Human Respiratory Epithelial Cells Research Articles

CD23-Dependent Transcytosis of IgE and Immune Complex across the Polarized Human Respiratory Epithelial Cells

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the IC was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed Ag-IgE complexes were competent in inducing degranulation of the cultured human mast cells. Because airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.

Effects of Receptor Binding Specificity of Avian Influenza Virus on the Human Innate Immune Response

Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.

Positive Signature-Tagged Mutagenesis in Pseudomonas aeruginosa: Tracking Patho-Adaptive Mutations Promoting Airways Chronic Infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.

TRPV4-Mediated Calcium Influx into Human Bronchial Epithelia upon Exposure to Diesel Exhaust Particles

BackgroundHuman respiratory epithelia function in airway mucociliary clearance and barrier function and have recently been implicated in sensory functions.ObjectiveWe investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying Ca2+ influx into human airway epithelia elicited by diesel exhaust particles (DEP).Methods and ResultsUsing primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates Ca2+-permeable TRPV4, which leads to activation of human respiratory disease–enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-Cβ3 in human airway epithelia and their DEP-enhanced protein–protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)–predisposing TRPV4P19S variant enhances Ca2+ influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease.ConclusionDEP evoked protracted Ca2+ influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism TRPV4P19S. This mechanism reprograms maladaptive inflammatory and extracellular-matrix–remodeling responses in human airways. The novel concept of air pollution–responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route.

Non-Typeable Haemophilus influenzae Invasion and Persistence in the Human Respiratory Tract

Non-typeable Haemophilus influenzae (NTHI) is an opportunistic bacterial pathogen of the human respiratory tract and is a leading cause of respiratory infections in children and adults. NTHI is considered to be an extracellular pathogen, but has consistently been observed within and between human respiratory epithelial cells and macrophages, in vitro and ex vivo. Until recently, few studies have examined the internalization, trafficking, and fate of NTHI in host cells. It is important to clarify this interaction because of a possible correlation between intracellular NTHI and symptomatic infection, and because NTHI infections frequently persist and recur despite antibiotic therapy and the development of bactericidal antibodies, suggesting a possible intracellular state or reservoir for NTHI. How does NTHI enter host cells? Can NTHI survive intracellularly and, if so, for how long? Strides have been made in the identification of host receptors, signaling, endocytosis, and trafficking pathways involved in the entry and persistence of NTHI in the respiratory tract.

Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells

Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed

Characterization of influenza virus sialic acid receptors in minor poultry species

It is commonly accepted that avian influenza viruses (AIVs) bind to terminal α2,3 sialic acid (SA) residues whereas human influenza viruses bind to α2,6 SA residues. By a series of amino acid changes on the HA surface protein, AIVs can switch receptor specificity and recognize α2,6 SA positive cells, including human respiratory epithelial cells. Animal species, like pigs and Japanese quail, that contain both α2,3 and α2,6 SA become ideal environments for receptor switching. Here, we describe the SA patterns and distributions in 6 common minor domestic poultry species: Peking duck, Toulouse geese, Chinese ring-neck pheasant, white midget turkey, bobwhite quail, and pearl guinea fowl. Lectins specific to α2,3 and α2,6 SA (Maakia amurensis agglutinin and Sambuca nigra agglutinin, respectively) were used to detect SA by an alkaline phosphotase-based method and a fluorescent-based method. Differences in SA moieties and their ability to bind influenza viruses were visualized by fluorescent labeling of 4 different H3N2 influenza viruses known to be specific for one receptor or the other. The geese and ducks showed α2,3 SA throughout the respiratory tract and marginal α2,6 SA only in the colon. The four other avian species showed both α2,3 and α2,6 SA in the respiratory tract and the intestines. Furthermore, the turkey respiratory tract showed a positive correlation between age and α2,6 SA levels. The fact that these birds have both avian and human flu receptors, combined with their common presence in backyard farms and live bird markets worldwide, mark them as potential mixing bowl species and necessitates improved surveillance and additional research about the role of these birds in influenza host switching.

Pandemic H1N1 Influenza A Viruses Are Resistant to the Antiviral Activities of Innate Immune Proteins of the Collectin and Pentraxin Superfamilies

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Effect of acidic pH on the invasion efficiency and the type III secretion system of Burkholderia thailandensis

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.

