Interferon-gamma enhances rhinovirus-induced RANTES secretion by airway epithelial cells

Respiratory viruses, including rhinoviruses, infect respiratory epithelium and induce a variety of cytokines and chemokines that can initiate an inflammatory response. Cytokines, such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, could enhance epithelial cell activation by inducing virus receptors. To test this hypothesis, effects of IFN-gamma or TNF-alpha on expression of intercellular adhesion molecule (ICAM)-1, rhinovirus binding, and virus-induced chemokine secretion on A549 and human bronchial epithelial cells (HBEC) were determined. The results varied with the type of cell. IFN-gamma was a stronger inducer of ICAM-1 and viral binding on HBEC, whereas TNF-alpha had greater effects on A549 cells. In addition, IFN-gamma, but not TNF-alpha, synergistically enhanced regulated on activation, normal T cells expressed and secreted (RANTES) mRNA expression and protein secretion induced by RV16 or RV49. To determine whether IFN-gamma could enhance RANTES secretion independent of effects on ICAM-1 and RV binding, HBEC were transfected with RV16 RNA in the presence or absence of IFN-gamma. RV16 RNA alone stimulated RANTES secretion, and this effect was enhanced by IFN-gamma. These results demonstrate that IFN-gamma can enhance rhinovirus-induced RANTES secretion by increasing viral binding, and through a second receptor-independent pathway. These findings suggest that IFN-gamma, by upregulating RANTES secretion, could be an important regulator of the initial immune response to rhinovirus infections.     

Human airway submucosal glands augment eosinophil chemotaxis during rhinovirus infection

BACKGROUND: Asthma exacerbations are frequently associated with rhinovirus (RV) infections. However, the contribution of airway submucosal gland (SMG) to exacerbations of asthma in RV respiratory infection has not been studied. OBJECTIVE: This study was undertaken to examine whether RV-infected human respiratory SMG cells produce pro-inflammatory cytokines and chemokines for eosinophils, and augment eosinophil transmigration across human airway epithelium. METHODS: We infected cultured human tracheal SMG cells with RV14, collected culture media at 1, 3, and 5 days after infection, and measured the chemotactic activity for eosinophils in the culture supernatant using a 48-well microchemotaxis chamber and a (51)Cr-labelled eosinophil transmigration assay. RESULTS: Exposing a confluent human tracheal SMG cell monolayer to RV14 consistently led to infection. Human SMG cells with RV infection secreted soluble factors activating human eosinophil chemotaxis into the culture supernatant in a time-dependent manner, and the culture supernatant significantly augmented the transmigration of (51)Cr-labelled eosinophils through human airway epithelial cell layers from the basal to mucosal side. These effects were completely abolished by a mixture of a monoclonal antibody regulated on activation, normal T cells expressed and secreted (RANTES) and an antibody to granulocyte macrophage-colony stimulating factor (GM-CSF). CONCLUSION: These results suggest that human respiratory SMG cells may augment eosinophil transmigration across the airway epithelium through the secretion of RANTES and GM-CSF after RV infection, and may contribute to exacerbations of asthma.     

Modulation of the epithelial inflammatory response to rhinovirus in an atopic environment

Background Immune responses to rhinovirus (RV) as well as direct effects of RV on respiratory epithelium may contribute to the induction of asthma exacerbations. Objective To evaluate the effect of the environment resulting from an atopic immune response on RV‐induced epithelial inflammation, replication and cytotoxicity. Methods Peripheral blood mononuclear cells (PBMC) from atopic asthmatic subjects and matched controls (12 pairs) were isolated and stimulated by RVs. Human bronchial epithelial (BEAS‐2B) cells were infected with RV in the presence of conditioned media from RV‐stimulated PBMC cultures. IL‐6, IL‐8, RANTES and TGF‐β1 levels were measured by ELISA, RV‐induced cytotoxicity by a colorimetric method and RV titres on Ohio‐HeLa cells. Results RV‐induced epithelial production of IL‐6, IL‐8 and RANTES was significantly lower, while TGF‐β1 was higher when cells were exposed to conditioned media from atopic asthmatic subjects compared with those from normal controls. Exposure to the ‘atopic’ environment also resulted in elevated RV titres and increased RV‐induced cytotoxicity. Conclusions Under the influence of an atopic environment, the epithelial inflammatory response to RV is down‐regulated, associated with increased viral proliferation and augmented cell damage, while TGF is up‐regulated. These changes may help explain the propensity of atopic asthmatic individuals to develop lower airway symptoms after respiratory infections and indicate a mechanism through which viral infections may promote airway remodelling.     

