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Abstract |
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Abstract
Introduction
Materials & Methods
Results
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Neuropeptides released from sensory nerve endings are potential mediators of airway inflammation in asthma and lung injury induced by inhalation of respiratory irritants. To develop an in vivo model for assessing the contribution of neurogenic inflammation in these processes, we have generated transgenic mice with altered innervation of the lung. To generate mice with an increased innervation of the airways, we placed the gene that encodes nerve growth factor (NGF) under control of the lung-specific Clara-cell secretory protein (CCSP) promoter. Two lineages of CCSP-NGF transgenic mice overexpressed NGF in the lung and developed a hyperinnervation of the airways. Immunohistochemistry for substance P, a substance P enzyme immunoassay, and catecholamine histofluorescence indicated that both tachykinin-containing sensory fibers and sympathetic fibers were increased around the airways of CCSP-NGF mice. Treatment of CCSP-NGF mice with the sympathetic-specific neurotoxin 6-hydroxydopamine (6-OHDA) eliminated the sympathetic component of the airway innervation, leaving a specific hyperinnervation by tachykinin-containing sensory fibers. CCSP-NGF mice were more sensitive than normal mice to capsaicin-induced increases in respiratory system resistance, demonstrating that the increased sensory innervation led to a change in airway function. We conclude that NGF overexpression from a lung-specific promoter produces anatomic and functional changes in lung innervation, and that CCSP-NGF mice will be useful for studying the role of neurogenic inflammation in airway disease.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Asthma is characterized by reversible airway obstruction, airway inflammation, and increased sensitivity to bronchoconstricting stimuli. Inhalation of respiratory irritants can produce persistent asthmalike symptoms or exacerbate existing asthma (1, 2). Through its ability to release inflammatory mediators and modulate smooth-muscle tone, the nervous system is likely to play an important role in the pathogenesis of asthma and irritant-induced lung injury. One potentially important mechanism in these processes is the release of tachykinin neuropeptides from airway sensory nerves (3). Tachykinins, including substance P, neurokinin A, and neurokinin B, are released from sensory nerve fibers known as C-fiber afferents by stimuli such as volatile organic compounds (4), ozone (5), and allergens (6). Tachykinins bind to neurokinin receptors present on a variety of cell types in the lung and mediate effects such as bronchoconstriction (7), mucus secretion (8), microvascular leakage (9), chemotaxis and activation of inflammatory cells (10), and stimulation of cytokine production (13). Paradoxically, tachykinins have also been reported to mediate bronchodilation and to limit irritant-induced airway inflammation (14).
Tachykinin levels may be altered in inflammatory states in human lung, and tachykinins have been implicated as mediators of inflammation in animal models. In humans, increased airway or serum levels of substance P have been detected in asthmatic individuals challenged with allergen (6) or hypertonic saline (17), in asthmatic individuals during spontaneous asthma attacks (18), and in normal individuals after ozone inhalation (5). In animal studies, tachykinins have been implicated in allergen- and ozone-induced plasma extravasation as well as airway reactivity to allergen and nonspecific bronchoconstrictors such as histamine (19). In other studies, however, depletion of sensory neuropeptides by chronic capsaicin treatment has been reported to either be without effect on these processes or to exacerbate inflammatory effects, suggesting a protective role for tachykinins (14, 25).
