interleukin-8 and Respiratory-Syncytial-Virus-Infections

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in young infants. However, it is also a significant pathogen in older adults. Validated biomarkers of RSV disease severity would benefit diagnostics, treatment decisions, and prophylactic interventions. This review summarizes knowledge of biomarkers for RSV disease in adults.. A literature review was performed using Ovid Medline, Embase, Global health, Scopus, and Web of Science for articles published 1946-October 2016. Nine articles were identified plus 9 from other sources.. From observational studies of natural infection and challenge studies in volunteers, biomarkers of RSV susceptibility or disease severity in adults were: (1) lower anti-RSV neutralizing antibodies, where neutralizing antibody (and local IgA) may be a correlate of susceptibility/severity; (2) RSV-specific CD8+ T cells in bronchoalveolar lavage fluid preinfection (subjects with higher levels had less severe illness); and (3) elevated interleukin-6 (IL-6), IL-8, and myeloperoxidase levels in the airway are indicative of severe infection.. Factors determining susceptibility to and severity of RSV disease in adults have not been well defined. Respiratory mucosal antibodies and CD8+ T cells appear to contribute to preventing infection and modulation of disease severity. Studies of RSV pathogenesis in at-risk populations are needed.

Topics: Antibodies, Neutralizing; Biomarkers; Bronchiolitis; CD8-Positive T-Lymphocytes; Humans; Immunity, Cellular; Inflammation; Interleukin-6; Interleukin-8; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Severity of Illness Index; Viral Load

Infants with the respiratory syncytial virus (RSV) and human rhinovirus respiratory infection (HRV) produce inflammatory interleukins (ILs) in the respiratory epithelium. The aim of this study was to evaluate the levels of interleukin-8 in RSV negative and RSV positive patients. This study search was conducted without a time limit until 2020 through the databases of PubMed, Wiley, Springer, ScienceDirect and Google Scholar search engines, by two researchers independently. The random-effects model was used to compare of interleukin-8 in RSV negative vs. RSV positive patients, using Revman software version 5 meta-analysis software. Totally, 921 patients were evaluated (207 RSV-negative and 714 RSV-positive). The mean concentration of IL8 in RSV positive patients was 15.02 pg/ml (95% CI: 13.68- 16.35%).  According to the meta-analysis results, the standardized mean difference (SMD) of IL8 concentration between RSV-positive and negative patients was 6.31 pg/ml) (95% confidence interval: 2.50- 10.13%). subtotal analysis of the IL8 laboratory assessment method revealed that there was no significant SMD deference in the studies that have used chemiluminescence (P=0.21). while IL8 concentrations were significantly higher in RSV positives in ELISA and Magnetic bead-based assays (P<0.05).  It appears that RSV positive patients may have greater levels of IL8 than RSV negative ones; whereas the synthesis of IL8 tends to be more secreted into the nasopharyngeal space; whereas the evaluation approach can also affect the results.

Topics: Bronchiolitis; Databases, Factual; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-8; Luminescent Measurements; Nasopharynx; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

Immunization of BALB/c mice with vaccinia virus expressing the G glycoprotein (vvG) of respiratory syncytial virus (RSV) or with formalin-inactivated alum-precipitated RSV (FI-RSV) predisposes for severe illness, type 2 cytokine production and pulmonary eosinophilia after challenge with live RSV. This similar disease profile has led to the proposal that the presence of the G glycoprotein in the FI-RSV preparation was the immunologic basis for the vaccine-associated enhancement of disease observed in the failed clinical trials of the 1960s. However, processes of disease pathogenesis observed in FI-RSV- and vvG-immunized mice suggest that FI-RSV and vvG immunizations induce immune responses of different compositions and requirements that converge to produce similar disease outcomes upon live virus challenge.. The potential role of RSV G present in FI-RSV preparations in increasing postimmunization disease severity was explored in mice.. The absence of RSV G or its immunodominant epitope during FI-RSV immunization does not reduce disease severity after RSV challenge. Furthermore although depletion of V beta 14+ T cells during RSV challenge modulates disease in G-primed mice, minimal impact on disease in FI-RSV-immunized mice is observed.. FI-RSV vaccine-enhanced illness is not attributable to RSV G. Furthermore formulation of a safe and effective RSV vaccine must ensure RSV antigen production, processing and presentation via the endogenous pathways. Thus gene delivery by vector, by DNA or by live attenuated virus are attractive vaccine approaches.

Topics: Animals; Antigens, Viral; Disease Models, Animal; Epitopes; Female; Glycoproteins; Immunity; Immunization; Interleukin-4; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Pulmonary Eosinophilia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Sensitivity and Specificity; Severity of Illness Index; Substance P; Viral Vaccines

Respiratory syncytial virus (RSV) bronchiolitis in infancy is a major risk factor for recurrent wheezing and asthma. Because azithromycin attenuated neutrophilic airway inflammation in a murine viral bronchiolitis model, demonstration of similar effects in human subjects might provide a strategy for the prevention of postbronchiolitis recurrent wheezing.. We sought to investigate whether azithromycin treatment during RSV bronchiolitis reduces serum and nasal lavage IL-8 levels and the occurrence of postbronchiolitis recurrent wheezing.. We performed a randomized, double-masked, placebo-controlled proof-of-concept trial in 40 otherwise healthy infants hospitalized with RSV bronchiolitis who were treated with azithromycin or placebo for 14 days. IL-8 levels were measured in nasal lavage fluid and serum on randomization, day 8, and day 15 (nasal lavage only). The occurrence of wheezing episodes was assessed monthly over the ensuing 50 weeks.. Compared with placebo, azithromycin treatment did not reduce serum IL-8 levels at day 8 (P = .6) but resulted in a greater decrease in nasal lavage fluid IL-8 levels by day 15 (P = .03). Twenty-two percent of azithromycin-treated participants experienced at least 3 wheezing episodes compared with 50% of participants in the placebo group (P = .07). Azithromycin treatment resulted in prolonged time to the third wheezing episode (P = .048) and in fewer days with respiratory symptoms over the subsequent year in comparison with placebo (36.7 vs 70.1 days, P = .01).. In this proof-of-concept study azithromycin treatment during RSV bronchiolitis reduced upper airway IL-8 levels, prolonged the time to the third wheezing episode, and reduced overall respiratory morbidity over the subsequent year.

Topics: Azithromycin; Bronchiolitis, Viral; Disease Progression; Female; Follow-Up Studies; Humans; Infant; Infant, Newborn; Interleukin-8; Male; Nasal Lavage Fluid; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Risk Factors; Treatment Outcome

Respiratory syncytial virus (RSV) bronchiolitis is the most common lower respiratory tract infection in infancy. To date, there is no effective therapy for RSV bronchiolitis. In order to investigate the efficacy of clarithromycin in the treatment of RSV bronchiolitis, the present authors conducted a randomised, double-blind, placebo-controlled trial comparing clarithromycin with placebo in 21 infants with a diagnosis of RSV bronchiolitis. The infants were randomised to receive clarithromycin or placebo daily for 3 weeks. Levels of interleukin (IL)-4, IL-8, eotaxin, and interferon-gamma were determined in plasma, before and after treatment, using ELISA. Six months after treatment, parents were surveyed as to whether their child had experienced wheezing within the previous 6 months. Treatment with clarithromycin was associated with a statistically significant reduction in the length of hospital stay, the duration of need for supplemental oxygen and the need for beta(2)-agonist treatment. There were significant decreases in plasma IL-4, IL-8 and eotaxin levels after 3 weeks of treatment with clarithromycin. Readmission to the hospital within 6 months after discharge was significantly lower in the clarithromycin group. In conclusion, clarithromycin has statistically significant effects on the clinical and laboratory findings in respiratory syncytial virus bronchiolitis. Therefore, clarithromycin treatment may be helpful in reducing the short-term effects of respiratory syncytial virus bronchiolitis.

Topics: Anti-Bacterial Agents; Bronchiolitis, Viral; Chemokine CCL11; Chemokines, CC; Clarithromycin; Double-Blind Method; Drug Administration Schedule; Female; Humans; Infant; Interferon-gamma; Interleukin-4; Interleukin-8; Length of Stay; Male; Respiratory Syncytial Virus Infections

Lower respiratory tract infection caused by respiratory syncytial virus (RSV) is in part an immune-mediated disease. For that reason corticosteroids might be effective, especially in patients with severe RSV lower respiratory tract infection. Our aim was to assess the effect of dexamethasone on tracheal viral load and airway inflammation in patients with RSV infection.. Mechanically ventilated children with proven RSV infection were randomized to receive dexamethasone (0.6 mg/kg/day in four doses for 48 h) or placebo. Daily tracheal aspirates were analyzed for viral load (by quantitative polymerase chain reaction), interleukin (IL)-8 and white blood cell count.. The RSV RNA concentrations decreased in a similar manner from baseline in the dexamethasone (9 patients) and in the placebo group (13 patients). IL-8 decreased from baseline in the dexamethasone group but increased in the placebo group during the first 48 h [change from baseline at 24 h, -2.3 vs. 0.9 ln ng/ml (95% confidence interval for difference, -4.2 to 0.3, P = 0.02) and at 48 h, -4.2 vs. 0.4 ln mg/ml (95% confidence interval for difference, -5.3 to -0.3; P = 0.03), respectively], without effect on the tracheal white blood cell count.. Dexamethasone does not cause an impaired decline of tracheal RSV but lowers IL-8 of children mechanically ventilated for RSV lower respiratory tract infection, potentially leading to less inflammation and reduced phagocyte activation.

Topics: Base Sequence; Dexamethasone; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Follow-Up Studies; Humans; Interleukin-8; Male; Molecular Sequence Data; Polymerase Chain Reaction; Probability; Respiration, Artificial; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; RNA, Viral; Severity of Illness Index; Statistics, Nonparametric; Trachea; Treatment Outcome; Viral Load

The goal of this study was to determine time courses of upregulation of several chemokines in nasal secretions after inoculation of human subjects with a low dose of live respiratory syncytial virus (RSV). Healthy, nonsmoking young adults were admitted to an inpatient clinical research unit. After baseline studies, subjects were nasally inoculated with approximately 10(3) plaque-forming units of RSV (strain A2), followed by daily nasal lavages. Nasal lavage fluid (NLF) was assayed for chemokines by specific ELISA. Of 10 subjects inoculated with RSV, 3 developed clinical symptoms of upper respiratory infection and also shed virus. Among infected subjects, there was a transient postinoculation increase in interleukin-8 (IL-8) in NLF to an average of 2.7-fold compared to baseline, followed by a prolonged increase (maximum mean 5.4-fold) during virus shedding. RANTES, MIP-1alpha, and MCP-1 all increased during virus shedding only (maximum mean increases of 5.3-fold, 13-fold, and 7.2-fold, respectively). Semiquantitative RT-PCR in brushed nasal epithelial cells on day 6 after inoculation suggested upregulation of RANTES, but not IL-8, mRNA during virus shedding. We conclude that chemokines IL-8, RANTES, MIP-1alpha, and MCP-1 are all increased in nasal secretions in human RSV infection at the time of virus shedding and symptomatic illness and that the epithelium lining the nasal turbinate contributes to the increase in RANTES.

Topics: Adolescent; Adult; Albumins; Chemokine CCL5; Chemokines; Epithelium; Humans; Interleukin-8; Leukocyte Count; Nasal Lavage Fluid; Nasal Mucosa; Neutrophils; Respiratory Syncytial Virus Infections; RNA, Messenger

The role of cellular immunity in disease severity in respiratory syncytial virus (RSV) bronchiolitis is largely unknown. This study investigated the association between disease severity and systemic cytokine responses in hospitalized ventilated and nonventilated RSV bronchiolitis patients. In whole blood cultures stimulated with phytohaemagglutinin (PHA), lymphoproliferative responses and interferon (IFN)-gamma and interleukin (IL)-4 production during acute illness were measured. In addition, plasma cytokines were measured. Measurements were repeated in the convalescent phase, 3-4 weeks after admission. Fifty patients were included. The median age in ventilaled patients was significantly lower than in nonventilated patients (1 versus 4 months, p<0.05). In comparison with nonventilated patients, the ventilated patients had significantly lower lymphoproliferative responses and a lower production of IFN-gamma and IL-4. In fact, IFN-gamma and IL-4 production in ventilated patients was almost completely undetectable. Plasma IL-8 levels in ventilated patients were significantly higher than in nonventilated patients. In the convalescent phase, lymphoproliferative and cytokine responses as well as plasma IL-8 levels were normal in both patient groups. Since RSV bronchiolitis is associated with the subsequent development of asthma, the possible skewing of the T-helper (Th1/Th2) cytokine balance was investigated. This was found neither in the acute nor in the convalescent phase. In conclusion, the data indicate that depressed lymphocyte function and elevated plasma interleukin-8 levels are markers of severe disease. It is suggested that age and maturation related immune mechanisms could explain the occurrence of severe respiratory syncytial virus bronchiolitis requiring mechanical ventilation in young infants.

Topics: Antibodies, Viral; Biomarkers; Bronchiolitis, Viral; Cells, Cultured; Female; Humans; Infant; Infant, Newborn; Interferon-gamma; Interleukin-12; Interleukin-4; Interleukin-8; Male; Phytohemagglutinins; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Severity of Illness Index; Th1 Cells; Th2 Cells

Viral respiratory infection causes inflammatory lung disease. Chitinase 3-like 1 (CHI3L1) contributes to airway inflammation, but its role in human airway epithelial cells following viral infection is unclear. Thus, we investigated whether CHI3L1 regulates inflammatory responses caused by viral infections in airway epithelial cells. Human bronchial epithelial cells, BEAS-2B, were stimulated with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)). To confirm the specific role of CHI3L1, CHI3L1 was knocked down in BEAS-2B cells using shRNA lentivirus. The expression of CHI3L1 and proinflammatory cytokines such as IL-8 and phosphorylation of mitogen-activated protein kinase (MAPK) pathways were analyzed. In addition to poly(I:C), BEAS-2B cells were infected with the human respiratory syncytial virus (RSV) A2 strain, and CHI3L1 and IL-8 expression was analyzed. Stimulating the cells with poly(I:C) increased CHI3L1 and IL-8 expression, whereas IL-8 expression was abrogated in CHI3L1 knockdown BEAS-2B cells. Poly(I:C) stimulation of BEAS-2B cells resulted in phosphorylation of MAPK pathways, and inhibition of MAPK pathways significantly abolished IL-8 secretion. Phosphorylation of MAPK pathways was diminished in CHI3L1 knockdown BEAS-2B cells. Infection with RSV increased CHI3L1 and IL-8 expression. IL-8 expression induced by RSV infection was abrogated in CHI3L1 knockdown cells. In conclusion, CHI3L1 may be involved in IL-8 secretion by regulating MAPK pathways during respiratory viral infections in airway epithelial cells.

