Inhibition by cigarette smoke of nuclear factor-κB-dependent response to bacteria in the airway.

Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on pulmonary defense are incompletely understood. Based on the observation that interactions between bacteria and host cells result in the expression of critical defense genes regulated by NF-κB, we hypothesized that cigarette smoke alters NF-κB function. In this study, primary human tracheobronchial epithelial cells were treated with cigarette smoke extract (CSE) and exposed to Haemophilus influenzae, and the effects of CSE on bacteria-induced signaling and gene expression were assessed. CSE inhibited high concentrations of induced NF-κB activation and the consequent expression of defense genes that occurred in airway epithelial cells in response to H. influenzae. This decreased activation of NF-κB was not attributable to cell loss or cytotoxicity. Glutathione augmentation of epithelial cells decreased the effects of CSE on NF-κB-dependent responses, as well as the effects on the inhibitor of κB and the inhibitor of κB kinase, which are upstream NF-κB regulators, suggesting the involvement of reactive oxygen species. The relevance of these findings for lung infection was confirmed using a mouse model of H. influenzae airway infection, in which decreased NF-κB pathway activation, keratinocyte chemoattractant (KC) chemokine expression, and neutrophil recruitment occurred in animals exposed to cigarette smoke. The results indicate that although cigarette smoke can cause inflammation in the lung, exposure to smoke inhibits the robust pulmonary defense response to H. influenzae, thereby providing one explanation for the increased susceptibility to respiratory bacterial infection in individuals exposed to cigarette smoke.

Inhibition of IFN-gamma-dependent antiviral airway epithelial defense by cigarette smoke.

BACKGROUND: Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on lung defense are incompletely understood. Because airway epithelial cell responses to type II interferon (IFN) are critical in regulation of defense against many respiratory viral infections, we hypothesized that cigarette smoke has inhibitory effects on IFN-gamma-dependent antiviral mechanisms in epithelial cells in the airway. METHODS: Primary human tracheobronchial epithelial cells were first treated with cigarette smoke extract (CSE) followed by exposure to both CSE and IFN-gamma. Epithelial cell cytotoxicity and IFN-gamma-induced signaling, gene expression, and antiviral effects against respiratory syncytial virus (RSV) were tested without and with CSE exposure. RESULTS: CSE inhibited IFN-gamma-dependent gene expression in airway epithelial cells, and these effects were not due to cell loss or cytotoxicity. CSE markedly inhibited IFN-gamma-induced Stat1 phosphorylation, indicating that CSE altered type II interferon signal transduction and providing a mechanism for CSE effects. A period of CSE exposure combined with an interval of epithelial cell exposure to both CSE and IFN-gamma was required to inhibit IFN-gamma-induced cell signaling. CSE also decreased the inhibitory effect of IFN-gamma on RSV mRNA and protein expression, confirming effects on viral infection. CSE effects on IFN-gamma-induced Stat1 activation, antiviral protein expression, and inhibition of RSV infection were decreased by glutathione augmentation of epithelial cells using N-acetylcysteine or glutathione monoethyl ester, providing one strategy to alter cigarette smoke effects. CONCLUSIONS: The results indicate that CSE inhibits the antiviral effects of IFN-gamma, thereby presenting one explanation for increased susceptibility to respiratory viral infection in individuals exposed to cigarette smoke.

Differential effects of cytokines and corticosteroids on toll-like receptor 2 expression and activity in human airway epithelia.

BACKGROUND: The recognition of microbial molecular patterns via toll-like receptors (TLRs) is critical for mucosal defenses. METHODS: Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-alpha and IFN-gamma) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints. RESULTS: Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-alpha and IFN-gamma), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines +/- dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-kappaB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects. CONCLUSION: While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-alpha and IFN-gamma may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.

Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1beta and TNFalpha acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface.

Recognizing the importance of respiratory syncytial virus in chronic obstructive pulmonary disease.

