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Department of Pediatrics, Catholic University School of Medicine
Institute of Neurobiology, Italian National Research Council, Rome, Italy
Batchelor Children's Research Institute
Departments of Pediatrics, Medicine
Molecular/Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida
ABSTRACT
Rationale: Nerve growth factor and its receptors are upregulated in the lungs of weanling rats with lower respiratory tract infection caused by the respiratory syncytial virus (RSV), and this is a major mechanism of the inflammatory response against the virus. However, no information is available in humans. Objectives: We sought to determine whether the expression of neurotrophic factors and receptors is increased in human infants infected with RSV. Methods: We used a highly sensitive immunoassay to measure the concentrations of nerve growth factor and brain-derived neurotrophic factor proteins in serum samples and in the supernatants and cell fractions of nonbronchoscopic bronchoalveolar lavage fluid. We also used immunofluorescence to detect expression in airway cells of the tyrosine kinase receptor TrkA, which binds nerve growth factor with high affinity. Samples were obtained at 24 and 48 hours postintubation from 31 mechanically ventilated infants: 15 patients with RSV infection, 5 patients with respiratory infection negative for RSV, and 11 postsurgical patients without respiratory infection. Main Results: Data show significantly higher concentrations of both neurotrophins in the cell fractions of bronchoalveolar lavage samples obtained from infants with RSV infection compared with control infants, whereas no significant difference was found in supernatants or serum samples. We also detected tyrosine kinase receptor immunoreactivity only in cells from airways infected with RSV. Conclusions: We conclude that neurotrophic factors and receptors are overexpressed in human airways infected by RSV, and may contribute to airway inflammation and hyperreactivity.
Key Words: asthma; brain-derived neurotrophic factor; bronchiolitis; nerve growth factor; pneumonia
Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infancy, accounting for approximately 80% of lower respiratory tract infections in this age group (1). Studies conducted in animal models suggest that dysregulated neuroimmune interactions and altered synthesis/release of proinflammatory neuropeptides play an important pathophysiologic role in the inflammatory events associated with RSV infection (2). In particular, early-life RSV infection is associated with increased expression in the developing lungs of the prototypic neurotrophin nerve growth factor (NGF) and its high-affinity tyrosine kinase receptor TrkA and low-affinity receptor p75 (3).
Under the influence of NGF, the nociceptive fibers innervating the lower respiratory tract increase both synthesis and release of the proinflammatory neurotransmitter substance P (4), and concomitant overexpression of the high-affinity receptor neurokinin 1 for substance P on target inflammatory cells leads to exaggerated neurogenic inflammation (5, 6). The extrapolation of these experimental observations to the pathophysiology of RSV disease in humans is complicated by the lack of information concerning the expression of NGF and other neurotrophins in infant lungs, and more importantly, the changes associated with human RSV infection.
This study sought to determine whether the neurotrophic factors NGF and brain-derived neurotrophic factor (BDNF) are overexpressed in the lower respiratory tract of infants infected with RSV, and whether this is reflected by measurable changes in serum levels. To this end, we measured the concentration of these neurotrophic factors in mechanically ventilated infants with RSV-positive lower respiratory tract infection (LRTI) in comparison with two control groups: (1) mechanically ventilated infants with RSV-negative LRTI and (2) infants without LRTI mechanically ventilated for postsurgical management. The measurements were performed in serum and nonbronchoscopic bronchoalveolar lavage (BAL) collected at 24 and 48 hours after intubation. In addition, we processed BAL cells from patients with LRTI for absolute and differential cell counts and for immunofluorescence with specific antibodies to study the expression of high-affinity NGF receptors.
Some of the results of these studies have been previously reported in the form of an abstract (7).
METHODS
Additional detail on the methods is provided in the online supplement.
Study Population
We conducted a prospective observational study of 31 intubated and mechanically ventilated infants. Of these patients, 15 had developed respiratory failure caused by severe LRTI after presenting with progressive respiratory distress, wheezing, cough, poor feeding, apnea, and radiologic signs of lung hyperinflation. All patients were admitted to the intensive care unit between 14 and 48 hours after the onset of LRTI symptoms (average, 26 hours). RSV infection was confirmed by direct immunofluorescence or viral culture performed on nasopharyngeal samples.