Topics: Cell Line; Chitinase-3-Like Protein 1; Cytokines; Epithelial Cells; Humans; Inflammation Mediators; Interleukin-8; Lung; MAP Kinase Signaling System; Phosphorylation; Poly I-C; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; RNA, Double-Stranded

Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV).. HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion was assessed and compared with that of RSV.. HRV-Cs infect and propagate in fully differentiated HBE cells and significantly increase the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus loads positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P = 0.048), IL-6 (P = 0.016), MCP-1 (P = 0.008), and IL-8 (P = 0.032), and similar secretion of IP10 (P = 0.214) and IFN-λ1 (P = 0.214) when compared with RSV.. HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV.

Topics: Chemokine CXCL10; Cytokines; Enterovirus; Epithelial Cells; Humans; Immunity; Interleukin-6; Interleukin-8; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Rhinovirus

Lower respiratory tract infections (LRTIs) produced by viruses are the most frequent cause of morbidity and mortality in children younger than 5 years of age. The immune response triggered by viral infection can induce a strong inflammation in the airways and cytokines could be considered as biomarkers for disease severity as these molecules modulate the inflammatory response that defines the outcome of patients. Aiming to predict the severity of disease during respiratory tract infections, we conducted a 1-year follow-up observational study in infants who presented upper or lower respiratory tract infections caused by seasonal respiratory viruses. At the time of enrollment, nasopharyngeal swabs (NPS) were obtained from infants to measure mRNA expression and protein levels of IL-3, IL-8, IL-33, and thymic stromal lymphopoietin. While all cytokines significantly increased their protein levels in infants with upper and lower respiratory tract infections as compared to control infants, IL-33 and IL-8 showed a significant increase in respiratory syncytial virus (RSV)-infected patients with LRTI as compared to patients with upper respiratory tract infection. We also found higher viral loads of RSV-positive samples with a greater IL-8 response at the beginning of the symptoms. Data obtained in this study suggest that both IL-8 and IL-33 could be used as biomarkers for clinical severity for infants suffering from LRTIs caused by the RSV.

Topics: Biomarkers; Child; Cytokines; Humans; Infant; Interleukin-3; Interleukin-33; Interleukin-8; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory Tract Infections; RNA, Messenger; Severity of Illness Index; Viruses

Topics: Female; Haemophilus; Haemophilus Infections; Humans; Immunity; Infant; Interleukin-6; Interleukin-8; Male; Nasopharynx; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Viral Load

We aimed to use serum metabolomics to discriminate infants with severe respiratory syncytial virus (RSV) bronchiolitis who later developed subsequent recurrent wheezing from those who did not and to investigate the relationship between serum metabolome and host immune responses with regard to the subsequent development of recurrent wheezing. Fifty-one infants who were hospitalized during an initial episode of severe RSV bronchiolitis at 6 months of age or less were included and followed for up to the age of 3 years. Of them, 24 developed subsequent recurrent wheezing and 27 did not. Untargeted serum metabolomics was performed by ultraperformance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-MS/MS). Cytokines were measured by multiplex immunoassay. Difference in serum metabolomic profiles was observed between infants who developed recurrent wheezing and those who did not. L-lactic acid level was significantly higher in infants with recurrent wheezing than those without. Pyrimidine metabolism, glycerophospholipid metabolism, and arginine biosynthesis were identified as the most significant changed pathways between the two groups. Moreover, L-lactic acid level was positively associated with serum CXCL8 level. This exploratory study showed that differential serum metabolic signatures during severe RSV bronchiolitis in early infancy were associated with the development of subsequent recurrent wheezing in later childhood.

Topics: Bronchiolitis; Child, Preschool; Chromatography, High Pressure Liquid; Cytokines; Female; Follow-Up Studies; Humans; Infant; Interleukin-8; Lactic Acid; Male; Metabolomics; Pyrimidines; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Serum; Tandem Mass Spectrometry

Early interactions between respiratory viruses and microbiota might modulate host immune responses and subsequently contribute to later development of recurrent wheezing and asthma in childhood. We aimed to study the possible association between respiratory microbiome, host immune response, and the development of recurrent wheezing in infants with severe respiratory syncytial virus (RSV) bronchiolitis.. Seventy-four infants who were hospitalized at Beijing Children's Hospital during an initial episode of severe RSV bronchiolitis at 6 months of age or less were included and followed up until the age of 3 years. Sputum samples were collected, and their microbiota profiles, LPS, and cytokines were analyzed by 16S rRNA-based sequencing, ELISA, and multiplex immunoassay, respectively.. Twenty-six (35.1%) infants developed recurrent wheezing by the age of 3 years, and 48 (64.9%) did not. The relative abundance of Haemophilus, Moraxella, and Klebsiella was higher in infants who later developed recurrent wheezing than in those who did not (LDA score >3.5). Airway levels of LPS (P = .003), CXCL8 (P = .004), CCL5 (P = .029), IL-6 (P = .004), and IL-13 (P < .001) were significantly higher in infants who later developed recurrent wheezing than in those who did not. Moreover, high airway abundance of Haemophilus was associated with CXCL8 (r = 0.246, P = .037) level, and that of Moraxella was associated with IL-6 level (r = 0.236, P = .046) and IL-10 level (r = 0.266, P = .024).. Our study suggests that higher abundance of Haemophilus and Moraxella in airway microbiome might modulate airway inflammation during severe RSV bronchiolitis in infancy, potentially contributing to the development of subsequent recurrent wheezing in later childhood.

Topics: Asthma; Beijing; Bronchiolitis; Child, Preschool; Female; Humans; Immunity; Infant; Interleukin-10; Interleukin-13; Interleukin-8; Male; Microbiota; Prospective Studies; Recurrence; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory System; RNA, Ribosomal, 16S; Sputum

Mechanisms influencing severity of acute lower respiratory infection (ALRI) in children are not established. We aimed to assess the role of inflammatory markers and respiratory viruses in ALRI severity.. Concentrations of interleukin(IL)-33, soluble suppression of tumorigenicity (sST)2, IL-1ß, tumor necrosis factor α, IL-4, IL-6 and IL- 8 and types of respiratory viruses were evaluated in children at the first and fifth days after hospital admission. Disease severity was defined as need for mechanical ventilation.. Seventy-nine children <5 years-old were included; 33(41.8%) received mechanical ventilation. No associations between virus type, viral load or co-detections and severity of disease were observed. Detection of IL-33 and sST2 in nasopharyngeal aspirates (NPA) on admission were associated with higher risk for mechanical ventilation (RR = 2.89 and RR = 4.57, respectively). IL-6 and IL-8 concentrations were higher on Day 5 in mechanically ventilated children. IL-6 NPA concentrations decreased from Day 1 to Day 5 in children who did not receive mechanical ventilation. Increase in sST2 NPA concentrations from Day 1 to Day 5 was associated with longer hospital length of stay (p < 0.01).. An exacerbated local activation of the IL-33/ST2 axis and persistently high sST2 concentrations over time were associated with severity of viral ALRI in children.

Topics: Biomarkers; Child, Preschool; Female; Hospitalization; Humans; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Interleukin-6; Interleukin-8; Male; Prospective Studies; Respiratory Syncytial Virus Infections; Respiratory Tract Infections; Severity of Illness Index

Baicalein, an isolate of secutellaria baicalensis is known for its anti-inflammatory activity. In the present study, 12-triazole derivatives of baicalein were synthesized and evaluated against RSV infected BEAS-2B cells in vitro and in mice model in vivo. The preventive effect of most active compound 5f against RSV infection was studied in detail. The compound 5f treatment increased IFN-β1 expression in BEAS-2B cells infected with RSV. In BEAS-2B cells treatment with compound 5f inhibited RSV-induced secretion of interleukin-6 and -8 cytokines. It decreased RSV-induced nitric oxide & malondialdehyde production and inhibited the RSV-mediated activation of NF-κB, COX-2, Stat3 and MAPK. The p38 phosphorylation was enhanced significantly in RSV infected cells by compound 5f pre-treatment. RT-qPCR showed that compound 5f treatment of the RSV-infected mice significantly (P < 0.05) decreased viral load through reduction in the viral replication. In the mice model of RSV-infection compound 5f treatment decreased interleukin-6, -8 and tumor necrosis factor-α expression. The level of MPO, nitric oxide and malondialdehyde was also decreased significantly by compound 5f in the RSV infected mice BALF. It also reduced the infiltration of neutrophils and lymphocytes in the BALF of RVS-infected mice. In summary, compound 5f inhibits RSV-infection and prevents pulmonary airway inflammation through the activation of IFN signalling pathway.

Topics: Animals; Cytokines; Female; Flavanones; Interferon-beta; Interleukin-6; Interleukin-8; Lymphocytes; Mice; Mice, Inbred BALB C; Neutrophils; NF-kappa B; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pneumonia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory Tract Infections; Ribavirin; STAT3 Transcription Factor; Triazoles; Tumor Necrosis Factor-alpha; Virus Replication

Maternally expressed gene 3 (MEG3), a long noncoding RNA (lncRNA) has been dysregulated in various tumors. However, the expression level and functional role of MEG3 in the progression of respiratory syncytial virus (RSV) infection remains to be elucidated. The present study quantified the expression level of MEG3 in the nasopharyngeal (NPA) samples of RSV‑infected patients and in BEAS‑2B cells infected with RSV. The findings of the present study demonstrated that the expression level of lncRNA MEG3 was reduced in the NPA samples of RSV‑infected patients and in BEAS‑2B cells infected with RSV. In vitro transfection revealed increased mRNA expression levels of toll‑like receptor 4 (TLR4), tumor necrosis factor‑α (TNFα) and interleukin (IL)‑8 following RSV infection in BEAS‑2B cells. Additionally, ectopic expression of MEG3 reduced the expression level of TLR4, subsequently suppressing the mRNA expression levels of TNFα and IL‑8, indicating the protective role of MEG3 in the process of RSV infection. It is of note, that RSV infection‑induced p38 mitogen activated protein kinase (MAPK) and nuclear factor‑κB (NF‑κB) activation was partly abolished by overexpression of MEG3. In conclusion, to the best of our knowledge, the present study provided the first evidence that lncRNA MEG3 expression level was reduced in the NPA samples of patients with RSV infection and RSV‑infected cells. Additionally, it was demonstrated that MEG3 protected human airway epithelial cells from RSV infection, primarily by suppressing TLR4‑dependent p38 MAPK and NF‑κB signaling.

Topics: Bronchiolitis, Viral; Cell Line; Child; Child, Preschool; Epithelial Cells; Female; Gene Expression Regulation; Host-Pathogen Interactions; Humans; Interleukin-8; Length of Stay; Male; Nasopharynx; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Long Noncoding; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

While almost all infants are infected with respiratory syncytial virus (RSV) before the age of 2 years, only a small percentage develops severe disease. Previous studies suggest that the nasopharyngeal microbiome affects disease development. We therefore studied the effect of the nasopharyngeal microbiome on viral load and mucosal cytokine responses, two important factors influencing the pathophysiology of RSV disease. To determine the relation between (i) the microbiome of the upper respiratory tract, (ii) viral load, and (iii) host mucosal inflammation during an RSV infection, nasopharyngeal microbiota profiles of RSV infected infants (< 6 months) with different levels of disease severity and age-matched healthy controls were determined by 16S rRNA marker gene sequencing. The viral load was measured using qPCR. Nasopharyngeal CCL5, CXCL10, MMP9, IL6, and CXCL8 levels were determined with ELISA.. Viral load in nasopharyngeal aspirates of patients associates significantly to total nasopharyngeal microbiota composition. Healthy infants (n = 21) and RSV patients (n = 54) display very distinct microbial patterns, primarily characterized by a loss in commensals like Veillonella and overrepresentation of opportunistic organisms like Haemophilus and Achromobacter in RSV-infected individuals. Furthermore, nasopharyngeal microbiota profiles are significantly different based on CXCL8 levels. CXCL8 is a chemokine that was previously found to be indicative for disease severity and for which we find Haemophilus abundance as the strongest predictor for CXCL8 levels.. The nasopharyngeal microbiota in young infants with RSV infection is marked by an overrepresentation of the genus Haemophilus. We present that this bacterium is associated with viral load and mucosal CXCL8 responses, both which are involved in RSV disease pathogenesis.

Topics: DNA, Bacterial; DNA, Ribosomal; Female; Haemophilus; Hospitalization; Humans; Infant; Infant, Newborn; Interleukin-8; Male; Nasopharynx; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Up-Regulation; Viral Load

Hospitalization with bronchiolitis is linked to the development of early childhood chronic wheeze and asthma. Viral etiology and severity of inflammation are potential contributing factors. Previously we observed reduced airway neutrophil infiltration in breastfed bronchiolitic infants, with a corresponding reduction in disease severity. This study aimed to examine whether respiratory viral etiology and co-infection alters the pattern of neutrophil influx, and the inflammatory mediator profile, resulting in epithelial damage in bronchiolitis.. Nasopharyngeal aspirates (NPAs) collected from hospitalized infants were assessed for viruses, soluble protein, cellular infiltrate, interleukin (IL)-6, -8, and myeloperoxidase (MPO).. NPAs were collected from 228 bronchiolitic and 14 non-bronchiolitic infants. In the bronchiolitic cohort, human rhinovirus was most prevalent (38%), followed by respiratory syncytial virus (36%), adenovirus (10%), and human metapneumovirus (6%), with 25% positive for viral co-infections and 25% negative for all screened viruses. Viral-induced bronchiolitis was associated with increased cellular infiltrate and protein, above control, and virus-negative infants (P < 0.05). Cellular infiltrate correlated to IL-6, -8, and MPO (r = 0.331, 0.669, and 0.661; P < 0.01). Protein, IL-6, -8, and MPO differed significantly between viral groups; however, the majority of marker values for all groups fall within an overlapping, indistinguishable range, precluding their use as biomarkers of viral etiology. No significant difference was found between single and viral co-infections for any parameter.. Bronchiolitic infants presenting with a detectable respiratory virus during hospitalization demonstrated elevated markers of airway tissue inflammation and injury. In this cohort, viral etiology did not discernibly modulate chemokine-mediated neutrophil infiltration and activation. Pediatr Pulmonol. 2017;52:238-246. © 2016 Wiley Periodicals, Inc.

