Respiratory syncytial virus infection alters surfactant protein A expression in human pulmonary epithelial cells by reducing translation efficiency.

Infection of neonatal lung by respiratory syncytial virus (RSV) is a common cause of respiratory dysfunction. Lung alveolar type II and bronchiolar epithelial (Clara) cells secrete surfactant protein A (SP-A), a collectin that is an important component of the pulmonary innate immune system. SP-A binds to the virus, targeting the infectious agent for clearance by host defense mechanisms. We have previously shown that while the steady-state level of SP-A mRNA increases approximately threefold after RSV infection, steady-state levels of cellular and secreted SP-A protein decrease 40-60% in human type II cells in primary culture, suggesting a mechanism where the virus alters components of the innate immune response in infected cells. In these studies, we find that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results in SP-A expression observed in primary lung cells. While SP-A protein is normally ubiquitinated, there is no change in the level of SP-A protein ubiquitination or proteasome activity during RSV infection, suggesting that the reduced levels of SP-A protein are not due to degradation by activated proteasomes. SP-A mRNA is appropriately processed and exported from the nucleus to the cytoplasm during RSV infection. As evidenced by polysome analysis of SP-A mRNA and pulse-chase analysis of newly synthesized SP-A protein, we find a decrease in translational efficiency that is specific for SP-A mRNA. Therefore, the decrease in SP-A protein levels observed after RSV infection of pulmonary bronchiolar epithelial cells results from a mechanism that affects SP-A mRNA translation efficiency.

Role of prophylactic azithromycin to reduce airway inflammation and mortality in a RSV mouse infection model.

BACKGROUND: Respiratory syncytial virus (RSV) infection is an important cause of morbidity and mortality in vulnerable populations. Macrolides have received considerable attention for their anti-inflammatory actions beyond their antibacterial effect. We hypothesize that prophylactic azithromycin will be effective in reducing the severity of RSV infection in a mouse model. METHODS: Four groups of BALB/c mice were studied for 8 days: Control (C), RSV-infected (R), early prophylaxis with daily azithromycin from days 1 to 8, (E), and late prophylaxis with daily azithromycin from days 4 to 8 (L). Mice were infected with RSV on day 4, except for the control group. All groups were followed for a total of 8 days when bronchoalveolar lavage cell count and cytokines levels were measured. Mouse weight, histopathology, and mortality data were obtained. RESULTS: Prophylactic azithromycin significantly attenuated post-viral weight loss between group R and both groups E and L (P = 0.0236, 0.0179, respectively). IL-6, IL-5, and Interferon-Gamma were significantly lower in group L (P = 0.0294, 0.0131, and 0.0056, respectively) compared with group R. The total cell count was significantly lower for group L as compared with group R (P < 0.05). Mortality was only observed in group R (8%). Lung histology in the prophylactic groups showed diminished inflammatory infiltrates and cellularity when compared with group R. CONCLUSION: Prophylactic azithromycin effectively reduced weight loss, airway inflammation, cytokine levels and mortality in RSV-infected mice. These results support the rationale for future clinical trials to evaluate the effects of prophylactic azithromycin for RSV infection.

Lack of long-term effects of respiratory syncytial virus infection on airway function in mice.

Epidemiological data suggests lower respiratory infections (LRI) with respiratory syncytial virus (RSV) are capable of causing long-term abnormalities in airway function. To directly test the effects of RSV LRI, we infected adult and weanling BALB/c mice with RSV (A2) or vehicle. Respiratory system impedance was used to assess baseline airway function and responses to iv methacholine (MCh) at 4, 8, 24 and 34 weeks post infection. In vitro airway responses were measured 24 weeks post infection using electrical field stimulation and MCh. Mice infected as adults showed no alterations in airway function. Mice infected as weanlings had increased MCh responses 24 weeks post infection. However, the increased response was not present 34 weeks post infection nor accompanied by alterations in in vitro responses or airway morphometry. This study did not detect long-lasting changes in airway function following RSV infection in mice. These data do not provide support for alterations in airway structure or function being responsible for the observed relationship between RSV infection in infants and asthma in later life.

