Human Respiratory Epithelial Cells Research Articles

Differences in receptor specificity between the influenza viruses of duck, chicken, and human

The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158–160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6"-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.

Cytokine responses to group B streptococci induce nitric oxide production in respiratory epithelial cells.

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis. Early-onset GBS pneumonia is characterized by marked pulmonary epithelial and endothelial cell injury. Innate proinflammatory responses to GBS infection that may contribute to the respiratory pathology include the synthesis and release of cytokines, prostaglandins, and nitric oxide (NO). The hypothesis that NO is directly induced in lung epithelial cells by invading GBS or indirectly induced by cytokines released by GBS-infected mononuclear cells was tested. A549 transformed human respiratory epithelial cells were directly cultured with GBS, cocultured with GBS-infected human mononuclear cells or purified macrophages, or exposed to conditioned culture medium from human mononuclear cells infected by GBS. The culture medium of A549 cultures was assayed for NO secretion, and the cell lysates were tested for presence of inducible nitric oxide synthase (iNOS) mRNA by reverse transcriptase PCR (RT-PCR). GBS-treated A549 cells neither secreted detectable NO nor expressed iNOS mRNA. GBS interaction with human mononuclear cells, however, stimulated release of soluble factors that readily induced iNOS mRNA expression and NO secretion by A549 cells. Inflammatory mediator-induced nitric oxide (NO) production by alveolar epithelium may exceed that of other lung cell types such as macrophages, and induction during GBS infection may play a significant role in pulmonary defense or free-radical-mediated lung injury.

Differential invasion of respiratory epithelial cells by members of the Burkholderia cepacia complex

To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Physiomer reduces the chemokine interleukin-8 production by activated human respiratory epithelial cells.

The authors have recently shown that the transcription factor nuclear factor-kappaB (NF-kappaB) is a central mediator in the NaCl-mediated interleukin (IL)-8 production by human airway epithelial cells. In this study, it was investigated whether Physiomer, an isotonic sea water-derived solution commercialized for cleaning the nasal mucosa, impaired the chemokine IL-8 expression and secretion by human respiratory epithelial cells compared with that obtained with an isotonic 9% NaCl solution. Primary human bronchial gland (HBG) epithelial cells were incubated either in Physiomer or in a NaCl 9% solution and activated either with 20 ng x mL(-1) tumour necrosis factor-alpha, or IL-1beta, respectively. Physiomer significantly reduced the IL-8 protein release in basal and activated HBG cells in comparison with that obtained with the 9% NaCl solution. In contrast to the effects of Physiomer observed on resting HBG cells, Physiomer did not significantly reduce the level of phosphorylation of the NF-kappaB inhibitor protein IkappaBalpha or the steady-state IL-8 messenger ribonucleic acid levels in activated HBG cells, suggesting that Physiomer would have a post-transcriptional effect on IL-8 expression in activated HBG cells. The authors conclude that Physiomer is potentially useful in the reduction of airway mucosal inflammation.

The characterisation of human respiratory epithelial cells cultured on resorbable scaffolds: first steps towards a tissue engineered tracheal replacement

In this study we have used lectin histochemistry and scanning electron microscopy (SEM) to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen, was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells of the REC monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This study indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.

Geldanamycin inhibits NF-kappaB activation and interleukin-8 gene expression in cultured human respiratory epithelium.

Geldanamycin is a benzoquinone ansamycin with multiple pharmacologic properties. Recent data demonstrated that geldanamycin conferred protection in an animal model of inflammation-associated acute lung injury. In the current study, we investigated the effects of geldanamycin on interleukin (IL)-8 gene expression and nuclear factor (NF)-kappaB activation. Geldanamycin inhibited tumor necrosis factor (TNF)-alpha-mediated IL-8 gene expression in A549 human respiratory epithelial cells as measured by enzyme-linked immunosorbent assay and Northern blot analyses. In cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid, geldanamycin inhibited TNF-alpha-mediated luciferase activity. Geldanamycin inhibited TNF-alpha-mediated NF-kappaB activation as measured by electromobility shift assays and transient transfections with a NF-kappaB-dependent luciferase reporter plasmid. In contrast, geldanamycin did not affect TNF-alpha-mediated degradation of the NF-kappaB inhibitory protein IkappaBalpha and did not block nuclear translocation of the NF-kappaB p65 subunit as measured by Western blot analyses. Geldanamycin added directly to nuclear extracts of TNF-alpha-treated cells reduced the formation of the NF-kappaB/DNA complex. These results demonstrate that geldanamycin inhibits TNF-alpha-mediated IL-8 gene expression in A549 cells by inhibiting activation of the IL-8 promoter. The mechanism of inhibition involves inhibition of NF-kappaB activation, which is independent of IkappaBalpha degradation or p65 nuclear translocation. Geldanamycin appears to directly inhibit the ability of NF-kappaB to bind DNA. The observed in vitro effects could account, in part, for the anti-inflammatory properties of geldanamycin observed in vivo.

