Respiratory syncytial virus-infected pulmonary epithelial cells induce eosinophil degranulation by a CD18-mediated mechanism

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.     

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.     

Respiratory syncytial virus bronchiolitis

Respiratory syncytial virus (RSV) bronchiolitis is associated with the clinical signs and symptoms of small airway obstruction. A major public health problem throughout the world, this condition is responsible for significant morbidity and mortality. Management is primarily preventive, through strict hand washing, avoidance of exposure during the respiratory illness season and intravenously administered prophylactic anti-RSV Immune globulin, especially in selected small infants with underlying cardiopulmonary disease. Supportive measures, including fluid hydration, good nutrition, aerosolized bronchodilators and steroids, may be helpful. Ribavirin may be useful in severely ill children or those with underlying cardiopulmonary disease. A significant number of patients have recurrent episodes of bronchiolitis and wheezing, and may develop asthma later in life. Avoidance of exposure to tobacco smoke, cold air and air pollutants is also beneficial to long-term recovery from RSV bronchiolitis. A number of vaccines to prevent this infection are currently being studied.     

Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus

Two neutralizing, fusion-inhibiting bovine monoclonal antibodies (MAbs; B4 and B13) directed at different epitopes on the fusion protein of respiratory syncytial virus (RSV) protected the lungs of gnotobiotic calves from RSV infection. The MAbs were administered intratracheally 24 h before the calves were challenged with bovine RSV. A third, nonneutralizing, non-fusion-inhibiting but complement-fixing MAb, B1, provided no significant protection against infection, and the disease was not exacerbated. Pneumonic consolidation and mean virus titer in lung 7 days after challenge were significantly lower in calves given the fusion-inhibiting MAbs than in either control calves or those given MAb B1. The proliferative bronchiolitis with syncytial formation and widespread distribution of RSV antigen in the lower respiratory tract of the B1-treated and control calves were indistinguishable and typical of experimental bovine RSV infection. Syncytia were markedly absent, and little or no viral antigen was detected in either the B4- or B13-treated calves.     

A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection.     

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies

Infections with respiratory syncytial virus (RSV) are characterized by frequently occurring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.     

Diabetes mellitus and lipid metabolism (author''s transl)

Fifteen infants with pneumonia caused by respiratory syncytial virus (RSV) and 19 infants with bronchiolitis caused by RSV were studied for the influence of homologous, circulating neutralizing antibody on the severity of their illness. All infants were under nine months of age. Although maternal neutralizing antibody did not prevent infection with RSV and illness, the severity of pneumonia caused by RSV was inversely related to the level of neutralizing antibody. The severity of bronchiolitis caused by RSV was unrelated to maternal antibody levels. Chest roentgenograms showed pneumonia to be slightly more severe than bronchiolitis. Neither the severity of illness nor the presence of maternal neutralizing antibody was related to the development of complement-fixing antibody.     

Interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6

Inflammation in nasal and airway tissue caused by allergens, microbial infection, and air pollution are likely to be regulated by inflammatory mediators produced by airway epithelial cells. We have therefore investigated the baseline expression of a number of cytokine genes known to be important inducers and modulators of inflammation, in freshly isolated human nasal epithelium. Cells were obtained by superficial scraping of turbinate tissue, and cDNA for polymerase chain reaction (PCR) amplification was reverse-transcribed directly from lysates of 3 x 10(3) to 5 x 10(3) epithelial cells using random hexamers. Constitutive expression of relatively high levels of interleukin-8 (IL-8) mRNA but undetectable levels (< 1 mRNA copy/cell) of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-6, IL-1, or tumor necrosis factor (TNF) mRNA were found after PCR amplification of the cDNA. IL-8 protein, but not IL-6, was identified in the nasal epithelial cells by immunocytochemistry. Infection with respiratory syncytial virus (RSV) or stimulation of nasal epithelium for 4 h with TNF or IL-1 in vitro resulted in a 4- to 10-fold increase in IL-8 mRNA expression but not in the expression of detectable levels of mRNA for the other cytokines. IL-8 was secreted by RSV-, IL-1-, and TNF-stimulated as well as unstimulated nasal epithelial cells after 6 to 20 h of culture. Neither IL-6, GM-CSF, nor TNF activity/immunoreactivity was detectable in the culture supernatants. Thus, it appears that IL-8 is a major cytokine of human nasal epithelium, constitutively expressed and readily secreted upon virus infection or stimulation with IL-1 and TNF.     