In vitro anti-influenza A H1N1 effect of extract of Bupleuri Radix

This study investigated the effect of the extract of Bupleuri Radix (BRE) on the infection of Madin-Darby Canine Kidney (MDCK) cells by anti-H1N1 virus. The effect of BRE on RANTES (the chemokine regulated on activation, normal T cells expressed and secreted) secretion in H1N1-infected A549 cells (human bronchial epithelial cells) was evaluated via quantative measurement of the changes in the cytopathic effects and by the ultraviolet (UV) absorbance at 600?nm. It was found that BRE was toxic to MDCK cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Results also showed that BRE possessed more than 50% suppressing effect on RANTES secretion in H1N1-infected A549 cells at a concentration of 100 and 200 μg/ml. Our findings show that BRE has a significant protective effect on MDCK cells infected in a dose-dependent manner with an excellent suppressing effect on RANTES secretion, suggesting that BRE can be developed as an antivirus agent.     

Parainfluenza virus infection of cultured airway epithelial cells

Studies of the cellular effects of respiratory viruses have generally used cultures of non-airway (particularly renal) epithelial cells. This requires the assumption that, despite the marked differences between renal epithelium and airway epithelium, the virus-host cell interactions in cultures of renal epithelium will be relevant to those in airway epithelium. To study viral infection of airway epithelial cells, we removed the epithelial cells from ferret tracheas using 0.1% pronase solution, and plated them at a density of 5 X 10(5) cells/cm2 in collagen-coated plastic tissue culture wells. Cultures grew to confluence after 5-7 days. Viral inocula, consisting of supernatants from parainfluenza type 1-infected rhesus monkey kidney cell monolayers, were added to the culture medium in a concentration 10(3) times that sufficient to produce infection in 50% of rhesus monkey kidney monolayers (TCID50). Cytopathic changes, consisting of cellular elongation and detachment, became apparent after 3-6 days, at which time the medium contained 5 X 10(8) TCID50/ml. The monolayer appeared to be uniformly infected as revealed by adsorption of guinea pig erythrocytes. Specific immunofluorescence revealed uniformly positive staining for parainfluenza type 1 antigens. The ability to infect pure cultures of airway epithelial cells with viruses will allow us to examine the effects of these viruses on epithelial cell function, and to study virus-host cell interactions in cell cultures derived from the natural host cell.     

Effect of loratadine on nitrogen dioxide-induced changes in electrical resistance and release of inflammatory mediators from cultured human bronchial epithelial cells.