Topics: Adenoviridae; Adenoviridae Infections; Breast Feeding; Bronchiolitis; Bronchiolitis, Viral; Coinfection; Female; Humans; Immunoassay; Infant; Inflammation; Interleukin-6; Interleukin-8; Male; Metapneumovirus; Nasopharynx; Neutrophil Infiltration; Neutrophils; Paramyxoviridae Infections; Peroxidase; Picornaviridae Infections; Polymerase Chain Reaction; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Rhinovirus; Severity of Illness Index

Primary respiratory syncytial virus (RSV) infections are characterized by high levels of IL-8 and an intense neutrophilia. Little is known about the cytokine responses in secondary infections. Preschool children experiencing RSV secondary infections were recruited from the siblings of infants admitted to hospital with RSV acute bronchiolitis.. Fifty-one infants with acute bronchiolitis (39 RSV positive, 12 RSV negative) and 20 age-matched control infants were recruited. In addition, seven older siblings of infants from the RSV-positive cohort and confirmed RSV infection were recruited. Samples of nasal secretions were obtained using a flocked swab, and secretions extracted using centrifugation. Cytokine bead array was used to obtain levels of interleukin (IL)-17A, IL-8, IL-6, IL-21, and tumor necrosis factor-α.. Levels of IL-8 and IL-6 were significantly lower in the RSV-positive siblings compared with the RSV-positive infants. There were no significant differences between levels of the other cytokines in the primary and secondary infections.. The very high levels of IL-8 and IL-6 response characteristic of the primary RSV infection was not observed in secondary RSV-positive infections and this did not appear to be due to a global reduction in cytokine production.

Topics: Bronchiolitis; Case-Control Studies; Cohort Studies; Cytokines; Epidemics; Female; Humans; Infant; Infant, Newborn; Interleukin-6; Interleukin-8; Male; Neutrophils; Patient Admission; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Seasons; Siblings

To explore the mechanisms for an increase in susceptibility of asthma induced by respiratory syncytial virus (RSV), to observe the expression of interleukin-8 (IL-8) in human bronchial epithelial cells (HBECs) after RSV infection and to invesigate the regulatory effect of IL-8 on Th17/Treg differentiation.
. HBECs were divided into a control group and a RSV infected group. The RSVE-infected model of HBECs was established and examined. The expression of IL-8 mRNA was detected by real-time PCR, and the levels of IL-8 were measured by ELISA. Peripheral blood lymphocytes in healthy people were extracted and divided into a control group and an IL-8 treatment group. Based on concentration of IL-8 in RSV-infected HBECs, lymphocytes were treated by a matched concentration of human recombinant IL-8 for 24 h. The distribution of Th17 and Treg subsets in lymphocytes were examined by flow cytometry.
. The RSV-infected HBECs model was successfully established. The infected HBECs were still able to split and passage. The RSV could be detected in every passage in the infected cells. Virus particles indicated by bright yellow green fluorescence were seen under fluorescence microscope. Edema of mitochondrias, expansion of endoplasmic reticulum, fissure around nucleus and intracellular virus particles were all observed under electron microscope. The expression IL-8 mRNA were significantly enhanced in the RSV-infected group, and the level of IL-8 in the RSV-infected group was higher than that in the control group (P<0.05). After IL-8 treatment for 24 h, the ratio of Th17 subsets in lymphocytes were dramatically increased compared to the control group (P<0.05), but there was no difference in the ratio of Treg subsets between the 2 groups (P>0.05).
. Over-secretion of IL-8 by the RSV-infected HBECs may promote the differentiation of Th17 subsets and maintain the Th17/Tred imbalance.. 目的：探讨呼吸道合胞病毒(respiratory syncytial virus，RSV)感染导致哮喘易感性增加的机制，观察人支气管上皮细胞感染RSV后IL-8的表达，以及IL-8对Th17/调节性T淋巴细胞(regulatory T cells，Treg)分化的调节作用。
方法：将人支气管上皮细胞(human bronchial epithelial cells，HBECs)分为对照组和RSV感染组，构建并验证RSV持续感染HBECs模型，real-time PCR检测对照组和RSV感染组中IL-8 mRNA的表达；ELISA检测感染细胞上清液中IL-8的浓度。提取健康人外周血淋巴细胞并将其分为空白对照组和IL-8作用组，参照ELISA检测到的浓度将IL-8作用于淋巴细胞24 h，采用流式细胞仪检测淋巴细胞中Th17和Treg亚群的分布情况。结果：本实验成功构建RSV持续感染HBECs模型，感染后的细胞仍能够继续分裂传代，并可检测到RSV持续存在的证据。免疫荧光显示细胞内有RSV致病蛋白的荧光表达；电镜下观察到感染细胞的线粒体水肿和内质网扩张，出现核周裂隙以及合胞现象，细胞核、胞浆内有病毒颗粒分布。Real-time PCR和ELISA分别检测到RSV感染组细胞内IL-8 mRNA表达和上清液中IL-8的分泌水平均高于对照组(均P<0.05)。进一步将IL-8作用于淋巴细胞，流式细胞学结果表明IL-8作用组淋巴细胞中Th17亚群比率增高(P<0.05)，但Treg亚群比率与对照组比较，差异无统计学意义(P>0.05)。结论：RSV感染气道上皮细胞后可通过过度分泌IL-8而引起Th17/Treg亚群分化异常。.

Topics: Cell Differentiation; Cells, Cultured; Epithelial Cells; Flow Cytometry; Humans; Interleukin-8; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; T-Lymphocytes, Regulatory; Th17 Cells

Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD.

Topics: Cell Proliferation; Chemokine CXCL12; Epithelial Cells; Fetal Blood; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-beta; Interferon-gamma; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; Lung; Mesenchymal Stem Cells; Microscopy, Fluorescence; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

Nontypeable Haemophilus influenzae (NTHI), a common colonizer of lungs of patients with chronic obstructive pulmonary disease (COPD), can enhance expression of the cellular receptor intercellular adhesion molecule 1 (ICAM-1), which in turn can be used by major group human rhinoviruses (HRVs) for attachment. Here, we evaluated the effect of NTHI-induced up-regulation of ICAM-1 on viral replication and inflammatory responses toward different respiratory viruses. Therefore, human bronchial epithelial cells were pretreated with heat-inactivated NTHI (hi-NTHI) and subsequently infected with either HRV16 (major group), HRV1B (minor group), or respiratory syncytial virus (RSV). Pretreatment with hi-NTHI significantly up-regulated ICAM-1 in BEAS-2B cells and primary bronchial epithelial cells. Concomitantly, release of infectious HRV16 particles was increased in cells pretreated with hi-NTHI. Pretreatment with hi-NTHI also caused a significant increase in HRV16 RNA, whereas replication of HRV1B and RSV were increased to a far lesser extent and only at later time points. Interestingly, release of IL-6 and IL-8 after RSV, but not HRV, infection was synergistically increased in hi-NTHI-pretreated BEAS-2B cells. In summary, exposure to hi-NTHI significantly enhanced sensitivity toward HRV16 but not HRV1B or RSV, probably through ICAM-1 up-regulation. Furthermore, hi-NTHI pretreatment may enhance the inflammatory response to RSV infection, suggesting that preexisting bacterial infections might exaggerate inflammation during secondary viral infection.

Topics: Bronchi; Cells, Cultured; Disease Susceptibility; Epithelial Cells; Haemophilus Infections; Haemophilus influenzae; Humans; Immunoblotting; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Polymerase Chain Reaction; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Viral; Virus Replication

Respiratory syncytial virus (RSV) is a negative-strand RNA virus that is an important cause of bronchiolitis and pneumonia. We investigated the effect of RSV infection on the expression patterns of cellular proteins involved in regulating mRNA translation and degradation, and found that a processing-body protein involved in mRNA degradation, decapping protein 1a (DCP1), was phosphorylated rapidly following infection. UV-inactivated and sucrose-purified RSV were sufficient to mediate DCP1 phosphorylation, indicating that it occurs as a consequence of an early event in RSV infection. Analysis using kinase inhibitors showed that RSV-induced DCP1 phosphorylation occurred through the ERK1/2 pathway. The DCP1 phosphorylation sites were limited to serine 315, serine 319, and threonine 321. Overexpression of wt DCP1 led to a decrease in RSV-induced IL-8 production, but this effect was abrogated in cells overexpressing phosphorylation-deficient DCP1 mutants. These results suggest that DCP1 phosphorylation modulates the host chemokine response to RSV infection.

Topics: Amino Acid Motifs; Endoribonucleases; Extracellular Signal-Regulated MAP Kinases; Hep G2 Cells; Humans; Interleukin-8; Phosphorylation; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Trans-Activators

Topics: Azithromycin; Bronchiolitis, Viral; Female; Humans; Interleukin-8; Male; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

Topics: Azithromycin; Bronchiolitis, Viral; Female; Humans; Interleukin-8; Male; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

Panax ginseng has been known to have a number of immuno-modulatory effects. In this study, we investigated whether Panax Korean red ginseng extract (KRGE) has in vitro and in vivo antiviral effects on respiratory syncytial virus (RSV) infection. KRGE improved the survival of human lung epithelial cells against RSV infection and inhibited RSV replication. In addition, KRGE treatment suppressed the expression of RSV-induced inflammatory cytokine genes (IL-6 and IL-8) and the formation of reactive oxygen species in epithelial cell cultures. Oral administration of mice with KRGE resulted in lowering lung viral loads after RSV infection. Additionally, the in vivo effects of KRGE showed an enhanced level of interferon-γ (IFN-γ) producing dendritic cells subsequent to RSV infection. Taken together, these results suggested that KRGE has antiviral activity against RSV infection.

Topics: Administration, Oral; Animals; Antiviral Agents; Cell Line; Cell Survival; Dendritic Cells; Epithelial Cells; Female; Host-Pathogen Interactions; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Lung; Mice; Mice, Inbred BALB C; Panax; Plant Extracts; Reactive Oxygen Species; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Viral Load; Virus Replication

This study aimed to determine the immunohistochemical expression of interleukin (IL)-1β, tumour necrosis factor alpha (TNF)-α, interferon (IFN)-γ, IL-4, IL-6, IL-8, IL-10 and IL-12 and to measure the concentrations of these cytokines in lung tissue from lambs infected experimentally with bovine respiratory syncytial virus (BRSV). Lambs (n = 15) were inoculated at 2 days of age with 20 ml of viral inoculum (1.26 × 10(6) TCID50 per ml) or sterile medium (n = 15). Rectal temperature, pulse and respiratory rates were monitored daily in control and infected lambs. Lambs were killed and subject to necropsy examination at 1, 3, 5, 7 and 15 days post inoculation (dpi). There was a temporal association between pulmonary expression of these cytokines and lung pathology in BRSV-infected lambs. The cytokines IL-4 and IL-10 were not elevated, but there was a significant increase in IL-1β, TNF-α, IFN-γ and IL-6 proteins and labelled cells, suggesting that these cytokines may play a role in the biological response to BRSV infection and contribute to the development of lung lesions. There was also a significant increase in the cytokine concentration and number of immunolabelled cells expressing IL-8 and IL-12 in infected lungs, suggesting that these cytokines might be used as therapeutic targets in the management of BRSV, in conjunction with measures to combat the causative pathogen and prophylactic methods aimed at preventing infection.

Topics: Animals; Cytokines; Interleukin-12; Interleukin-8; Lung; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Bovine; Sheep; Sheep Diseases

Respiratory syncytial virus (RSV) is a leading cause of respiratory duct infection that can result in severe clinical symptoms, particularly among children under 3 years of age. In the current study, the effect of RSV on airway epithelial cell function and the potential signaling pathways involved were investigated. A549 human airway epithelial cells were infected with RSV at a multiplicity of infection of 1. After 24 h, interleukin (IL)‑8 secretion in the cell supernatant was analyzed. A microarray assay of RSV‑infected A549 cells was conducted in order to identify any potential pathways involved, and quantitative polymerase chain reaction was performed to examine mRNA expression levels in these pathways. Electrophoretic mobility shift assays of nuclear transcription factors were conducted for further verification. IL‑8 levels increased significantly in the supernatant of RSV‑infected A549 cells compared with levels in non‑infected cells. Microarray data suggested the involvement of the Toll‑like receptor 4 (TLR4) pathway, and mRNA expression levels of genes (MYD88, TRAM and TRIF) involved in this pathway were higher in infected cells. Enhanced synthesis of activator protein‑1 (AP‑1) was observed. RSV infection of A549 cells may promote IL‑8 secretion. In conclusion, the results of the present study indicate that the TLR4 signaling pathway, in conjunction with MYD88, TRAM, TRIF and the transcription factor AP‑1, may activate immune responses to RSV infection in airway epithelial cells.

Topics: Activating Transcription Factor 1; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Cell Line, Tumor; Epithelial Cells; Humans; Interleukin-8; Microarray Analysis; Myeloid Differentiation Factor 88; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Messenger; Signal Transduction

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 μg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60-120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.

Topics: Administration, Intranasal; Analysis of Variance; Animals; Antiviral Agents; Apolipoprotein A-I; Bronchoalveolar Lavage Fluid; Cell Line; Drug Delivery Systems; Female; Humans; Interleukin-8; Lipid Bilayers; Lung; Mice; Mice, Inbred BALB C; Molecular Dynamics Simulation; Nanostructures; Phosphatidylglycerols; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Virus Attachment; Virus Cultivation

Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and significant morbidity and mortality in the elderly, immunosuppressed, and immunocompromised patients and in patients with chronic lung diseases. Recently, we reported that the pulmonary surfactant phospholipid palmitoyl-oleoyl-phosphatidylglycerol (POPG) inhibited RSV infection in vitro and in vivo by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice markedly attenuated infection and associated inflammatory responses. Based on these findings, we expanded our studies to determine whether POPG is effective for prophylaxis and postinfection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2-1.0 mg/ml at 24 h after RSV infection of HEp-2 cells suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2-6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making postinfection application impractical. Intranasal administration of POPG (0.8-3.0 mg), 45 min before RSV inoculation in mice reduced viral infection by 1 log unit, suppressed inflammatory cell appearance in the lung, and suppressed virus-elicited interferon-γ production. These findings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection and that it has potential for application in humans.

Topics: Animals; Cells, Cultured; Chemoprevention; Humans; Interleukin-8; Mice; Mice, Inbred BALB C; Phosphatidylglycerols; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Structure-Activity Relationship; Time Factors

Current tools to predict the severity of respiratory syncytial virus (RSV) infection might be improved by including immunological parameters. We hypothesized that a combination of inflammatory markers would differentiate between severe and mild disease in RSV-infected children.. Blood and nasopharyngeal samples from 52 RSV-infected children were collected during acute infection and after recovery. Retrospectively, patients were categorized into three groups based on disease severity: mild (no supportive treatment), moderate (supplemental oxygen and/or nasogastric feeding), and severe (mechanical ventilation). Clinical data, number of flow-defined leukocyte subsets, and cytokine concentrations were compared.. Children with severe RSV infection were characterized by young age; lymphocytopenia; increased interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF), and IL-6 concentrations; and decreased chemokine (C-C motif) ligand (CCL-5) concentrations in plasma. The combination of plasma levels of IL-8 and CCL-5, and CD4+ T-cell counts, with cutoff values of 67 pg/ml, 13 ng/ml, and 2.3 × 10(6)/ml, respectively, discriminated severe from mild RSV infection with 82% sensitivity and 96% specificity.. This study demonstrates that the combination of CD4+ T-cell counts and IL-8 and CCL-5 plasma concentrations correlates with disease severity in RSV-infected children. In addition to clinical features, these immunological markers may be used to assess severity of RSV infection and guide clinical management.