Double-stranded rna dependence of nitric oxide synthase 2 expression in human bronchial epithelial cell lines BET-1A and BEAS-2B.

The human airway epithelium expresses abundant nitric oxide synthase 2 (NOS2) in vivo. Although NOS2 is easily induced by cytokines in primary cultured human airway epithelial cells and lung adenocarcinoma cell line A549, the human bronchial epithelial cell lines BEAS-2B and BET-1A do not express NOS2 in response to cytokines. Mechanisms regulating NOS2 expression in human respiratory epithelial cells are complex, but we have recently shown that NOS2 expression in primary human airway epithelial cells occurs in response to double-stranded RNA (dsRNA) through activation of signaling proteins including nuclear factor (NF)-kappaB and interferon (IFN) regulatory factor (IRF)-1. In this context, we hypothesized that BEAS-2B and BET-1A cells may express NOS2 in response to dsRNA. Here, we show that although cytokines (IFN-gamma, tumor necrosis factor-alpha and interleukin-1beta) do not induce NOS2 expression in BEAS-2B or BET-1A cells, addition of dsRNA to this cytokine mix enables BEAS-2B cells to express NOS2. IFN-gamma and dsRNA induction of NOS2 in BET-1A cells occurs in a serum concentration-dependent manner, with a minimum of 3 d of serum treatment necessary for BET-1A cells to acquire the potential to induce NOS2. Importantly, dsRNA strongly activates NF-kappaB and IRF-1 in BEAS-2B cells, transcription factors essential for NOS2 gene expression in other cell lines. On the basis of these results, dsRNA-activated signaling pathways are clearly important for NOS2 expression in human respiratory epithelial cells. With conditions for NOS2 expression characterized, these cell lines are a convenient in vitro system to investigate the mechanisms regulating NOS2 expression in human respiratory epithelial cells.

Invasion of human respiratory epithelial cells by Bordetella pertussis: Possible role for a filamentous hemagglutinin Arg-Gly-Asp sequence and α5β1 integrin

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human α5β1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell α5β1 integrin.

Expression of C-reactive protein in the human respiratory tract.

C-reactive protein (CRP) is a normal constituent of human sera synthesized by hepatocytes and induced by proinflammatory cytokines. The function of this acute-phase reactant includes activation of complement and enhancement of opsonophagocytosis. CRP binds to phosphorylcholine (ChoP), a constituent of eukaryotic membranes that is also found on the cell surface of major bacterial pathogens of the human respiratory tract, including Streptococcus pneumoniae and Haemophilus influenzae. The presence of CRP on mucosal surfaces and role in innate immunity in the human respiratory tract where ChoP-containing organisms reside have not been previously studied. We have shown using a monoclonal antibody to CRP that CRP is present in inflamed (0.17 to 42 microg/ml) and uninflamed (<0.05 to 0.88 microg/ml) secretions from the human respiratory tract in sufficient quantities for an antimicrobial effect. In addition, the CRP gene was expressed in epithelial cells of the human respiratory tract using in situ hybridization on nasal polyps and reverse transcriptase PCR of pharyngeal cells in culture. The complement-dependent bactericidal activity of normal nasal airway surface fluid and sputum against ChoP-expressing H. influenzae was abolished when the secretions were pretreated to remove CRP. In summary, the results indicate that CRP is present in secretions of the human respiratory tract, that human respiratory epithelial cells are capable of CRP expression, and that this protein may contribute to bacterial clearance in the human respiratory tract.

Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo

Background: Human rhinovirus (HRV) infections are the predominant cause of the common cold and are associated with exacerbations of asthma. Nitric oxide (NO) may play an important role in host defense by means of its potent antiviral properties. Objective: We sought to determine whether epithelial expression of type 2 nitric oxide synthase (NOS 2), which produces NO, is induced on rhinovirus infection in vitro and in vivo. Methods: Primary cultures of human airway epithelial cells were infected with HRV-16, and NOS 2 mRNA expression was assessed by conventional and real-time RT-PCR and NOS 2 protein by using Western blot analysis. Human subjects were also infected with HRV-16 in vivo, and mRNA for NOS 2 was assessed in nasal epithelial scrapings obtained before and after infection. Results: NOS 2 mRNA levels increased within 8 hours after HRV-16 infection of cultured cells and remained elevated up to 48 hours after infection. NOS 2 protein was elevated at 24 hours. Induction of NOS 2 did not occur with UV-inactivated HRV-16 but could be reproduced by using double-stranded RNA, indicating that induction was dependent on viral replication. Increased NOS 2 expression was also observed in nasal epithelial scrapings during symptomatic colds. Conclusion: Increased epithelial expression of NOS 2 mRNA occurs as part of the host response to HRV infection in vitro and in vivo. Given the antiviral effects of NO, we speculate that increased host production of NO may play an important role in host defense during HRV infections. (J Allergy Clin Immunol 2001;107:235-43.)

Expression of the Human Glucocorticoid Receptor α and β Isoforms in Human Respiratory Epithelial Cells and Their Regulation by Dexamethasone

Two isoforms of the human glucocorticoid receptor (hGR) have been described, hGRalpha and hGRbeta. We analyzed the expression and regulation of both hGR isoforms in human respiratory epithelial cells (BEAS-2B, A549, and primary nasal epithelial cells). In BEAS-2B cells, the expression of hGRalpha messenger RNA (mRNA) was much higher than that of hGRbeta mRNA. Dexamethasone (DEX) (10(-6) M) downregulated hGRalpha mRNA at 6 and 24 h (55 +/- 8 and 58 +/- 5% of control, respectively; P < 0.01), whereas it decreased hGRbeta mRNA only at 6 h (55 +/- 7% of control; P < 0.01). Downregulation of hGRalpha and hGRbeta mRNAs occurred even in the presence of cycloheximide. Actinomycin-D studies revealed that DEX enhanced the stabilization of hGRalpha and hGRbeta messages. hGRalpha but not hGRbeta protein was detected in BEAS-2B, A549, and nasal epithelial cells. After 24 h of incubation, 10(-6) M DEX decreased the expression of hGRalpha protein in BEAS-2B, A549, and nasal epithelial cells (16 +/- 4, 14 +/- 4, and 28 +/- 7% of control, respectively; P < 0.01). These results suggest that in respiratory epithelial cells: (1) hGRalpha is much more expressed than hGRbeta at both the mRNA and protein levels; (2) hGRalpha is downregulated by corticosteroids both in cell lines (BEAS-2B, A549) and in nasal primary cells; and (3) transcriptional, post-transcriptional, and post-translational mechanisms appear to be involved in the regulation of hGR expression by corticosteroids.

Biocompatible hydrogel supports the growth of respiratory epithelial cells: Possibilities in tracheal tissue engineering

Extensive tracheal defect reconstruction is a major challenge in plastic and reconstructive surgery. The lack of an epithelial lining on the luminal surfaces of tracheal prostheses is among the major causes of their failure. Chitosan-gelatin hydrogels were synthesized for the development of biocompatible, growth-supportive substrata for respiratory epithelial cells. We employed J774 macrophages to test the immunocompatibility of this gel. The hydrogel did not exert a cytotoxic effect on macrophages, as confirmed by tetrazolium reduction and neutral red uptake assay. Flow cytometric analysis of macrophages cultured on the hydrogel showed a comparable expression of activation markers CD11b/CD18, CD45, and CD14 to the control. Semiquantitative RT-PCR results showed an absence of upregulation of interleukin-6 (IL-6) and TNF-alpha in these macrophages with respect to the controls. Primary human respiratory epithelial cells cultured on the hydrogel showed proper attachment, normal morphology, and growth. A small proportion of cells on the hydrogel showed synchronously beating cilia. RT-PCR analysis showed that cells on the hydrogel expressed mucins 2 and 5 and cytokeratin 13, which are markers for secretory goblet and squamous cells, respectively. All these results demonstrate that the hydrogel supports the growth of a mixed population of differentiated epithelial cells. This hydrogel is suitable as a culture substratum for respiratory epithelial cells and could be used as a potential candidate for coating tracheal prostheses.

