Showing papers on "Respiratory epithelium published in 1995"

A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis

Abstract: Background Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. Methods An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (P...

Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo

Abstract: Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.

Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium.

Abstract: The mechanisms by which Aspergillus fumigatus colonizes the respiratory mucosa are unknown. Culture filtrates of eight of nine clinical isolates of A. fumigatus slowed ciliary beat frequency and damaged human respiratory epithelium in vitro. These changes appeared to occur concurrently. Culture filtrates of two clinical isolates of Candida albicans had no effect on ciliated epithelium. We have purified and characterized cilioinhibitory factors of a clinical isolate of A. fumigatus. The cilioinhibitory activity was heat labile, reduced by dialysis, and partially extractable into chloroform. The activity was associated with both high- and low-molecular-weight factors, as determined by gel filtration on Sephadex G-50. A low-molecular-weight cilioinhibitory factor was further purified by reverse-phase high-performance liquid chromatography and shown by mass spectrometry to be gliotoxin, a known metabolite of A. fumigatus. Gliotoxin significantly slowed ciliary beat frequency in association with epithelial damage at concentrations above 0.2 microgram/ml; other Aspergillus toxins, i.e., fumagillin and helvolic acid, were also cilioinhibitory but at much higher concentrations. High-molecular-weight (> or = 35,000 and 25,000) cilioinhibitory materials had neither elastolytic nor proteolytic activity and remain to be identified. Thus, A. fumigatus produces a number of biologically active substances which slow ciliary beating and damage epithelium and which may influence colonization of the airways.

Progenitor cells of the adult human airway involved in submucosal gland development.

Abstract: A bronchial xenograft model of the human airway was used to identify submucosal gland progenitor cells within the surface airway epithelium. Lineage analysis using recombinant retroviruses has demonstrated considerable diversity in the cellular composition of expanded clones within reconstituted xenograft airway epithelium. These findings provide evidence for the existence of multiple progenitors in the airway with either limited or pluripotent capacity for differentiation. Furthermore, the development of transgene-expressing submucosal glands was associated with a single subset of surface airway epithelial clones. This gland progenitor cell demonstrated two discernible characteristics consistent with the identification of an airway stem cell including: (1) pluripotent capacity for airway differentiation and (2) a two-fold higher proliferative rate than other observed clone types. The number of progenitor cells involved in gland development was also assessed by clonal analysis using alkaline phosphatase and beta-galactosidase transgenes. These studies demonstrated that more than one airway progenitor cell is involved in the initial stages of gland development. A second explanation for the high prevalence of non-clonality in developing glands was suggested from three-dimensional reconstruction of transgene marked glands. These reconstruction experiments demonstrated that 27% of glands contained more than one duct to the surface airway epithelium. This observation suggests a novel mechanism of gland morphogenesis by which independently formed glands interact to join glandular lumens. Such a mechanism of glandular development and morphogenesis may play an important role in normal submucosal gland development and/or the progression of hypersecretory diseases of the adult human airway as seen in cystic fibrosis, chronic bronchitis and asthma. The identification of progenitor cells with the capacity to form submucosal glands has implications on the targets for gene therapy in cystic fibrosis.

Plasticity of airway cell proliferation and gene expression after acute naphthalene injury

Abstract: The goal of this study was to determine the temporal and spatial sequence of events that accompany lung injury and repair after parenteral administration of the Clara cell-specific cytotoxicant, naphthalene. Changes in airway epithelial cells were evaluated by measuring alterations in the expression of markers for differentiated Clara cells (CYPIIF and Clara cell 10-kDa secretory protein, CC10), distal airway/alveolar type II cells (surfactant protein B; SP-B) and for cycling/proliferating cells (cyclin dependent kinase 1;CDK1). Naphthalene-induced Clara cell cytotoxicity resulted in the exfoliation of epithelial cells containing CC10 protein. This was accompanied by a dramatic reduction in the abundance of mRNA for CC10 and CYPIIF. Large numbers of CDK1 mRNA-positive cells were identified in and around bronchioles and terminal bronchioles 48 h after treatment. This cellular proliferation resulted in the population of airways by immature epithelial cells lacking normal levels of CC10 mRNA but overexpressing SP-B mRNA. Seventy-two hours after naphthalene treatment a reduction in CDK1 mRNA-positive cells was noted within bronchioles and terminal bronchioles at all locations, with the exception of airway bifurcations. At airway bifurcations CDK1 mRNA appeared to be more abundant at the 72-h time point than at 48 h. Comparison of these sections with serial sections probed for CC10 mRNA demonstrated a correlation between the expression of CDK1 and CC10 mRNA at bifurcations. Temporal increases in the abundance of CC10 mRNA observed at later time points were largely accounted for by the processive maturation of newly repopulated cells neighboring bifurcations in bronchioles. These studies identify spatially distinct populations of cells that act in concert to repopulate naphthalene-injured airways and support the notion that branch point cells play an important role in the maturation of newly regenerated airway epithelial cells after acute injury.