Control groups included the following: (1) five infants with respiratory failure caused by severe LRTI but with negative RSV immunofluorescence and (2) 11 infants without respiratory pathology and with negative RSV immunofluorescence, mechanically ventilated for postsurgical management.
Specimen Collection
NGF and BDNF were measured in blood and BAL collected at 24 and 48 hours after intubation. Blood samples (1 ml each) were centrifuged at 1,000 x g for 3 minutes and the serum was immediately removed and frozen at eC70°C. BAL was collected following the recommendations of the European Respiratory Society Task Force (8) by instillation of two sequential and separate aliquots of 1 ml/kg 0.9% NaCl each into the endotracheal tube, followed immediately by suctioning. The first BAL specimen from each patient was centrifuged at 1,000 x g for 3 minutes, and the supernatant was immediately removed and frozen at eC70°C for later neurotrophin assay. The cell pellet was lysed in 0.25 ml of buffer, then centrifuged at 10,000 rpm for 30 minutes, and the supernatant processed for neurotrophin assay.
The second BAL specimen was split into two aliquots. The cell pellet from one aliquot was resuspended in 250 e of 0.9% NaCl and absolute cell counts were obtained with an automatic analyzer. Two slides were prepared from this aliquot spinning 25 e of suspended cells onto glass slides, and a differential count was performed on 500 nucleated cells per slide. The cell pellet from the other aliquot was processed for TrkA immunofluorescence.
Neurotrophin Assays
NGF concentration was measured in serum, BAL supernatants, and BAL cell pellets using a modification of the highly sensitive and specific enzyme-linked immunoassay originally described by Weskamp and Otten (9). With this assay, NGF recovery exceeded 90%, sensitivity was 3 pg/ml, and cross-reactivity with other neurotrophins was less than 3%. BDNF concentration was measured using the enzyme-linked immunoassay from Promega (Madison, WI). The sensitivity of this assay is 15 pg/ml and cross-reactivity with other neurotrophins is less than 3%. NGF and BDNF concentrations were expressed as ng/ml for serum and supernatants, and as ng/g for cell fractions.
TrkA Immunofluorescence
As specific antiserum, we used rabbit antihuman TrkA antibody (10, 11). Specific binding to the primary antibody was detected by incubation with a fluorescein isothiocyanateeCconjugated antirabbit/-mouse F(ab')2 fragment.
Statistical Analysis
Data are expressed as mean ± SD, unless otherwise indicated. Clinical characteristics were compared by 2 Yates' corrected test, whereas continuous variables were compared by analysis of variance (12). Differences with p values less than 0.05 were considered significant. Statistical analysis was performed using the software EpiInfo version 6 (Centers for Disease Control and Prevention, Atlanta, GA).
RESULTS
There was no significant difference in mean chronologic age, weight, or sex ratio between infants with RSV-positive LRTI, infants with RSV-negative LRTI, and infants without LRTI (Table 1). Also, gestational age at birth was similar for the three groups: 38.5 ± 1.5 weeks, 37.8 ± 1.1 weeks, and 38.5 ± 1.1 weeks, respectively. Oxygen supplementation for infants without LRTI was shorter compared with infants with LRTI (p < 0.001), whereas no significant difference was found between RSV-positive and RSV-negative LRTI. Also, there was a trend for higher FIO2 requirement, longer mechanical ventilation, and longer hospitalization in infants with LRTI compared with infants without LRTI, but these differences did not reach statistical significance. Of the infants with RSV-negative LRTI, one had BAL cultures positive for adenovirus and another was positive for parainfluenza virus; the other cultures were negative.
Analysis of serum NGF and BDNF concentrations at 24 and 48 hours after intubation showed no significant differences between RSV-infected infants and either control group (Figure 1). Also, no significant differences in NGF or BDNF concentrations were found in the supernatants of BAL samples collected from RSV-infected and control infants at 24 and 48 hours (Figure 2).
