Nerve growth factor (NGF) controls sensorineural development and responsiveness and modulates immunoinflammatory reactions. Respiratory syncytial virus (RSV) potentiates the proinflammatory effects of sensory nerves in rat airways by upregulating the substance P receptor, neurokinin 1 (NK1). We investigated whether the expression of NGF and its trkA and p75 receptors in the lungs is age dependent, whether it is upregulated during RSV infection, and whether it affects neurogenic inflammation. Pathogen-free rats were killed at 2 (weanling) to 12 (adult) wk of age; in addition, subgroups of rats were inoculated with RSV or virus-free medium. In pathogen-free rats, expression of NGF and its receptors in the lungs declined with age, but RSV doubled expression of NGF, trkA, and p75 in weanling and adult rats. Exogenous NGF upregulated NK1 receptor expression in the lungs. Anti-NGF antibody inhibited NK1 receptor upregulation and neurogenic inflammation in RSV-infected lungs. These data indicate that expression of NGF and its receptors in the lungs declines physiologically with age but is upregulated by RSV and is a major determinant of neurogenic inflammation.
- airway inflammation
- substance P
respiratory syncytial virus (RSV) is the most common respiratory pathogen in infancy, infecting nearly all children within the first 2 years of life (9). Furthermore, there is growing evidence that early RSV infection is an important risk factor for the development of recurrent wheezing and asthma in children (20). However, the risk of post-RSV wheezing declines with age and it is no longer significant after the first decade of life (30). Despite intense research efforts, the specific pathogenic mechanisms that underlie the link between RSV and childhood asthma remain elusive.
Studies performed on animal models suggest that abnormal neural control leads to airway hyperreactivity and inflammation after early-life RSV infection (20). RSV has been shown (15) to potentiate the neural pathways that favor bronchoconstriction (cholinergic and excitatory noncholinergic, nonadrenergic) and to hinder the pathways that favor bronchodilation (inhibitory noncholinergic, nonadrenergic). Our previous work has shown exaggerated neurogenic inflammatory responses upon stimulation of sensory nerves in the respiratory tract of rats during RSV infection (25), which are mediated by selective upregulation of the high-affinity substance P receptor (neurokinin 1 receptor, NK1), and we have also found that the distal airways in young rats are far more affected by neurogenic mediated inflammation than adult rats (14).
The prototypical neurotrophin nerve growth factor (NGF; Ref.16) is a key regulatory element of neuronal development and responsiveness (13) and controls the expression of genes that encode the precursors of substance P and other peptide neurotransmitters in sensory neurons (17). In addition, NGF modulates immune responses and has been associated with allergic inflammation and airway hyperresponsiveness in animal models as well as humans (1, 2), which suggests that neurotrophic factors may play an important pathophysiological role in asthma (3).
We first hypothesized that the different manifestations of RSV infection in infants, children, and adults result in part from age-related differences in the expression of neurotrophic factors that control the sensorineural pathways. Therefore, we studied whether the expression of NGF and its receptors in the lung tissues of rats varies at different developmental stages. Extending these studies, we sought to determine whether NGF and/or its receptors are upregulated during RSV lower respiratory tract infection. To answer these questions, we analyzed the expression of NGF, the high-affinity tyrosine kinase receptor trkA, and the low-affinity receptor p75 in the lungs of pathogen-free and RSV-infected rats at ages ranging from 2 wk (weanlings) to 12 wk (adults). In addition, we studied the effects of RSV inactivated by ultraviolet (UV) light irradiation as a negative control for the infection with replicating virus and compared the histopathological changes caused by the respiratory infection at different ages. Finally, we studied the role played by NGF in the mechanism of neurogenic inflammation in the lungs of weanling and adult rats during RSV infection by measuring the expression of the NK1 receptor and the exudative response to pharmacological stimulation of sensory nerves after stimulation with exogenous NGF or inhibition with anti-NGF antibody.