In vivo restitution of airway epithelium.

Abstract: Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-μm wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [3H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 μm/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.

Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness

Abstract: A murine model for antigen-induced bronchial hyperreactivity (BHR) and airway eosinophilia, two hallmarks of asthma, was developed using ovalbumin-immunized mice, which produce large amounts of IgE (named BP2, "Bons Producteurs 2," for High Line of Selection 2). A single intranasal ovalbumin challenge failed to modify the bronchial responses, despite the intense eosinophil recruitment into the bronchoalveolar lavage fluid and airways. When mice were challenged twice a day for 2 days or once a day for 10 days, BHR in response to i.v. 5-hydroxytryptamine or to inhaled methacholine was induced in BP2 mice but not in BALB/c mice. Histological examination showed that eosinophils reached the respiratory epithelium after multiple ovalbumin challenges in BP2 mice but remained in the bronchial submucosa in BALB/c mice. Total IgE titers in serum were augmented significantly with immunization in both strains, but much more so in BP2 mice. Interleukin 5 (IL-5) titers in serum and bronchoalveolar lavage fluid of BP2 mice were augmented by the antigenic provocation, and a specific anti-IL5 neutralizing antibody suppressed altogether airway eosinophilia and BHR, indicating a participation of IL-5 in its development. Our results indicate that the recruitment of eosinophils to the airways alone does not induce BHR in mice and that the selective effect on BP2 mice is related to their increased IgE titers associated with antigen-driven eosinophil migration to the epithelium, following formation and secretion of IL-5.

An immunochemical, ultrastructural, and developmental characterization of the horizontal basal cells of rat olfactory epithelium

Abstract: The olfactory epithelium, which retains a capacity for neurogenesis throughout life, contains two categories of basal cells, dark/horizontal and light/globose, neither of which is fully characterized with respect to their function during the processes of neurogenesis and epithelial reconstitution after injury The aim of this study was to define the potential biological role(s) of dark/horizontal basal cells (D/HBCs) in the epithelium by performing immunochemical, electron microscopic, and developmental analyses of this cell population The D/HBCs express several specific immunochemical characteristics, which include the rat homologues of human cytokeratins 5 and 14, which were identified on the basis of staining with subunit- specific monoclonal antibodies and two-dimensional immunoblot analysis of the immunoreactive proteins Indeed, the D/HBCs are the only cells in the olfactory mucosa that express these specific cytokeratins The D/HBCs also express an α-galactose or α-N galactosamine moiety to which the Is4 isolectin from Bandeiraea simplicifolia binds Moreover, the D/HBCs are heavily labeled by two different antibodies against the EGF receptor and by a monoclonal antibody that binds to phosphotyrosine These characteristics are also common to the basal cells of respiratory epithelium The electron microscopic analysis of the basal region of the olfactory epithelium and the light microscopic immunofluorescence observations demonstrate that the D/HBCs provide a bridge between the basal processes of some sustentacular cells and the basal lamina The most striking ultrastructural feature of the D/HBCa is their enfolding of virtually all bundles of olfactory axons within tunnels formed where D/HBCs arch over the basal lamina The intimacy of the arrangement between D/HBCs and olfactory axons suggests that signals may pass from axons to D/HBCs or vice-versa With respect to the development of D/HBCs, cells that express cytokeratins 5 and 14 and the EGF receptor first appear near the boundary with respiratory epithelium late in development, but do not extend throughout the olfactory epithelium until the middle of the first postnatal week Taken together, the present findings and previously published data suggest that D/HBCs help to maintain the structural integrity of the olfactory epithelium, participate in its recovery from injury, and may also function to signal the status of the neuronal population of the epithelium © 1995 Wiley-Liss, Inc

Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice

Abstract: Clara cells, progenitors for bronchiolar epithelium, are also primary targets for metabolically activated pulmonary cytotoxicants and have an abundance of the cytochrome P-450 monooxygenases required for xenobiotic metabolism. To define the repair pattern after massive Clara cell injury, mice were treated with naphthalene, and lungs evaluated 1-14 days postinjury (DPI). Clara cells of terminal bronchioles were vacuolated and swollen 1 DPI, exfoliated 2 DPI, and resembled controls at 14 DPI. The volume fraction of vacuolated cells was highest 1 and 2 DPI and minimal at 5-7 DPI. The volume fraction of normal nonciliated cells decreased 40% at 1 DPI. Cell proliferation increased within epithelium and interstitium at 1 DPI, was maximal at 2 DPI, and at all other time points was similar to baseline levels. Expression of Clara cell differentiation markers was barely detectable in terminal bronchiolar epithelium at 1 and 2 DPI, clearly detectable at 4 DPI, and gradually returned to control levels at 5-14 DPI. We conclude that bronchiolar epithelial repair after naphthalene injury involves distinct phases of proliferation and differentiation, proliferation of cells that are not differentiated Clara cells, and interaction of multiple cell types including nontarget cells.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Abstract: Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis.

Abstract: PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.

Persistence of Replication-Deficient Adenovirus-Mediated Gene Transfer in Lungs of Immune-Deficient (nu/nu) Mice

Abstract: To evaluate the role of cell-mediated immunity during gene transfer to the respiratory epithelium, the time course of luciferase activity was assessed after intratracheal administration of...

Effect of oxidant air pollutants on the respiratory system: insights from experimental animal research.

Abstract: In the present paper, we have reviewed experimental animal studies on the effects of the two most important oxidant airborne pollutants, nitrogen dioxide and ozone, on the respiratory system. The toxic effects depend on concentration and length of exposure, and are generally similar for both oxidants, with ozone operative at lower concentrations. High doses of both oxidants cause death due to lung oedema. Exposure to sublethal levels causes functional alterations such as airflow limitation and airway hyperresponsiveness to bronchoconstrictor stimuli. These effects, which are generally reversible, are associated with epithelial injury, oedema and airway and parenchymal infiltration by inflammatory cells. Loss of cilia of airway epithelium and necrosis of type I alveolar epithelial cells are the most prominent consequences at the epithelial level. Inflammation is characterized by early neutrophilic infiltration, followed by an increased number of mononuclear cells, predominantly alveolar macrophages. After long-term exposure, whilst nitrogen dioxide causes predominantly emphysema, ozone produces mainly pulmonary fibrosis. Biochemical effects include lipid peroxidation, increased antioxidant metabolism, and alteration of enzyme activity. Nitrogen dioxide and ozone may also alter the immunological response and reduce the defence against infections, increasing the susceptibility of exposed animals to infections.

Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells

Abstract: Secretory leukocyte protease inhibitor (SLPI) is the predominant antiprotease of the conducting airways and may play a role in reducing airway inflammation. In this study, the effect of corticoster...

Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals.

Abstract: A variety of studies suggest that the increased susceptibility of neonates to allergic and infectious respiratory diseases is due to delayed postnatal maturation of local mucosal immune function. We have recently demonstrated that the postnatal development of the major resident APC population in the respiratory tract (RT), class II MHC (Ia)-bearing dendritic cells (DC), is delayed relative to that in other tissues, and that both the intensity of Ia expression on these RTDC and their density within respiratory epithelia remain low until after weaning. The present study focuses on the functional capacity of neonatal RTDC and their responses to exogenous stimuli, and demonstrates that 1) infant Ia+ RTDC respond poorly to GM-CSF, under conditions that stimulate high levels of Ia expression and concomitant APC activity in adult cells; 2) both infant and adult RTDC contain a subpopulation of Ia- cells recognized by mAb OX62 that also respond poorly to GM-CSF; 3) inhalation of microbial stimuli or parenteral administration of IFN-gamma triggers rapid recruitment of DC into the airway epithelium and lung parenchyma of adults; this response is markedly attenuated in newborns and does not attain levels of competence until after weaning; and 4) endogenous macrophage-mediated suppression of the RTDC response to GM-CSF, the principal mechanism limiting in situ DC functional maturation in the adult lung, is highly active in the neonates. Taken together with earlier evidence of the relatively rapid postnatal development of T and B cell function in these animals, the present findings suggest that the sluggish performance of respiratory mucosal immune function(s) during infancy is attributable primarily to delayed maturation of local DC populations.

Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium.

Abstract: Increasing evidence indicates that cationic liposomes are capable of safely transferring foreign genes to pulmonary epithelium in vitro and in vivo. To transfer reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) to mammalian respiratory epithelium we used two cationic lipid formulations: N-[1-(2,3-dioleoyloxy)propyl] N,N,N-triethylammonium chloride (DOTMA), and 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). Lipid-DNA conjugates containing either CFTR or LacZ were instilled directly into the airways of Sprague-Dawley rats. Rats treated with LacZ cDNA in vivo demonstrated expression in 30-50% of the large and medium-sized airways, with some airways showing high efficiency gene transfer and expression (in the most proximal airways, 70-80% of surface epithelial cells were positive for expression of a nuclear targeted LacZ). While control and LacZ treated tracheas mounted in Ussing chambers showed minimal stimulation of transepithelial chloride (Cl)-currents by cAMP (suggesting low levels of endogenous rat CFTR activity), tracheas taken from animals receiving CFTR exhibited significant forskolin-stimulated currents at 72 h after gene transfer. Human CFTR gene expression was also detected by polymerase chain reaction (PCR) analysis of reverse transcribed lung RNA. These results, together with previous studies using lipid-mediated gene transfer in mice, help confirm the potential for cationic lipid-mediated gene transfer in the gene therapy of cystic fibrosis in humans.

TNF-alpha and IL-1 beta upregulate nitric oxide-dependent ciliary motility in bovine airway epithelium

Abstract: Airway epithelial cells can be modulated by cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta that are released from inflammatory cells. Since ciliary motility is an important host defense function of airway epithelium, we hypothesized that cytokines, released from lung macrophages, upregulate ciliary motility. To test this hypothesis, ciliary beat frequency (CBF) was measured by video microscopy in cultured ciliated bovine bronchial epithelial cells (BBECs) incubated for 24 h with bovine alveolar macrophage-conditioned medium (AM-CM). Exposure to AM-CM resulted in a delayed (> or = 2 h) increase in CBF that was maximal after 24 h exposure (13.70 +/- 0.43 for AM-CM vs. 9.44 +/- 0.24 Hz for medium; P < 0.0001) and which was largely blocked by either anti-TNF-alpha or anti-IL-1 beta antibodies. rTNF-alpha or rIL-1 beta similarly increased CBF, which could be blocked by preincubation with either anti-rTNF-alpha or anti-rIL-1 beta antibodies. Preincubation of BBECs with actinomycin D or dexamethasone also blocked rTNF-alpha- and rIL-1 beta-induced cilia stimulation, suggesting that new protein synthesis is required for cytokine-induced upregulation of CBF. Since NO is known to upregulate ciliary motility and cytokines can induce NO synthase (NOS), we hypothesized that TNF-alpha and IL-1 beta increase CBF by inducing NOS in BBECs. The cilia stimulatory effects of TNF-alpha or IL-1 beta were inhibited by NG-monomethyl-L-arginine, a competitive NOS inhibitor, and restored by the addition of either L-arginine, an NOS substrate, or sodium nitroprusside, an NO donor.(ABSTRACT TRUNCATED AT 250 WORDS)

Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells.

Abstract: Haemophilus influenzae penetrates the respiratory epithelium during carriage and invasive disease, including respiratory tract infections. We developed an in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports to study the passage of H. influenzae through lung epithelial cell layers. The NCI-H292 cells formed tight layers with a Ca(2+)-dependent transepithelial resistance of around 40 omega.cm2. H. influenzae passed through the cell layers without affecting the viability of the cells and [3H]inulin penetration. The passage time was independent of the inoculum of H. influenzae in the apical compartment and was not influenced by the presence of capsule or fimbriae on H. influenzae or by the ability of the bacteria to adhere to the epithelial cells. However, highly adherent strains showed greater paracytosis. Different strains passed through the cell layer independently. The passage time was shorter for rapidly growing strains than for slowly growing strains (10 to 18 h and 30 h, respectively). Microscopic examination revealed the presence of clusters of H. influenzae bacteria between the epithelial cells, indicating that bacterial passage was due to paracytosis. After the addition of chloramphenicol, no bacteria were cultured from the basolateral side, and no bacterial clusters between the epithelial cells were seen, suggesting that de novo bacterial protein synthesis was needed for the bacteria to reach the intercellular space. We conclude that H. influenzae passes through viable cell layers of the human lung epithelial cell line NCI-H292 by paracytosis, requiring bacterial protein synthesis.