Acute exacerbations of chronic obstructive pulmonary disease (COPD) are responsible for a large proportion of the health care dollar expenditure, morbidity, and mortality related to COPD. Respiratory infections are the most common cause of acute exacerbations, but recent evidence indicates that the importance of respiratory syncytial virus (RSV) infection in COPD is under-appreciated. Improved detection of RSV using techniques based on the polymerase chain reaction accounts for much of the increased recognition of the importance of this virus in COPD patients. Furthermore, COPD patients may be more susceptible to RSV infection, possibly due to RSV-or immune response-induced pulmonary effects that are altered by age, environmental exposures, genetics, COPD itself, or a combination of these. However, although RSV infection occurs throughout life, viral and host factors that place COPD patients at increased risk are unclear. The complexities of RSV effects in COPD present opportunities for research with the goal of developing approaches to selectively modify damaging viral effects (e.g., altered airway function), while retaining beneficial immunity (e.g., clearance of virus) in COPD patients. This review explores the role RSV plays in acute exacerbations of COPD, the potential for RSV disease in chronic stable COPD, and newer concepts in RSV diagnosis, epidemiology, and host defense.

Cigarette smoke alters respiratory syncytial virus-induced apoptosis and replication.

Individuals exposed to cigarette smoke have a greater number and severity of viral infections, including respiratory syncytial virus (RSV) infections, than do nonsmokers, but the cellular mechanism is unknown. Our objective was to determine the mechanism by which cigarette smoke augments viral infection. We hypothesize that cigarette smoke causes necrosis and prevents virus-induced cellular apoptosis, and that this is associated with increased inflammation and viral replication. Primary airway epithelial cells were exposed to cigarette smoke extract for 2 days, followed by 1 day of RSV exposure. Western blot detection of cleaved caspases 3 and 7 showed less apoptosis when cells were treated with cigarette smoke before viral infection. This finding was confirmed with ELISA and TUNEL detection of apoptosis. Measures of cell viability, including propidium iodide staining, ATP assay, and cell counts, indicated that cigarette smoke causes necrosis rather than virus-induced apoptosis. Using plaque assay and fluorescently-labeled RSV, we showed that although there were less live cells in the cigarette smoke-pretreated group, viral load was increased. The effect was inhibited by pretreatment of cells with N-acetylcysteine and aldehyde dehydrogenase, suggesting that the effect was primarily mediated by reactive aldehydes. Cigarette smoke causes necrosis rather than apoptosis in viral infection, resulting in increased inflammation and enhanced viral replication.

Regulation of bacteria-induced intercellular adhesion molecule-1 by CCAAT/enhancer binding proteins.

Direct interaction between bacteria and epithelial cells may initiate or amplify the airway response through induction of epithelial defense gene expression by nuclear factor-kappaB (NF-kappaB). However, multiple signaling pathways modify NF-kappaB effects to modulate gene expression. In this study, the effects of CCAAT/enhancer binding protein (C/EBP) family members on induction of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) was examined in primary cultures of human tracheobronchial epithelial cells incubated with nontypeable Haemophilus influenzae. Increased ICAM-1 gene transcription in response to H. influenzae required gene sequences located at -200 to -135 in the 5'-flanking region that contain a C/EBP-binding sequence immediately upstream of the NF-kappaB enhancer site. Constitutive C/EBPbeta was found to have an important role in epithelial cell ICAM-1 regulation, while the adjacent NF-kappaB sequence binds the RelA/p65 and NF-kappaB1/p50 members of the NF-kappaB family to induce ICAM-1 expression in response to H. influenzae. The expression of C/EBP proteins is not regulated by p38 mitogen-activated protein kinase activation, but p38 affects gene transcription by increasing the binding of TATA-binding protein to TATA-box-containing gene sequences. Epithelial cell ICAM-1 expression in response to H. influenzae was decreased by expressing dominant-negative protein or RNA interference against C/EBPbeta, confirming its role in ICAM-1 regulation. Although airway epithelial cells express multiple constitutive and inducible C/EBP family members that bind C/EBP sequences, the results indicate that C/EBPbeta plays a central role in modulation of NF-kappaB-dependent defense gene expression in human airway epithelial cells after exposure to H. influenzae.

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins.

In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.

Human adenovirus modulates surfactant phospholipid trafficking.