Serum KL-6 and surfactant proteins A and D in pediatric interstitial lung disease.

OBJECTIVE: To determine if serum KL-6, surfactant protein A (SP-A), and surfactant protein D (SP-D) levels are elevated in pediatric interstitial lung disease (ILD) and associated with pulmonary function and disease severity score. METHODS: Serum KL-6, SP-A, and SP-D levels were measured by enzyme-linked immunosorbent assay in 10 children with ILD and in 10 healthy volunteers. In the ILD group, FEV1 percentage of predicted, FVC percentage of predicted, and ILD disease severity score were measured and correlated with serum KL-6, SP-A, and SP-D levels. RESULTS: For the ILD and control groups, respectively, mean serum KL-6 was 4,523 U/mL and 206 U/mL (p = 0.007), mean serum SP-A was 133 ng/mL and 21 ng/mL (p = 0.003), and mean serum SP-D was 304 ng/mL and 75 ng/mL (p = 0.004). There was an inverse relationship between SP-A and FVC (p = 0.05), and between SP-D and FEV1 (p = 0.05). There was a direct relationship between SP-D and ILD score (p = 0.05). CONCLUSIONS: Serum KL-6, SP-D and SP-D levels are elevated in children with ILD. SP-A and SP-D levels appear to correlate with some measures of disease severity.

Hyperresponsiveness to inhaled but not intravenous methacholine during acute respiratory syncytial virus infection in mice.

BACKGROUND: To characterise the acute physiological and inflammatory changes induced by low-dose RSV infection in mice. METHODS: BALB/c mice were infected as adults (8 wk) or weanlings (3 wk) with 1 x 10(5) pfu of RSV A2 or vehicle (intranasal, 30 microl). Inflammation, cytokines and inflammatory markers in bronchoalveolar lavage fluid (BALF) and airway and tissue responses to inhaled methacholine (MCh; 0.001-30 mg/ml) were measured 5, 7, 10 and 21 days post infection. Responsiveness to iv MCh (6-96 microg/min/kg) in vivo and to electrical field stimulation (EFS) and MCh in vitro were measured at 7 d. Epithelial permeability was measured by Evans Blue dye leakage into BALF at 7 d. Respiratory mechanics were measured using low frequency forced oscillation in tracheostomised and ventilated (450 bpm, flexiVent) mice. Low frequency impedance spectra were calculated (0.5-20 Hz) and a model, consisting of an airway compartment [airway resistance (Raw) and inertance (Iaw)] and a constant-phase tissue compartment [coefficients of tissue damping (G) and elastance (H)] was fitted to the data. RESULTS: Inflammation in adult mouse BALF peaked at 7 d (RSV 15.6 (4.7 SE) vs. control 3.7 (0.7) x 10(4) cells/ml; p < 0.001), resolving by 21 d, with no increase in weanlings at any timepoint. RSV-infected mice were hyperresponsive to aerosolised MCh at 5 and 7 d (PC200 Raw adults: RSV 0.02 (0.005) vs. control 1.1 (0.41) mg/ml; p = 0.003) (PC200 Raw weanlings: RSV 0.19 (0.12) vs. control 10.2 (6.0) mg/ml MCh; p = 0.001). Increased responsiveness to aerosolised MCh was matched by elevated levels of cysLT at 5 d and elevated VEGF and PGE2 at 7 d in BALF from both adult and weanling mice. Responsiveness was not increased in response to iv MCh in vivo or EFS or MCh challenge in vitro. Increased epithelial permeability was not detected at 7 d. CONCLUSION: Infection with 1 x 10(5) pfu RSV induced extreme hyperresponsiveness to aerosolised MCh during the acute phase of infection in adult and weanling mice. The route-specificity of hyperresponsiveness suggests that epithelial mechanisms were important in determining the physiological effects. Inflammatory changes were dissociated from physiological changes, particularly in weanling mice.

