Normal Airway Epithelial Cells Research Articles

Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

Basement Membrane Matrix Modifies Cytokine Interactions between Lung Cancer Cells and Fibroblasts

Objective: Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated. Methods: The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel™. Results: All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel. Conclusion: These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.

Abnormalities in the Pulmonary Innate Immune System in Cystic Fibrosis

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.

Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.

Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells

To transfer genes into airway epithelial cells, we have generated auxotrophic dap Escherichia coli BM2710 mutant that expresses the invasin of Yersinia pseudotuberculosis and the listeriolysin of Listeria monocytogenes. E. coli BM2710 harboring a plasmid carrying the gfp gene was incubated with immortalized normal or cystic fibrosis (CF) airway epithelial cells or with primary bronchial epithelial cells grown as an explant-outgrowth cell culture model. Approximately 2% of immortalized cells expressed GFP. Few primary cells were transfected that were always poorly differentiated and located at the edge of the outgrowth. This was consistent with the expression of β1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with β1-integrins. The subsequent intracellular trafficking of E. coli BM2710 studied by confocal and electronic microscopy showed that the E. coli-containing phagosomes rapidly matured into phagolysosomes. This is the first demonstration that recombinant bacteria are able to transfer genes into primary airway epithelial cells, provided that they are able to invade the cells.

Toll-like receptors in normal and cystic fibrosis airway epithelial cells.

Toll-like receptors (TLRs) mediate cellular responses to diverse microbial ligands. The distribution and function of TLRs in airway cells were studied to identify which are available to signal the presence of inhaled pathogens and to establish if differences in TLR expression are associated with the increased proinflammatory responses seen in cystic fibrosis (CF). Isogenic, polarized CF and control bronchial epithelial cell lines, human airway cells in primary culture, and cftr null and wild-type mice were compared. TLRs 1-10, MD2, and MyD88 were expressed in CF and normal cells. Only TLR2 transcription was modestly increased in CF as compared with normal epithelial cells following bacterial stimulation. TLR2 was predominantly at the apical surface of airway cells and was mobilized to cell surface in response to bacteria. TLR4 was present in a more basolateral distribution in airway cells, but appeared to have a limited role in epithelial responses. Lipopolysaccharide failed to activate nuclear factor-kappaB in these cells, and TLR2 dominant negative but not TLR4 dominant negative mutants inhibited activation by both Gram-negative and Gram-positive bacteria. Increased availability of TLR2 at the apical surfaces of CF epithelial cells is consistent with the increased proinflammatory responses seen in CF airways and suggests a selective participation of TLRs in the airway mucosa.

Interleukin-1β and Tumor Necrosis Factor-α Induce MUC5AC Overexpression through a Mechanism Involving ERK/p38 Mitogen-activated Protein Kinases-MSK1-CREB Activation in Human Airway Epithelial Cells

Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-response element-binding protein and cAMP-response element signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and enhance our understanding on mucin hypersecretion during inflammation.

Sustained Calcium Entry through P2X Nucleotide Receptor Channels in Human Airway Epithelial Cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma.

To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heat-shocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique.

BackgroundSFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex.MethodsIn this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM).ResultsWe demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio.ConclusionsThese studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.

Glucocorticoid effects on the β-adrenergic receptor–adenylyl cyclase system of human airway epithelium

Background: Activation of the β 2-adrenergic receptor (β 2AR) system expressed by human airway epithelial cells elicits a variety of cyclic adenosine monophosphate (cAMP)-dependent processes that help determine airway caliber and the intensity of airway inflammation in asthma. Glucocorticoids, mainstays in the treatment of asthma, profoundly affect the expression and function of the β 2-adrenergic receptor-adenylyl cyclase (β 2AR-AC) system in a variety of cell types. However, the effects of glucocorticoids on the β 2AR-AC system expressed by human airway epithelial cells are unstudied. Objective: We examined the effects of dexamethasone (DEX) on β 2AR gene expression and the function of the β 2AR-AC system in cultured human airway epithelial cells. Methods: Studies were performed in normal airway epithelial cells and BEAS-2B cells. β 2AR gene expression was assessed from measurements of β-adrenergic receptor density, β 2AR mRNA, and the activity of a full-length β 2AR promoter-luciferase reporter construct. The function of the β 2AR-AC system was assessed from cAMP production in response to the β 2-agonist isoproterenol and the expression of the stimulatory G protein Gαs. Results: DEX had no effect on β-adrenergic receptor density or on the β 1/β 2 ratio over a wide range of concentrations and exposure times. However, DEX significantly but transiently enhanced β 2AR mRNA levels (~1.5-fold) and β 2AR promoter activity (~1.5-fold), indicating increased β 2AR gene transcription. DEX also dose-dependently enhanced cAMP responses to isoproterenol but not to forskolin, a direct activator of adenylyl cyclase. DEX-induced changes in cAMP production were associated with small (~15%) increases in Gαs expression. Conclusions: These data indicate that glucocorticoids only transiently enhance β 2AR gene transcription and fail to increase steady-state levels of β 2AR protein in human airway epithelial cells. Nonetheless, glucocorticoid-induced effects on the β 2AR-AC system of human airway epithelial cells contribute to the beneficial effects of corticosteroids in asthma by enhancing the functional response to β 2-agonists. (J Allergy Clin Immunol 2002;109:491-7.)