We used rats of Fischer 344 (F-344) strain of both sexes born to pathogen-free, timed-pregnant dams (Charles River Breeding Laboratories, Raleigh, NC) at 2, 4, 8, and 12 wk of age. Because previous studies have shown a profound effect of respiratory infections on neurogenic control and inflammatory responses in the respiratory tract (23), all animals used in this study were maintained under strict barrier conditions from birth to death to prevent any microbial contamination. Each dam or up to two litters were housed in polycarbonate cages isolated by polyester filter covers. These cages were placed on racks that provided positive individual ventilation with class-100 air to each cage at the rate of ∼1 cage change of air/min (Maxi-Miser, Thoren Caging System, Hazleton, PA; Refs. 14,22, 25). We used separate rooms for housing infected and pathogen-free rats, both of which were serviced by specifically trained husbandry technicians. All manipulations were conducted inside class-100 laminar flow hoods. Bedding, water, and food were autoclaved before use and unpacked only under laminar flow. Cages and water bottles were run through a tunnel washer after every use and disinfected with both chemicals and heat. The Division of Veterinary Resources of the University of Miami School of Medicine approved all experimental procedures followed in this study.
Total RNA was extracted from homogenized lung tissues with Tri-Reagent solution (Molecular Research Center, Cincinnati, OH). cDNA was synthesized from 2 μg of RNA using dT priming and Moloney murine leukemia virus RT (GIBCO-BRL, Grand Island, NY). mRNA levels were determined by semiquantitative PCR based on previously published protocols (2, 29). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. cDNA samples of 5 μl were added to 45 μl of PCR mixture that contained 5 μl of 10× PCR buffer, 1 μl of 10 mM deoxynucleotide triphosphate mixture, 1 unit of Taq DNA polymerase (Perkin-Elmer, Foster City, CA), and 50 pmol of 3′- and 5′-specific primers (Table 1). The primer pairs were designed to differentiate cDNA-generated PCR products from genomic DNA contamination.
Amplification was initiated with 10 min of denaturation at 94°C followed by 35 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min using a thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer). Amplified PCR products were size-fractionated by electrophoresis through 2% agarose gels and stained with ethidium bromide. The intensity of DNA bands was analyzed by computerized densitometry and expressed as the ratio of the densitometric score measured for each target gene normalized by the GAPDH control.
NGF protein levels in the lungs were measured with a commercial kit (Promega, Madison, WI) using the antibody-sandwich technique. In brief, 100 mg of lung-tissue samples were homogenized in 5 volumes of lysis buffer that contained 20 mM Tris · HCl, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 0.5 mM sodium vanadate. The supernatants of homogenized tissue samples were incubated for 18 h at 4°C in 96-well plates coated with 100 μl of anti-NGF polyclonal antibody (1.5 μg/ml in 100 mM carbonate coating buffer; pH 9.7) to bind NGF from the homogenates. After washing, a specific rat monoclonal antibody was applied (0.6 μg/ml in buffer, 100 μl/well) and incubated for 18 h at 4°C to bind the captured NGF. The plates were again thoroughly washed, and horseradish peroxidase-conjugated antibody to rat IgG was added to each well and incubated for 3 h at room temperature to detect the amount of specifically bound monoclonal antibody. After final washing to remove unbound antibody conjugate, the chromogenic substrate was added, and the color change generated by the reaction was read at a 450-nm wavelength. Test samples and NGF standards (100 μl/well) were measured in duplicate. With this assay, NGF can be quantified with a lower detection limit of 15.6 pg/ml and <2% cross reactivity with other neurotrophic factors.
Preparation and inoculation of RSV.
RSV suspensions were prepared as described previously (14, 22,25). HEp-2 cells from the American Type Culture Collection (ATCC, Rockville, MD) were grown in MEM (GIBCO-BRL) supplemented with 10% fetal bovine serum (GIBCO-BRL). Confluent monolayers of HEp-2 cells were infected with 0.1 plaque-forming units of human RSV strain ALong (ATCC), and the infection was allowed to proceed at 37°C in a 5% CO2 atmosphere until >75% of the cells exhibited a cytopathic effect. Cell debris was removed by centrifugation at 9,500 g for 20 min at 4°C in a refrigerated centrifuge. Aliquots of the virus stock were snap-frozen in liquid nitrogen and stored at −80°C. Before inoculation, the virus stock was titrated and diluted as needed for a final titer of 5 × 104 TCID50 (50% of the tissue-culture infective dose) in 0.1 ml. Supernatants and cell lysates from virus-free flasks of HEp-2 cells in MEM were harvested, centrifuged, and aliquoted following the same protocol to obtain the virus-free medium used as a negative control. Aliquots of RSV suspended in a buffer that contained 33 μg/μl of 8-methoxypsoralen were irradiated with a 365-nm UV light source for 30 min to inactivate the viral nucleic acid (4, 24).