Because of the controversy about the function of tachykinins during periods of lung injury and inflammation, a method for manipulating the amount of C-fiber afferent innervation that the lung receives would be valuable. As a novel approach to examining the role of tachykinins in airway inflammation and hyperreactivity, we have manipulated the innervation of the airways in transgenic mice by overexpressing nerve growth factor (NGF). NGF is secreted by neuronal target tissues and affects the development of a subset of peripheral nervous system neurons, including tachykinin-containing sensory neurons (28). During development, NGF has multiple effects on responsive neurons, including promotion of survival, induction of axonal outgrowth and branching, and chemoattraction of growing axons (28, 29). Transgenic mice overexpressing NGF from tissue-specific promoters exhibit localized hyperinnervation around the sites of NGF expression (29, 30). Here we report that lung-specific expression of NGF in transgenic mice, driven by the Clara-cell secretory protein (CCSP) promoter, results in a hyperinnervation of the airways. These transgenic mice represent a novel model for studying the role of neurogenic inflammation in irritant- and allergen-induced airway disease.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
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Animals
Mice were housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)- accredited facility according to National Institutes of Health (NIH) guidelines, and were specific-pathogen-free. Mice were maintained on a 14 h light/10 h dark schedule and given food and water ad libitum. Transgenic mice were generated and maintained on a mixed genetic background derived from the C57BL/6 and SJL inbred strains.
Generation of CCSP-NGF Transgenic Mice
A 2.4-kb DNA fragment from the 5' flanking region of the
rat CCSP gene was isolated with the polymerase chain reaction (PCR). The upstream PCR primer of sequence 5'-CGCGGATCCTCTGTGCAGCAGAGGGTGCACAC-3' contained bases 
2301 to 2278 of the CCSP gene (numbered
as in [31]) and a flanking BamHI site. The downstream
PCR primer of sequence 5'-GGATCGTCGACGATGTGGGCTGATGTTGTAATGTGAGG-3' contained CCSP
bases 15 to 41 and a flanking Sal I site. These primers were used to amplify from rat DNA a 2.4-kb fragment that was
cloned into the BamHI and Sal I sites of Bluescript II KS+
(Stratagene, La Jolla, CA). The NGF coding sequence is
derived from the human NGF gene and consists of bases
9084 (Genbank numbering) to the EcoRI site at the 3' end
of clone 
h
N8 (32). Insulin sequences were derived from
the rat insulin II gene, and consisted of bases +6 to +177
(33). The CCSP-NGF DNA construct was assembled in the Bluescript vector, and then linearized and cut free
from vector sequences for microinjection.
Transgenic mice were generated by microinjection of the linear CCSP-NGF DNA fragment into fertilized mouse eggs derived from matings between B6SJLF1 hybrids, as described (34). Mice that developed from injected eggs were screened for the presence of the transgene by hybridization of tail-biopsy DNA with a transgene-specific probe consisting of sequences from the human NGF gene. Five transgenic founder mice carrying the CCSP-NGF gene were identified in this manner and were bred to B6SJLF1 hybrids to generate transgenic offspring for analysis.
RNA Analysis
RNA was prepared from tissue samples by acid phenol extraction or by ultracentrifugation through cesium chloride (35). RNA was subjected to Northern blot analysis (36) with a probe derived from the human NGF gene that does not hybridize with mouse NGF sequences under the conditions used, or from the rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which does hybridize with mouse sequences and serves as a loading control.
Histologic, Histochemical, and Immunohistochemical Analysis
For histologic analysis, lungs were fixed by intratracheal instillation of 10% neutral buffered formalin at a pressure of 25 cm H2O for 30 min at room temperature, followed by fixation by immersion overnight at 4°C. Lungs were embedded in paraffin and sectioned at 5 µm. Immunohistochemistry was done with an immunoperoxidase technique similar to that recently described (37). Rabbit polyclonal antibody to synaptophysin (Dako, Carpinteria, CA) was used at a dilution of 1:100, and rat monoclonal antibody to substance P (clone YMC-1021; Accurate, Westbury, NY) was used at a dilution of 1:50. Bound antibodies were detected with biotinylated goat antirabbit or antirat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:4,000, followed by streptavidin-conjugated horseradish peroxidase (Jackson ImmunoResearch) diluted 1:2,000 (500 ng/ml). After incubation with diaminobenzidine (DAB) as a chromogen, slides were counterstained with hematoxylin. Catecholamine histofluorescence was produced by reacting frozen tissue sections with glyoxylic acid as described (29, 38).