Keratinocyte growth factor increases mRNAs for SP-A and SP-B in adult rat alveolar type II cells in culture

Abstract: The production of pulmonary surfactant, a complex of phospholipids and lung-specific surfactant proteins, is a primary function of alveolar type II cells. Although previous studies have demonstrated a role for cell-extracellular matrix interactions and normal cell shape in the maintenance of differentiated function in primary cultures of adult rat type II cells, a positive role for growth factors in surfactant protein gene expression in isolated normal adult type II cells has not been reported. In the present study, we have examined the effects of a panel of hormones, growth factors, and cytokines on the expression of mRNAs for surfactant proteins A, B, and C (SP-A, SP-B, and SP-C). Our results show that keratinocyte growth factor (KGF) induced a two- to threefold increase in steady-state levels of mRNAs for SP-A and SP-B, but had no effect on or decreased SP-C mRNA. The increase in SP-A mRNA was accompanied by an increase in SP-A protein. The effects of KGF were both dose and time dependent, and they could be neutralized by a monoclonal antibody against KGF. The effects of KGF were mimicked by acidic fibroblast growth factor, which will bind the KGF receptor. We conclude that KGF can support differentiation of alveolar type II cells as well as act as a mitogen, thus suggesting an important role for KGF in maintenance of the alveolar epithelium.

Stimulatory effects of hepatocyte growth factor on normal and neoplastic human bronchial epithelial cells.

Abstract: We examined the mitogenic, chemoinvasive, and chemotactic effects of hepatocyte growth factor (HGF) toward normal and neoplastic human epithelial cells derived from the bronchial mucosa. Primary cultures of human bronchial epithelial cells (HBE cells), immortalized bronchial epithelial cells (IB3-1 cells), and cells derived from a squamous cell carcinoma of the lung (128-88T cells) were used as targets. HGF was mitogenic for all three cell types as measured by bromodeoxyuridine (BrdU) labeling and colony-forming efficiency (CFE). With the use of BrdU labeling, 9.8-16.8% of nuclei were labeled in controls vs. 56.9-65.6% labeled nuclei in cells treated with HGF. HGF stimulated colony formation 3.6-6.2-fold over untreated control. Analysis by reverse transcription-polymerase chain reaction demonstrated the presence of the c-met gene, the receptor for HGF, in all three cell types. Cell lysates from all three cell types contained proteins that were recognized by a c-met antibody as determined by Western blotting. The gene for HGF was not expressed in any of the cell types, although it was expressed in control MRC5 fibroblasts. No HGF protein could be detected by Western blotting in the conditioned medium from epithelial cells, although it was readily detectable in medium conditioned by lung fibroblasts. HGF proved to be a powerful chemotactic agent for all three cell types and also stimulated invasion into Matrigel, an artificial basement membrane. The results indicate HGF acts mainly as a paracrine growth factor for cells derived from the human bronchus, and may play a role in the growth and progression of lung tumors.

Distribution of integrins alpha v beta 6 and alpha 9 beta 1 and their known ligands, fibronectin and tenascin, in human airways.

Abstract: We have previously identified two integrins, alpha 9 beta 1 and alpha v beta 6, from guinea pig airway epithelium. The extracellular matrix protein tenascin is a ligand for both of these receptors, and fibronectin is also a ligand for alpha v beta 6. In the present study, we used immunohistochemistry to examine the expression and spatial distribution of the alpha 9 subunit, alpha v beta 6, tenascin, and fibronectin in the proximal airways of 10 normal nonsmoking subjects and eight patients undergoing lung resection for cancer. We also performed the same analyses on sections of peripheral lung obtained from an additional seven subjects undergoing lung resection. alpha 9 was highly expressed throughout the airway epithelium (but not on alveolar epithelium) irrespective of clinical status. In contrast, alpha v beta 6 was expressed on proximal airway epithelial cells in four of eight smokers undergoing lung resection, but in none of the normal subjects and none of the distal airways examined. On bronchial epi...