A volume of 0.4 ml/kg body wt of RSV suspension was inoculated in each nostril while the rat was under pentobarbital sodium anesthesia (50 mg/kg ip). Control rats were administered the same volume of virus-free medium. Average weights at the time of inoculation were 21.9 ± 0.2 g for the 2-wk-old weanlings and 218.7 ± 4.2 g for the 12-wk-old adults.
Virus detection and histopathology.
Immunoperoxidase staining for RSV detection and hematoxylin and eosin staining for histopathological analysis were performed on formalin-fixed, 3-μm-thick lung sections as described previously (14, 22, 25). In brief, the sections were incubated with a pool of monoclonal antibodies composed of four clones specific for the matrix (M2) protein, phosphoprotein (P), fusion (F) protein, and nuclear (N) protein of human RSV (Vector Laboratories, Burlingame, CA). This technique has been shown to maximize the sensitivity of RSV detection (27). The localization of RSV antibodies was delineated with the streptavidin-biotin peroxidase complex method using an immunostaining kit (DAKO, Carpinteria, CA) and developed with the 3,3′-diaminobenzidine tetrahydrochloride chromogen. With this technique, cells expressing viral antigens are stained with a dark-brown precipitate lining the cell membrane and cytoplasm. All slides were coded and were interpreted by a pathologist who did not know whether the section corresponded to a RSV- or medium-inoculated animal.
The increase in vascular permeability in response to sensory nerve stimulation with capsaicin (8-methyl-N-vanilly-6-nonenamide; Sigma, St. Louis, MO) was measured 5 days after the inoculation of RSV or virus-free medium. The rats were reanesthetized with pentobarbital sodium and injected with Evans blue dye (30 mg/kg iv over 5 s) to measure the extravasation of albumin from airway blood vessels (14, 22, 25). Immediately after the injection of the tracer, RSV-infected and pathogen-free rats received an intravenous infusion of capsaicin (75 μg/kg over 2 min) or its vehicle (0.75% ethanol, 0.375% Tween 80, and 0.85% NaCl in aqueous solution; 1 ml/kg over 2 min). All chemicals were delivered in a volume of 1 ml/kg of body wt. Five minutes after the injection of the tracer, the chest was opened and a 22-gauge cannula was inserted into the ascending aorta through the left ventricle. After incision of the left atrium, the circulation was perfused for 2 min with PBS using a syringe pump set at the rate of 25 ml/min for weanling rats and 50 ml/min for adult rats. The lungs were dissected, blotted, weighed, and incubated in 1 ml of formamide (Sigma) at 50°C for 18 h to extract the extravasated Evans blue dye. The extravasation of Evans blue-labeled albumin was quantified by measuring the optical density of the formamide extracts at a wavelength of 620 nm. Evans blue extravasation expressed in nanograms per milligram of wet-tissue weight was interpolated from a standard curve of Evans blue concentrations (0.5–10 μg/ml).
As neurotrophic factors are highly conserved in different species (28), in our rat model we used exogenous murine NGF (NGF-7S; Sigma) and blocked endogenous NGF activity using a polyclonal rabbit anti-mouse antibody (bioactivity: a 1:10,000 dilution blocks NGF-induced growth of PC-12 cells; Sigma). NGF-7S (8 ng/kg) or its vehicle (PBS, 5 ml/kg) was injected intraperitoneally in pathogen-free weanling and adult rats 30 min or 24 h before death of the animals and dissection of the lung tissues. Anti-NGF (1:2,000 dilution, 4 ml/kg), vehicle (PBS, 4 ml/kg), or control antibody (rabbit purified IgG, 1:2,000 dilution, 4 ml/kg; Sigma) was injected intraperitoneally 3 h before the inoculation of RSV. Timing and doses of NGF and anti-NGF treatment were chosen on the basis of previous studies (2, 5).