BACKGROUND: Recent studies have demonstrated that some antihistamines can attenuate histamine-induced release of inflammatory mediators from bronchial epithelial cells. OBJECTIVE: The purpose of study was to test the hypothesis that loratadine may influence pollution-induced inflammation of the airways by modulating epithelial membrane integrity and the synthesis and/or release of inflammatory mediators from airway epithelial cells. METHODS: We have cultured human bronchial epithelial cell (HBEC) cultures from surgical explants and investigated the effect of loratadine on NO2-induced changes in both electrical resistance of HBEC cultures and release of IL-8, RANTES, and soluble intercellular adhesion molecule-1 (sICAM-1) from these cells after exposure for 6 hours to either air or 400 ppb NO2. RESULTS: Exposure for 6 hours to NO2 significantly decreased the electrical resistance of HBEC cultures by 18.1% from baseline (P <.05). Incubation with 0.25 to 25 micromol/L loratadine did not alter the NO2-induced decrease in the electrical resistance of HBEC cultures. NO2 also significantly increased the release of IL-8 from a control value of 52.5 pg/microgram cellular protein to 81.9 pg/microgram cellular protein (P <.05), RANTES from a control value of 0.023 pg/microgram cellular protein to 0.062 pg/microgram cellular protein (P <.05), and sICAM-1 from a control value of 7.7 pg/microgram cellular protein to 16.3 pg/microgram cellular protein (P <.05). The NO2-induced release of all 3 mediators was significantly attenuated by incubation of HBECs with 25 micromol/L loratadine. Incubation with 2.5 micromol/L loratadine also significantly attenuated the NO2-induced release of RANTES and sICAM-1, but not IL-8. CONCLUSIONS: These results suggest that loratadine has the potential to reduce airway inflammation by modulating the release of inflammatory cytokines from airway epithelial cells.     

Respiratory syncytial virus infection of human primary nasal and bronchial epithelial cell cultures and bronchoalveolar macrophages.

In adults, clinical symptoms caused by respiratory syncytial virus (RSV) are usually confined to the upper respiratory tract, whereas RSV infection in infants frequently causes bronchiolitis and pneumonia. The preferential localization of RSV infection to the upper airways may partially be due to protective immunity, but may also depend on a difference in susceptibility of epithelial cells from upper and lower airways, or on antiviral activities of bronchoalveolar macrophages (AM). In this study, we have compared the susceptibility of primary adult human nasal epithelium, primary adult human bronchial epithelium, a human bronchial epithelial cell line (BEAS-2B), and adult human AM to infection with RSV. The cell cultures were infected with multiplicities of infection (moi) of 1 and 0.1. Virus release into the supernatants was assayed at days 1, 2, 4, and 7, and the percentage of virus-positive cells determined by immunofluorescence at the same time points. Similar proportions of nasal epithelial cells (NE) and bronchial epithelial cells (BE) were infected with RSV. Approximately 50 to 75% (with moi 1) and 2 to 10% (with moi 0.1) of the cells were infected by 24 h; almost all the cells were RSV positive by day 4. However, BE released less infectious RSV than do NE. With moi 0.1, 10-fold less virus was released over 4 days of culture. By days 4 to 7, cytopathic effects (CPE) were maximal in all epithelial cell cultures, but CPE developed latest in BE infected with moi 0.1. AM were also productively infected with RSV, with peak virus production at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhinovirus and asthma

Rhinoviruses (RVs) cause the majority of common colds, which often provoke wheezing in patients with asthma. The precise mechanisms responsible for the RV infection-induced exacerbations of bronchial asthma are still uncertain. However, several reports reveal airway hyperresponsiveness, increases in chemical mediators in airway secretions such as kinin and histamine, and airway inflammation in patients with bronchial asthma after RV infection. RV infection induces an accumulation of inflammatory cells in airway mucosa and submucosa including neutrophils, lymphocytes and eosinophils. RV affects the barrier function of airway epithelial cells, and activates the airway epithelial cells and other cells in the lung to produce pro-inflammatory cytokines, including various kinds of interleukins, GM-CSF and RANTES, and histamine. RV also stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptors in the airway epithelium, receptors for major and minor RVs. On the other hand, RV infection is inhibited by treatment with soluble ICAM-1, and by reduction of ICAM-1 expression in the airway epithelial cells after treatment with erythromycin. Both soluble ICAM-1 and erythromycin were reported to reduce the frequency of common colds. Here, we review the pathogenesis and management of RV infection-induced exacerbation of bronchial asthma.     