Topics: Age Factors; Biomarkers; Bronchiolitis, Viral; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Chemokine CCL5; Chi-Square Distribution; Female; Humans; Infant; Infant, Newborn; Inflammation Mediators; Interleukin-8; Male; Predictive Value of Tests; Prognosis; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Retrospective Studies; Severity of Illness Index; Viral Load

Most patients with respiratory syncytial virus (RSV) bronchiolitis requiring admission to the pediatric intensive care unit (PICU) have no risk factors for severe disease. We sought to investigate the relationship between serum cytokine concentrations, innate immune responsiveness, and RSV disease severity.. Previously healthy infants (median age, 2.6 months) with RSV bronchiolitis (PICU, n = 20; floor, n = 46) and healthy matched controls (n = 14) were enrolled, and blood samples were obtained within 24 hours of admission to measure plasma tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10) concentrations and, whole blood lipopolysaccharide-stimulated cytokine production capacity.. Plasma IL-6, IL-8, and IL-10 concentrations were comparable between PICU and floor patients, but higher than in healthy controls (P < .05). In contrast, TNF-α, IL-6, and IL-8 production capacity was significantly decreased in PICU compared with both floor patients and healthy controls. In adjusted analyses, only impaired TNF-α and IL-8 production capacity were associated with longer length of stay (P = .035) and greater disease severity scores (P = .001).. Infants with severe RSV bronchiolitis had increased plasma cytokine concentrations and yet impaired innate immunity cytokine production capacity, which predicted worse disease outcomes. Immune monitoring of otherwise healthy infants with RSV lower respiratory tract infection could help identify patients at risk for severe disease at the time of hospitalization.

Topics: Bronchiolitis, Viral; Cytokines; Female; Humans; Immunity, Innate; Infant; Interleukin-6; Interleukin-8; Male; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Severity of Illness Index; Tumor Necrosis Factor-alpha

Respiratory syncytial virus (RSV) is the most common pathogen responsible for lower respiratory diseases in children. So far, there is no effective treatment or preventative vaccine available for RSV infection, although ribavirin and dexamethasone are commonly prescribed. Resveratrol has been shown to inhibit the replication of several other viruses, thus the effect of resveratrol on RSV-induced inflammatory mediators in 9HTEo cell cultures was evaluated, and possible mechanisms of action were explored and compared with dexamethasone and ribavirin. Incubation with resveratrol resulted in decreased IL-6 production and partial inhibition of RSV replication. Resveratrol treatment also inhibited virus-induced TIR-domain-containing adapter-inducing interferon-β (TRIF) and TANK binding kinase 1 (TBK1) protein expression. These data demonstrate the ability of resveratrol to inhibit cytokine production by RSV in airway epithelial cells, indicating that it might be a therapeutic agent with both anti-inflammatory and antiviral potential for the treatment of RSV infection.

Topics: Adaptor Proteins, Vesicular Transport; Antiviral Agents; Cell Line; Dexamethasone; Down-Regulation; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Protein Serine-Threonine Kinases; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory System; Resveratrol; Ribavirin; Stilbenes; Virus Replication

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFNγ, IL-8, MCP-1, and PD-L1, and decreased IFNβ, IL-10, and TGF-β. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.

Topics: Animals; Animals, Newborn; Child; Humans; Infant, Newborn; Infant, Newborn, Diseases; Inflammation; Interleukin-8; Lung; Macrophages; Receptors, CCR2; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Sheep; Transcription, Genetic; Transforming Growth Factor beta

Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants. Although antiinflammatory in nature, glucocorticoids have been shown to be ineffective in the treatment of RSV-induced bronchiolitis and wheezing. In addition, the effectiveness of glucocorticoids at inhibiting RSV-induced proinflammatory cytokine production in cell culture has been questioned. In this study, we have investigated the effect of RSV infection on glucocorticoid-induced gene activation in lung epithelium-derived cells. We show that RSV infection inhibits dexamethasone induction of three glucocorticoid receptor (GR)-regulated genes (glucocorticoid-inducible leucine zipper, FK506 binding protein, and MAPK phosphatase 1) in A549, BEAS-2B cells, and primary small airway epithelial cells. UV irradiation of the virus prevents this repression, suggesting that viral replication is required. RSV is known to activate the nuclear factor κB (NFκB) pathway, which is mutually antagonistic towards the GR pathway. However, specific inhibition of NFκB had no effect on the repression of GR-induced genes by RSV infection, indicating that RSV repression of GR is independent of NFκB. RSV infection of A549 cells does not alter GR protein levels or GR nuclear translocation but does reduce GR binding to the promoters of the glucocorticoid responsive genes analyzed in this study. Repression of GR by RSV infection may account for the apparent clinical ineffectiveness of glucocorticoids in RSV bronchiolitis therapy. In addition, this data adds to our previously published data suggesting that GR may be a general target for infectious agents. Identifying the mechanisms through which this suppression occurs may lead to the development of novel therapeutics.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cells, Cultured; Chromatin Immunoprecipitation; Dexamethasone; Enzyme-Linked Immunosorbent Assay; Gene Silencing; Humans; Immunohistochemistry; Interleukin-1beta; Interleukin-6; Interleukin-8; Polymerase Chain Reaction; Receptors, Glucocorticoid; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Small Interfering; Transcriptional Activation

Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma, and severe lower respiratory tract disease in infants and young children. The airway epithelium, which has a well-developed barrier regulated by tight junctions, is the first line of defense during respiratory virus infection. In upper airway human nasal epithelial cells (HNECs), however, the primary site of RSV infection, the mechanisms of replication and budding of RSV, and the epithelial cell responses, including the tight junctional barrier, remain unknown. To investigate the detailed mechanisms of replication and budding of RSV in HNECs and the epithelial cell responses, we established an RSV-infected model using human telomerase reverse transcriptase--transfected HNECs. We first found that the expression and barrier function of tight junction molecules claudin-4 and occludin were markedly induced together with production of proinflammatory cytokines interleukin 8 and tumor necrosis factor-α in HNECs after RSV infection, and the induction of tight junction molecules possibly contributed to budding of RSV. Furthermore, the replication and budding of RSV and the epithelial cell responses in HNECs were regulated via a protein kinase C δ/hypoxia-inducible factor-1α/nuclear factor-κB pathway. The control of this pathway in HNECs may be useful not only for prevention of replication and budding of RSV, but also in therapy for RSV-induced respiratory pathogenesis.

Topics: Claudin-4; Claudins; Epithelial Cells; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Nasal Mucosa; NF-kappa B; Protein Kinase C-delta; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Signal Transduction; Tight Junctions; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Up-Regulation; Virus Replication

To study the association of haplotypes of IL-8 -251T/A and 781 C/T single nucleotide polymorphisms (SNPs) with the susceptibility of respiratory syncytial virus (RSV).. This study included 101 hospitalized patients under 2 years who suffered from RSV pneumonia and 108 hospitalized patients under 2 years who suffered only from pneumonia without RSV infection. Genotypes of two SNP loci in all enrolled persons were defined by allele-specific polymerase chain reaction. The allele's frequencies of SNPs were analyzed with case-control study, linkage of two loci and haplotypes composed by the two loci were also studied.. (i) The frequency of IL-8 -251T in cases was dramatically high (OR = 2.08, p = 0.0002). (ii) Haplotype of TC was significantly high in cases (p = 0.01).. These findings support that haplotype of TC composed by IL-8 -251T and 781C is associated with the susceptibility of RSV.

Topics: Case-Control Studies; Child, Preschool; Female; Gene Frequency; Genetic Predisposition to Disease; Haplotypes; Humans; Infant; Interleukin-8; Linkage Disequilibrium; Logistic Models; Male; Polymorphism, Single Nucleotide; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Sequence Analysis, DNA

Human respiratory syncytial virus (RSV) is a ubiquitous respiratory virus that causes serious lower respiratory tract disease in infants and young children worldwide. Studies have shown that RSV infection modulates chemokine expression patterns, suggesting that particular cytokine expression profiles may be indicators of disease severity. In this study, we show that RSV F or G protein treatment of fully differentiated primary normal human bronchial epithelial cells induces apical and basolateral secretion of interleukin 8 (IL-8), interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted). Purified RSV G (attachment) protein was shown to stimulate the secretion of interleukin 1alpha and RANTES, whereas purified F (fusion) protein elicited the production of IL-8, IP-10, and RANTES. Studies of ultraviolet-inactivated RSV showed that treatment of normal human bronchial epithelial cells induces apical IL-8, IP-10, and MCP-1 secretion independent of infection, suggesting that RSV proteins alone modify the chemokine response pattern, which may affect the early immune response before infection.

Topics: Adolescent; Bronchi; Cells, Cultured; Chemokine CXCL10; Chemokines, CC; Chemokines, CXC; Epithelium; Gene Expression Regulation, Viral; Humans; Interleukin-1alpha; Interleukin-8; Male; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Viral Fusion Proteins

Alveolar macrophages (AMvarphis) secrete regulatory molecules that are believed to be critical in maintaining normal lung homeostasis. However, in response to activating signals, AMvarphis have been shown to become highly phagocytic cells capable of secreting significant levels of pro-inflammatory cytokines. There is evidence to suggest that susceptibility of Mvarphi subpopulations to viral infection, and their subsequent cytokine/chemokine response, is dependent on age of the host. In the present study, we compared bovine respiratory syncytial virus (BRSV) replication and induction of cytokine responses in neonatal ovine AMvarphis to those cells isolated from adult animals. While neonatal AMvarphis could be infected with BRSV, viral replication was limited as previously shown for AMvarphis from mature animals. Interestingly, following BRSV infection, peak mRNA levels of IL-1beta and IL-8 in neonatal AMvarphi were several fold higher than levels induced in adult AMvarphis. In addition, peak mRNA expression for the cytokines examined occurred at earlier time points in neonatal AMvarphis compared to adult AMvarphis. However, the data indicated that viral replication was not required for the induction of specific cytokines in either neonatal or adult AMvarphis. TLR3 and TLR4 agonists induced significantly higher levels of cytokine transcripts than BRSV in both neonatal and adult AMvarphis. It was recently proposed that immaturity of the neonatal immune system extends from production of pro-inflammatory cytokines to regulation of such responses. Differential regulation of cytokines in neonatal AMvarphis compared to adult AMvarphis in response to RSV could be a contributory factor to more severe clinical episodes seen in neonates.

Topics: Animals; Base Sequence; Cattle; Cytokines; DNA Primers; Gene Expression; In Vitro Techniques; Interleukin-1beta; Interleukin-8; Macrophages, Alveolar; Polymerase Chain Reaction; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Bovine; RNA, Messenger; Sheep; Sheep Diseases; Toll-Like Receptors; Virus Replication

Breastfeeding during the first 12 months of life confers demonstrable immunologic benefit against infective pathogens, including those of the respiratory tract. However, the mechanism by which the ingestion of human milk modifies immunologic defense against such pathogens remains elusive. Bronchiolitis, caused predominantly by respiratory syncytial virus, is the most common clinical presentation of severe upper respiratory illness requiring hospitalization in infants and remains one of the developed world's leading causes of infant mortality and morbidity over both the short and long term. The mechanism by which an early, severe case of bronchiolitis can result in the development of recurrent childhood wheeze or asthma is unclear; however, mucosal inflammation and pulmonary neutrophilia are believed to play a significant role. The aim of this study was to examine the immune response of breastfed infants hospitalized with severe bronchiolitis, compared with formula-fed controls. Nasopharyngeal aspirates (NPA) were collected from 18 infants (aged

Topics: Acute Disease; Breast Feeding; Bronchiolitis; Cell Degranulation; Chemokine CCL2; Disease Progression; Female; Humans; Immunity, Maternally-Acquired; Infant; Infant, Newborn; Interleukin-8; Male; Neutrophils; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Risk Factors; Sputum

To study the nutritional status of children with Respiratory Syncitial virus infection.. One hundred and twenty six children with acute respiratory infection, between the age of 4-24 months, were investigated for RSV infection with bronchiolitis, pneumonia and upper respiratory tract infection. Nasopharyngeal aspirates were collected and cytokine responses were determined by ELISA. Upper respiratory tract infections were detected in 16.66%, bronchiolitis in 30.15% and Pneumonia in 53.17% children.. Of the 126 patients, 46.66% children were positive for RSV while 58.33% were negative for RSV. Children with bronchiolitis were more commonly positive for RSV compared to URTI and pneumonia. RSV was almost equally distributed among boys (42.5%) and girls (48.7%). More children were RSV positive when the mean age lesser (8.4 mo) was compared to RSV negative (9.93 mo). Well nourished children and children with normal birth weight had more RSV positives, though not statistically significant. In a sub sample analysis of cytokines done (n=25), Interleukin-2 and Interleukin-8 levels were higher in the RSV positive children and these levels declined after 5 days of illness.. RSV is more commonly associated with bronchiolitis in younger infants with normal birth weight or more weight for age (WFA). Proinflammatory cytokine IL-8 was secreted at high concentrations in the nasopharyngeal aspirate in all the children.

Topics: Bronchiolitis, Viral; Child, Preschool; Female; Humans; India; Infant; Interleukin-2; Interleukin-8; Male; Nutritional Status; Pneumonia, Viral; Prevalence; Respiratory Syncytial Virus Infections; Respiratory Tract Infections; Risk Factors

Respiratory syncytial virus (RSV), a negative-strand RNA virus, is the most common cause of epidemic respiratory disease in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-κB (NF-κB). In this study we have investigated the role of the non canonical IκB kinase (IKK)ε in modulating RSV-induced NF-κB activation. Our results show that inhibition of IKKε activation results in significant impairment of viral-induced NF-κB-dependent gene expression, through a reduction in NF-κB transcriptional activity, without changes in nuclear translocation or DNA-binding activity. Absence of IKKε results in a significant decrease of RSV-induced NF-κB phosphorylation on serine 536, a post-translational modification important for RSV-induced NF-κB-dependent gene expression, known to regulate NF-κB transcriptional activity without affecting nuclear translocation. This study identifies a novel mechanism by which IKKε regulates viral-induced cellular signaling.