Chinks in the armor of the airway: Pseudomonas infection in the cystic fibrosis lung.

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway lumen and submucosal gland epithelial cells leads to persistent and damaging lung infection that begins early in life and causes the majority of the morbidity and mortality in patients with cystic fibrosis (CF) (1). Respiratory infections do not appear to be a consequence of altered pulmonary development because lungs of neonates with CF are structurally normal (with the exception of acinar distension of submucosal glands) as well as sterile (2). However, at some point after birth, the respiratory tract becomes colonized with characteristic bacterial species. Initially, Staphylococcus aureus , Haemophilus influenzae , and Escherichia coli are frequently isolated from young patients with CF, but these organisms eventually surrender the lung to Pseudomonas aeruginosa (3). The isolation of mucoid strains of P. aeruginosa from the respiratory tract is virtually pathognomonic for this disease, and its early acquisition is associated with a poorer prognosis (4). Once acquired, P. aeruginosa is virtually impossible to eradicate from the CF lung, even with aggressive use of inhaled or systemic antibiotics. Unfortunately, the precise mechanisms by which abnormal CFTR leads to persistent pulmonary infection and the almost singular vulnerability to P. aeruginosa in patients with CF remain unclear. The vulnerability of CF patients to chronic pulmonary infection with P. aeruginosa has focused research on identification of Pseudomonas -specific interactions with the CF airway that explain this predilection. One possibility for the predisposition to infection with P. aeruginosa could be increased adherence of this organism to airway epithelial cells of CF patients (5). P. aeruginosa bacilli adhere in greater numbers to epithelial cells of patients homozygous for the F508 CFTR mutation (6), and this appears to be secondary to increased asialylated glycoconjugate receptors at the epithelial surface (7). Bacterial adhesins that are components of P. aeruginosa pili attach specifically to asialylated glycosphingolipid receptors containing an N-acetylgalactosamine-galactose binding moiety, such as gangliotetraosylceramide (asialo-GM 1 ), which are increased on respiratory epithelial cells from CF patients (8). Increased attachment of the Pseudomonas bacilli to the cystic fibrosis airway leads to impaired clearance, and binding via pili and other bacterial structures promotes inflammation by provoking airway epithelial cells to release mediators such as interleukin-8 (9). However, the clinical significance of this mechanism for Pseudomonas -epithelial cell interaction is uncertain because increased binding to asialylated receptors is relatively modest and not bacteria-specific (10). Another proposal to explain the predilection of P. aeruginosa for the CF lung focuses on the capacity of respiratory epithelial cells to participate in clearance of the bacterium. In this model, normal airway epithelial cells internalize P. aeruginosa and then desquamate to eliminate the organism from the airway (11). Moreover, CFTR may actually serve as the receptor for binding and internalization of P. aeruginosa in this system, and airway clearance through this process may be decreased by CFTR mutation (12). Accordingly, decreased ingestion of P. aeruginosa at the epithelial surface may permit establishment of Pseudomonas infection in the CF lung, but the actual role of epithelial cells as “phagocytes” and its relevance to innate defense of the airway in vivo is unclear (13). Since the neutrophil is the predominant inflammatory cell in the CF airway and central to bacterial killing (14, 15), others have concentrated on Pseudomonas interactions with these professional phagocytes. P. aeruginosa is a potent stimulus for neutrophil influx into the lungs, yet the bacteria has virulence factors, such as a quorum sensing system for biofilm generation, that allow it to evade neutrophil killing (16). Epithelial cells containing a mutant CFTR may amplify neutrophil recruitment through constitutive and bacteria-induced release of inflammatory mediators that is disproportionate to the infectious stimulus (17–19). Neutrophil proteases that normally degrade outer membrane proteins of bacteria are released into the CF airway (14, 20, 21). High extracellular concentrations of proteases such as neutrophil elastase damage the lung and can interfere with antibacterial defenses through digestion of proteins that promote opsonic phagocytosis such as immunoglobulins, complement components, and receptors (22, 23). Taken together, it is possible that P. aeruginosa is best adapted for the CF airway because it not only survives, but also can persist and thrive in the face of established pulmonary inflammation. Still, these virulence factors do not clearly explain why Pseudomonas colonization occurs in the first place. Because the CFTR is a chloride channel, several investigators have worked to more directly link abnormal airway salt and water transport with respiratory infection. ( Received in original form May 31, 2001 )

