Neutrophil Activation And Protease Imbalance In The Respiratory Tract Of Infants With Respiratory Syncytial Virus Bronchiolitis

BackgroundRespiratory syncytial virus (RSV) is one of the major respiratory pathogens which cause the lower respiratory tract infections in children less than 2 years of age and RSV bronchiolitis is the most common disease in infants. The precise pathogenesis of RSV bronchiolitis has not been clearly characterized. Epidemiological studies have showed that at least 1/3 of infants with acute viral bronchiolitis due to RSV have subsequent episodes of wheezing and asthma. This leads to the suggestion that there may be a close link between RSV bronchiolitis and asthma . Airway inflammation in RSV bronchiolitis is undoubtedly a multicellular process in which epithelial cells, macrophages, cytotoxic T cells, eosinophils and neutrophils are involved. In recent years, there is increasing direct evidence supporting that neutrophil- mediated inflammation play an important role in the pathogenesis of the respiratory disease with viral infection. However, the activation of neutrophils and the severity of their inflammation in human RSV bronchiolitis have not yet been evaluated in detail. It has been proposed, that interleukin (IL-8) and neutrophil elastase (NE) are key factors in this process. IL-8 induces a mass of neutrophil recruitment and activation and neutrophils can contribute tothe inflammation by releasing neutrophil elastase to destroy pathogen , at the same time, the structures of lung. The organism have enough protease inhibitors, which protects lung tissue from proteolytic damage by NE and α₁-antitrypsin(α₁-AT)is the major. Therefore proteinase-antiproteinase balance acts an important role to maintain the stability of lung tissue which has been studied in detail but no documents in RSV bronchiolitis . To reveal the level of airway inflammation and protease disequilibrium, RSV bronchiolitis was compared with the bacterial pneumonia in which neutrophil inflammation and protease disequilibrium is clearly implicated. To assess neutrophil activation and protease imbalance is significant for exploring the pathogenesis of RSV bronchiolitis and may be useful for seeking better therapeutic strategy.ObjectiveTo better understand neutrophil activation and protease imbalance in RSV bronchiolitis, and to explore its role in the pathogenesis.MethodsWe studied the activation of neutrophils and the concentration of IL-8 and the imbalance of protease in the nasopharyngeal aspirates (NPA) from 30 infants with RSV bronchiolitis, 20 infants with RSV bronchiolitis of convalescent stages, 24 infants with bacterial pneumonia and 15 infants without respiratory infections as control.1. NPA were collected from patients with a fine catheter into a mucus trap under negative pressure. Aspirates was diluted with saline. Recorded the volume and dilution power and detected within 30 minutes.2. Direct immunofluorescence technique was utilized to identify RSV antigen in the respiratory epithelium cells.3. The samples were centrifuged at lOOOrpm for 10 minutes and the cell-free supernatant and cell-pellet were then separated.4. Cytology was analyzed by Wright-Giemsa stain and the percentage of the different cell types determined by counting at least 500 cells, including neutrophil, lymphocyte and eosinophil and other karyote.5. The supernatants were assayed for the concentration of NE and IL-8 using enzyme-linked immunosorbent assay(ELISA).6. Elastase inhibition capacity(EIC) / free neutrophil elastase activity was detected using a colorimetric assay, measuring the activity of elastase upon the synthetic substrate Meo-Suc-Ala-Ala-Pro-Val-p-nitroanilide.7. The smear of cell residue were assayed for the expression of a l^antitrypsin(a 1-AT) using cellular immunohistochemistry.8. SPSS 11. 5 for Windows was used for data handling and analysis. Data were showed with the mean±SD or medians and range. Differences in results among groups were examined with one-way analysis of variance (ANOVA) or f-test. Non-normally distributed data were log transformed or non-parametric tests performed as appropriate. P<0.05 was considered significant.Result1. Neutrophil: As compared with control and convalescent infants, RSV bronchiolitis showed an elevated percentage of neutrophil(M=52. 8% vs 7. 4%. 30. 6%, Z=6. 100, P<0. 01;t=2. 208, F<0. 05), but no difference between RSV bronchiolitis and pneumonia group (M=37. 6%, ï¿¡=0. 706, P>0. 05) was found. The percentage of neutrophil in convalescent infants were higher than in the control(t=2. 479, P<0. 05). Severe patients in bronchiolitis group had a higher percentage of neutrophil than mild (M=60 % vs 30.6%, t=2. 220, P<0. 05).2. Eosinophil: The percentage of eosinophil in RSV bronchiolitis (M=0. 9%) was highest within four groups, but no statistical significance was found.3. IL-8: As compared with control and convalescent infants, RSV bronchiolitis showed an elevated concentration of IL⁸(M=13622. 3pg/ml vs 2639. 7pg/ml,5951. lpg/ml, ï¿¡=14.051,6.436, P<0. 01), but no difference with pneumonia group (M=l3226. 8pg/ml, ï¿¡=1.619, P>0. 05) was found. The concentration of IL-8 in the convalescent infants was higher than that in the control(t=2. 925, P<0. 01). Severe patients in bronchiolitis group had higher concentration of IL-8 than mild(M=14047. 9pg/ml vs 12938. Opg/ml, f=2. 251, P<0.05).4. NE: The concentration of NE was highest in RSV bronchiolitis group when compared with control and convalescent infants (M=639. 2ng/ml vs 252.5 ng/ml, 123. 3ng/ml, ï¿¡=2.205,2.897, P<0. 05), producing significant differences. But no difference between RSV bronchiolitis and the pneumonia group (M=405ng/ml, ï¿¡=0.424, P>0. 05) was found. There was no difference between convalescent infants and control either (ï¿¡=0. 861, P >0.05). NE in severe cases was not higher than that in mild group(M= 715. 8ng/ml vs 562. 5ng/ml, ï¿¡=0.873, F>0. 05).5. EIC: Free elastase activity was found in 6 of the 30 infants(20 %) in RSV bronchiolitis group, without significant difference with 29. 2 % in pneumonia group(7/24) ( x*=0. 613, P>0. 05), and it was only one in convalesent infants and no found in control.6. a ,-AT: Increased expression of a ,-AT in RSV bronchiolitis was found when compared with convalesent infants (M=0. 49 vs 0.09, ï¿¡=2.732, P<0. 05), but no difference with the control (M=0. 26, ï¿¡=1.167, P>0. 05) was found. The expression score of a ^AT in the pneumonia group(M=0. 89)was higher than that in the control significantly(ï¿¡=2. 863, P<0. 05).Conclusion1. Mass of neutrophil was aggregated and activated, and interleukin-8 was released in a large amount in the respiratory tract in response to RSV bronchiolitis, being similar to bacterial pneumonia.2. Local neutrophil aggregation and IL-8 concentration were associated with the clinical severity and both of them recovered slowly.3. No obvious local eosinophil elevation in RSV bronchiolitis was found.4. There existed protease system imbalance and elevated NE in RSV bronchiolitis being similar to bacterial pneumonia, but the mechanism to redress the disequilibrium might be different.