To determine age-dependent changes in the mRNA levels encoding NGF and the neurotrophin receptors trkA and p75 in lung tissues, we extracted total RNA from the left lung of pathogen-free rats killed at 2, 4, 8, and 12 wk of age (n = 3 rats/group) and performed semiquantitative RT-PCR amplification using the primers listed in Table1. Changes in NGF protein concentration were measured in the right lung from the same animals using a highly sensitive and specific immunoassay.
To determine whether lower respiratory tract infection with RSV modifies the expression of NGF and/or its receptors in lung tissues, 5 weanling rats (2 wk of age) and 5 adult rats (12 wk of age) were inoculated intranasally with RSV. Age-matched control rats were dosed with virus-free medium (n = 5 rats/group). In a separate experiment, a group of adult rats inoculated with active RSV was compared to another group inoculated with RSV from the same aliquot inactivated by exposure to UV light and psoralen (n = 5 rats/group). All rats were killed 5 days after inoculation and the lungs were removed and homogenized for semiquantitative RT-PCR analysis of NGF, trkA, and p75 mRNA levels and for NGF protein immunoassay.
To determine whether NGF upregulates the expression of the high-affinity substance P receptor, groups of weanling rats (2 wk of age) and adult rats (12 wk of age) were injected with exogenous NGF-7S or its vehicle (n = 6 weanling and 6 adult rats/each treatment group). Half of these rats were killed 30 min after the injection of NGF, the other half 24 h later. NK1receptor mRNA levels were measured in homogenized lung tissues using semiquantitative RT-PCR analysis.
To determine the role of NGF in the mechanism of neurogenic inflammation, groups of weanling rats (2 wk of age) and adult rats (12 wk of age) were injected with anti-NGF, vehicle, or control antibody (n = 5 rats/group) before the inoculation of RSV. Control groups of weanling and adult rats were inoculated with virus-free medium and injected with vehicle (n = 5 rats/group). Five days after inoculation, all rats were injected with capsaicin to stimulate sensory nerves in the respiratory tract (10). After vascular perfusion, the left lung was prepared for the extraction of extravasated Evans blue-labeled albumin, whereas the right lung was homogenized for RNA extraction and RT-PCR analysis of NK1 receptor mRNA.
Results are presented as mean values ± SE. Data obtained by densitometry analysis of RT-PCR products or by immunoassay and mean values of Evans blue extravasation were compared by ANOVA (35), and multiple comparisons between means were performed with the Fischer protected least-significant-difference test (32). The interactive effect between age and RSV infection was tested using ANOVA of log-transformed variables. Statistical analysis was performed using StatView software version 5.0.1 (SAS Institute, Cary, NC). Differences having a P value < 0.05 were considered significant.
Age-dependent expression of NGF and neurotrophin receptors.
In the lungs of pathogen-free rats, the expression of NGF and neurotrophin receptors declined progressively with age (Fig.1). Significantly decreased levels of the mRNAs encoding NGF, the high-affinity receptor trkA, and the low-affinity receptor p75 were measured by 8 wk of age (P < 0.05), whereas all levels measured at 4 wk of age were not significantly different compared with those measured at 2 wk of age (P = 0.2, 0.5, and 0.1, respectively).
NGF and trkA mRNA levels in the lungs of adult rats at 12 wk of age were reduced approximately by half compared with weanling rats at 2 wk of age (P = 0.009 and 0.01, respectively). The p75 receptor mRNA levels declined more rapidly, and the difference between weanling rats and adult rats was more than fourfold (P< 0.0001).
Consistently, the concentration of NGF protein measured in lung homogenates by immunoassay exhibited the same age-related decline (Fig.2). Compared with the concentration measured at 2 wk of age, the NGF decrease was significant by 8 wk of age (P < 0.0001) but not at 4 wk of age (P = 0.1). An approximately twofold difference was found between weanling rats at 2 wk of age and adult rats at 12 wk of age (P < 0.0001).
NGF and neurotrophin receptors in RSV-infected lungs.