Topics: Animals; Cell Line; Humans; I-kappa B Kinase; Interleukin-8; Mice; Mice, Knockout; Mutation; NF-kappa B; Phosphorylation; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA Interference; Transcription Factor RelA; Transcription, Genetic

To better understand the neutrophil activation and protease imbalance in respiratory syncytial virus (RSV) bronchiolitis.. Pediatric patients with RSV bronchiolitis were collected, 11 with the Lowell scores > or = 10 (severe group), and 19 with the Lowell scores < 10 (mild group). 20 RSV bronchiolitis patients in convalescent group, 24 patients with bacterial pneumonia, and 15 patients as control group. The percentages of neutrophils were detected. ELISA was used to detect the concentration of neutrophil elastase (NE) and interleukin (IL)-8, and the elastase inhibition capacity (EIC)/free neutrophil elastase activity were detected by colorimetry. Immunohistochemistry was used to examine the expression of alpha(1) antitrypsin (alpha(1)AT).. The neutrophil percentage, the NE and IL-8 concentration of the RSV bronchiolitis group (0.528, 6.39 x 10(7) kg/L and 13.62 x 10(9) kg/L)were all significantly higher than those of the control (0.074, 2.53 x 10(7) kg/L and 2.64 x 10(9) kg/L) and convalescent groups (0.306, 1.23 x 10(7) kg/L and 5.95 x 10(9) kg/L, all P < 0.05). The neutrophil percentage and IL-8 concentration of the convalescent group were both significantly higher than those of the control group (both P < 0.05). Increased expression of alpha(1)AT in RSV bronchiolitis was found when compared with convalescent infants (0.49 vs 0.09, P < 0.05). The free elastase activity level of the bronchiolitis group was 6/30, not different with 7/24 of pneumonia group, and it was only one in convalescent infants.. Mass of neutrophil aggregation and activation exist in RSV bronchiolitis, as well as protease system imbalance, and may play an important role in the inflammatory response and pathogenesis of RSV bronchiolitis.

Topics: alpha 1-Antitrypsin; Bronchiolitis, Viral; Humans; Infant; Interleukin-8; Leukocyte Elastase; Neutrophil Activation; Neutrophils; Respiratory Syncytial Virus Infections

To observe the epithelial Toll like receptor (TLR)4 expression changes and the signaling pathway function after respiratory syncytial virus (RSV) infection and to explore the mechanisms of RSV-induced airway inflammation.. 9HTEo-human tracheal epithelial cell line was infected by RSV (MOI = 10), and TLR1-10 mRNA were detected by RT-PCR assay at 3 h post RSV infection. TLR4 mRNA was detected by real time Q-PCR assay at 3 h, 6 h and 9 h post RSV infection, and TLR4 protein expression and cell apoptosis were determined by flow cytometry at 24 h post RSV infection. IL-8 in supernatant was detected by ELISA after RSV-infected cells exposed to lipopolysaccharide (LPS). A normal control group and a RSV infection group were set up for the RT-PCR and flow cytometry experiments, and the data were analyzed by paired t test using GraphPad 4.0 software. A normal group, a RSV group and a UV-inactivated RSV group were set up for the real time Q-PCR, experiments, and the data were analyzed by Kruskal-Wallis test. The ELISA experiments were divided into 4 groups including a normal control, a RSV, a LPS stimulation, and a RSV plus LPS co-stimulation groups, and the data were analyzed by One-way ANOVA test.. (1) TLR2-10 mRNA level was significantly up-regulated (t value of TLR2-10: 3.49 -14.47, P < 0.05), especially TLR-2, 6 enhanced expression, compared with the normal epithelial cells. Real time Q-PCR assay showed that TLR4 mRNA started to increase at 3hr (Kruskal-Wallis test value = 8.82, P < 0.05, n = 6) and significantly elevated at 9 hour (Kruskal-Wallis test value = 6.62, P < 0.05, n = 6). UV inactivated-RSV had no effect on the TLR4 mRNA level. (2) Flow cytometry showed that membrane TLR4 mean fluorescence intensity (MFI) increased (RSV: 1.27 +/- 0.48, normal: 0.97 +/- 0.25; t = 2.39, P > 0.05, n = 10) while cytoplasmic TLR4 MFI simultaneously decreased (RSV: 3.08 +/- 1.38, normal: 3.36 +/- 1.31, t = 2.92, P = 0.225, n = 10). Percentage of membrane TLR4-positive cells was higher in RSV infected population [RSV: (11.99 +/- 7.74)%, normal: (1.16 +/- 0.47)%, Mann-Whitney t value = 0.001, P < 0.01, n = 8], most (93.32 +/- 1.7)% of which were Annexin V positive. IL-8 was significantly induced in the RSV plus LPS costimulation group compared with RSV group (F = 59.29, P < 0.01, n = 3).. RSV induced epithelial TLR4 up-regulation, localization changes from cytoplasm to membrane, IL-8 secretion through TLR4 signaling pathway and epithelial cell apoptosis in membrane TLR4 positive population. These results indicate TLR4 is involved in RSV-induced acute or chronic epithelial-dependent inflammation, which might contribute to acute or chronic airway inflammation.

Topics: Apoptosis; Cell Line; Epithelial Cells; Humans; Inflammation; Interleukin-8; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Messenger; Toll-Like Receptor 4; Up-Regulation

Granzymes (Grs), serine proteases present in granules of effector lymphocytes, are involved in several host immune responses, including the activation of cell death and inflammatory pathways. The main goal of this study was to determine whether the local cell-mediated Gr pathway is activated during severe respiratory syncytial virus (RSV) lower respiratory tract illness (LRTI) in children. Tracheal aspirates (TA) from 23 children with RSV-LRTI and 12 controls without pulmonary disease were analyzed for Gr A and B. Bronchoalveolar lavage fluid samples from seven children with RSV-LRTI were analyzed for cellular expression of GrB. Levels of GrA and GrB in TA were significantly increased in RSV patients compared with controls and both Grs showed preserved activity. Gr levels correlated with the total leukocyte counts and IL-8 levels in the airways at several time points. However, no correlation between Gr levels and release of caspase-cleaved cytokeratin-18 was found. There was evidence for marked expression of GrB by both CD8(+) and CD4(+) T cells and natural killer cells in the respiratory tract. These findings suggest activation of the cell-mediated Gr pathway during severe RSV-LRTI in children.

Topics: Bronchoalveolar Lavage Fluid; Case-Control Studies; Female; Granzymes; Humans; Immunity, Cellular; Infant; Infant, Newborn; Interleukin-8; Keratin-18; Leukocyte Count; Male; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Severity of Illness Index; Signal Transduction; T-Lymphocytes; Time Factors; Up-Regulation

To explore the association of interleukin 8 (IL-8)-251T/A and 781 C/T single nucleotide polymorphisms (SNPs) with the susceptibility of infants and young children to respiratory syncytial virus (RSV) infection.. This study included 101 hospitalized patients under 2 years of age who suffered from RSV pneumonia, 108 hospitalized patients under 2 years of age with non-RSV pneumonia and 35 core families with a child hospitalized for RSV pneumonia. Genotypes of 2 SNP loci in all enrolled persons were defined by allele specific polymerase chain reaction (AS-PCR), and confirmed by gene sequencing. The allele's frequencies of SNPs were analyzed with case-control study and transmission disequilibrium test (TDT), linkage of 2 loci and haplotypes composed of the 2 loci were also studied.. (1) The frequency of IL-8-251T in cases was dramatically high (OR = 2.08, P = 0.0002, case-control study; LRT = 14.31, P = 0.0008, TDT). (2) IL-8-251T and 781C was linkaged (D' = 0.607 +/- 0.03, r(2) = 0.2861, P = 0.0000). (3) Haplotype of TC was significantly high in cases (P = 0.01).. These findings support that haplotype of TC composed of IL-8-251T and 781C is associated with the susceptibility to RSV, namely, some RSV predisposing genes are located in the gene fragment including TC haplotype or linked tightly with this gene fragment.

Topics: Case-Control Studies; Child, Preschool; Female; Gene Frequency; Genetic Predisposition to Disease; Haplotypes; Humans; Infant; Interleukin-8; Linkage Disequilibrium; Male; Polymorphism, Single Nucleotide; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Sequence Analysis, DNA

Topics: Acute Disease; Bronchiolitis, Viral; C-Reactive Protein; Humans; Infant, Newborn; Inflammation; Interferon-gamma; Interleukin-4; Interleukin-8; Leukocytes; Macrolides; Respiratory Syncytial Virus Infections; Treatment Outcome

Respiratory syncytial virus (RSV) infects nearly all children under two years of age. It is not understood why some develop serious bronchiolitis. Whether there is a genetic component is not known. The nature of the association between RSV bronchiolitis and subsequent wheezing remains unknown. interleukin-8 (IL-8) is a potent neutrophil chemokine and activator, which plays a role in virus-induced wheezing diseases. The purpose of this study was to assess the genetic association between the IL-8 gene promoter -251A/T polymorphism and RSV bronchiolitis and post-bronchiolitis wheezing in children.. Totally 320 children who were hospitalized for bronchiolitis together with positive immunofluorescence for RSV were recruited in this study from Jan. 2002 to Jan. 2004. A group of 272 healthy children were enrolled as controls. The age of these children ranged from 1 to 12 months. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to identify the polymorphism at position-251 of the IL-8 promoter in RSV bronchiolitis and control groups. The total IL-8 and IgE concentrations in serum samples were measured by enzyme-linked immunosorbent assay (ELISA). The patients with RSV bronchiolitis were followed up in order to analyze the occurrence of wheezing post-bronchiolitis.. (1) Both A allele and T allele were detected at -251 of the IL-8 promoter; the prevalence of the A allele in RSV bronchiolitis group was 45.6%, as compared with 37.7% in normal group. The prevalence of IL-8-251A allele was significantly different between the two groups (P < 0.05). (2) For genotypes T/T, A/T, A/A in RSV bronchiolitis, level of serum IL-8 were (17 +/- 6) ng/L, (21 +/- 7) ng/L, (24 +/- 9) ng/L, respectively, the difference was significant among the three genotypes (P < 0.01). (3) The prevalence of the A allele in the group who wheezed after the episode of RSV bronchiolitis was 54.6%, as compared with 35.8% in the group who had bronchiolitis but did not go on to wheeze. The prevalence of IL-8-251A allele was significantly different between the two groups (P < 0.05).. Polymorphism of IL-8 promoter-251A/T was associated with susceptibility to RSV bronchiolitis in children. The association of IL-8-251A with severe RSV bronchiolitis is most marked in the children who go on to wheeze.

Topics: Adolescent; Alleles; Bronchiolitis; Child; Child, Preschool; Chromosome Mapping; Enzyme-Linked Immunosorbent Assay; Female; Genetic Predisposition to Disease; Genotype; Humans; Infant; Interleukin-8; Male; Polymorphism, Genetic; Respiratory Sounds; Respiratory Syncytial Virus Infections

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.

Topics: Cell Line; Cells, Cultured; DNA; eIF-2 Kinase; Humans; Interleukin-8; NF-kappa B; Poly I-C; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Double-Stranded; RNA, Viral; Toll-Like Receptor 3

Topics: Bronchial Diseases; Case-Control Studies; Female; Humans; Immunity, Innate; Infant; Infant, Newborn; Interleukin-1beta; Interleukin-6; Interleukin-8; Interleukins; Male; Nasopharynx; Respiratory Syncytial Virus Infections

3-nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the COOH-terminal tyrosine of alpha-tubulin, posttranslationally altering microtubular functions. Because infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of alpha-tubulin. A human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor-3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated alpha-tubulin, and NO2Tyr colocalized with microtubules. RSV-infected cells released viral particles, RANTES, and IL-8 in a time- and dose-dependent manner, and intracellular RSV proteins coprecipitated with alpha-tubulin. NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles by 50-90% and decreased alpha-tubulin-associated RSV proteins. 3-chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO(2) (0.15 ppm, 4 h), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8, or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were cocultured with the preinfected cells in NO2Tyr-containing medium. These results indicate that NO2Tyr, by replacing the COOH-terminal tyrosine of alpha-tubulin, attenuated RSV infection, and the inhibition appeared to occur at the early stages of RSV infection.

Topics: Antiviral Agents; Bronchi; Cell Line; Chemokine CCL5; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Interferon Regulatory Factor-3; Interferon-gamma; Interleukin-8; Microtubules; Nitrogen; Nitrogen Dioxide; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Signal Transduction; Transcription Factors; Tubulin; Tumor Necrosis Factor-alpha; Tyrosine

Respiratory syncytial virus (RSV) is an important cause of upper respiratory infections and is known to play a causal role in the pathogenesis of rhinitis, sinusitis, acute otitis media, and pneumonia. RSV appears to prime the respiratory tract to secondary inciting events, such as bacterial or antigen challenges. To study the proinflammatory priming effects of RSV infection, cytokine expression was measured in well-differentiated human nasal epithelial cells (WD-NE) after RSV infection alone or after subsequent tumor necrosis factor (TNF)-alpha stimulation.. In vitro investigation.. Human nasal epithelial cells were obtained from surgical specimens and allowed to differentiate in air-liquid interface cultures until ciliation and mucus production were evident. Two experimental paradigms were used. First, accumulation of cytokines in the media was measured by real-time, quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay after RSV infection alone. In the second set of experiments, cytokines were also measured after TNF-alpha stimulation in both RSV-infected and uninfected cultures.. RSV infection of WD-NE resulted in significant accumulations of interleukin (IL)-6, IL-8, and RANTES when compared with findings in control samples. Real-time, quantitative RT-PCR demonstrated significant increases in IL-8 gene expression following RSV infection when compared to controls. Secondary TNF-alpha stimulation following well-established (i.e., 72 h) RSV infection induced marked increases in IL-6, IL-8, and RANTES when compared with both RSV infection alone and TNF-alpha stimulation alone.. These findings suggest that RSV infection primes nasal epithelial cells to secondary proinflammatory challenge, resulting in a hyperimmune response. RSV-induced priming of a hyperimmune response may be important in the pathogenesis of sinusitis, acute otitis media, and pneumonia.

Topics: Chemokine CCL5; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Nasal Mucosa; Otitis Media; Pneumonia; Respiratory Syncytial Virus Infections; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Respiratory syncytial virus infects almost all children by 2 years of age. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and they are activated in the presence of infection. However it is not clear whether RSV can directly signal to activate neutrophil cytotoxic function. To investigate this we have used a preparation of RSV washed using a new centrifugal diafiltration method to rapidly remove inflammatory molecules produced by the epithelial cells used to propagate the RSV stock. Human neutrophils were isolated from peripheral blood and activated with either the unwashed crude RSV preparations or the purified intact RSV. Neutrophils were also challenged with purified RSV G-glycoprotein. The effect of challenging human neutrophils with these preparations of intact RSV, or the RSV G-glycoprotein, was assessed by measuring the cell surface expression of CD11b and CD18b, the phagocytic oxidative burst, and intracellular release of calcium pools. Neutrophils challenged with the washed RSV exhibited significantly lower activation of surface marker expression (P < 0.001) and oxidative burst (P < 0.001) than those challenged with unwashed virus or with virus free supernatant. There was no increase in intracellular calcium release on exposure to the washed RSV. Purified G glycoprotein did not stimulate neutrophils, whilst the use of a blocking antibody to the F protein did not prevent unwashed RSV from activating cytotoxic responses. These results suggest that neutrophils have no innate signalling system that recognizes RSV but they are activated at sites of RSV infection as a result of the cytokines and inflammatory molecules released by virally infected cells.