Substance-P Assays
Substance P was measured in lung homogenates of CCSP-NGF and normal mice with an enzyme immunoassay. Lungs were collected, weighed, minced, and boiled for 10 min in 2 ml of 4% acetic acid. After boiling, lung tissue was homogenized and the homogenate was centrifuged at 1,900 × g for 20 min. Supernatants were applied to Sep-Pak C18 reversed phase columns (Waters Associates, Milford, MA) and washed with 6 ml 4% acetic acid. Substance P was eluted from the columns with 3 ml ethanol:water:acetic acid (36:64:0.4). The eluates were evaporated to dryness under vacuum and redissolved in 0.4 ml water. Substance P was measured in aliquots of the samples, using a commercially available enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.
6-Hydroxydopamine Treatment
Mice were treated with 6-hydroxydopamine (6-OHDA) as described previously for rats (39). 6-OHDA was dissolved in normal saline containing 1 mg/ml ascorbic acid, and was injected subcutaneously at 100 mg/kg. The drug was injected on the day of birth (postnatal day 1) and on postnatal days 3, 5, 8, and 13. Parallel sets of animals received injections of saline containing 1 mg/ml ascorbic acid (vehicle-treated controls). After the treatment, mice were allowed to grow to adulthood before killing and analysis of lung innervation.
Respiratory Measurements
Total respiratory system resistance (Rrs) was measured in conscious, spontaneously breathing mice placed in a calibrated, dual-chamber plethysmographic system (Buxco Electronics, Troy, NY) in which an airtight seal was applied at the level of the neck of the animal. Ventilatory measurements corresponding to the thoracic and nasal compartments were acquired separately, using the methods described by Bartlett and Tenney (40) and Pappenheimer (41). At least 15 min prior to the start of each protocol, animals were allowed to acclimate to the chamber, through which room air was passed at a rate of 1 liter/min using a precision-flow pump-reservoir system. Pressure changes in each chamber caused by inspiratory and expiratory temperature changes were measured with a high-gain differential pressure transducer (Model MP45-1; Validyne, Inc., Northridge, CA). Analog signals were continuously digitized and analyzed on-line by a microcomputer software program (Buxco Electronics). A rejection algorithm included in the breath-by-breath analysis routine allowed for accurate rejection of motion-induced artifacts. The phase shift between the thoracic and nasal displacement flows was measured from the Lissajous loop presentation, and Rrs was computed in a breath-by-breath mode using a modification of the method described by Pennock and colleagues (42). Computed Rrs values were stored for subsequent off-line analysis. After stable Rrs baseline measurements were obtained for each animal, increasing doses of aerosolized capsaicin (0, 10, 33, 100, 330, and 1,000 µg/ml) were delivered for 2 min, using a Devilbiss 646 nebulizer (Devilbiss, Somerset, PA) at a constant airflow of 5 liters/ min, and Rrs was measured over a 5-min period after each dose. Capsaicin dose-response curves for CCSP-NGF (n = 7) and control (n = 5) mice were significantly different (P < 0.01, analysis of variance [ANOVA]), and were used to determine the concentration of capsaicin inducing a 50% increase in Rrs (PC50).
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Results |
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Introduction
Materials & Methods
Results
Discussion
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Generation of CCSP-NGF Transgenic Mice
The DNA construct used to generate CCSP-NGF transgenic mice contained a 2.4-kb fragment from the 5' flanking region of the rat CCSP gene fused to the human NGF coding region (Figure 1). The rat CCSP promoter fragment has been previously shown to direct expression of transgenes specifically to airway epithelial cells in mice (31, 43). The coding sequence of NGF contains the entire translated sequence and 1 kb of 3' untranslated sequence, but lacks most of the 5' untranslated sequence (29). The CCSP-NGF construct also has an intron-containing 5' untranslated sequence from the rat insulin II gene upstream from the NGF fragment. The insulin sequence was included because the presence of introns increases the efficiency with which DNA constructs are expressed in transgenic mice (33, 44). Microinjection of the CCSP-NGF DNA construct yielded five founder transgenic mice, four of which produced progeny carrying the transgene, which were used to establish transgenic mouse lineages.