Transcellular Passage of Neisseria meningitidis across a Polarized Respiratory Epithelium

Neisseria meningitidis is a major cause of sepsis and meningitis but is also a common commensal, present in the nasopharynx of between 8 and 20% of healthy individuals. During carriage, the bacterium is found on the surface of the nasopharyngeal epithelium and in deeper tissues, while to develop disease the meningococcus must spread across the respiratory epithelium and enter the systemic circulation. Therefore, investigating the pathways by which N. meningitidis crosses the epithelial barrier is relevant for understanding carriage and disease but has been hindered by the lack of appropriate models. Here, we have established a physiologically relevant model of the upper respiratory epithelial cell barrier to investigate the mechanisms responsible for traversal of N. meningitidis. Calu-3 human respiratory epithelial cells were grown on permeable cell culture membranes to form polarized monolayers of cells joined by tight junctions. We show that the meningococcus crosses the epithelial cell barrier by a transcellular route; traversal of the layer did not disrupt its integrity, and bacteria were detected within the cells of the monolayer. We demonstrate that successful traversal of the epithelial cell barrier by N. meningitidis requires expression of its type 4 pili (Tfp) and capsule and is dependent on the host cell microtubule network. The Calu-3 model should be suitable for dissecting the pathogenesis of infections caused by other respiratory pathogens, as well as the meningococcus.

Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials

Selecting a scaffold that facilitates ciliary differentiation of respiratory epithelial cells (RECs) is crucial in developing tissue engineered respiratory epithelium. Hyaluronan derivative membranes, consisting of an esterified form of hyaluronan (HYAFF ®), have been proved to promote ciliary differentiation of RECs in the presence of retinoic acid (RA). However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. In addition to investigating the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA, this study elucidates the role of the receptor for hyaluronan-mediated motility (RHAMM) in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially “replace” RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs.

Gold nanorod delivery of an ssRNA immune activator inhibits pandemic H1N1 influenza viral replication

The emergence of the pandemic 2009 H1N1 influenza virus has become a world-wide health concern. As drug resistance appears, a new generation of therapeutic strategies will be required. Here, we introduce a nanotechnology approach for the therapy of pan-demic and seasonal influenza virus infections. This approach uses gold nanorods (GNRs) to deliver an innate immune activator, pro-ducing a localized therapeutic response. We demonstrated the utility of a biocompatible gold nanorod, GNR-5'PPP-ssRNA nanoplex, as an antiviral strategy against type A influenza virus. In human respiratory bronchial epithelial cells, this nanoplex activated the retinoic acid-inducible gene I (RIG-I) pathogen recognition pathway, resulting in increased expression of IFN-beta and other IFN-stimulated genes (ISGs) (e.g., PKR, MDA5, IRF1, IRF7, and MX1). This increase in type I IFN and ISGs resulted in a decrease in the replication of H1N1 influenza viruses. These findings suggest that further evaluation of biocompatible nanoplexes as unique antivirals for treatment of seasonal and pandemic influenza viruses is warranted.

Murine complement deficiency ameliorates acute cigarette smoke-induced nasal damage

Objective Exposure to cigarette smoke is a risk factor for chronic rhinosinusitis. Current literature confirms complement fragments are activated in human nasal mucosa. The mechanism(s) responsible for this activation is unclear. We investigated the effects of cigarette smoke on nasal mucosa in vitro and via a model of cigarette smoke exposure by using animals deficient in complement components. Study Design Prospective, controlled animal and in vitro human cell line study. Setting University laboratory. Subjects and Methods Human respiratory epithelial cells were exposed to five, 10, and 20 percent cigarette smoke extract (CSE) in vitro in the presence or absence of human serum. Complement activation was assessed by enzyme-linked immunosorbent assay and immunofluorescent techniques. Complement-deficient (C3 –/–, n = 6; factor B –/–, n = 50) and sufficient mice (wild type, n = 10) were exposed to the smoke of four cigarettes per exposure for two exposures per day for three days. Mice were sacrificed 12 hours after the last exposure, and the nasal cavity was surgically removed. Histological characteristics were analyzed by the use of a subjective scale and quantitative image analysis scoring systems. Results In vitro analysis of respiratory cell cultures demonstrated that exposure of serum to CSE resulted in complement activation. Furthermore, immunofluorescent staining for C3d could only be demonstrated in CSE-exposed cultures. In vivo analysis demonstrated that complement deficiency, either C3 or factor B deficiency, resulted in a significant reduction in histological evidence of damage as compared with wild-type control mice (wild type vs C3 –/–, P = 0.02; wild type vs factor B –/–, P = 0.07; no significant difference between C3 –/– vs factor B –/–). Conclusion These data demonstrate that cigarette smoke activates the complement system. Furthermore, complement deficiency protected against smoke-induced mucosal damage in this small series.