Mechanisms determining cholinergic neural responses in airways of young and mature rabbits.

Neural pathways help control airway caliber and responsiveness. Yet little is known of how neural control changes as a function of development. In rabbits, we found electrical field stimulation (EFS) of airway nerves led to more marked contractile responses in 2- vs. 13-week-old animals. This enhanced response to EFS may be due to prejunctional, junctional, and/or postjunctional neural mechanisms. We assessed these mechanisms in airways of 2- and 13-week-old rabbits. The contractile responses to methacholine did not differ in the groups, suggesting postjunctional neural events are not primarily responsible for differing responses to EFS. To address junctional events, acetylcholinesterase (AChE) was measured (spectrophotometry). AChE was elevated in 2-week-olds. However, this should lead to less and not greater responses. Prejunctionally, EFS-induced acetylcholine (ACh) release was assessed by HPLC. Airways of 2-week-old rabbits released significantly more ACh than airways from mature rabbits. Choline acetyltransferase, a marker of cholinergic nerves, was not different between groups, suggesting that more ACh release in young rabbits was not due to increased nerve density. ACh release in the presence of polyarginine increased significantly in both groups, supporting the presence of functional muscarinic autoreceptors (M2) at both ages. Because substance P (SP) increases release of ACh, SP was measured by ELISA. This neuropeptide was significantly elevated in airways of younger rabbits. Nerve growth factor (NGF) increased SP and was also significantly increased in airways from younger rabbits. This work suggests that increases in EFS-induced responsiveness in young rabbits are likely due to prejunctional events with enhanced release of ACh. Increases in NGF and SP early in life may contribute to this increased responsiveness.

Enhanced release of thromboxane A(2) after exposure of human airway epithelial cells to meconium.

Meconium aspiration syndrome (MAS) is a cause of significant morbidity and mortality in the perinatal period. Despite the clinical relevance of MAS, its pathogenesis is poorly understood. Epithelial cell-derived prostanoids are involved in the regulation of several cellular functions within the lung, including the control of tone and reactivity of airway and vascular smooth muscle. In this study, we evaluated whether exposure to meconium affects the metabolic function of human airway epithelial cells. Monolayers of A549 cells, a transformed human epithelial cell line, were incubated with various concentrations of meconium. Control cells were incubated with serum-free medium in a similar manner. The supernatant fluid was removed at various time points and assayed for thromboxane A(2) (TXA(2)) production. The latter was accomplished by measuring its immediate and stable metabolite thromboxane B(2), using an enzyme-linked immunosorbent assay (ELISA). In selected experiments, the modulatory effects of indomethacin (10(-6) M), dexamethasone (10(-6) M), and L-nitroarginine methyl ester (L-NAME, 10(-6) M) on TXA(2) production were evaluated. Results were expressed in terms of pg/mg protein (mean +/- SE). We found that exposure to meconium produced a significant release of TXA(2) from A549 cells. Indomethacin, dexamethasone, and in part, L-NAME inhibited meconium-induced release of TXA(2). Our findings demonstrate that meconium enhances the production of thromboxanes from A549 cells, suggesting that airway epithelial cells and their metabolic products may play an important role in the pathogenesis of MAS.

Immunocytochemical detection of milk proteins in tracheal aspirates of ventilated infants: a pilot study.