Topics: Antibodies, Viral; Calcium; CD11b Antigen; CD18 Antigens; Epithelial Cells; Filtration; HeLa Cells; Humans; Interleukin-8; Neutrophil Activation; Neutrophils; Phagocytosis; Respiratory Burst; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Viral Envelope Proteins; Viral Fusion Proteins

The aim of this study was to determine whether the regulatory immune response (interleukin (IL)-10 response) differed between children hospitalized with acute respiratory infections and wheezing.. Infants with signs and symptoms of acute viral respiratory infection, admitted during winter 2000 to Princess Margaret Hospital for Children, Perth, WA, Australia, were enrolled in this study. Nasopharyngeal aspirates were collected in the first 48 h of admission. Total cell count and differential cell counts were assessed. Samples were tested for the presence of respiratory viruses. The concentrations of the anti-inflammatory cytokine IL-10, and pro-inflammatory cytokines IL-8, interferon-gamma, and IL-11 were determined by ELISA.. Children with acute bronchiolitis (AB; n = 36), recurrent wheeze (RW; n = 17) and upper respiratory infection (URI; n = 18) were enrolled. Respitory syncytial virus was the most commonly detected virus in all groups. IL-10 concentrations were significantly increased in AB (median, 0.019 ng/mL) when compared to URI (median, 0.006 ng/mL) or to RW (median, 0.007 ng/mL; P < 0.05). Neutrophils were the predominant cells in the cytological analysis in all subjects.. These data argue that host-response factors are important in determining the clinical phenotype, independent of the causative virus.

Topics: Acute Disease; Biomarkers; Cell Count; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Inpatients; Interferon-gamma; Interleukin-10; Interleukin-11; Interleukin-8; Male; Nasal Lavage Fluid; Nasal Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Retrospective Studies; Severity of Illness Index

Susceptibility to viral bronchiolitis, the commonest cause of infant admissions to hospital in the industrialised world, is associated with polymorphism at the IL8 locus. Here we map the genomic boundaries of the disease association by case-control analysis and TDT in 580 affected UK infants. Markers for association mapping were chosen after determining patterns of linkage disequilibrium across the surrounding region of chromosome 4q, a 550-kb segment containing nine genes, extending from AFP to PPBP. The region has three major clusters of high linkage disequilibrium and is notable for its low haplotypic diversity. We exclude adjacent chemokine genes as the cause of the association, and identify a disease-associated haplotype that spans a 250-kb region from AFM to IL8. In between these two genes there is only one structural feature of interest, a novel gene RASSF6, which is predicted to encode a Ras effector protein.

Topics: Base Sequence; Bronchiolitis, Viral; Case-Control Studies; Chromosome Mapping; Chromosomes, Human, Pair 4; Gene Frequency; Genes; Genetic Markers; Genetic Predisposition to Disease; Haplotypes; Humans; Infant; Interleukin-8; Linkage Disequilibrium; Polymorphism, Single Nucleotide; Respiratory Syncytial Virus Infections; United Kingdom; White People

Interleukin-8 (IL-8) has been implicated in the pathogenesis of RSV-induced bronchiolitis. Previously, we have described an association between bronchiolitis disease severity and a specific IL-8 haplotype comprising six single-nucleotide polymorphisms (SNPs) (-251A/+396G/+781T/+1238delA/+1633T/+2767T, haplotype 2). Here we investigated the functional basis for this association by measuring haplotype-specific transcription in vivo in human primary cells. We found a significant increase in transcript level derived from the IL-8 haplotype 2 relative to the mirror haplotype 1 (-251T/+396T/+781C/+1238insA/+1633C/+2767A) in respiratory epithelial cells but not in lymphocytes. A promoter polymorphism, -251A, present on the high producer haplotype, had no significant affect on the allele-specific level of transcription when analyzed in reporter gene experiments in human respiratory epithelial A549 cells. We proceeded to systematically screen for allele-specific protein-DNA binding in this functional haplotype, which revealed significant differential binding at the +781T/C polymorphism. C/EBP beta was identified as being part of a transcription factor binding complex that preferentially bound in the presence of the +781 T allele. These results suggest that the mechanism for disease susceptibility to RSV-induced bronchiolitis may occur through a haplotype-specific increase in IL-8 transcription, which may be mediated by functional polymorphisms within that haplotype.

Topics: Cell Nucleus; Genetic Predisposition to Disease; Haplotypes; Humans; Interleukin-8; Nuclear Proteins; Promoter Regions, Genetic; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Sequence Analysis, DNA; Tumor Necrosis Factor-alpha

The nature of the association between severe respiratory syncytial virus (RSV) bronchiolitis and subsequent wheezing remains unknown. In a previous study, we showed that genetic variation in the IL-8-promoter region is associated with susceptibility to severe bronchiolitis.. The purpose of this study was to assess the association between wheezing post-bronchiolitis and the genetic variant of IL-8 gene.. We collected data from 134 children who had suffered from bronchiolitis, enrolled in our previous study. The occurrence of wheezing post-bronchiolitis was recorded from a questionnaire sent by post. The association between the genotype and wheezing phenotype was assessed by family-based and case-control approaches.. Family-based association showed that the IL-8 variant was transmitted significantly more often than expected in the children who wheezed after the episode of bronchiolitis (transmission=56%, P=0.02). This effect was not observed in the group of children who had bronchiolitis but did not go on to wheeze. Moreover, the variant was significantly more frequent in post-bronchiolitis wheezers compared with the general population (odds ratio=1.6, 95% confidence interval 1.0-2.6).. These preliminary results suggest that there is a genetic predisposition to wheeze following severe RSV bronchiolitis.

Topics: Alleles; Bronchiolitis, Viral; Case-Control Studies; Chi-Square Distribution; Child; Female; Follow-Up Studies; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Male; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

IL-8 is a strong inductor of inflammation. Accordingly, it plays a pivotal role in acute inflammatory responses during respiratory syncytial virus (RSV) infections and in chronic inflammatory diseases such as bronchial asthma and juvenile idiopathic arthritis. Recently, 2 studies have found association of the polymorphism -251A of IL8 with RSV bronchiolitis. Furthermore, epidemiologic studies have demonstrated an increased risk for the development of asthma after RSV bronchiolitis, and a common genetic background for the 2 diseases is currently being discussed.. This study investigated whether IL-8 is in association with asthma and/or arthritis and whether the results can confirm a common genetic background of RSV bronchiolitis and asthma.. The polymorphisms -A251T, C781T, C1633T, and A2767T within IL8 were genotyped in the following 4 populations: children with asthma, atopic children, children with juvenile idiopathic arthritis, and control subjects. Statistical analysis made use of the Armitage trend test and the software program Arlequine.. Association of all polymorphisms was found with asthma ( P =.008 to P =.03). Surprisingly -251T was associated with asthma, which is the opposite allele as described in association with RSV bronchiolitis. Furthermore, all polymorphisms were significantly more common in children with arthritis than in asthmatic children ( P =.006 to P =.02). No association was seen with the diagnosis of arthritis per se or with atopy.. This is the first study to describe association of IL-8 with asthma and a significant inverse distribution of the polymorphisms in juvenile idiopathic arthritis. In addition, the results of this study might suggest that RSV bronchiolitis and bronchial asthma have at least some different genetic factors.

Topics: Adolescent; Adult; Arthritis, Juvenile; Asthma; Bronchiolitis, Viral; Child; Child, Preschool; Genetic Predisposition to Disease; Genotype; Haplotypes; Humans; Interleukin-8; Polymorphism, Genetic; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

The airway epithelium is the first cellular component of the lung to be encountered by the particles and pathogens present in inhaled air. In addition to its role as a physical barrier, the immunological activity of the airway epithelium is an essential part of the pulmonary immune system. This means that the symptoms of lung diseases that involve immunological mechanisms are frequently exacerbated by infection of the airway epithelium with respiratory viruses. The virus-induced enhancement of immunological activity in infected epithelial cells is well characterized. However, the effects that contaminants of inhaled air have upon the infectivity and replication of respiratory viruses and the inflammation they cause, are comparatively unknown. In this study, we have shown that pre-exposure of airway epithelial cells to bacterial lipopolysaccharides or a proteolytically active house dust mite allergen, is able to, respectively, inhibit or enhance the level of cellular infection with respiratory syncytial virus and similarly alter virus-induced expression of the inflammatory chemokine interleukin-8. These results suggest that respiratory syncytial virus infection and the inflammation caused by respiratory syncytial virus may be modified by the biologically active contaminants of indoor air.

Topics: Air Pollutants; Air Pollution, Indoor; Antigens, Dermatophagoides; Arthropod Proteins; Cell Culture Techniques; Cell Line; Cysteine Endopeptidases; Disease Susceptibility; Epithelial Cells; Humans; Interleukin-8; Lipopolysaccharides; Pneumonia, Viral; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Virus Replication

Surfactant protein A (SP-A) and lactoferrin (LF) play important roles in innate immune systems in the respiratory mucous membranes. We investigated how SP-A and LF act against respiratory syncytial virus (RSV) infection. The present study indicated that RSV-induced IL-8 secretion from HEp-2 cells was up-regulated by SP-A (170% of control) but down-regulated by LF (23% of control). RSV infectivity determined by viral titers and the uptake of FITC-labeled RSV were also increased by SP-A, but decreased by LF. To clarify the mechanism of these opposite effects, we examined the interactions of SP-A and LF with RSV F protein, the most important surface glycoprotein for viral penetration. RSV F protein was found to be the ligand for both SP-A and LF, but the manners of binding were different. LF directly interacted with the F(1) subunit, which involved antigenic sites of F protein. Contrarily, SP-A associated with the F(2) subunit, which was highly glycosylated. SP-A but not LF failed to interact with deglycosylated F protein. Moreover, SP-A initiated the hemolyzing fusion activity of F protein. These results suggest that SP-A and LF modulate RSV infection by different binding specificity to F protein.

Topics: Cell Line; Hemolysis; Humans; Interleukin-8; Lactoferrin; Pulmonary Surfactant-Associated Protein A; Respiratory Syncytial Virus Infections; Viral Proteins

This study measured chemokines in nasal lavage fluids (NLF) from infants with respiratory syncytial virus (RSV) bronchiolitis, defined by lung hyperinflation and wheezing. Comparison was made to RSV-positive infants without bronchiolitis and RSV-negative infants with acute respiratory illnesses. RSV-positive illnesses were associated with increased epithelial shedding, increased RANTES/protein ratios, and increased IL-8/protein ratios in NLF compared to RSV-negative illnesses. Among RSV-positive infants, bronchiolitics had greater total cell counts and percentage epithelial cells in NLF than nonbronchiolitics. Bronchiolitics also had roughly twice the NLF RANTES/IL-8 ratio than nonbronchiolitics (P =.043). Semiquantitative reverse transcriptase-polymerase chain reaction of nasal epithelium suggested similar RANTES/IL-8 ratio increases among bronchiolitics. A more mildly affected, RSV-positive outpatient population showed none of these differences. We conclude that RSV bronchiolitis is associated with a shift toward relatively more RANTES in nasal secretions of infants sick enough to require hospitalization, and mucosal epithelium may contribute to this process. Similar processes in the lower airways may enhance inflammation due to RANTES-responsive cell types and affect clinical manifestations.

Topics: Blood Proteins; Bronchiolitis, Viral; Chemokine CCL5; Eosinophil Granule Proteins; Epithelial Cells; Humans; Infant; Interleukin-8; Leukocyte Count; Leukocytes, Mononuclear; Nasal Lavage Fluid; Neutrophils; Outpatients; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Ribonucleases; RNA, Messenger

Large numbers of neutrophils in the airway of infants infected by respiratory syncytial virus (RSV) are recruited by chemokines, such as interleukin-8, and specific inflammatory molecules can delay apoptosis increasing their longevity. The aim of this study was to investigate whether airway secretions in RSV bronchiolitis contain factors that influence neutrophil apoptosis. Nasal lavage fluid (NLF) was obtained from 24 infants with RSV bronchiolitis (31 infant controls and 12 adults). Neutrophils isolated from healthy adult volunteers were incubated with the NLF in Dulbecco modified Eagle medium (DMEM) for 24 h, and apoptosis and necrosis were quantified using Hoechst 33342 and propidium iodide viability dyes. The presence of putative factors that delay neutrophil apoptosis was investigated using inhibitors to leukotriene-B4, lipopolysaccharide and the IL-8 receptor CXCR2, and blocking antibodies to granulocyte-monocyte colony-stimulating factor. Characterisation of NLF involved tests of thermal instability, proteolysis, deoxyribonuclease digestion and molecular filtration. NLF from infants with RSV bronchiolitis and controls significantly delayed neutrophil apoptosis, whereas NLF from healthy adults did not. None of these inhibitor molecules blocked this delay in apoptosis but activity was heat liable and >3 kDa. The study showed that nasal lavage fluid from infants significantly delays neutrophil apoptosis. The speculation is that the prolonged survival of neutrophils in the infant airway contributes to the characteristic accumulation of neutrophils in the airways of infants with respiratory infections.

Topics: Adult; Apoptosis; Bronchiolitis, Viral; Cell Survival; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Infant; Interleukin-8; Leukotriene B4; Nasal Lavage Fluid; Neutrophils; Polymyxin B; Receptors, Interleukin-8B; Respiratory Syncytial Virus Infections; Respiratory System

The differences between respiratory syncytial virus (RSV) and influenza A virus (IFAV) in the pathogenesis of wheezing in young children have not been clearly defined. The aim of this study was to assess the contributions of RSV vs IFAV in the pathogenesis of upper airway inflammation in wheezy young children. We compared interleukin (IL)-6, IL-8, IL-11, and interferon-gamma (IFN-gamma) levels in nasopharyngeal aspirates (NPA) from non-asthmatic children with respiratory virus infections (RSV in 17 children and IFAV in 13 children), asthmatic children with viral infections (RSV in nine children, IFAV in 10 children), and 22 unaffected healthy children (controls). Levels of IL-11 in NPA from asthmatic children were significantly higher than those from non-asthmatic children with RSV infection, and RSV infection enhanced the IL-11 production in NPA significantly compared to IFAV infection. Nasopharyngeal epithelium from children with RSV infection secreted more IL-6 than that of children with IFAV infection. There was little difference in the IL-8 and IFN-gamma levels between asthmatic and non-asthmatic children with RSV or IFAV infection. In conclusion, asthma enhanced IL-11 production in RSV infection rather than IFAV infection in early childhood. There was a trend towards greater IL-6 production in RSV infection compared with IFAV infection.