Immunoperoxidase staining with specific monoclonal antibodies performed on lung sections from weanling and adult F-344 rats killed 5 days after the inoculation of RSV revealed the presence of RSV antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells (Fig.3). No virus was detected in the airways of rats dosed with virus-free medium or with UV-inactivated virus. The histopathological changes caused by RSV consisted of a predominantly mononuclear cell infiltration in the bronchiolar mucosa. Comparative analysis by a pathologist revealed no qualitative or quantitative difference between weanling and adult rats (Fig.4).
RT-PCR analysis of NGF, trkA, and p75 mRNA levels in lung homogenates revealed consistently a much stronger signal 5 days after the inoculation of RSV in both weanling rats (Fig.5) and adult rats (Fig.6) compared with age-matched pathogen-free control rats dosed with virus-free medium. In weanling rats, the levels of NGF, trkA, and p75 mRNA during RSV infection increased, respectively, to 214% (P = 0.002), 171% (P = 0.01), and 245% (P = 0.02) of pathogen-free controls. In adult rats, the levels of NGF, trkA, and p75 mRNA during RSV infection increased, respectively, to 194% (P = 0.002), 174% (P = 0.006), and 172% (P = 0.003) of pathogen-free controls. Thus RSV infection in adult rats increased the expression of NGF and neurotrophin receptors to levels comparable with those measured in pathogen-free weanling rats. UV-inactivated RSV did not upregulate the expression of NGF and its receptors to the degree of active RSV. In the lungs of rats dosed with inactive virus, mRNA levels of NGF (0.39 ± 0.04 vs. 0.85 ± 0.08; P = 0.0007), trkA (0.12 ± 0.02 vs. 0.39 ± 0.07; P = 0.004), and p75 (0.23 ± 0.01 vs. 0.62 ± 0.09; P = 0.004) were, respectively, 45, 30, and 38% of controls inoculated with active RSV from the same aliquot.
Immunoassay analysis confirmed that NGF protein concentration was significantly increased in the lungs of RSV-infected rats compared with age-matched pathogen-free controls (Fig.7). NGF protein concentration during RSV-infection increased to 211% of pathogen-free controls in weanling rats (P < 0.0001) and to 155% of pathogen-free controls in adult rats (P < 0.0001). Analysis of the interaction between age and infection confirmed that the magnitude of this increase was significantly larger in weanling rats than in adult rats (P < 0.0001).
Effect of NGF on NK1 receptor expression.
NK1 receptor mRNA had relatively stable and low-level expression in the lungs of the pathogen-free rats from 2 to 12 wk of age (Fig. 8). After the administration of exogenous NGF-7S (Fig. 9), NK1 receptor expression in the lungs of weanling rats increased to 368% of vehicle-treated controls at 30 min (P= 0.006) and to 254% at 24 h (P = 0.007). In adult rats, NK1 receptor expression increased to 240% of vehicle-treated controls at 30 min (P = 0.0009) and to 190% at 24 h (P = 0.01).
Pretreatment with anti-NGF antibody (Fig.10 A) reduced NK1 receptor expression to 51% of nontreated RSV-infected weanling rats (P = 0.03) and to 62% of nontreated RSV-infected adult rats (P = 0.004). After NGF blockade, NK1 receptor mRNA levels in the lungs of weanling and adult rats infected with RSV were not significantly different from age-matched pathogen-free controls (P = 0.2 and 0.08, respectively). Treatment with the control antibody had no effect on NK1 receptor expression.
Effect of NGF blockade on neurogenic inflammation.
Pharmacological stimulation of sensory nerve fibers with capsaicin 5 days after the inoculation of RSV (Fig. 10 B) increased significantly the extravasation of Evans blue-labeled albumin in the lungs of infected rats compared with age-matched pathogen-free controls. Evans blue extravasation during RSV-infection increased to 260% of pathogen-free controls in weanling rats (P = 0.01) and to 184% of pathogen-free controls in adult rats (P < 0.0001). Pretreatment with anti-NGF antibody reduced Evans blue extravasation to 27% of nontreated RSV-infected weanling rats (P = 0.004) and to 69% of nontreated RSV-infected adult rats (P = 0.002). After NGF blockade, Evans blue extravasation in the lungs of weanling and adult rats infected with RSV was not significantly different from age-matched pathogen-free controls (P = 0.6 and 0.1, respectively). Analysis of the interaction between age and infection revealed that the inhibitory effect of anti-NGF was significantly larger in weanling rats than in adult rats (P = 0.007). Treatment with the control antibody had no effect on capsaicin-induced plasma extravasation.
