensitized group had a productive cough and sputum. The neutrophil counts in induced sputum were significantly higher in subjects with abnormal HRCT than in those with normal HRCT. IL-8 and ENA-78 concentrations following stimulation with 10(4) and 10(6) spores were significantly increased compared with PBS only on day 9. IL-8 mRNA expression due to spore stimulation was significantly increased compared with control. IL-8 mRNA expression with 10(6) spore stimulation was significantly increased on days 6 and 12 compared with 10(4) spores. The inhalation of spores directly produces toxic inflammatory effects in the airways, independent of the degree of sensitization.

Regulated Airway Goblet Cell Mucin Secretion

Major advances in understanding regulated mucin secretion from airway goblet cells have been made in the past decade in the areas of pharmacology and basic cell biology. For instance, it is now appreciated that nucleotide agonists acting locally through P2Y purinoceptors on apical membranes of surface goblet cells provide the major regulatory system for mucin secretion. Similarly, Clara cells, the primary secretory cell in the mouse airways (and human small airways), are now recognized as major mucin-secreting cells. In Clara cells, the relative lack of staining for mucosubstances reflects essentially equal baseline rates of mucin synthesis and secretion, with little to no accumulation of mucin granules in storage pools. During mucous metaplasia induced under inflammatory conditions, mucin synthesis is massively upregulated in Clara cells, and stored mucin granules come to dominate the secretory cell phenotype. More importantly, we have seen a transition in the past few years from a pharmacological focus on regulated mucin secretion to a more molecular mechanistic focus that has great promise going forward. In part, these advances are occurring through the use of well-differentiated primary human bronchial epithelial cell cultures, but recent work in mouse models perhaps has had the most important impact. Emerging data from Munc13-2- and synaptotagmin 2-deficient mouse models represent the first direct, molecular-level manipulations of proteins involved in regulated secretory cell mucin secretion. These new data indicate that Munc13-2 is responsible for regulating a baseline mucin secretory pathway in the airways and is not essential for purinergic agonist-induced mucin secretion. In contrast, synaptotagmin 2, a fast Ca2+ sensor for the SNARE complex, is essential for regulated secretion. Interestingly, these early results suggest that there are two pathways for excocytic mucin release from goblet cells.

A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus

The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing, (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park MS, et al. Proc Natl Acad Sci USA 2006;103(21):8203-8). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus, respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine.

Evaluation of the Acute Toxicity and Genotoxicity of Orange, Red, Violet and Yellow Pyrotechnic Smokes In Vitro

AbstractThe acute toxicity and the genotoxicity of four colored smokes were studied by an in vitro method based on the exposure of human bronchial epithelial cell cultures to the smokes. All smoke formulations consisted of the oxidizer fuel mixture (potassium chlorate/lactose), talcum and the following dyes: 1,4‐dihydroxy anthraquinone (orange), 1‐(p‐tolylamino)‐4‐hydroxy anthraquinone (violet), 1‐methylamino anthraquinone (red) and 4,4′‐methylidyne‐bis‐3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one (yellow). The experiments were carried out in a laboratory scale chamber and in a large container. The toxicity was compared to that of hexachloroethane (HC)‐based reference smoke with known toxicity. All the colored smokes displayed acute toxicity. The order of toxicity in the laboratory scale tests was orange&gt;violet≈red&gt;HC&gt;yellow and in the container tests orange&gt;violet≈yellow&gt;red. The orange smoke appeared genotoxic in all the tests. With the yellow and the violet smokes, the genotoxicity could not be totally excluded. The red smoke showed evidence of weak genotoxicity only in one test series at the highest concentration level.

Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis

BackgroundCigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis.MethodsMicroarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin (3.1-fold, p < 0.002), an iron-binding nuclear protein and transcription cofactor. In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE) and an adenovirus vector encoding the pirin cDNA (AdPirin) were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis.ResultsQuantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p < 0.02). CSE applied to primary human bronchial epithelial cell cultures demonstrated that pirin mRNA levels increase in a time-and concentration-dependent manner (p < 0.03, all conditions compared to controls).Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay (5-fold, p < 0.01) and ELISA for cytoplasmic nucleosomes (19.3-fold, p < 0.01) compared to control adenovirus vector.ConclusionThese observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases.