Topics: Asthma; Biomarkers; Bronchiolitis, Viral; Child Welfare; Child, Preschool; Cytokines; Female; Humans; Infant; Infant Welfare; Influenza A virus; Influenza, Human; Inhalation; Interferon-gamma; Interleukin-11; Interleukin-6; Interleukin-8; Male; Nasopharynx; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Statistics as Topic

Both virus-mediated damage to airway tissues and induction of pro-inflammatory cytokines such as interleukin-8 (IL-8) could contribute to symptom severity during viral respiratory infections in children. To test the hypothesis that IL-8 contributes to the pathogenesis of respiratory symptoms during naturally acquired respiratory viral infections in children, nasal wash samples collected from infants with acute viral infections (n = 198) or from healthy uninfected infants (n = 31) were analysed for IL-8. Nasal wash IL-8 was positively related to age in uninfected children (rs = 0.36, p < 0.05). Respiratory syncytial virus (RSV) infection caused more severe respiratory symptoms compared to infections with influenza A, parainfluenza viruses, or rhinoviruses. In addition, RSV, parainfluenza and rhinovirus infections increased levels of IL-8 in nasal lavage fluid, and there were some differences in the ability of the viruses to induce IL-8 production (RSV>influenza, p < 0.05). Finally, there were significant correlations between nasal wash IL-8 levels and symptom scores during infections with rhinovirus (rs = 0.56, p < 0.001) or influenza A (rs = 0.45, p < 0.05), but not with parainfluenza virus or RSV. These findings provide evidence of a close relationship between the generation of IL-8 and symptoms during acute community-acquired infections with rhinovirus or influenza A. In contrast, for RSV and parainfluenza infections, factors in addition to IL-8 production appear to contribute to the generation of clinical symptoms.

Topics: Age Factors; Fluorescent Antibody Technique, Direct; Humans; Infant; Infant Welfare; Infant, Newborn; Influenza A virus; Influenza, Human; Interleukin-8; Nasal Lavage Fluid; Picornaviridae Infections; Prospective Studies; Respiratory Hypersensitivity; Respiratory Sounds; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Rhinovirus; Severity of Illness Index; Statistics as Topic

In infants with respiratory syncytial virus (RSV) bronchiolitis, we investigated whether disease severity is associated with the genotype of the infecting virus, or with the infant's immunological response to the infection, as determined by measurement of interleukin-8 mRNA in the nasopharyngeal aspirate. This was a cross-sectional observational study, performed in the Accident and Emergency Department, wards, and Intensive Care Unit of a large pediatric hospital. Participants included 276 infants with respiratory syncytial virus infection. Outcome variables included: disease severity (infants requiring oxygen or ventilation were classified as having severe disease); RSV virus genotype (determined according to typing scheme based on the nucleoprotein and G glycoprotein genes); and amount of interleukin-8 mRNA in the nasopharyngeal aspirate, as measured by a semiquantitative polymerase chain reaction assay. This was corrected for the amount of cellular material in the sample by expressing it relative to mRNA for a constitutively expressed gene, HGPRT. We found a highly significant association between the ratio of interleukin-8 mRNA/HGPRT mRNA in the nasopharyngeal aspirate and the occurrence of severe disease. Odds ratio per unit increase of interleukin-8 mRNA/HGPRT mRNA was 1.15 (95% CI, 1.06, 1.24), P = 0.0004. There was no association between virus genotype and either disease severity or amount of interleukin-8 mRNA/HGPRT mRNA. In conclusion, there is a strong, dose-related association between interleukin-8 mRNA produced locally in the airways and disease severity, and a lack of association with virus genotype. This suggests that clinical manifestations of respiratory syncytial virus bronchiolitis are determined by local immunological responses to infection, rather than by characteristics of the infecting virus.

Topics: Bronchiolitis; Cross-Sectional Studies; Genotype; Humans; Infant; Interleukin-8; Respiratory Syncytial Virus Infections; Severity of Illness Index

Topics: Animals; Bronchiolitis, Viral; Humans; Infant, Newborn; Interferon-gamma; Interleukin-8; Rats; Respiration, Artificial; Respiratory Syncytial Virus Infections

Interleukin-8 (IL8) is believed to play a role in the pathogenesis of bronchiolitis, a common viral disease of infancy, and a recent U.K. family study identified an association between this disease and the IL8-251A allele. In the present study we report data, from a different set of families, which replicate this finding; combined analysis of 194 nuclear families through use of the transmission/disequilibrium test gives P = .001. To explore the underlying genetic cause, we identified nine single-nucleotide polymorphisms (SNPs) in a 7.6-kb segment spanning the IL8 gene and its promoter region and used six of these SNPs to define the haplotypic structure of the IL8 locus. The IL8-251A allele resides on two haplotypes, only one of which is associated with disease, suggesting that this may not be the functional allele. Europeans show an unusual haplotype genealogy that is dominated by two common haplotypes differing at multiple sites, whereas Africans have much greater haplotypic diversity. These marked haplotype-frequency differences give an F(ST) of.25, and, in the European sample, both Tajima's D statistic (D = 2.58, P = .007) and the Hudson/Kreitman/Aguade test (chi(2) = 4.9, P = .03) reject neutral equilibrium, suggesting that selective pressure may have acted on this locus.

Topics: Africa; Alleles; Animals; Bronchiolitis, Viral; Gene Frequency; Genetic Predisposition to Disease; Genetic Variation; Haplotypes; Humans; Infant; Interleukin-8; Introns; Molecular Sequence Data; Mutation; Pan troglodytes; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Prospective Studies; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Selection, Genetic; United Kingdom

To investigate the role of cell-mediated immunity during respiratory syncytial virus (RSV) infection, interferon (IFN)-gamma and interleukin (IL)-10 levels in nasopharyngeal secretions were measured in infants with lower respiratory tract infection (LRTI) caused by RSV. A novel technique was used to measure in vivo cytokine levels in nasopharyngeal aspirates (NPAs). Cytokine levels in the NPAs of 17 mechanically ventilated infants and 43 nonventilated hospitalized infants were compared. As expected, mechanically ventilated infants were significantly younger than nonventilated infants (7 vs. 14 weeks). IFN-gamma levels were above the limit of detection in the NPAs of 3 (18%) mechanically ventilated infants and in the NPAs of 26 (60%) nonventilated infants. IL-10 levels in the NPAs of mechanically ventilated and nonventilated infants were comparable. It is hypothesized that maturation-related mechanisms have a key role in the development of RSV LRTI that results in mechanical ventilation.

Topics: Age Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukin-8; Male; Nasopharynx; Netherlands; Predictive Value of Tests; Respiration, Artificial; Respiratory Syncytial Virus Infections; Respiratory Tract Infections

In this work we continue our study of the biochemical responses of respiratory epithelial cells to infection with human paramyxovirus pathogens. In our earlier studies, we detected elevated concentrations of the proinflammatory chemokines MIP-1alpha and IL-8 in upper and lower respiratory tract secretions from patients infected with respiratory syncytial virus (RSV). Here we demonstrate the same trend for individuals infected with parainfluenza virus (PIV), with elevated concentrations of MIP-1alpha and IL-8 (means of 309 +/- 51 and 2280 +/- 440 pg/ml/mg protein, respectively) detected in nasal wash samples from 17 patients with culture-positive PIV. Similar to our findings with RSV, cells of the HEp-2 epithelial line and primary cultures of human bronchial epithelial cells respond to PIV infection with production and release of both MIP-1alpha and IL-8. Addition of the glucocorticoid anti-inflammatory agent hydrocortisone (200-1000 ng/ml) attenuated the production of MIP-1alpha and IL-8 in PIV-infected cells while having minimal to no effect on the production of these mediators from cells infected with RSV. Neither virus infection resulted in a change in the total cellular concentration of glucocorticoid receptors, nor did hydrocortisone exert any differential effect on viral replication. As repression of chemokine production by epithelial cells is likely to result in diminished recruitment of proinflammatory leukocytes, these results may explain in part why glucocorticoid therapy reduces the symptoms associated with acute PIV infection, but have little to no effect in the overall outcome in the case of RSV.

Topics: Anti-Inflammatory Agents; Cells, Cultured; Chemokine CCL3; Chemokine CCL4; Child, Preschool; Culture Media; Drug Resistance; Epithelial Cells; Female; Gene Expression; Humans; Hydrocortisone; Infant; Interleukin-8; Macrophage Inflammatory Proteins; Male; Nasal Lavage Fluid; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Tumor Cells, Cultured; Virus Replication

Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 +/- 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 +/- 2,160 pg/ml; RSV alone, 12,170 +/- 300 pg/ml; RSV-CM plus RSV, 27,040 +/- 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IkappaBalpha within 5 min but did not affect IkappaBbeta. RSV-CM activated transient nuclear binding of NF-kappaB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-kappaB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-kappaB-dependent, NF-IL6-requiring mechanism.

Topics: Animals; Blotting, Northern; Blotting, Western; Bronchi; Cell Line; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Monocytes; NF-kappa B; Promoter Regions, Genetic; Respiratory Syncytial Virus Infections

Respiratory syncytial virus (RSV) infects nearly all children by the end of their second winter. Why some develop bronchiolitis is poorly understood; it is not known whether there is a genetic component. The pathological features include neutrophil infiltration and high levels of interleukin 8 (IL-8), a potent neutrophil chemoattractant.. Common genetic variants of the promoter region of the IL-8 gene were identified by sequencing DNA from 36 healthy individuals. Genetic correlates of IL-8 production were assessed using whole blood from 50 healthy subjects. To investigate genetic correlates of disease severity 117 nuclear families were recruited in which a child had required hospital admission for RSV bronchiolitis.. A common single nucleotide polymorphism (allele frequency 0.44) was identified 251 bp upstream of the IL-8 transcription start site. The IL8-251A allele tended to be associated with increased IL-8 production by lipopolysaccharide stimulated whole blood (p=0.07). Using the transmission disequilibrium test, the frequency of this allele was significantly increased in infants with bronchiolitis (transmission = 62% (95% confidence interval (CI) 53 to 71), p=0.014) and particularly in those without known risk factors (transmission = 78% (95% CI 62 to 93), p=0.004).. Disease severity following RSV infection appears to be determined by a genetic factor close to the IL-8 gene. Further analysis of this effect may elucidate causal processes in the pathogenesis of RSV bronchiolitis.

Topics: Alleles; Bronchiolitis; Cell Culture Techniques; Female; Genetic Predisposition to Disease; Genotype; Humans; Infant; Infant, Newborn; Interleukin-8; Male; Polymorphism, Genetic; Promoter Regions, Genetic; Respiratory Syncytial Virus Infections

Characterization of chemokine expression patterns in virus-infected epithelial cells provides important clues to the pathophysiology of such infections. The aim of this study was to determine the chemokine response pattern of respiratory epithelium when infected with respiratory syncytial virus (RSV). Macrophage inflammatory protein-1-alpha (MIP-1-alpha), interleukin-8 (IL-8), and RANTES concentrations were measured from RSV-infected HEp-2, MRC-5, and WI-38 cell culture supernatants daily following infection. Additionally, MIP-1-alpha, IL-8, and RANTES concentrations were measured from lower respiratory secretions obtained from 10 intubated infants (0-24 mo) with RSV bronchiolitis, and from 10 control subjects. Our results indicate that respiratory epithelial cells respond to RSV infection by producing MIP-1-alpha, IL-8, and RANTES. Production of MIP-1-alpha required ongoing viral replication, whereas RANTES and IL-8 could be elicited by inactivated forms of the virus. MIP-1-alpha, RANTES, and IL-8 were also present in lower airway secretions obtained from patients with RSV bronchiolitis. Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), the eosinophil secretory ribonucleases, were detected in lower airway secretions from RSV-infected patients; ECP concentrations correlated with MIP-1-alpha concentrations (r = 0.93). We conclude that MIP-1-alpha is present in the lower airways during severe RSV disease. The correlation between MIP-1-alpha and ECP concentrations suggests a role for eosinophil degranulation products in the pathogenesis of RSV bronchiolitis.

Topics: Blood Proteins; Cell Degranulation; Cell Line; Chemokine CCL4; Chemokine CCL5; Chemokines; Eosinophil Granule Proteins; Eosinophil-Derived Neurotoxin; Eosinophils; Epithelial Cells; Humans; Interleukin-8; Macrophage Inflammatory Proteins; Peroxidase; Proteins; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory System; Ribonucleases; Virus Activation

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes that cause farmer's lung disease. The early response to Ag is an increase in neutrophils in the lung, whereas the late response is a typical Th1-type granulomatous disease. Many patients who develop disease report a recent viral respiratory infection. These studies were undertaken to determine whether viruses can augment the inflammatory responses in HP. C57BL/6 mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) for 3 consecutive days per wk for 3 wk. Some mice were exposed to SR at 2 wk after infection with respiratory syncytial virus (RSV), whereas others were exposed to SR after exposure to saline alone or to heat-inactivated RSV. SR-treated mice developed a typical, early neutrophil response and a late granulomatous inflammatory response. Up-regulation of IFN-gamma and IL-2 gene expression was also found during the late response. These responses were augmented by recent RSV infection but not by heat-inactivated RSV. Mice with a previous RSV infection also had a greater early neutrophil response to SR, with increased macrophage inflammatory protein-2 (MIP-2, murine equivalent of IL-8) release in bronchoalveolar lavage fluid. These studies suggest that viral infection can augment both the early and late inflammatory responses in HP.

Topics: Alveolitis, Extrinsic Allergic; Animals; Bronchoalveolar Lavage Fluid; Female; Granuloma, Respiratory Tract; Interleukin-8; Leukocyte Count; Mice; Mice, Inbred C57BL; Neutrophils; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Saccharopolyspora

The aim of this study was to determine whether interleukin (IL)-8 is released within the upper respiratory tract of infants during respiratory syncytial virus (RSV) bronchiolitis and whether the large number of polymorphonuclear neutrophils (PMNs) present in the respiratory tract of these infants are contributing to the inflammation through release of inflammatory mediators. Twenty-seven infants with acute bronchiolitis were recruited during one winter epidemic and 20 infant control subjects were recruited from a cohort participating in a community-based vaccine study. Samples of airways fluid were obtained using nasal lavage. The lavage fluid was spun to remove the cells, and the supernatant was stored at -70 degrees C. The supernatants were subsequently assayed for the presence of IL-8, total human neutrophil elastase (HNE) and neutrophil elastase activity. In the children with bronchiolitis compared with control infants, elevated levels of IL-8 (median (range) 1.53 (0-153) versus 0 (0-5.6) ng x mL(-1)) HNE (136 (32-694) versus 14 (0-516) ng x mL(-1)) and elastase activity (4 (1-220) versus 1 (0-339) mU x mL(-1)) were found. These results indicate that interleukin-8 is released in the upper respiratory tract in response to respiratory syncytial virus infection and suggest that polymorphonuclear neutrophil products are playing an important role in the inflammatory response to respiratory syncytial virus infection in infants with acute bronchiolitis. This contrasts with the predominantly eosinophilic response evident in atopic upper and lower respiratory tract disease.