Respiratory syncytial virus infection in childhood

Respiratory syncytial virus is the most frequent cause of respiratory tract infections in infants and is responsible for annual winter epidemics of acute bronchiolitis. Over the last decades medical therapy has remained unchanged and controversial, despite intensive research. Inhaled bronchodilators are often not effective and should be discontinued if no beneficial response can be documented. Steroids and ribavirin are not indicated in previously healthy infants with acute RSV bronchiolitis. There is some evidence, however, that certain risk groups may benefit from their use. With good supportive care the mortality from RSV infection is now low. Postinfectious alterations in lung function are usually transient and reversible. High-risk infants can be protected from severe RSV infections by monthly infusions of RSV immune globulins. This treatment modality has, however, not gained wide acceptance because of the benign nature of the disease and the high costs and side effects of regular immune globulin infusions. An international consensus statement on the treatment of RSV bronchiolitis may help to reduce the wide differences in clinical practice.     

Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.     

Efficacy of bronchodilators in the treatment of bronchiolitis

Bronchiolitis is a common respiratory infection affecting young children. Much controversy revolves around the efficacy of bronchodilators in the treatment of bronchiolitis. This study was conducted to address this issue. AIM: To determine the efficacy of bronchodilators in the treatment of bronchiolitis. METHOD: All children less than 2 years old with bronchiolitis were randomly assigned to receive nebulisations of Salbutamol, Ipratropium bromide or normal saline. A fourth group given only humidified oxygen without nebulisation were used as a control. RESULTS: Data were obtained for 120 patients. Fifty-one (42%) had respiratory syncytial virus (RSV) isolated from their nasopharyngeal aspirates. The demographic characteristics of the 4 groups were similar. There was no significant difference between the groups in terms of severity score, number of nebulisations required in the nebulised groups and the outcome as measured by the length of hospitalisation. CONCLUSION: The use of bronchodilators did not alter the course of the disease and is therefore not effective in the treatment of bronchiolitis.     

Epidemiology of respiratory syncytial virus infection requiring hospitalization in East Denmark

BACKGROUND: Prophylaxis against infection caused by respiratory syncytial virus (RSV) with high titered RSV immunoglobulin or humanized antibody may soon be available in Europe. OBJECTIVE: To study the epidemiology of RSV infections requiring hospitalization in infants <6 months in East Denmark to provide a rational basis for decisions concerning prophylaxis against RSV. METHOD: Populat ion-based retrospective review of case records of infants <6 months admitted to pediatric departments with RSV infection in East Denmark from November 1, 1995, to April 30, 1996. RESULTS: Data were obtained from 459 infants. Seventy-three had predisposing conditions: prematurity, 49; pulmonary disease, 2; congenital heart disease, 7; neurologic disease, 6; others, 9. One preterm infant had bronchopulmonary dysplasia. The incidence of RSV infection requiring hospitalization in East Denmark among infants <6 months was estimated to be 34/1000/season. It was 32/1000/season among term infants and 66/ 1000/season among preterm infants (P<0.001). Infants with predisposing conditions and/or nosocomial infection (n = 24) had significantly more severe courses than otherwise healthy infants (P<0.01). One-hundred thirty infants received respiratory support by nasal continuous positive airway pressure, but only six required mechanical ventilation. No infants died. CONCLUSION: The course of RSV disease in East Denmark was milder than reported elsewhere, possibly as a result of the low prevalence of bronchopulmonary dysplasia in Denmark. However, RSV constitutes a considerable burden to the Danish pediatric health care system, and therefore prophylaxis against RSV is desirable.     