Expression of Eotaxin by Normal Airway Epithelial Cells after Influenza Virus A Infection

Background: Viral infection is known to cause lung inflammatory disease, including bronchial asthma. The mechanisms of inflammatory cell accumulation into the airways after viral infection are not well understood. Eotaxin is a CC chemokine which is a potent and specific agonist for CC chemokine receptor 3 (CCR3). CCR3 is expressed on eosinophils, basophils and T lymphocytes. These cells are known to be key cells in the pathogenesis of asthma. Although it has recently been demonstrated that airway epithelial cells express eotaxin in vivo and in vitro, there are few data about its epxression in viral infection. We hypothesized that eotaxin may play an important role in attracting inflammatory cells to the airways after viral infection, and analyzed whether viral infection attracts eotaxin in bronchial epithelial cells in vitro. Methods: Human airway epithelial cells obtained from bronchial tissue at lobectomy for lung cancer were infected with influenza virus A (subtype H3N2). The cells and cultured media were collected 8, 24, and 48 h after infection. Eotaxin mRNA was analyzed with reverse transcriptase-polymerase chain reaction. Eotaxin protein levels in the culture media were analyzed by enzyme-linked immunosorbent assay. We also studied a blocking assay to analyze the intervention of proinflammatory cytokines in its production induced by influenza virus. Results: Eotaxin mRNA appeared to be expressed constitutively in uninfected cells but was expressed more clearly in infected cells. Eotaxin protein release into culture media significantly increased after infection. Anti-TNF-α and anti-IL-1β antibodies did not alter the eotaxin protein levels after viral infection. Conclusions: These results suggest that influenza virus A infection in airway epithelial cells activates the expression of eotaxin and that eotaxin may participate in the pathogenesis of airway inflammatory disease caused by viral infection, such as infectious type asthma.

Gene expression profiling of advanced lung cancer.

Lung cancer is a highly aggressive neoplasm with 85% mortality. To identify new tumor-associated genes, we compared the expression profile of a primary metastasizing adenocarcinoma with normal airway epithelial cells. Two cDNA libraries of up- and downregulated genes were generated, comprising 253 and 299 clones, respectively. The sequence analysis revealed 205 different known genes and 314 cDNA fragments of unknown functions. Northern-blot analysis of 167 clones confirmed differential expression in 58%, and indicated a similar expression pattern in additional lung-cancer cell lines for selected clones, strengthening the value of this model for the identification of new candidate genes in lung carcinogenesis.

Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine.

1. Exposure to epinephrine has been shown to have a range of effects on cells and tissues. A recent study suggested that the proliferative ability of CF epithelial cells, exposed to high concentrations of epinephrine (200 - 300 microM), was reduced when compared to that of normal cells. This approach could potentially provide a means to effectively separate cells with functional cyclic AMP-dependent Cl-ion transport from those defective in this pathway. 2. The sensitivity to killing by epinephrine is reported here for four different CF cell lines, three normal cell lines, and two CF epithelial cell lines complemented with wild-type (wt) CF transmembrane conductance regulator (CFTR) cDNA. 3. While each cell line exhibited varying sensitivity to 200 microM epinephrine, no predictable pattern was observed between the expression of wt-CFTR and cell survival following epinephrine exposure. Overall, normal cell lines did exhibit a greater resistance to epinephrine-induced cell death although, the most resistant cell line was derived from CF tracheal epithelium (SigmaCFTE29o-). 4. The expression of exogenous wt-CFTR increased the survival of one cell line (CFDEo-) when compared to the parent line, but in another complemented line, survival was reduced. 5. These findings suggest that while epinephrine induces cell killing, it is not consistently effective for preferential selection of normal over CF cells. Although CFTR may play a role in the mechanism(s) of epinephrine killing, other factors such as cell density, proliferative ability, cell type origin and phenotype are involved.