In contrast, significantly higher NGF and BDNF concentrations were measured in the BAL cell fractions from infants with RSV LRTI compared with both control groups (Figure 3). At 24 hours, NGF in the RSV-positive LRTI group was 7.8-fold higher compared with the RSV-negative LRTI group (p < 0.001) and 5.8-fold higher compared with the group without LRTI (p < 0.001). At 48 hours, NGF in the RSV-positive LRTI group was 7.4-fold higher compared with the RSV-negative LRTI group (p < 0.05) and 5.4-fold higher compared with the group without LRTI (p < 0.01). All differences in NGF concentration between control infants with RSV-negative LRTI and control infants without LRTI were not statistically significant.
BDNF concentrations were one (in BAL samples) to two (in serum samples) orders of magnitude larger than NGF concentrations. At 24 hours, BDNF in the RSV-positive LRTI group was 3.4-fold higher compared with the RSV-negative LRTI group (p < 0.001) and 4.4-fold higher compared with the group without LRTI (p < 0.001). At 48 hours, BDNF in the RSV-positive LRTI group was 11.7-fold higher compared with the RSV-negative LRTI group (p < 0.05) and 5.4-fold higher compared with the group without LRTI (p < 0.01). Again, all differences in BDNF concentration between control infants with RSV-negative LRTI and control infants without LRTI were not statistically significant.
Cytologic analysis of the BAL (see Table E1 in the online supplement) showed no significant differences in absolute and differential leukocyte counts between RSV-positive and RSV-negative infants with LRTI.
TrkA receptor localization by immunofluorescence was performed on BAL cells from 13 infants with LRTI, 10 who were RSV-positive and three who were RSV-negative. These preparations were examined by a pathologist blind to the illness groups, who detected immunofluorescence staining in all specimens from RSV-positive patients, whereas all specimens from RSV-negative patients were read as negative. TrkA immunofluorescence was detected both in desquamated epithelial cells and inflammatory cells recovered from RSV-infected airways (Figure 4).
DISCUSSION
Our data show that the concentrations of the neurotrophic factors NGF and BDNF in the lower respiratory tract of human infants increase during RSV infection. Both neurotrophic proteins were significantly increased in the cell fractions obtained by nonbronchoscopic BAL from RSV-infected infants compared with matched control infants with RSV-negative LRTI or control infants without LRTI. BDNF was more abundant than NGF in all samples analyzed and exhibited a sharper increase during the infection. Absolute and differential cell counts from the BAL of RSV-positive and RSV-negative patients with LRTI were not different, suggesting that the increased synthesis of neurotrophic factors derives from RSV-induced activation rather than recruitment of inflammatory cells. In addition, epithelial and inflammatory cells from the airways of RSV-infected infants expressed TrkA receptor immunoreactivity, whereas cells from RSV-negative airways did not.
All patients underwent BAL at the same time points after intubation; therefore, exposure to mechanical ventilation cannot explain our findings. In addition, oxygen requirement was quite similar in RSV-positive and RSV-negative patients with LRTI despite the large difference in neurotrophin levels measured in their BAL; thus, hyperoxia alone, at least with FIO2 levels up to 0.5 for 48 hours, is unlikely to be a critical determinant of neurotrophin expression in the airways.
We did not detect significant changes in serum samples and BAL supernatants collected during the first 48 hours after intubation. A possible explanation is that these specimens reflect only indirectly the actual concentrations of neurotrophic factors present in the cells and tissues where they are produced, secreted, and involved in biological functions. However, in a subgroup of RSV-infected infants requiring mechanical ventilation for more than 48 hours, we detected significantly higher NGF concentrations in serum samples obtained at 72 hours (see Figure E1).
This subgroup was too small to support any definitive conclusion, but suggests that the transfer of locally secreted neurotrophins to the systemic circulation is gradual, and becomes measurable only later in the course of the infection and/or only in infants with more prolonged and severe RSV LRTI. This interpretation of our data would also explain why higher NGF levels were found in the serum (13) and BAL fluid (14) of patients with chronic asthma; as in our study, serum neurotrophin levels may have been low because the sampling was done early in the course of the illness.
Our study provides the first evidence of a strong increase of neurotrophic factors and receptors in infected human airways. Probable sources of neurotrophic factors are infected structural cells (14), as well as inflammatory cells recruited and stimulated by the virus, such as activated CD4+ T lymphocytes (15) that produce NGF and express TrkA receptors (16). Whatever the source, NGF plays an important role in the mechanism of the inflammatory response during acute infection, as shown by previous studies in animal models (3), and can also exert long-term effects on airway reactivity by remodeling neuronal networks in the respiratory tract (17).