A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

A critical issue has been the observation that vaccination of children with a formalin-inactivated respiratory syncytial virus (RSV) vaccine is associated with disease enhancement. We have taken advantage of bovine RSV and our experience with this disease in calves to develop a natural model that parallels human disease. Using formalin-inactivated bovine RSV vaccine calves were either sham-vaccinated/infected, vaccinated/infected, or vaccinated/sham-infected and their clinical signs, pulmonary function, and histological lung lesions quantitatively scored. Interestingly there was significantly greater disease in vaccinated/infected calves and histological lesions in calves were similar to those of affected children. Finally, we note that vaccination did not induce neutralizing antibodies, but IgG antibodies were detected by ELISA. Our model of RSV enhanced disease is important because it provides quantifiable evidence of disease severity that can be applied to evaluate the mechanisms of immunopathology and the safety of candidate RSV vaccines.     

Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis

BACKGROUND: Obliterative bronchiolitis is characterized histologically by inflammation, epithelial cell damage and loss, fibrosis, and eventual obliteration of airways. Production of high levels of the potential cytotoxin nitric oxide by inducible nitric oxide synthase has been implicated in several inflammatory diseases. The damaging effects of nitric oxide are mediated by peroxynitrite, are formed from nitric oxide and superoxide, and can be demonstrated by the detection of nitrotyrosine. Our previous finding of high inducible nitric oxide synthase expression in inflamed airway epithelium led us to hypothesize that release of nitric oxide in obliterative bronchiolitis mediates the characteristic epithelial damage. METHODS: Immunocytochemistry was carried out to seek expression of inducible nitric oxide synthase and nitrotyrosine in transplant samples from patients with obliterative bronchiolitis (n=10) and, as controls, unused donor lungs (n=5). RESULTS: Inducible nitric oxide synthase was strongly expressed in the damaged airway epithelium in obliterative bronchiolitis and in inflammatory cells, where its distribution was matched by that of nitrotyrosine. Normal controls showed little or no immunoreactivity for any of the antigens studied. CONCLUSIONS: Our findings suggest that nitric oxide may play a role in the pathogenesis of obliterative bronchiolitis and indicate that further work is essential to fully understand the processes and mechanisms involved.     

Successful treatment of severe dysrhythmias in infants with respiratory syncytial virus infections: two cases and a literature review

OBJECTIVES: To describe severe myocardial manifestations in two infants with respiratory syncytial virus infection and to review published literature reporting cardiac involvement in patients with respiratory syncytial virus disease. DESIGN: Case report and literature review. SETTING: Tertiary care pediatric intensive care unit (ICU). PATIENTS: Two infants admitted to the pediatric ICU for dysrhythmias and severe myocardial dysfunction and infected with respiratory syncytial virus. INTERVENTIONS: Conventional cardiovascular, antidysrhythmic, and respiratory support, as well as extracorporeal membrane oxygenation and high-frequency oscillatory ventilation. MEASUREMENTS AND MAIN RESULTS: Both patients had respiratory syncytial virus infections and clinical evidence of severe myocarditis, with dysrhythmias, cardiomegaly, and cardiogenic shock. Both infants survived their hospitalizations. To our knowledge, these two patients are the first reported cases of myocarditis in infants with respiratory syncytial virus infection. CONCLUSIONS: Severe myocardial dysfunction and dysrhythmias may accompany respiratory syncytial virus infection in some infants and may be reversible with aggressive supportive therapy.     

Approaching the RSV season with a nursing plan of action

As the statistics show with year-in, year-out regularity, during November through March in the United States, approximately 90,000 infants and young children will be hospitalized with a severe lower respiratory infection attributable to the respiratory syncytial virus (RSV). This virus, discovered only as recently as 1956, appears to be ubiquitous, infecting virtually 100% of children by age 4. For most of them the resulting illness will be mild and easily vanquished by an intact immune system. For some, however, RSV infection confers considerable morbidity, and these infants and children are the concern of the symposium held in conjunction with Pediatric Nursing's 11th annual conference. The symposium addressed several aspects of RSV infection: Who is at risk and should be hospitalized? How can nurses contribute to the care of hospitalized patients? Are there environmental risks to health-care personnel from ribavirin aerosol, the antiviral treatment approved for RSV infection? Are there special considerations for mechanically ventilated patients? Speakers generally concluded that symptomatic treatment and antiviral therapy with ribavirin aerosol can reduce severe morbidity in severely infected patients with minimal occupation risk to health-care personnel.