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Analysis of Transgene Expression
Mice from the four CCSP-NGF lineages were screened with Northern blot analysis to determine those in which the transgene was expressed. Three lineages, designated 8-2, 14-3, and 20-6, exhibited a transgene-specific band by Northern blot analysis of lung RNA with a human NGF probe (Figure 2A). No transgene message could be detected in lungs from the fourth CCSP-NGF lineage. Mice from the 20-6 lineage displayed the expected 2.2-kb band for the CCSP-NGF message. Mice from lineages 8-2 and 14-3 displayed a message of smaller size (1.4 kb), suggesting that the transgene was truncated or that the message was not processed properly in mice from these lineages. Mice of the 20-6 lineage had the expected tissue distribution of CCSP-NGF message, since the transgene was expressed in the lung but not in spleen, liver, small intestine, kidney, testis, heart, brain, or skeletal muscle (Figure 2B).
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To examine expression of the NGF polypeptide, we analyzed homogenates of CCSP-NGF mouse lungs with an enzyme-linked immunosorbent assay (ELISA). An initial screening of one mouse from each lineage revealed that two of the lineages in which transgene message was detected (14-3 and 20-6) exhibited high levels of NGF production in the lung, whereas the remaining two lineages (8-2 and 20-4) produced the same amount of NGF as nontransgenic mice (not shown). The transgene message in lineage 8-2 is apparently not translated efficiently, since no overexpression of NGF was detected by ELISA. Although lineage 14-3 produces a transcript that is smaller than expected, it could still generate functional NGF if the missing sequence was derived from the 3' untranslated region. These results indicated that the 14-3 and 20-6 lineages expressed high levels of NGF polypeptide in the lung, and these lineages were studied in more detail. To obtain a more accurate determination of the extent of NGF overexpression in CCSP-NGF mice, NGF levels in lung homogenates were measured in three mice from each of the expressing lineages and in three nontransgenic mice (Figure 3). These results indicated that NGF was overexpressed 28-fold and 31-fold in the 14-3 and 20-6 lineages, respectively, over the levels found in nontransgenic mice of similar genetic background. Mice from these two lineages displayed the histologic changes described subsequently, whereas mice from the two lineages in which NGF overexpression was not detected with ELISA displayed normal lung histology.
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Histologic and Immunohistochemical Analysis of CCSP-NGF Mouse Lungs
CCSP-NGF transgenic mice were analyzed with histologic and immunohistochemical techniques to determine the effects of NGF overexpression on the innervation of the airways. Hematoxylin and eosin (H&E)-stained sections revealed clear histologic abnormalities that were confined to the airways of CCSP-NGF mice. Mice from both the 14-3 and 20-6 lineages displayed a subepithelial thickening around the airways that was not seen in nontransgenic littermates (Figures 4a-b, 5 a-b). CCSP-NGF mice also contained abundant large nerves adjacent to larger airways. The thickening around the airways was observed in bronchi as well as in large and small bronchioles, but was minimal in the trachea. Alveolar structure in CCSP-NGF mice was normal, and no evidence of inflammation was observed. On the basis of the known functions of NGF, we hypothesized that the airway thickening in CCSP-NGF mice was a result of ingrowth of excessive peripheral nerve fibers. To ascertain the neuronal origin of the material surrounding the airways, immunohistochemistry with antibodies against the neuronal marker synaptophysin was performed on lung sections from CCSP-NGF transgenic mice. Staining with this antibody was observed throughout the subepithelial material around the airways in the transgenic mice (Figures 5c and 5e). In nontransgenic mice, synaptophysin staining was absent from small airways (Figure 5d), or was present in larger airways in isolated nerve fibers at much lower density than in the transgenic mice (Figure 5f). This result confirms the postulate that the CCSP-NGF gene results in a hyperinnervation of the airways. Similar results were obtained with neurofilament immunohistochemistry (not shown). To examine alterations in tachykinin-containing sensory fibers, lung sections from CCSP-NGF and nontransgenic mice were stained with antibodies to substance P. Substance P is stored in varicosities of extremely fine sensory nerve endings. The varicosities containing substance P can be visualized with light microscopy as a series of beadlike structures. Clusters of varicosities that contained substance P immunoreactivity and appeared to be derived from multiple nerve fibers were observed adjacent to the airways in CCSP-NGF transgenic mice (Figure 5g). In nontransgenic mice, immunoreactive varicosities could be seen, but always appeared to be derived from single fibers. Typically, only a few varicosities could be seen before the fiber turned out of the plane of section (Figure 5h). Clusters of immunoreactive varicosities like those in CCSP-NGF mice were not observed in nontransgenic mice. The results of the staining for substance P indicated that innervation of the airways by tachykinin-containing sensory fibers is increased in CCSP-NGF mice.