In this study, we evaluated immunocytochemical staining for milk proteins (alpha-lactalbumin and beta-lactoglobulin) in tracheal aspirates of mechanically ventilated infants, and assessed whether this staining technique supported a clinical diagnosis of aspiration in infants receiving orogastric feedings. All newborns requiring mechanical ventilation in the neonatal intensive care unit of a major tertiary care hospital were potential subjects for this study. Tracheal aspirates were obtained prior to the introduction of enteral feeding and at various time points thereafter in newborns requiring mechanical ventilation. Cells were obtained and processed for immunocytochemical staining of alpha-lactalbumin and beta-lactoglobulin. In total, 88 specimens recovered from 34 infants were adequate for staining. Alveolar macrophages recovered from most of the infants who were never fed (true negative controls) did not display immunoreactivity for milk proteins: 4/34 or 12% of infants' aspirates demonstrated presence of milk proteins before enteral feeding was commenced. Tracheal aspirates obtained from 12 infants after introduction of enteral feedings appeared to support clinical and radiological findings suggestive of aspiration events, with positive immunostaining on several occasions. These observations support our work in a murine model and demonstrate that immunocytochemical staining of tracheal aspirates for milk proteins may enhance the ability to diagnose pulmonary aspiration. Further studies are needed to define the clinical significance of our findings and the effects of single and repeated aspiration events on respiratory status.

Influence of lipopolysaccharide exposure on airway function and allergic responses in developing mice.

Exposure to endotoxin has been associated with an exacerbation of asthmatic responses in humans and animal models. However, recent evidence suggests that microbial exposure in early life may protect from the development of asthma and atopy. In this study, we sought to evaluate the effects of lipopolysaccaride (LPS) on airway function in developing mice. In addition, we evaluated the influence of LPS on subsequent allergen sensitization and challenge. Under light anesthesia, 2-3-week-old Balb/c mice received a single intranasal instillation of LPS or sterile physiologic saline. Measurements of airway function were obtained in unrestrained animals, using whole-body plethysmography. Airway responsiveness was expressed in terms of % enhanced pause (Penh) increase from baseline to aerosolized methacholine (Mch). In additional studies, we assessed the functional and cellular responses to ovalbumin sensitization and challenge following prior exposure to LPS. We found that exposure to LPS induced transient airway hyperresponsiveness to Mch. These functional changes were associated with the recruitment of neutrophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid. Airway responsiveness after allergen sensitization and challenge was decreased by prior exposure to LPS. The analysis of BAL cells and cytokines (interferon-gamma and interleukin-4) did not reveal alterations in the overall Th1/Th2 balance. Our findings suggest that LPS leads to airway hyperresponsiveness in developing mice, and may protect against the development of allergen-driven airway dysfunction.

Dysanaptic growth of lung and airway in children with post-infectious bronchiolitis obliterans.

RATIONALE: Post-infectious bronchiolitis obliterans (PBO) is a rare form of chronic obstructive lung disease associated with small airway fibrosis following a severe insult to the lower respiratory tract. It has been suggested that PBO is a non-progressive disease. However, evidence supporting this statement is limited. In this case series, we sought to determine the changes of pulmonary function tests (PFT) over time in children with PBO. METHODS: Seven children with PBO, ages 6-15 years old, were retrospectively studied between 1994 and 2012. Spirometry and lung volumes tests were performed in accordance with American Thoracic Society (ATS) guidelines and were monitored over time. The average rate of change was calculated using generalized linear mixed models. RESULTS: The median baseline values for forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), the FEV1/FVC ratio and forced expiratory flow 25%-75% (FEF25%-75%) were 57%, 50%, 87% and 29%, respectively. FVC increased at a rate of 1.8% per year (P = 0.008). There was no significant change in FEV1 over time (P = 0.112). However, the FEV1/FVC ratio decreased by 2.6% per year (P < 0.001). CONCLUSION: PFT in childhood PBO was characterized by significant airway obstruction. Over time, FVC (lung parenchyma) increased and FEV1 (airway) remained stable, but FEV1/FVC ratio declined more than expected, suggesting a mismatch in the growth of the airway and lung parenchyma (dysanaptic growth). Further studies in larger populations are needed to validate these observations.