It remains to be defined by future studies whether other pathogens have similar neurotrophic effects, although the finding that RSV-negative LRTI was not associated with changes in neurotrophic pathways suggests that this effect may be unique to RSV, or at least shared by specific pathogens only.
After uptake by the peripheral nerve fibers, neurotrophic factors reach their nucleus by axonal transport, and there modulate the expression of genes ultimately responsible for neuronal growth, survival, and apoptosis (18). Specific receptors for NGF and BDNF are also expressed on multiple nonneural cell types (mast cells, eosinophils, T lymphocytes, epithelial and endothelial cells), where they modulate diverse biological functions (18eC21).
A number of recent studies have shown that NGF is overexpressed in human and animal models of lung inflammation (22), but the exact role played by NGF in these conditions has not been established conclusively, and there is circumstantial evidence supporting both protective and pathologic effects (23eC27). As an example of the former, NGF has been shown to be involved in cell protection within the nervous and immune systems during viral infections, and may confer resistance against the herpes simplex virus (28). Thus, the possibility exists that overexpression of these neurotrophic factors in RSV-infected cells might serve innate protective mechanisms, either alone or synergistically with other neurokines and cytokines.
On the other hand, NGF, through its multiple actions on resident airway cells, immune and inflammatory cells, and neurons, is likely to play an important role in the pathologic manifestation of RSV infection. Studies in animal models suggest that this neurotrophin and its high-affinity (TrkA) and low-affinity (p75) receptors are markedly upregulated in RSV-infected lungs (3). NGF turns on airway inflammation during the acute infection by increasing the expression of high-affinity NK1 receptors for the proinflammatory neuropeptide substance P (3). In addition, NGF leads to structural and functional remodeling of the nociceptive fibers innervating the respiratory mucosa, increasing both synthesis and release of substance P from C-type nociceptive fibers (4). Thus, NGF represents an essential link between RSV-infected epithelial cells and the dense subepithelial neural networks and can contribute to the development of airway inflammation and hyperreactivity (29).
The studies in animal models of RSV infection outlined above have explored primarily the expression and inflammatory effects of NGF and its receptors. Thus, the concurrent large increase of BDNF levels in infants with RSV bronchiolitis was a somewhat unexpected finding, which has set off a new series of experiments in animals looking at the specific modulation and biological effects of this neurotrophin and its high-affinity receptor TrkB. Indeed, it is possible that, in addition to the absolute expression of individual neurotrophic factors and receptors, the relative expression of these molecules also may have important pathophysiologic significance for airway inflammation and remodeling.
The increased concentration of NGF from BAL cells is complemented by overexpression of high-affinity TrkA receptors on a variety of target cell types (epithelium, macrophages, mast cells), suggesting that locally secreted NGF, and most likely also BDNF, act in an autocrine and/or paracrine fashion to amplify and propagate inflammation in the infected respiratory tract.
In conclusion, we have shown that the neurotrophic proteins NGF and BDNF are markedly overexpressed in cells recovered from the lower airways of infants with severe RSV LRTI. Cells from RSV-infected airways also display strong immunoreactivity for the high-affinity NGF receptor, which suggests that autocrine and/or paracrine mechanisms are involved. The increased production of neurotrophic proteins is not reflected in blood samples or BAL supernatants collected during the first 48 hours after intubation, but may become measurable later in the course of the disease or in particularly severe infections. These findings are consistent with previous animal data suggesting an important role for neurotrophins in the pathogenesis of airway inflammation and hyperreactivity during RSV infection. Selective blockade of neurotrophic factors or receptors may offer a promising new strategy for the management of this common pediatric respiratory disease, although additional basic and clinical research work is needed.
Acknowledgments
The authors thank the staff of the Pediatric Intensive Care Unit of the Catholic University School of Medicine, Rome, Italy, for their invaluable help with the collection of specimens for this study. They also thank Silvia Misiti, M.D., Ph.D. (University of Rome "La Sapienza" School of Medicine), for reading and interpreting the TrkA immunofluorescence preparations.
Some of the findings reported in this article were presented at the 2004 Pediatric Academic Societies' Annual Meeting in San Francisco, CA.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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