IL-12 p80-dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection

A protective immune response to a respiratory viral infection requires a series of coordinated cellular and molecular responses. We have previously demonstrated that increased expression of airway epithelial cell interleukin (IL)-12 p80, a macrophage chemoattractant, is associated with human respiratory viral infection and mediates post-viral mortality in the mouse. To better understand the role of IL-12 p80-dependent macrophage chemotaxis in mediating viral immunity, we generated a transgenic mouse strain utilizing a promoter to drive IL-12 p40 gene expression in the airway epithelium. This transgenic strain secreted biologically active IL-12 p80 in a lung-specific manner, and demonstrated a selective increase in the number of resident, unactivated airway macrophages at baseline. Following infection with a sublethal dose of mouse parainfluenza virus type 1 (Sendai virus), the transgenic mice demonstrated an earlier peak and decline in the number of airway inflammatory cells. The transgenic mice were resistant to a lethal dose of virus and this viral resistance was dependent on the increased number of airway macrophages at baseline as partial depletion prior to infection abrogated this phenotype. The survival advantage in the transgenic mice was independent of viral load but was associated with a more rapid decline in the number of airway inflammatory cells and concentrations of multiple chemokines including the CC chemokine ligand 2 (CCL2)/JE, CCL3/macrophage inflammatory protein (MIP)-1alpha, CCL4/MIP-1beta, and CCL5/RANTES. Collectively, these results suggest that IL-12 p80-driven increases in the number of resident airway macrophages prime the host for a protective immune response that can confer increased survival following a lethal respiratory viral infection.     

Rhinovirus-induced PBMC responses and outcome of experimental infection in allergic subjects

BACKGROUND: The immune response to rhinovirus (RV) infections is considered to contribute to upper respiratory symptoms and may also be an important contributor to lower airway dysfunction in patients with asthma. OBJECTIVE: This study was conducted to determine the relationship of RV-specific responses in PBMCs to the outcome of experimentally induced infection with RV16. METHODS: Twenty-two subjects with either allergic rhinitis or asthma were inoculated with RV16: virus-induced proliferation and cytokine production were determined on PBMCs obtained before and then again 7 and 28 days after inoculation. RESULTS: Several subjects had proliferative responses to RV16 before inoculation, and precold RV-specific proliferative responses were inversely correlated (r(s) = -0.62, P <. 005) with RV shedding after inoculation. In addition, there was a negative correlation (r(s) = -0.58, P = 0.01) between precold RV-induced IFN-gamma secretion ex vivo and peak RV shedding during the cold. CONCLUSIONS: Certain RV-specific lymphocyte responses before the cold (vigorous proliferation or IFN-gamma secretion) were associated with reduced viral shedding after inoculation. These findings suggest that variations in mononuclear cell responses to RV could contribute to the individual variability in viral shedding during experimentally induced, and perhaps naturally acquired, RV infections in subjects with respiratory allergy or asthma.     

Secretion of rubella virions and virus-like particles in cultured epithelial cells.

Rubella virus (RV) is an enveloped RNA virus that causes systemic infections in humans. More importantly, first trimester in utero infection leads to a collection of devastating birth defects known as congenital rubella syndrome. Epithelial cells are the first line of defense against viruses and consequently, the polarity of virus secretion is an important factor affecting viral spread. As a first step toward understanding how RV interacts with epithelial cells, we have examined the release of RV-like particles and virions from polarized cells in culture. RV structural proteins were targeted to the Golgi complex and virus particle formation occurred on intracellular membranes in three different polarized epithelial cells. Polarized cells could be infected from the apical and basal membranes, indicating that receptors are not confined to one surface. The secretion of virus-like particles and infectious virions varied according to cell type. In two of the three polarized cell lines examined, virus was released primarily from the apical surface, but significant quantities were also secreted from the basolateral membrane. Release of virus from the apical surface may facilitate virus spread from person to person, whereas basolateral secretion could be important for establishing a systemic infection and/or crossing the placenta prior to fetal infection.     

Double-stranded RNA induces the synthesis of specific chemokines by bronchial epithelial cells

Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1alpha. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-kappaB and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.     

Infection of cultured human tracheal epithelial cells by human parainfluenza virus types 2 and 3.