Airway obstruction in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) is the failure of the autonomic system to control adequate ventilation while asleep with preserved ventilatory response while awake. We report a case of a patient with CCHS who presented with intrathoracic and extrathoracic airway obstruction after tracheostomy tube decannulation and phrenic nerve pacer placement. Nocturnal polysomnography (NPSG) revealed hypoxia, hypercapnia and obstructive sleep apnoea, which required bilevel positive airway pressure titration. Airway endoscopy demonstrated tracheomalacia and paretic true vocal cords in the paramedian position during diaphragmatic pacing. Laryngeal electromyography demonstrated muscular electrical impulses that correlated with diaphragmatic pacer settings. Thus, we surmise that the patient's upper and lower airway obstruction was secondary to diaphragmatic pacer activity. Thorough airway evaluation, including NPSG and endoscopy, may help identify the side effects of diaphragmatic pacing, such as airway obstruction, in patients with CCHS.

Effects of infant formula on cell homeostasis and cytokine levels in an in vivo and in vitro murine aspiration model.

RATIONALE: The role of infant formula aspiration in lung injury has not been studied extensively. We evaluated the effects of a single infant formula aspiration into the lungs of mice and the effect of infant formula exposure on cell lines representing murine alveolar macrophages and type II epithelial cells. OBJECTIVES: To study the effects of exposure to infant formula on cell count histology and cytokine levels in an in vivo and in vitro model of aspiration. METHODS: In vivo: Juvenile mice received 2.5 µl/g of 50% infant formula intranasally. Bronchoalveolar lavage samples were collected at 1, 2, and 7 days after aspiration and evaluated for cell count and differential. In vitro: RAW 264.7 and MLE-15 cells were exposed to 1% infant formula for 6 hr. Extracellular levels of IL-6, TNF-α, MIP-2, and KC were measured in lavage fluid and cell media using ELISA assays. RESULTS: In vivo: An increase in neutrophils, IL-6 and KC levels were noted 24 hr after infant formula exposure. In vitro: An increase in TNF-α levels from RAW 264.7 and MIP-2 and KC levels from MLE-15 cells was noted after infant formula exposure. CONCLUSIONS: A single aspiration of infant formula into the lungs leads to an acute inflammatory response involving both lung macrophages and epithelial cells.

Modulation of airway responses by prostaglandins in young and fully grown rabbits.

Maturational changes have been noted in neurally mediated contractile and relaxant responses in airways from New Zealand White rabbits. In this study, we focused on prostaglandins with bronchoprotective properties as potential modulators of airway tone in maturing rabbits. Tracheal rings from 1-, 2-, and 13-wk-old rabbits were assessed for neurally mediated contractile and relaxant responses produced by electrical field stimulation (EFS) of nerves in the presence and absence of the prostaglandin inhibitor, indomethacin (Indo). We also measured EFS-induced release of prostaglandin E(2) (PGE(2)) and the stable metabolite of prostacyclin, 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)). In the presence of Indo, EFS produced significant increases in contractile responses in segments from 1- and 2-wk-old animals but not in segments from 13-wk adult rabbits. Tracheal rings from 1- and 2-wk-old animals precontracted with neurokinin A (NKA) relaxed 100% in response to EFS when Indo was not in the bath. In rings from 13-wk-old animals, relaxation was 40%. With Indo, relaxation was abolished in 1-wk-old animals and reduced to 30% in the 2- and 13-wk-old groups. Buffer from baths collected after EFS had significant increases in PGE(2) and 6-keto-PGF(1alpha) released from tissues from 1- vs. 2- and 13-wk-old animals. Dose response curves to PGE(2) using tissues precontracted to NKA showed significant increases in relaxant responses in 1- and 2- vs. 13-wk-old rabbits. In rabbit airways, this study demonstrates enhanced modulation of airway tone by PGE(2) and greater release of the bronchoprotective prostaglandins PGE(2) and prostacyclin early in life.

Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection.

Virus respiratory infections often precede bacterial pneumonia in healthy individuals. In order to determine the potential role of respiratory syncytial virus (RSV) in bacterial secondary infections, a mouse sequential pulmonary infection model was developed. Mice were exposed to RSV then challenged with Streptococcus pneumoniae (StPn). Exposure of BALB/c mice to 10(6)-10(7) plaque forming units (pfu) of virus of RSV significantly decreased StPn clearance 1-7 days following RSV exposure. This finding was not restricted to StPn alone: exposure to RSV followed by Staphylococcus aureus (SA) or Pseudomonas aeruginosa(PA) resulted in similar decreases in bacterial clearance. Both bronchoalveolar lavage (BAL) cell counts and pulmonary histopathology demonstrated that RSV-StPn exposed mice had increased lung cellular inflammation compared to mice receiving StPn or RSV alone. The effect of RSV infection on bacterial clearance was dependent on the mouse genetic background: C57BL/6J mice (relatively resistant to RSV infection) demonstrated a modest change in StPn clearance following RSV exposure, whereas FVBN/J mice (similar to the BALB/cJ mice in RSV susceptibility) demonstrated a similar degree of RSV-associated decrease in StPn clearance 7 days following RSV exposure. Neutrophils from the RSV-StPn sequentially exposed BALB/cJ mice were functionally altered-produced greater levels of peroxide production but less myeloperoxidase (MPO) compared to mice receiving StPn alone. These data demonstrate that RSV infection decreases bacterial clearance, potentially predisposing to secondary bacterial pneumonia despite increased lung cellular inflammation, and suggest that functional changes occur in the recruited neutrophils that may contribute to the decreased bacterial clearance.

Respiratory syncytial virus infection in a murine model of cystic fibrosis.

Viral respiratory infections play an important role in the development and progression of pulmonary disease in cystic fibrosis (CF). The CF mouse model provides a tool to examine the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) defect and lung disease. This work investigates the cellular response to a common viral pathogen, respiratory syncytial virus (RSV) in the lung of CF mice. RSV was administered by intranasal inoculation of CFTR(tm1Unc)-Tg(FABPCFTR)1Jaw/J (CFTR-/-) and control mice. At day 5 post infection, viral titers, bronchoalveolar fluid nitrate levels (BALF) cell and differential counts, histology and studies on airway mechanics were performed. CFTR-/- mice had an impaired ability to clear RSV. This was associated with an exaggerated inflammatory response (increased lymphocytes and neutrophils) in BALF of RSV-infected CFTR-/- mice and a decreased ability to generate nitric oxide (NO) (measured as BAL nitrate). Lung histopathology of RSV-infected CFTR-/- mice demonstrated increased inflammation compared to RSV (-) CFTR-/- and control mice (regardless of RSV treatment). The airway response to methacholine was increased by RSV infection in CF mice when compared to controls. The CFTR-/- mouse exhibits an aberrant response to RSV infection. This model should be useful in providing further mechanistic information on the biology of respiratory viruses in mammalian models, and provide new insights into the pathogenesis of airway inflammation in patients with CF.

Phosphodiesterase IV and neutral endopeptidase in airways from developing and allergen sensitized rabbits.