Beyond Our Pages

Beyond Our Pages

IL-13 and Epidermal Growth Factor Receptor Have Critical but Distinct Roles in Epithelial Cell Mucin Production

Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13-induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-alpha. However, there was constitutive release of TGF-alpha from normal human bronchial epithelial cells, and inhibition of TGF-alpha or EGFR reduced both constitutive and IL-13-induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production.

Glucocorticoids Increase Repair Potential in a Novel in vitro Human Airway Epithelial Wounding Model

Airway epithelial damage is a cardinal feature of chronic asthma. Agents which enhance epithelial repair without triggering uncontrolled fibrosis of the mesenchyme would be predicted to be useful in the management of asthma. We have developed a repeat wound model using mucociliated human bronchial epithelial cell (HBEC) cultures to define the key pathways involved in airway epithelial repair, and to study the effects of potential therapeutic agents on epithelial repair in a chronic setting. We show that repair occurs primarily by cell migration to close a defect; this process requires activation of the EGF receptor (EGFR) and subsequent tyrosine kinase signalling. Migration is accompanied by up-regulation of CD44 in motile cells at the wound margins with proliferation of non-migrating cells adjacent to the wound area. In long-term studies beta2 adrenoceptor agonists and phosphodiesterase (PDE) inhibitors have no effect on repair potential, in contrast chronic treatment with the glucocorticoid dexamethasone extends the lifespan of repeatedly wounded differentiated cultures. We suggest part of the beneficial effects of glucocorticoids in asthma is related to this ability to prolong repair potential following repeated episodes of epithelial injury.

Role of epidermal growth factor receptor in the proliferation of human airway epithelial cells

SummaryA novel airway trypsin‐like serine proteinase (ATSP) has been found in the sputum of asthma patients and might play a role in the pathogenesis of allergic airway inflammation, but the physiological significance of this enzyme is unknown. In this study we examined human primary bronchial epithelial cell cultures to determine whether ATSP affects the proliferation of airway epithelium and the mechanism of action involved. Incubation of cells in serum‐free medium with recombinant ATSP increased the incorporation of [3H]‐thymidine and [3H]‐leucine into cells in a time‐ and dose‐dependent manner. This effect was not altered by secretory leucocyte proteinase inhibitor (SLPI), but completely inhibited by pretreatment with the serine proteinase inhibitors leupeptin and 4‐amidinophenylmethane‐sulphonyl fluoride (p‐APMSF), the epidermal growth factor receptor (EGF‐R) tyrosine kinase inhibitors AG1478 and BIBX1382, and the mitogen‐activated protein kinase kinase (MEK) inhibitor PD98059. Transfection of a dominant negative mutant of H‐Ras likewise abolished the effect of ATSP. Stimulation with ATSP also induced processing of membrane anchored pro‐heparin binding (HB) EGF, association of phosphorylated EGF‐R and Shc with glutathione‐S‐transferase–Grb2 fusion protein, and phosphorylation of extracellular signal‐regulated kinase (ERK). Pretreatment with leupeptin abolished ATSP‐induced HB‐EGF processing, and pretreatment with each of AG1478, [Glu52]Diphtheria toxin, a specific inhibitor of HB‐EGF, and neutralizing HB‐EGF antibody inhibited ATSP‐induced ERK activation. These results suggest that ATSP stimulates the proliferation of airway epithelium through the activation of ERK cascade, involving processing of HB‐EGF and concomitant activation of EGF‐R.