Despite growing information of the effects of human respiratory virus infection on airway physiology, little information is available on the mechanisms of pathology and pathophysiology in these infections. The human respiratory pathogens, parainfluenza virus types 2 and 3 (hPIV2, hPIV3, respectively), clinically cause laryngotracheobronchitis (infection of the large proximal airways). In order to examine the pathobiology of these viruses in airway cells of human origin, we exposed primary cultures of human tracheal epithelial cells. Primary cultures of human tracheal epithelial cells were readily infected by these agents: cells exposed to hPIV2 and hPIV3 expressed viral antigens (demonstrated by indirect immunofluorescence assay), produced infectious virus, and demonstrated cytopathic effects (including early syncytium formation). Peak viral titers of 2 x 10(7) plaque-forming units per milliliter were obtained, similar to titers from permissive CV-1 cells. Trypan blue staining and direct cell counts demonstrated no difference in the viability of the control and infected cells until the infected cells began to detach from the culture substrate. However, infected cells release significantly more LDH than control cells by 48 h following infection at a multiplicity of infection of 1 virus/target cell. This system provides a model for studying the effects of infection of the human tracheal epithelium by human respiratory viral pathogens without confounding interactions with other cell and tissue types.     

Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format.

Despite growing evidence that respiratory virus infections precipitate episodes of airway obstruction and airway hyper-responsiveness in young children and in asthma, little information is available on the mechanisms by which virus infections alter the airway physiology. Airway inflammatory changes (including influx of inflammatory cells such as neutrophils) have been described during episodes of airway hyper-responsiveness in both animal models and human subjects. Neutrophil damage to several cell types has been shown to require adhesion as a primary step. In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we have studied the ability of neutrophils to adhere to virus-infected airway epithelial cell cultures. Neutrophil adherence was determined indirectly, using myeloperoxidase as a marker for adherent neutrophils in an assay system described here. Airway epithelial cell cultures (both primary human tracheal epithelial cells, and two permanent cell lines, A549 and BEAS-2B) were grown in 96-well tissue culture plates and infected with human parainfluenza virus type 2. Infected airway epithelial cell cultures supported significantly enhanced levels of neutrophil adherence (up to 50-75% of neutrophils added to the wells) compared to uninfected control cultures. Moreover, this adherence occurred in a virus dose-dependent fashion, with increasing levels of adherence noted at increasing viral multiplicities of infection. The assay system described allows the detection of small numbers of adherent neutrophils (as few as 1000 neutrophils) in a 96-well format.     

Pseudomonas aeruginosa suppresses interferon response to rhinovirus infection in cystic fibrosis but not in normal bronchial epithelial cells

Despite increased morbidity associated with secondary respiratory viral infections in cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infection, the underlying mechanisms are not well understood. Here, we investigated the effect of P. aeruginosa infection on the innate immune responses of bronchial epithelial cells to rhinovirus (RV) infection. CF cells sequentially infected with mucoid P. aeruginosa (MPA) and RV showed lower levels of interferons (IFNs) and higher viral loads than those of RV-infected cells. Unlike results for CF cells, normal bronchial epithelial cells coinfected with MPA/RV showed higher IFN expression than RV-infected cells. In both CF and normal cells, the RV-stimulated IFN response requires phosphorylation of Akt and interferon response factor 3 (IRF3). Preinfection with MPA inhibited RV-stimulated Akt phosphorylation and decreased IRF3 phosphorylation in CF cells but not in normal cells. Compared to normal, unstimulated CF cells or normal cells treated with CFTR inhibitor showed increased reactive oxygen species (ROS) production. Treatment of CF cells with antioxidants prior to MPA infection partially reversed the suppressive effect of MPA on the RV-stimulated IFN response. Together, these results suggest that MPA preinfection inhibits viral clearance by suppressing the antiviral response particularly in CF cells but not in normal cells. Further, increased oxidative stress in CF cells appears to modulate the innate immune responses to coinfection.     

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.