Phosphodiesterase IV (PDE IV) and neutral endopeptidase (NEP) may modulate the neurally mediated nonadrenergic noncholinergic inhibitory (NANCi) response. This response is not present in normal rabbits until 2 weeks of age. Allergen sensitization and challenge of fully grown 13-week old rabbits decreases the NANCi response. Our goal was to assess NANCi as a function of age and allergen sensitization. Tracheal smooth muscle (TSM) rings from normal 1-, 2-, and 13-week old rabbits plus ragweed immune as well as ragweed immune/challenged (I/C) 13-week old rabbits were assessed. Colorimetric assays of PDE IV and NEP activity were conducted on TSM from each group. NANCi responses were obtained in the presence and absence of Ro 20-1724 (PDE IV inhibitor) and/or phosphoramidon (Phos; NEP inhibitor) after contraction of TSM with neurokinin A. In normal TSM, there was no difference in PDE IV as a function of age. Conversely, NEP decreased significantly from 1 to 13 weeks of age. The immune and I/C groups had decreases in NEP and increases in PDE IV that were significant. Neither Ro 20-1724 nor Phos alone or together increased NANCi responses in TSM from 1- or 2-week old rabbits. However, both enhanced relaxation in TSM from normal, immune, and I/C 13-week old rabbits with an additive effect when drugs were combined. This work demonstrates (1) normal maturational changes in NEP but not PDE IV within TSM of this species; (2) modulation of the NANCi response by inhibitors of PDE IV and NEP in 13- but not 2-week old rabbits; (3) increased PDE IV and decreased NEP levels in the immune and I/C groups with reconstitution of NANCi responses by the combination of inhibitors. We conclude that mediation of the NANCi response is different in normal 2- and 13-week old rabbits. Both PDE IV and NEP modulated relaxation in fully grown rabbits, but had no effect at the younger age. Furthermore, both ragweed sensitization alone and ragweed challenge of immune rabbits altered NANCi via increases in PDE IV and decreases in NEP.

Antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung inflammation.

RATIONALE: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in children. No treatment has been shown to significantly improve the clinical outcome of patients with this infection. Recent evidence suggests that oxidative stress could play an important role in the pathogenesis of acute and chronic lung inflammatory diseases. We do not known whether RSV induces pulmonary oxidative stress and whether antioxidant treatment can modulate RSV-induced lung disease. OBJECTIVES: To investigate the effect of antioxidant administration on RSV-induced lung inflammation, clinical disease, and airway hyperreactivity (AHR). METHODS: BALB/c mice were infected with 10(7) plaque-forming units of RSV, in the presence or absence of orally administered butylated hydroxyanisole (BHA), an antioxidant. Malondialdehyde and 4-hydroxynonenal were measured in bronchoalveoar lavage (BAL) by colorimetric assay. Cytokines and chemokines were measured in BAL by Bio-Plex and leukotrienes were measured by enzyme-linked immunosorbent assay. AHR to methacholine challenge was measured by whole-body plethysmography. RESULTS: BHA treatment significantly attenuated RSV-induced lung oxidative stress, as indicated by the decrease of malondialdehyde and 4-hydroxynonenal content in BAL of RSV-infected mice. RSV-induced clinical illness and body weight loss were also reduced by BHA treatment, which inhibited neutrophil recruitment to the lung and significantly reduced pulmonary cytokine and chemokine production after RSV infection. Similarly, antioxidant treatment attenuated RSV-induced AHR. CONCLUSION: Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation and potentially prevent long-term consequences associated with RSV infection, such as bronchial asthma.

Effects of RSV infection on pulmonary surfactant protein SP-A in cultured human type II cells: contrasting consequences on SP-A mRNA and protein.

Respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory illness in infants and children. Surfactant proteins A (SP-A) and D (SP-D) play critical roles in lung defense against RSV infections. Alterations in surfactant protein homeostasis in the lung may result from changes in production, metabolism, or uptake of the protein within the lung. We hypothesized that RSV infection of the type II cell, the primary source of surfactant protein, may alter surfactant protein gene expression. Human type II cells grown in primary culture possess lamellar bodies (a type II cell-specific organelle) and the ability to express surfactant protein mRNA. These cells were infected with RSV (by morphology and antibody binding). Surfactant protein mRNA levels determined by quantitative RT-PCR indicated a marked increase in SP-A mRNA levels (3-fold) 24 h after RSV exposure, whereas SP-D mRNA levels were unaffected. In contrast to mRNA levels, total SP-A protein levels (determined by Western blot analysis) were decreased 40% after RSV infection. The percentage of secreted SP-A was 43% of the total SP-A in the RSV-infected cells, whereas the percentage of secreted SP-A was 61% of the total SP-A in the uninfected cells. These changes in SP-A transcript levels and protein secretion in cultured human cells were recapitulated in RSV-infected mouse lung. Our findings suggest that type II cells are potentially important targets of RSV lower respiratory infection and that alterations in surfactant protein gene expression and SP-A protein homeostasis in the lung may arise via direct effects of RSV.