This is the first study to show that the expression of NGF and its receptors trkA and p75 in lung tissues decreases progressively with age. Weanling rats have approximately twofold higher levels of NGF mRNA and protein than adult rats, and the adult expression pattern for these molecules is reached by 8 wk of age. The expression of the high-affinity receptor trkA parallels that of its ligand, whereas the decline in the expression of the low-affinity p75 receptor is steeper and its mRNA level is more than fourfold lower in adult rats than in weanling rats. These findings are consistent with previous reports of minimal neurotrophin receptor expression in adult lung tissues (18) and show a different profile of developmental maturation in the lungs compared with other nonneuronal tissues, e.g., the thymus, where the expression of neurotrophin receptors peaks at 12 wk of age in rats (8).
This study also provides the first evidence of a strong increase of NGF and neurotrophin receptor expression in lungs infected with RSV 5 days after intranasal inoculation of the virus. The source of this increase could be the infected respiratory epithelium (7, 12), inflammatory cells recruited and/or stimulated by the virus such as activated CD4+ T lymphocytes that produce NGF and express the trkA receptor (6), or both. Whatever the source, NGF plays an important role in the mechanism of the inflammatory response during acute infection as is shown by the marked reduction in neurogenic plasma extravasation measured in the lungs of RSV-infected rats after selective blockade of NGF activity. NGF may also exert long-term effects on airway reactivity by remodeling the neuronal network in the respiratory tract. UV inactivation of the viral nucleic acid hinders the effect of RSV on the expression of NGF and its receptors in the lungs, which suggests that the changes observed in the neurotrophin pathways are linked to viral replication and expression of the viral genome in the respiratory epithelium. In light of this finding, it is also unlikely that nonviral factors released from the cells used for the preparation of RSV suspension may have contributed significantly to the changes in the expression of NGF or its receptors.
Although the relative magnitude of the changes caused by RSV in the neurotrophin system is comparable in weanling and adult rats, absolute mRNA levels that encode NGF and its receptors are much higher in RSV-infected weanling rats due to the different baseline. Furthermore, the increase in NGF protein concentration and the inhibitory effect of anti-NGF on neurogenic plasma extravasation are greater in the lungs of weanling rats, which suggests that early-life RSV infection may have a more profound influence on neurotrophin systems and lung development.
NGF is the first discovered component of the neurotrophin family, which includes the brain-derived neurotrophic factor and the neurotrophins 3 and 4/5. All of these peptides bind with low affinity to the p75 receptor. The high-affinity receptors are the tyrosine kinase receptors, which show some degree of selectivity. NGF binds with high affinity to the trkA subtype. By binding different receptors, NGF and the other neurotrophins activate different signal transduction pathways (13). Depending on the relative expression of different receptor subtypes and the predominance of specific signal transduction pathways, neuronal cells receive trophic support, grow and differentiate, or start the process of cell death.
NGF is synthesized in several nonneuronal cell types including epithelial cells and inflammatory cells (18). This function may target the innervation of specific tissues, but there is also growing evidence that NGF is a potent and eclectic neuroimmunomodulator, which releases and is released by a variety of inflammatory mediators.
NGF and neurogenic inflammation.
Our previous work has shown that the neurogenic inflammatory responses mediated by substance P-containing sensory nerves (21) are markedly potentiated in the airways of rats infected with RSV (25) via the selective upregulation of the high-affinity substance P (NK1) receptor. The present study shows that these characteristic changes in NK1 receptor expression are mimicked by the administration of exogenous NGF in the absence of infection, and that the blockade of NGF activity with a selective antibody inhibits both NK1 receptor upregulation and the potentiation of neurogenic plasma extravasation in infected lungs, which suggests that NGF plays an important role in the mechanism of airway inflammation during RSV infection. This novel observation can also explain the recent report of NGF-induced airway hyperresponsiveness in guinea pigs, which is completely blocked by selective pharmacological antagonism of NK1 receptors (5).