Adhesion of activated eosinophils to respiratory epithelial cells is enhanced by tumor necrosis factor-alpha and interleukin-1 beta.

Abstract: Eosinophilic infiltration and damage to airway epithelium are characteristic features of asthma. To assess possible interactions between eosinophils and airway epithelium, Percoll-purified human peripheral blood eosinophils were evaluated for their ability to adhere to respiratory epithelial cell (REC) cultures. REC (an immortalized cell line, A549, and primary bronchial epithelial cells) were grown in 96-well tissue culture plates, treated with proinflammatory cytokines (TNF-alpha or IL-1 beta), and eosinophil adhesion to these tissues was determined. Cytokine treatment of the REC cultures significantly increased expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.01). Eosinophils demonstrated a variable baseline adhesion to untreated REC which was then significantly increased following activation with phorbol myristate acetate (PMA) (P < 0.01). Furthermore, treatment of REC monolayers with TNF-alpha or IL-1 beta significantly increased adhesion of PMA-stimulated eosinophils (P < 0.01). To delineate the adhesion proteins involved in the cell-cell interactions, assays were performed in the presence of specific blocking monoclonal antibodies to eosinophil CD18, CD11a, or CD11b, and REC ICAM-1 molecules. Blocking antibodies to ICAM-1 had no significant effect on levels of eosinophil adhesion. In contrast, antibodies to CD18, CD11a, and CD11b significantly decreased (P < 0.01) eosinophil adhesion, thus demonstrating pivotal roles for the CD11/CD18 (beta 2) integrins, but not necessarily for ICAM-1, in interactions between the REC and eosinophils. These data demonstrate that TNF-alpha and IL-1 beta increase eosinophil adhesion to human respiratory epithelial cell cultures by induction of ligands recognized by eosinophil beta 2 integrins.

Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease.

Abstract: The CC10 gene encodes the Clara cell 10-kDa protein, which is expressed in airway epithelial cells. Quantification of CC10 gene expression in freshly isolated human proximal airway epithelial cells...

Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro.

Abstract: Aspergillus species frequently colonize lower respiratory tracts and lungs with localized underlying conditions (healed tuberculous cavity, cystic fibrosis, bronchiectasis, etc.) even in subjects without systemic predisposing factors. We investigated the in vitro effects of culture filtrates of Aspergillus species and sputum sols from patients with pulmonary aspergillosis on ciliary beat frequency (CBF) and epithelial integrity of human respiratory ciliated epithelium. Culture filtrates of 25 clinically isolated fungi (16 Aspergillus fumigatus, three Aspergillus niger, one Aspergillus flavus, three Candida albicans, and two Cryptococcus neoformans) were obtained by culturing the fungi in Medium-199 at 37 degrees C for 7 days, and five sputum sols were obtained from patients with pulmonary aspergillosis infected by A. fumigatus. During 6 h experiments using a photometric technique, 14 out of 16 A. fumigatus culture filtrates caused progressive and significant reduction in CBF associated with marked epithelial disruption, whilst the culture filtrates of A. niger and A. flavus caused minor epithelial damage without slowing of CBF, and Medium-199 alone (Control) showed neither epithelial damage nor slowing of CBF. All of the sputum sols also caused significant slowing of CBF as well as epithelial disruption. Culture filtrates of C. albicans and Cr. neoformans had no effects on human respiratory epithelium. We conclude that Aspergillus species, especially A. fumigatus release a factor (or factors) which causes damage to respiratory epithelium and slows CBF, and that these factors may contribute to the colonization of the lower respiratory tracts by the Aspergillus species and may possibly contribute to the further proliferation and spread of the lesions in pulmonary aspergillosis.