Smoking is associated with increased telomerase activity in short-term cultures of human bronchial epithelial cells

Telomerase plays an important role in the maintenance of telomere ends in normal and tumor cells and ectopic expression can immortalize human bronchial epithelial (HBE) cells. We assessed telomerase activation, growth properties and methylation status in the hTERT promoter in a panel of HBE cell cultures in relation to smoking and previous lung cancer history. HBE cells were obtained from a total of 26 subjects, six of whom were lifelong non-smokers, while 20 subjects had a smoking history, including seven who had lung carcinoma. Telomerase activity was determined using the telomeric repeat amplification protocol (TRAP). Maximum passage number and time to senescence were also determined through extended culturing. The distribution of the telomerase activity between ever-smokers and never-smokers was significantly different ( P=0.03, F-test), and there was a strong correlation between telomerase activity and the number of pack-years smoked ( P=0.0012, F-test for slope). A small difference in telomerase activity was observed according to lung cancer status ( P=0.02, F-test). Telomerase activity was not correlated with maximum passage number after extended culturing or with time to senescence. None of the HBE cultures demonstrated methylation of the hTERT promoter. Our results indicate an association between tobacco carcinogen exposure and telomerase activity in normal bronchial epithelium, although a causative role of tobacco smoking in the (re)activation of telomerase can not be proven. An increase in telomerase activity in normal bronchial epithelium might extend the lifespan of cells at risk for malignant transformation, and thus contribute to lung carcinogenesis.

Chromosomal abnormalities in bronchial epithelium from smokers, nonsmokers, and lung cancer patients

The identification of individuals who are at greatest risk of developing lung cancer would greatly improve diagnosis and possibly lead to early treatment. To study the use of karyotypes for this purpose, we used short-term human bronchial epithelial (hBE) cell cultures from nonsmokers, smokers, and lung cancer patients. Twenty-five metaphases were scored for hBE cell cultures obtained from 32 patients: 8 were nonsmokers, and 24 had a history of smoking (of whom 11 had had lung cancer surgery). The number of abnormal metaphases ranged from 0 to 4 per cell culture. No overall differences in the number of abnormal metaphases were observed between nonsmokers and smokers or between lung cancer patients and non–lung cancer patients. The most commonly observed abnormalities were structural changes in chromosome 1 (six cultures), loss of chromosome 17 (six cultures), and trisomy of chromosome 20 (three cultures). These specific alterations were found almost exclusively in patients with a history of tobacco smoking. The results did not indicate that general chromosomal abnormalities are a useful marker for tobacco smoke exposure or cancer risk.

Effect of immune response to rhinovirus (RV) on RV-induced bronchial epithelial inflammation, cytotoxicity, and viral load

RATIONALE: Since the immune response to RV is different between atopic and healthy individuals we examined the influence of this different response on RV-induced bronchial epithelial cell inflammation. METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from whole blood of atopic asthmatic and healthy individuals (n=13) and were exposed in-vitro to RVs. Supernatants were harvested at 48hrs and IFN-γ and IL-10 levels were measured by ELISA. These supernatants were added to human bronchial epithelial (BEAS-2B) cell cultures, infected or not by RV. Epithelial cell supernatants were harvested at 6 and 48hrs later and IL-6, IL-8, RANTES, and TGF-b1 were measured. At the same time points BEAS-2B cells were lysed and RV titer assessed by serial dilutions on Ohio-Hela cells. Finally RV-induced cytotoxity was evaluated by staining the infected BEAS-2B cells with crystal violet, distaining by methanol, and measuring their optical density at 620 nm with spectrophotometer. RESULTS: The production of IL-6, IL-8, and RANTES by the infected bronchial epithelium was greater when exposed to infected pbmc supernatant from healthy compared to atopic subjects while the production of TGF-b1 was lower (p<0.05). In addition, the defective immune response of atopic asthmatic individuals reduced viral clearance, evidenced by the elevated viral titers compared to the ones of healthy subjects 6hs but mostly 48hs after infection (p<0,05) and had also impaired protection from virus- induced cytotoxicity. CONCLUSIONS: The atopic environment modulates the epithelial response to RV by impairing its inflammatory response and reducing viral clearance, inducing the existing remodeling and having impaired protection from virus-mediated cytotoxicity.