Topics: Acute Disease; Antibodies, Viral; Biomarkers; Bronchiolitis, Viral; Humans; Infant; Infant, Newborn; Interleukin-8; Leukocyte Elastase; Nasal Lavage Fluid; Neutrophils; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory System; Retrospective Studies; Severity of Illness Index

We investigated the expression of interleukin-8 (IL-8) in pneumonic pasteurellosis of cattle because neutrophils are important mediators of tissue injury in this disease and because IL-8 is a major neutrophil chemoattractant in other species. We also compared IL-8 expression in bacterial and viral pneumonia, since the latter lacks the severe neutrophil exudation typical of pneumonic pasteurellosis. IL-8 expression was assessed by northern analysis, in situ hybridization, enzyme-linked immunosorbent assay, and in vivo bioassay. IL-8 mRNA expression was elevated dramatically in lesions of pneumonic pasteurellosis compared to unaffected lung from the same calves. In situ hybridization revealed intense expression of IL-8 mRNA in alveolar macrophages and neutrophils and milder expression in bronchiolar and alveolar epithelium, interstitial cells, and pleural mesothelium. Bronchoalveolar lavage fluid from lesional lung contained 16.06+/-4.00 ng/ml IL-8, whereas those from nonlesional and normal lung contained 0.34+/-0.11 and 0.01+/-0.002 ng/ml, respectively. We detected IL-8 expression at only minimal levels in bovine respiratory syncytial viral pneumonia. Lung extracts from lesions of pneumonic pasteurellosis induced vigorous neutrophil infiltration following injection into bovine skin, and 89% depletion of IL-8 from the extract reduced this neutrophil influx by 60%. These results demonstrate consistent upregulation of IL-8 expression in lesions of pneumonic pasteurellosis, implying a role for IL-8 in the ongoing recruitment of neutrophils to established lesions of pneumonic pasteurellosis. Because neutrophil-mediated tissue injury is critical to the pathogenesis of pneumonic pasteurellosis, these data suggest that neutralization of IL-8 activity could ameliorate the severe clinical signs and lesions of this disease.

Topics: Animals; Blotting, Northern; Bronchoalveolar Lavage; Cattle; Chemotaxis, Leukocyte; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; In Situ Hybridization; Interleukin-8; Intradermal Tests; Lung; Macrophages, Alveolar; Mannheimia haemolytica; Neutrophils; Pasteurellosis, Pneumonic; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Bovine; RNA, Messenger

Respiratory syncytial virus (RSV) is an important cause of bronchiolitis in infants, is an important trigger of asthma exacerbation, and stimulates chemokine production by human respiratory epithelial cells in vitro. We tested the effect of the corticosteroid fluticasone propionate (FP) on RSV-stimulated production of the chemokines interleukin 8 (IL-8) and RANTES (regulated upon activation, normal T cell expressed and secreted) by a human bronchial epithelial cell line, BEAS-2B. Confluent BEAS-2B cultures were inoculated with RSV at approximately 1 plaque-forming unit/cell, and media were collected at 24 h intervals. Concentrations of IL-8 and RANTES were measured in supernatants using ELISA. The effect of FP at varying concentrations on RSV-induced chemokine release was determined. RSV stimulated increased release of both IL-8 and RANTES, particularly at 24-48 h after virus inoculation. Significant but incomplete inhibition of RSV-stimulated increases for both chemokines was found when cultures were treated with FP at > or = 10(-8) M (for IL-8) or > or = 10(-7) M (for RANTES). There was no significant effect of FP on release of RSV itself from infected BEAS-2B cells. We conclude that a possible mechanism for the efficacy of inhaled corticosteroids in reducing the frequency or severity of asthma exacerbations is inhibition of virus-induced chemokine production by airway cells.

Topics: Androstadienes; Anti-Inflammatory Agents; Bronchi; Cell Line; Chemokine CCL5; Epithelial Cells; Fluticasone; Humans; Interleukin-8; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human

Respiratory epithelial cells infected with respiratory syncytial virus (RSV) produce interleukin-8 (IL-8); however, the mechanisms of RSV-induced regulation of IL-8 are poorly understood. In the present study, the regulation of IL-8 by RSV was evaluated using pulmonary type II-like epithelials (A549). Live purified RSV (pRSV) induced a significant increase in IL-8 after 8 hr of exposure, while conditioned supernatants from pRSV-infected A549 cells (cRSV) induced IL-8 production in fresh A549 cultures within 4 hr of infection. Furthermore, cRSV that had been rendered non-infectious by ultraviolet-irradiation (UV-cRSV) or ribavirin treatment also induced an increased production of IL-8 in fresh A549 cells, suggesting that RSV induced the synthesis of a soluble mediator(s) which in turn enhanced the synthesis of IL-8. We have previously shown that RSV-infected A549 cells produce IL-1alpha, IL-1-beta and tumour necrosis factor-alpha (TNF-alpha), which by themselves are known to induce the synthesis of IL-8. Preincubation of UV-cRSV or simultaneous incubation of pRSV with recombinant IL-1 receptor antagonist almost completely blocked (95-98%) the production of IL-8 by A549 cells. Furthermore, incubation with neutralizing antibodies against IL-1alpha, IL-1beta and TNF-alpha showed that IL-1alpha was the predominant soluble mediator that enhanced the mRNA expression and synthesis of IL-8. IL-1beta and TNF-alpha induced the synthesis of IL-8 at 24 hr, but partially inhibited the synthesis at 48 hr. In summary, these experiments provide direct evidence for an autocrine mechanism of enhanced IL-8 production in RSV-infected epithelial cells that is primarily mediated by IL-1alpha. In clinical settings, inhibitors of IL-1alpha may be useful in suppressing inflammation due to IL-1alpha as well as IL-8.

Topics: Antibodies, Monoclonal; Autocrine Communication; Cell Line; Epithelium; Humans; Interleukin-1; Interleukin-8; Lung; Protein Isoforms; Respiratory Syncytial Virus Infections; RNA, Messenger; Tumor Necrosis Factor-alpha; Virus Cultivation

Respiratory syncytial virus (RSV) infections in children precipitate acute episodes of respiratory obstruction that are associated with influx of inflammatory cells into the airway. Since RSV can induce the expression of adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), by the respiratory epithelium, the hypothesis has been proposed that ICAM-1 expression contributes to airway inflammation by supporting adhesion and retention of infiltrating inflammatory leukocytes. To test this hypothesis, A549 cells (an immortalized human alveolar epithelial type II cell-like fine) were infected with RSV, and the ability of these infected monolayers to support adhesion by human neutrophils (NEUT) and eosinophils (EOS) was measured. RSV infection significantly increased ICAM-1 expression by A549 monolayers (p < 0.001). Although NEUT adhesion to A549 monolayers was significantly enhanced following RSV infection (p < 0.001), infection alone resulted in little change in EOS adherence. However, if EOS were first activated with phorbol ester (PMA), adhesion to both control and RSV-infected A549 cells was enhanced, with greater levels of adhesion supported by RSV-infected cultures (p < 0.001). The requirement for EOS activation (but not for NEUT activation) before adhesion remained when NEU and EOS were prepared and compared from the same donor. Despite this difference, NEUT and EOS adhesion was reduced by blocking Abs to epithelial ICAM-1 or granulocyte CD18 adhesion proteins (p < 0.01). However, only NEUT adhesion was blocked by Ab to CD11a. Our results show that RSV infections of respiratory epithelial monolayers can promote inflammatory cell adherence which could, in turn, potentially contribute to the airway injury and obstruction that accompanies bronchiolitis.

Topics: Adult; CD11 Antigens; CD18 Antigens; Cell Adhesion; Cells, Cultured; Eosinophils; Female; Histocompatibility Antigens Class I; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Male; Neutrophils; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses

Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5-10 after 18-24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 microg/5 X 10(5) PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 microg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.

Topics: Base Sequence; Cells, Cultured; HN Protein; Humans; Interleukin-8; Kinetics; Molecular Sequence Data; Neutrophils; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; Viral Envelope Proteins; Viral Proteins; Virus Replication

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

Topics: DNA Mutational Analysis; Gene Expression Regulation; Humans; Interleukin-8; NF-kappa B; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Tumor Cells, Cultured; Virus Replication

During the initial phase of respiratory syncytial virus (RSV) infection, when a low virus-cell ratio is most probable, signs of inflammation are detectable in the infected respiratory tissue. Therefore we analysed the release of the proinflammatory cytokines interleukin-6 (IL-6), IL-8, tumour necrosis factor-alpha (TNF-alpha), and the soluble form of the TNF receptor-I (sTNFR-I), from peripheral blood mononuclear cells (PBMC) after exposure to low infectious RSV doses (multiplicity of infection, MOI, 0.001-1) and incubation times of up to 24 hr. The PBMC secreted IL-8 in a time- and virus dose-dependent fashion. As was verified by Northern blot analysis, the increased IL-8 secretion rate was accompanied by an enhanced IL-8 mRNA steady-state level. The infection of the PBMC after 4 hr post-RSV exposure was verified by detection of RSVSH genomic RNA and mRNA after reverse transcription and polymerase chain reaction (PCR) amplification. In addition, after 24 hr post-infection we determined the percentage of infected cells by specific immunofluorescence using monoclonal antibodies directed against the F- and G-proteins. After exposure of PBMC to inactivated RSV, we observed only RSVSH genomic RNA and a reduced IL-8 release. Thus, even the binding and/or phagocytosis of RSV by PBMC induced an IL-8 synthesis to some extent. Following an incubation time of 24 hr, PBMC exposed to small RSV doses synthesized and released high amounts of IL-6 into the cell supernatant. In contrast, only low amounts of TNF-alpha were released from PBMC. In addition to the release of the proinflammatory cytokines, an enhanced level of the sTNFR-I was measured in the cell supernatants at a MOI of 0.1. However, there was no correlation between TNFR-I membrane expression and cell supernatant concentration. Co-culture experiments performed with PBMC and human epithelial cells (A549) revealed that the enhanced IL-8 secretion profile observed in the coculture was partially dependent on the cytokines TNF-alpha, IL-1 beta and TNF-beta/lymphotoxin released by the cells themselves.

Topics: Antigens, CD; Base Sequence; Blotting, Northern; Cells, Cultured; Cytokines; Epithelium; Humans; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Respiratory Syncytial Virus Infections; Tumor Necrosis Factor-alpha

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway, and causes a local inflammatory response. Although it has been previously demonstrated that RSV-infected airway epithelial produce cytokines, including interleukin-8 (IL-8), which contributes to the inflammatory response, the regulation of this effect of RSV is unknown. To further characterize the mechanisms by which RSV infection triggers release of IL-8, we first exposed cultured A549 cells to RSV, and measured IL-8 release via enzyme-linked immunosorbent assays (ELISA), and IL-8 messenger RNA (mRNA) induction via Northern blot analysis. We observed a dose- and time-dependent release of IL-8 in response to RSV. The optimal dose of RSV was 10(4) TCID50/ml, and maximal release of IL-8 was measured at 72 to 96 h after infection. RSV induced a biphasic (early and late) increase in IL-8 mRNA. The early phase was independent of viral infection, whereas the more pronounced late phase required the presence of live virus and infection of the epithelium. Partial (< 50%) cytopathic effects were noted at 48 h and progressed to 75% at 96 h. The monolayer was still intact at 96 h. Inhibitors of nitric oxide, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and aminoguanidine had no effect on IL-8 release or IL-8 mRNA induction. We did, however, demonstrate a dose-dependent decrease in IL-8 release and IL-8 mRNA induction in RSV-infected epithelial treated with the antioxidants dimethyl sulfoxide (DMSO) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Peak effects were noted at a concentration of 2% DMSO and 50 microM DMPO. The antioxidants did not inhibit viral replication or infection. This data suggest that RSV-induced IL-8 production in airway epithelium is mediated via changes in oxidant tone. The data also suggest a potential therapeutic role for antioxidants in RSV infections.

Topics: Antioxidants; Blotting, Northern; Epithelium; Humans; Interleukin-8; Laryngeal Neoplasms; Nitric Oxide; Oxidants; Respiratory Syncytial Virus Infections; RNA, Messenger; Tumor Cells, Cultured; Virus Replication

The mechanism of respiratory syncytial virus (RSV)-induced inflammation in the airways of infants and children is not fully understood. We hypothesized that RSV directly induces interleukin (IL)-8 gene expression in airway epithelial cells, independent of IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) production. Exposure of A549 cells (an airway epithelial cell line) to RSV resulted in increased IL-8 mRNA expression and IL-8 protein release from the cells as early as 2 h after treatment. Neither IL-1 beta nor TNF-alpha (mRNA or protein) were detected. Viral replication was not necessary for the effects of RSV on IL-8 mRNA expression and protein release early in the infectious process. However, sustained levels of increased IL-8 production required RSV replication. A dose-response relationship was observed between the multiplicity of infection and IL-8 production with both active and nonreplicative RSV at the 2-h time point. Both active RSV and nonreplicative RSV increased the transcriptional activity of the 1.6-kb 5' flanking region of the IL-8 gene. Neither active RSV nor nonreplicative RSV increased the stability of the IL-8 mRNA in A549 cells. We conclude that RSV increases IL-8 gene expression in A549 cells in a biphasic pattern independent of viral replication early (2 h) but dependent on viral replication late (24 h).

Topics: Animals; Chlorocebus aethiops; Drug Stability; Gene Expression; Genes; Humans; Interleukin-8; Lung Neoplasms; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; RNA, Messenger; Time Factors; Transcription, Genetic; Tumor Cells, Cultured; Virus Replication

Topics: Bronchiolitis, Viral; Humans; Infant; Interleukin-8; Respiratory Syncytial Virus Infections; Tumor Necrosis Factor-alpha

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.

Topics: Blotting, Northern; Cell Line; Cytokines; Dose-Response Relationship, Immunologic; Epithelium; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Lung; Receptors, Interleukin; Receptors, Interleukin-8A; Receptors, Tumor Necrosis Factor; Respiratory Syncytial Virus Infections; RNA, Messenger