Surfactant, highly enriched with phosphatidylcholine (PC), is secreted into the airspace by a classic apical secretory route, thereby maintaining lung stability. Herein, we show that adenoviral infection decreases surfactant PC in lungs by inhibiting its apical secretion and redirecting its export in alveolar cells by a basolateral route. These effects were not observed with replication-deficient adenovirus (Ad), specifically lacking early region 1 (E1) gene products. Adenoviral stimulation of basolateral PC export from cells was not observed after pharmacologic inhibition of ATP-binding cassette proteins, after introduction of small interfering RNA to the lipid pump ATP-binding cassette transporter A1 (ABCA1) or in ABCA1-defective human Tangier disease fibroblasts. Adenovirus and its E1A gene product increased ABCA1 levels by transcriptionally activating the ABCA1 gene. Thus, Ad lowers surfactant, in part, by triggering ABCA1-directed basolateral PC export, thereby limiting the cellular pool of surfactant PC destined for apical secretion. The results support a novel pathway, whereby a viral pathogen disrupts surfactant trafficking.

Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells.

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.

Inhibition of Jak1-dependent signal transduction in airway epithelial cells infected with adenovirus.

Adenoviral evolution has generated mechanisms to resist host cell defense systems, but the biochemical basis for evasion of multiple antiviral pathways in the airway by adenoviruses is incompletely understood. We hypothesized that adenoviruses modulate airway epithelial responses to type I interferons by altering the levels and activation of specific Janus family kinase-signal transducer and activator of transcription (JAK-STAT) signaling components. In this study, specific effects of adenovirus type 5 (AdV) on selected JAK-STAT signal transduction pathways were identified in human tracheobronchial epithelial cells, with focus on type I interferon-dependent signaling and gene expression. We found that wild-type AdV infection inhibited IFN-alpha-induced expression of antiviral proteins in epithelial cells by blocking phosphorylation of the Stat1 and Stat2 transcription factors that are required for activation of type I interferon-dependent genes. These effects correlated with AdV-induced down-regulation of expression of the receptor-associated tyrosine kinase Jak1 through a decrease in Jak1 mRNA levels. Phosphorylation of Stat3 in response to IL-6 and oncostatin M was also lost in AdV-infected cells, indicating loss of epithelial cell responses to other cytokines that depend on Jak1. In contrast, IL-4- and IL-13-dependent phosphorylation of Stat6 was not affected during AdV infection, indicating that the virus modulates specific signaling pathways, as these Stat6-activating pathways can function independent of Jak1. Taken together, the results indicate that AdV down-regulates host epithelial cell Jak1 to assure inhibition of the antiviral effects of multiple mediators to subvert airway defense responses and establish a productive infection.

Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface.

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation. Using primary human airway epithelial cells differentiated at an air-liquid interface in culture, we found that responses to several cytokines required basolateral mediator application. In contrast, responses to Haemophilus influenzae occurred after either basolateral or apical interaction with airway epithelia. Experiments focused on IFN-gamma receptor polarity confirmed its predominant basolateral location in cultured airway epithelia as well as in normal human airway tissue. Furthermore, physical and pharmacologic disruption of barrier function in airway epithelia allowed responses to apical application of IFN-gamma and other cytokines. These in vitro studies directly correlated with experiments in mice in which an airway epithelial response to IFN-gamma injected into the airway lumen was seen only after disruption of barrier function. The results indicate that airway epithelia with intact barrier function restrict inflammatory responses by limitation of cell activation through requiring interaction of selected mediators with the basolateral surface. However, loss of barrier integrity allows epithelial responses to these mediators if located in the airway lumen to amplify airway defenses.

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth.

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus.

The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.

Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells.

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.

Respiratory syncytial virus nonstructural protein 2 specifically inhibits type I interferon signal transduction.

Human respiratory syncytial virus (RSV) inhibits type I interferon-induced gene expression by decreasing expression of signal transducer and activator of transcription (Stat)2. To identify the RSV protein that mediates effects on Stat2, airway epithelial cells were infected with vaccinia virus vectors that express single RSV proteins. Expression of RSV nonstructural (NS)2 protein alone was sufficient to decrease Stat2 levels. Furthermore, decreasing RSV NS2 levels using RNA interference in respiratory epithelial cells inhibited the RSV-mediated decrease in Stat2 expression. Airway epithelial cells were also infected with equivalent inoculums of RSV without or with single gene deletions of NS1 or NS2. RSV infection without NS2 expression did not result in decreased Stat2 levels or loss of type I interferon-dependent signaling, indicating that NS2 expression is necessary for RSV effects on Stat2. Taken together, our results indicate that NS2 regulates Stat2 levels during RSV infection, thereby modulating viral effects on interferon-dependent gene expression.

ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia.

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.