Retrovirus-mediated gene transfer in lungs of living fetal sheep

Abstract: In utero somatic gene transfer may be a useful therapeutic strategy for a variety of inherited disorders. In the present study, we demonstrate transgene expression in the airways of fetal lamb lungs, 2-3 weeks after injection of Moloney murine leukemia retrovirus based vectors containing cDNA for beta-galactosidase (lacZ) or human interleukin receptor antagonist protein (IRAP), into the fluid filled future airspace of fully catheterized twin fetal lambs (104-117 days gestational age; term 147 days). Expression of lacZ or IRAP was limited to the twin that received the respective vector and was apparent, at light microscopic level, in the epithelium and submucosal space of proximal airways, and to a lesser extent, in the respiratory epithelium of the distal airways. These data demonstrate for the first time that transfer of foreign DNA to fetal lung can be accomplished. These findings support the use of retroviral vectors for somatic lung DNA transfer and suggest that inherited disorders such as cystic fibrosis may be approached therapeutically via gene transfer, in utero.

Oxidant tone regulates IL-8 production in epithelium infected with respiratory syncytial virus.

Abstract: Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway, and causes a local inflammatory response. Although it has been previously demonstrated that RSV-infected airway epithelial produce cytokines, including interleukin-8 (IL-8), which contributes to the inflammatory response, the regulation of this effect of RSV is unknown. To further characterize the mechanisms by which RSV infection triggers release of IL-8, we first exposed cultured A549 cells to RSV, and measured IL-8 release via enzyme-linked immunosorbent assays (ELISA), and IL-8 messenger RNA (mRNA) induction via Northern blot analysis. We observed a dose- and time-dependent release of IL-8 in response to RSV. The optimal dose of RSV was 10(4) TCID50/ml, and maximal release of IL-8 was measured at 72 to 96 h after infection. RSV induced a biphasic (early and late) increase in IL-8 mRNA. The early phase was independent of viral infection, whereas the more pronounced late phase required the presence of live virus and infection of the epithelium. Partial (< 50%) cytopathic effects were noted at 48 h and progressed to 75% at 96 h. The monolayer was still intact at 96 h. Inhibitors of nitric oxide, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and aminoguanidine had no effect on IL-8 release or IL-8 mRNA induction. We did, however, demonstrate a dose-dependent decrease in IL-8 release and IL-8 mRNA induction in RSV-infected epithelial treated with the antioxidants dimethyl sulfoxide (DMSO) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Peak effects were noted at a concentration of 2% DMSO and 50 microM DMPO. The antioxidants did not inhibit viral replication or infection. This data suggest that RSV-induced IL-8 production in airway epithelium is mediated via changes in oxidant tone. The data also suggest a potential therapeutic role for antioxidants in RSV infections.

Modulation of Airway Intraepithelial Dendritic Cells Following Exposure to Steroids

Abstract: Recent studies from our laboratory have identified a network of constitutively class II major histocompatibility complex (MHC) (Ia)-bearing dendritic cells (DC) within the epithelium of the conducting airways of laboratory animal species and in humans. These studies have also demonstrated that the density of the DC network increases within the airway epithelium in response to inflammatory challenge. In the present report, we demonstrate that exposure of adult rats to inhaled steroids leads to a rapid but readily reversible decrease both in the number of airway intraepithelial DC, and in their surface Ia expression. Similar changes are also seen in response to high doses of systemic dexamethasone. In addition, we demonstrate that steroid inhalation reduces the rate of postnatal expansion of the airway intraepithelial DC network in rat pups, and prevents the rapid expansion of the DC network in adults which occurs during the acute inflammatory response following inhalation of microbial stimuli.

Replication-deficient adenovirus induces expression of interleukin-8 by airway epithelial cells in vitro.

Abstract: Preclinical studies with first-generation adenovirus (Ad) vectors administered in vivo to the respiratory tract have demonstrated a nonspecific host response consisting, in part, of parenchymal neutrophil accumulation followed by mononuclear cell and macrophage accumulation. We hypothesized that the mechanism for this host response might be the elaboration of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from the airway epithelium following the exposure to Ad. To evaluate this hypothesis, we infected A549 cells (a human-derived lung epithelial cell line) in vitro with an adenovirus type 5 (Ad5)-based vector expressing a nuclear targeted β-galactosidase enzyme (Av1LacZ4). We found that cellular transduction was efficient, resulting in gene delivery to 85.5% ± 3.9% of the cell monolayer after 96 hr. Importantly, IL-8 mRNA transcript levels in Av1LacZ4-transduced cells were significantly higher than uninfected controls by 24 hr and remained elevated for 96 hr. IL-8 protein secr...