Subinhibitory concentrations of erythromycin reduce pneumococcal adherence to respiratory epithelial cells in vitro.

We have investigated the influence of subinhibitory concentrations of erythromycin on the interaction between Streptococcus pneumoniae and human respiratory epithelial cells. Confluent in vitro cell cultures were inoculated with erythromycin-resistant S. pneumoniae and incubated for 24 h. Erythromycin significantly reduced adherence of the pneumococci after 4 h and 24 h: 4.0% +/- 0.7% (mean +/- S.E.M. ) of the pneumococci adhered to the epithelial cells in medium with erythromycin, compared with 7.7% +/- 0.8% in medium without erythromycin (P: = 0.002) after 4 h, and the corresponding values after 24 h were 24.2% +/- 5.3% and 38.4% +/- 5.0%, respectively (P: = 0.038). Disruption of epithelial integrity by S. pneumoniae, measured as the decrease in transepithelial electrical resistance, was delayed in the presence of erythromycin. Neither addition of erythromycin to the culture medium nor infection of the cell cultures with pneumococci significantly affected secretion of interleukin-8 by the epithelial cells. Addition of erythromycin to a pneumococcal suspension in cell culture medium without respiratory epithelial cells almost completely prevented the release of pneumolysin. We conclude that erythromycin at subinhibitory concentrations reduces the adherence to and disruption of respiratory epithelial cells by S. pneumoniae, possibly by interfering with pneumolysin release.

Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium.

Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.

Rhinovirus infection induces major histocompatibility complex class I and costimulatory molecule upregulation on respiratory epithelial cells.

Human respiratory epithelial cells may act as antigen-presenting cells during respiratory viral infections. In addition to major histocompatibility complex (MHC) molecules, antigen presentation requires participation of costimulatory molecules. Here the authors investigated class I and class II antigens and B7-1 and B7-2 costimulatory molecule expression in human A549 pulmonary epithelial cells and primary bronchial epithelial cells (HBECs) at baseline and after rhinovirus infection. Constitutive expression of MHC class I and B7-1 molecules was observed on both cell types. MHC class I molecules were up-regulated by rhinovirus infection, while B7-1 was up-regulated only on A549 cells. B7-2 molecules were constitutively expressed at a low level and were up-regulated by rhinovirus only on HBECs. Rhinovirus induction of antigen-presenting molecule expression on A549 cells was accompanied by cellular activation in terms of induction of release of the chemokines RANTES and Groalpha. These data show that respiratory epithelium expresses full antigen-presentation machinery and that rhinovirus infection up-regulates this expression.

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells.

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.

Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells

No modification of purine or pyrimidine bases was observed when isolated DNA was incubated with 1 mM nitrite at pH 7.4. However, exposure of human bronchial epithelial cells in culture medium at pH 7.4 to nitrite at concentrations of 100 μM or greater led to deamination of purine bases in cellular DNA. Deamination was more extensive in cells exposed to lower extracellular pH values and higher nitrite concentrations. Significant increases in the levels of xanthine and hypoxanthine, putative deamination products of guanine and adenine, respectively, were observed in DNA from nitrite-treated cells but no rise in any base oxidation products such as 8-hydroxyguanine. This pattern of damage suggests that exposure of cells to nitrite (even at pH 7.4) leads to intracellular generation of “reactive nitrogen species” capable of deaminating purines in DNA. In addition, significant DNA strand breakage occurred in nitrite-treated cells. The time course of base damage suggested that the repair of deaminated purine lesions in these cells is slow. By contrast, DNA isolated from cells exposed to hypochlorous acid (HOCl) has significant oxidation of pyrimidine bases and chlorination of cytosine but little oxidation of purines. Exposure of cells to both species (NO 2 − plus HOCl) potentiated the oxidative DNA base damage observed but decreased the extent of deamination. We hypothesize that this is due to the formation of nitryl chloride (NO 2Cl) from reaction of HOCl with •NO 2 −. The relevance of our observations to events in the stomach and respiratory tract, at sites of inflammation, and in ischemic tissues is discussed.