Role of cysteinyl leukotrienes in airway inflammation and responsiveness following RSV infection in BALB/c mice.

Cysteinyl leukotrienes (CysLTs) contribute to the development of airway obstruction and inflammation in asthma; however little information is available on the role of these molecules in the pathophysiology of respiratory syncytial virus (RSV) bronchiolitis. This study was designed to evaluate the effects of RSV infection on CysLTs production in a well-established mouse infection model. Furthermore, we assessed the effect of anti-inflammatory agents (a leukotriene receptor antagonist, MK-571, and dexamethasone) on the functional and immune changes induced by RSV infection. Six to 8-wk-old BALB/c mice were infected with human RSV (strain A2). Measurements of airway function were performed using whole body plethysmography. Lung inflammation was assessed by cell counts, measurement of cytokines and CysLTs in bronchoalveolar lavage fluid (BALF) in the absence and presence of treatment with MK-571 or dexamethasone. RSV infection produced a marked increase in CysLTs in the BALF and lung tissue, recruitment of neutrophils and lymphocytes into the airways, increased IFN-gamma levels and airway hyperresponsiveness (AHR). Treatment with MK-571 decreased RSV-induced AHR without affecting the cellular and inflammatory responses to RSV. Dexamethasone decreased AHR and markedly reduced the recruitment of inflammatory cells and production of IFN-gamma. Our findings suggest CysLTs play an important role in the pathogenesis of RSV-induced airway dysfunction. Treatment with MK-571 decreases RSV-induced AHR but does not appear to alter the lung inflammatory responses to RSV. In contrast, dexamethasone decreases RSV-induced AHR but interferes with recruitment of inflammatory cells, resulting in decreased Th1 cytokines (a potentially Th2-prone environment) in this model. These studies support recent reports on the beneficial effects of CysLT receptor antagonist in human trials and provide a model for investigating the role of CysLTs in RSV bronchiolitis.

Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection.

BACKGROUND: Respiratory syncytial virus (RSV) infection of respiratory epithelial cell cultures increases expression of inducible nitric oxide synthase (iNOS). The present study was designed to evaluate both the effect of RSV infection on expression of iNOS and the role of NO in the host responses to RSV infection in vivo. METHODS: RSV infection was performed by nasal inoculation of BALB/c mice (6-8 weeks old). Total cell and differential counts were measured in bronchoalveolar lavage (BAL) fluid. Lung nitrates were measured in BAL fluid by use of the Greiss reaction, and cytokines were measured by enzyme-linked immunosorbent assay. Lung hyperresponsiveness to methacholine was measured by use of a Buxco unrestrained whole-body plethysmograph. RESULTS: RSV infection increased levels of lung nitrites, levels of iNOS protein and activity, and levels of iNOS mRNA. RSV infection resulted in recruitment of neutrophils and lymphocytes into the lungs, enhanced levels of interferon (IFN)-gamma, and increased airway hyperresponsiveness (AHR). Treatment with iNOS inhibitors (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and N-nitro-L-arginine methyl ester) increased RSV titers in the lungs yet reduced lung inflammation and RSV-induced AHR. Inhibition of iNOS activity with either agent did not significantly alter levels of IFN-gamma or interleukin-4 in the lungs. CONCLUSIONS: The results of the present study suggest that RSV-induced production of NO participates in complex host responses and may mediate important aspects of the clinical disease.