In addition, because NGF is released from airway epithelial cells (7, 12), increases the production and release of substance P and other tachykinins from adult sensory neurons (17), and induces sensory hyperinnervation in the airways of transgenic mice (11), it represents the ideal link between virus-infected respiratory epithelium and the dense subepithelial network of unmyelinated sensory fibers. Overexpression of NGF and its low- and high-affinity receptors during RSV infection may exert a dual action by1) increasing the responsiveness of sensory fibers and the release of proinflammatory peptides during the acute phase and2) promoting long-term remodeling of the nonadrenergic, noncholinergic innervation to the airways by overgrowth of neurites with higher substance P content.
We have also shown that stimulation with the neurotoxin capsaicin, which selectively stimulates C-type sensory fibers, causes potent inflammatory responses in the intrapulmonary airways of RSV-infected weanling rats (14), whereas the distal airways of adult rats exhibit a smaller and less-consistent response to capsaicin (25), which suggests a lower density and/or reactivity of sensory terminals in the distal airways of adult rats. In the present study, the measurements of microvascular permeability in the intrapulmonary airways confirm that neurogenic inflammation is more prominent in the lungs of RSV-infected weanling rats and it is abolished by NGF blockade, whereas in the lungs of RSV-infected adult rats the magnitude of the neurogenic plasma extravasation and the inhibitory effect of NGF blockade are relatively small.
All of these findings are consistent with our hypothesis that NGF plays an important role in the mechanism of neurogenic inflammation during RSV infection and that the magnitude of its effect is age dependent. Because the histopathological changes caused by the infection were qualitatively and quantitatively undistinguishable in rats of different age and there was no difference in clinical manifestations or mortality deriving from the infection between weanling and adult animals, it is unlikely that our results derive from age-dependent differences in virus replication. Rather, the higher susceptibility of weanling rats appears to derive from a higher degree of plasticity of the peripheral nervous system of young animals. It is likely, however, that other mechanisms unrelated to the neutrophin system participate in the complex changes in vascular reactivity and inflammation during viral infections of the respiratory tract and contribute to the different clinical manifestations of the infection at different ages. Furthermore, several effects of tachykinins on airway inflammation and reactivity have been found to differ among species, and thus the translation of experimental findings from animal models to human disease must be performed with caution and confirmed by clinical investigation.
In conclusion, this study shows that aging is associated with a progressive decline in the expression of the neurotrophin NGF in the lungs, which is paralleled by a similar decline in the expression of its high-affinity (trkA) and low-affinity (p75) receptors. RSV infection interferes with this physiological decline, promoting a large increase in the expression of both NGF and neurotrophin receptors. The lungs of weanling rats not only have markedly higher baseline expression, but also seem to be more responsive to the perturbation introduced by the infecting virus. Furthermore, our data indicate that NGF upregulates expression of the NK1 receptor, which mediates the inflammatory effects of substance P and is responsible for the exaggerated neurogenic inflammation in RSV-infected airways. Accordingly, selective NGF blockade inhibits neurogenic mediated inflammation during RSV infection, and the magnitude of this inhibitory effect is more prominent in younger animals. RSV-induced release of NGF may lead to short- and long-term changes in the distribution and reactivity of sensory nerves across the respiratory tract, participating in exaggerated inflammatory reactions during and after the infection. NGF and its receptors may also amplify other immunoinflammatory and neuronal pathways that contribute to airway inflammation and hyperreactivity.
The authors thank Drs. Xiaobo Jiang and Mian Xu for invaluable technical assistance. The authors also thank Drs. Lisa Baumbach, Gary Berkowitz, and Paul Shapshak for intellectual contributions, and Edward Mager for contribution to the RT-PCR studies.
This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-61007 (to G. Piedimonte). Some of the findings reported in this paper were presented at the ATS 2001 International Conference in San Francisco, CA.
Address for reprint requests and other correspondence: G. Piedimonte, Batchelor Children's Research Institute, Pediatric Pulmonology and Cystic Fibrosis Center, Univ. of Miami School of Medicine, 1580 NW 10th Ave., Miami, FL 33136 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 8, 2002;10.1152/ajplung.00414.2001
- Copyright © 2002 the American Physiological Society