P120-Catenin Is Transcriptionally Downregulated by FOXC2 in Non–Small Cell Lung Cancer Cells

p120-catenin (p120ctn) plays a major role in cell adhesion and motility through the regulation of E-cadherin and interaction with RhoGTPase and Rac1. p120ctn is downregulated in several malignancies including non-small cell lung cancer (NSCLC). Here, we investigated transcriptional regulation of p120ctn in NSCLC. We cloned a 1,400-bp amplicon of chromosome 11 from position -1,082 to +320 relative to the transcription start site into a firefly luciferase reporter vector and prepared serial deletion constructs to pinpoint cis-acting elements involved in the regulation of p120ctn. We transfected NSCLC cell lines and immortalized normal human respiratory epithelial cells with the abovementioned constructs. We found reduced p120ctn promoter activity, protein level, and mRNA message in lung cancer cells compared with noncancerous immortalized lung epithelial cells. Serial deletion analysis of p120ctn promoter identified a region between positions +267 and +282, which mediated the transcriptional repression of p120ctn. This region harbored putative binding sites for FOXC2 and FOXL1 transcription factors. Direct binding of FOXC2 to the p120ctn promoter between positions +267 and +282 was confirmed by electromobility shift assay. RNAi-mediated silencing of FOXC2 in A549, H157, and H358 cells resulted in increasing p120ctn promoter activity as well as mRNA and protein levels. Finally, silencing FOXC2 in these NSCLC cells enhanced E-cadherin level, which was reversed by simultaneous silencing of p120ctn. In summary, our data support the notion that FOXC2 mediates the transcriptional repression of p120ctn in NSCLC.

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.

Modelling the effects of microgravity on the permeability of air interface respiratory epithelial cell layers

Although it has been suggested that microgravity might affect drug absorption in vivo, drug permeability across epithelial barriers has not yet been investigated in vitro during modelled microgravity. Therefore, a cell culture/diffusion chamber was designed specifically to accommodate epithelial cell layers in a 3D-clinostat and allow epithelial permeability to be measured under microgravity conditions in vitro with minimum alteration to established cell culture techniques. Human respiratory epithelial Calu-3 cell layers were used to model the airway epithelium. Cells grown at an air interface in the diffusion chamber from day 1 or day 5 after seeding on 24-well polyester Transwell cell culture inserts developed a similar transepithelial electrical resistance (TER) to cells cultured in conventional cell culture plates. Confluent Calu-3 layers exposed to modelled microgravity in the 3D-clinostat for up to 48 h maintained their high TER. The permeability of the paracellular marker 14C-mannitol was unaffected after a 24 h rotation of the cell layers in the 3D-clinostat, but was increased 2-fold after 48 h of modelled microgravity. It was demonstrated that the culture/diffusion chamber developed is suitable for culturing epithelial cell layers and, when subjected to rotation in the 3D-clinostat, will be a valuable in vitro system in which to study the influence of microgravity on epithelial permeability and drug transport.

Functional expression of the epithelial sodium channel delta subunit in human respiratory epithelial cells

The expression and function of the epithelial sodium channel δ subunit (δ‐ENaC) as well as its contribution to ion and fluid transport in human respiratory epithelial cells.Expression of δ‐ENaC was investigated in human alveolar (A549) and bronchial (Calu‐3, 16HBE14o‐) epithelial cell lines on genomic and protein levels. Reverse transcriptase and real‐time PCR were carried out as well as analysis by Western blot and immuno‐confocal laser scanning microscopy (CLSM). Ussing chamber studies were performed with Calu‐3 monolayers using Evans blue at 10 – 50 μM.Delta‐ENaC expression was confirmed in all cell lines on mRNA level with higher abundance in A549 and 16HBE14o‐ than in Calu‐3 cell monolayers. Generally, mRNA levels of δ‐ENaC were weaker than that of α, β and γ ENaC subunits. By Western blot and CLSM δ‐ENaC protein was detected in all cell lines, with the highest expression found in Calu‐3 and 16HBE14o‐ cells. CLSM also revealed the δ‐ENaC subunit located in the apical aspect of cellular membranes. Evans blue exhibited a concentration‐dependant increase of ISC up to 650% at 50 μM.These results indicate that ä‐ENaC is functionally expressed in human respiratory epithelial cell monolayers. Further experiments will investigate its role in ion and fluid transport processes across the respiratory epithelial barrier.ES is funded by an IRSCET postgraduate scholarship.