Low neutral endopeptidase levels in bronchoalveolar lavage fluid of lung cancer patients.

Neutral endopeptidase (NEP) is a cell surface enzyme found in normal human lung and which hydrolyzes small bioactive peptides, some of which act as growth factors for normal and malignant airway epithelial cells. Expression of NEP varies widely in human lung tissue from different individuals. NEP is often expressed at low or undetectable levels in both small-cell and non-small-cell lung cancer, and inhibits the growth of lung cancer cell lines. Variation in the expression of NEP could be a factor in susceptibility to lung cancer. We hypothesized that NEP could be measured in bronchoalveolar lavage fluid (BALF) and that airway levels of NEP would be low in lung cancer patients as compared with normal controls. We measured NEP and total protein in cell-free BALF supernatant, and expressed the respective concentrations as a ratio. NEP levels showed wide variation in BALF of healthy volunteers. Most patients with lung cancer had no NEP detectable in BALF. The mean NEP/total protein ratio was significantly lower in patients with lung cancer (0.87 +/- 0.7 ng NEP/mg protein) than in normal healthy subjects (14.0 +/- 4.3, p < 0.0003). We conclude that NEP levels are highly variable in BALF of normal volunteers, and are low or undetectable in most BALF specimens from patients with lung cancer. Low NEP levels in the airways may be a factor in the pathogenesis of carcinoma of the lung.

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments.

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non-transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.

Expression of RANTES by normal airway epithelial cells after influenza virus A infection.

The chemokine regulated on activation, normal T cells expressed and secreted (RANTES), is a C-C chemokine and a potent chemoattractant for monocytes, T lymphocytes, basophils, and eosinophils. Its expression by human airway epithelium has been demonstrated both in vitro and in vivo. We investigated whether RANTES is expressed by normal human airway epithelial cells after influenza viral infection and examined its bioactivity. Epithelial cells were obtained from bronchial tissue or nasal polyps of patients who had undergone lobectomy for lung cancer or polypectomy for nasal polyps. These cells were cultured by the outgrowth method. Cultured cells were infected with influenza virus A (subtype H3N2) after which the supernatants and the cells were collected 8 to 72 h after infection. RANTES mRNA (messenger RNA) was analyzed by the reverse transcriptase-polymerase chain reaction and Southern blot analysis of its product. Concentrations of RANTES in the supernatants were analyzed by enzyme-linked immunosorbent assay. RANTES protein and mRNA were not detected in the media of uninfected cells. PCR products for RANTES were clearly detected in nasal and bronchial epithelial cells 24 h after infection. Southern blot analysis confirmed that the PCR products were indeed specific for RANTES mRNA. Twenty-four to 72 h after infection, significant levels of RANTES protein were detected in culture media. We also investigated the chemotactic activity of the supernatant of cultured cells. The supernatant of the cells 48 h after infection had potent chemotactic activity for eosinophils, which was attenuated by the addition of anti-RANTES antibodies. These findings suggest that influenza virus infection may induce expression of bioactive RANTES by normal human bronchial and nasal epithelial cells.

Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies

The reactivity of two rat monoclonal antibodies was studied. These antibodies, A2R and A2C, bind a 32 kDa alveolar type II cell membrane receptor for surfactant protein A. A2R and A2C also bind apical cell membranes of ciliated and nonciliated cells of the conducting airways. Because this reactivity suggested possible utility in targeting those cells for therapeutic gene transfer, the binding activity of these two antibodies was examined in human tissues. In conducting airways, A2R and A2C bound apical epithelial cell membranes throughout the embryologic period studied: from 15 weeks of gestation, through maturity. Reactivity was more restricted to ciliated cells of the airways as maturation progressed. In the peripheral lung, A2C and A2R only bound most cells in the early developing lung, but mainly type II cells in mature lungs. Other normal tissues recognized by these antibodies included crypt lining cells of the adult and fetal stomach, large bile duct epithelium, and pancreatic acinar cells. All of these cells derive from embryonic foregut endoderm. Other normal tissues, both of endodermal and nonendodermal origin, were negative. Pulmonary carcinomas were studied. A2C and A2R recognized all non-small cell carcinomas of the lung tested. In contrast, none of the small cell carcinomas or carcinoid tumors of the lung were recognized by these antibodies. The function of p32 in these diverse cell types is not clear, but whatever its role in these tissues, antibodies versus p32 may potentially be used to target gene or drug therapy to the normal or malignant cells they recognize.