Human bronchial epithelial cells express and secrete MMP-12

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, which may be responsible for enlargement of alveoli in chronic obstructive pulmonary disease (COPD) and remodeling of pulmonary tissue associated with chronic asthma. Here, we provide novel evidence that MMP-12 is expressed and secreted by normal human bronchial epithelial cell cultures (NHBECs) and reveal the regulation of MMP-12 gene expression by tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), and interferon gamma (IFN-γ). Reverse transcription-polymerase chain reaction analyses demonstrated MMP-12 mRNA presence in unstimulated differentiated NHBEC cultures. Cultures stimulated independently with EGF or IFN-γ failed to alter MMP-12 mRNA abundance, while TNF-α, TNF-α+EGF, or TNF-α+IFN-γ elicited relatively early (6h) peak increases in MMP-12 mRNA levels. Western blot analyses specifically indicated the presence of MMP-12 in differentiated NHBEC-conditioned media. These findings indicate that the bronchial epithelium may be an important source of elastolytic activity in COPD and tissue remodeling in chronic asthma.

Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway.

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [(3)H]thymidine and [(3)H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione-S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.

Mucins and their O-Glycans from human bronchial epithelial cell cultures.

A longstanding question in obstructive airway disease is whether observed changes in mucin composition and/or posttranslational glycosylation are due to genetic or to environmental factors. We tested whether the mucins secreted by second-passage primary human bronchial epithelial cell cultures derived from noncystic fibrosis (CF) or CF patients have intrinsically different specific mucin compositions, and whether these mucins are glycosylated differently. Both CF and non-CF cultures produced MUC5B, predominantly, as judged by quantitative agarose gel Western blots with mucin-specific antibodies: MUC5B was present at approximately 10-fold higher levels than MUC5AC, consistent with our previous mRNA studies (Bernacki SH, Nelson AL, Abdullah L, Sheehan JK, Harris A, William DC, and Randell SH. Am J Respir Cell Mol Biol 20: 595-604, 1999). O-linked oligosaccharides released from purified non-CF and CF mucins and studied by HPLC mass spectrometry had highly variable glycan structures, and there were no observable differences between the two groups. Hence, there were no differences in either the specific mucins or their O-glycans that correlated with the CF phenotype under the noninfected/noninflammatory conditions of cell culture. We conclude that the differences observed in the mucins sampled directly from patients are most likely due to environmental factors relating to infection and/or inflammation.

IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation.

In addition to a direct proinflammatory role, IL-13 has been demonstrated to induce a goblet cell metaplastic phenotype in the airway epithelium in vivo. We have studied the direct effects of IL-13 (and IL-4) on well-differentiated, air-liquid interface cultures of human bronchial epithelial cells (HBEs) and provide a quantitative assessment of the development of a mucus hypersecretory phenotype induced by these cytokines. Using Alcian blue staining of goblet cells and immunohistochemical detection of MUC5AC, we found that IL-13 (and IL-4) induced increases in the goblet cell density (GCD) of the HBE cultures. The effects of these cytokines were critically dependent on concentration: 1 ng/ml routinely induced a 5- to 10-fold increase in GCD that was associated with a hypersecretory ion transport phenotype. Paradoxically, 10 ng/ml of either cytokine induced a profound reduction in GCD. Removal of EGF from the culture media or treatment of the cells with AG-1478 [a potent inhibitor of EGF receptor tyrosine kinase (EGFR-TK)] demonstrated that the EGFR-TK pathway was key to the regulation of the basal GCD but that it was not involved in the IL-13-driven increase. The IL-13-driven increase in GCD was, however, sensitive to inhibition of MEK (PD-98059, U-0126), p38 MAPK (SB-202190), and phosphatidylinositol (PtdIns) 3-kinase (LY-294002). These data support the concept that IL-13 is in part able to induce a mucus hypersecretory phenotype through a direct interaction with the airway epithelium and that MAP kinase and PtdIns 3-kinase signaling pathways are involved.