CD23 dependent transcytosis of IgE and immune complex across the polarized human respiratory epithelial cells (141.20)

Abstract IgE is frequently present in airway secretions and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we showed that CD23 (FceRII) was expressed in the established or primary human airway epithelial cells; its expression was significantly up-regulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, CD23 was capable of transporting IgE across the polarized human Calu-3 monolayer in a bidirectional manner. Furthermore, CD23 was able to transport IgE derived immune complexes. CD23 specific antibody or soluble CD23 significantly reduced the efficiency of IgE or immune complex transcytosis, suggesting a specific receptor-mediated transport by CD23. Exposure of Calu-3 monolayer to IL-4 also enhanced the transcytosis of either human IgE or the immune complex. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed immune complexes were competent in inducing degranulation of the cultured human mast cells. Since airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.

Highly pathogenic avian influenza A virus H5N1 NS1 protein induces caspase-dependent apoptosis in human alveolar basal epithelial cells

BackgroundIt is widely considered that the multifunctional NS1 protein of influenza A viruses contributes significantly disease pathogenesis by modulating a number of virus and host-cell processes, but it is highly controversial whether this non-structural protein is a proapoptotic or antiapoptotic factor in infected cells.ResultsNS1 protein of influenza A/chicken/Jilin/2003 virus, a highly pathogenic H5N1 strain, could induce apoptosis in the carcinomic human alveolar basal epithelial cells (A549) by electron microscopic and flow cytometric analyses. NS1 protein-triggered apoptosis in A549 cells is via caspase-dependent pathway.ConclusionsInfluenza A virus NS1 protein serves as a strong inducer of apoptosis in infected human respiratory epithelial cells and plays a critical role in disease pathogenesis.

The Stability of Salmon Calcitonin Against Trypsin, Chymotrypsin and Neutrophil Elastase in Human Lung Epithelial Cells

At least three enzymes (i. e., trypsin, chymotrypsin and neutrophil elastase) are responsible for the degradation of salmon calcitonin (sCT) in the human body [1]. Here, we investigated how these proteinases affect drug disposition of sCT in the lungs. Stability of sCT was studied in human respiratory epithelial cells. The presence of trypsin, chymotrypsin and neutrophil elastase in cell supernatant and homogenate was studied by Western blot. Enzymes’ activities were studied in cell supernatant by measuring UV absorption, caused by interaction with model substrates. In vitro stability studies were carried out by adding sCT to cell supernatant, homogenate, as well as on intact cell monolayers, followed by incubation at 37°C for 2 h. Samples were serially withdrawn and analysed by HPLC for sCT content. Degradation of sCT by pure trypsin, chymotrypsin and neutrophil elastase was investigated after incubation for 2 h at 37°C. We observed that sCT concentrations remained unchanged over the period of 2 h, when incubated in supernatant or on cell monolayers. When cell homogenates were studied, sCT concentrations were reduced to 82.5% (hBEpC), 45.9% (Calu-3), 45.1% (16HBE14o-), 4.3% (A549) and 18.5% (Caco-2), respectively. sCT was also degraded when incubated with pure enzymes. Western blot revealed strong signals for all proteinases in the cell homogenates and weaker, varying expression in supernatants. Calcitonin is rapidly degraded in homogenate, indicating high intracellular enzymatic activity. Epithelial proteases hence, appear to play a role in interactions of sCT with lung epithelium. Although trypsin, chymotrypsin and neutrophil elastase are secreted in vitro, sCT remained intact in supernatant for 2 h. The concentration of the relevant enzymes (indicated by very low measured activities) might be too low to cause degradation. This work was funded by a Strategic Research Cluster grant (07/SRC/B1154) under the National Development Plan co-funded by EU Structural Funds and Science Foundation Ireland and by an IRCSET Embark Postgraduate Scholarship.