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Substance-P Content of CCSP-NGF Mouse Lungs
To quantitate the increase in tachykinin-containing sensory innervation in CCSP-NGF mouse lung, substance P was measured in lung homogenates with an enzyme immunoassay (Figure 6). Lungs from CCSP-NGF transgenic mice contained approximately 5-fold more substance P than did lungs from nontransgenic mice. Since the immunohistochemical results indicated that substance P in the lungs of CCSP-NGF mice was confined to nerve fibers, the increase in substance P in CCSP-NGF mice resulted from an increase in tachykinin-containing nerve fibers.
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Treatment with 6-OHDA
The airways in CCSP-NGF transgenic mice contained abundant nerve fibers that did not stain with antibodies to substance P. Since NGF promotes the growth of sympathetic axons, we hypothesized that many of the nerve fibers observed in the airways of CCSP-NGF mice were of sympathetic origin. To test this hypothesis, CCSP-NGF and nontransgenic mice were treated with the sympathetic-specific neurotoxin 6-OHDA. Treatment of CCSP-NGF mice with 6-OHDA resulted in a dramatic loss of nerve fibers from the airways, as judged by staining with H&E (Figure 7a; compare with Figure 5a). To demonstrate directly the loss of sympathetic fibers, lung sections were stained with a catecholamine histofluorescence technique that detects sympathetic nerve fibers (29, 38). Vehicle-treated CCSP-NGF transgenic mice contained abundant sympathetic fibers innervating the airways (Figure 7b). The airways of vehicle-treated nontransgenic mice contained very few staining fibers, indicating a sparse innervation by sympathetic nerve endings in normal mice (Figure 7c). Treatment of CCSP-NGF mice with 6-OHDA eliminated the majority of staining fibers around the airways (Figure 7d). The sections showing this appeared similar to those from untreated nontransgenic mice. Sections from nontransgenic mice treated with 6-OHDA showed no detectable sympathetic fibers around the airways (Figure 7e). It is not clear why there are residual sympathetic fibers after 6-OHDA treatment in CCSP-NGF mice. This may relate to the large number of fibers initially present in these mice, or may result from some protective effect of high levels of NGF. Immunohistochemistry with antibodies to substance P revealed that 6-OHDA treatment did not alter the increased level of substance P-immunoreactive fibers characteristic of CCSP-NGF mice (Figure 7f). These results indicated that the majority of the neuronal fibers innervating the airways in CCSP-NGF mice were of sympathetic origin, and that 6-OHDA treatment could be used to generate CCSP-NGF mice that had increased tachykinin innervation but normal levels of sympathetic innervation of the airways.
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Sensitivity to Inhaled Capsaicin
To determine whether altered airway innervation in CCSP-NGF mice had functional consequences, we measured the effect of inhaled capsaicin on Rrs. Capsaicin stimulates sensory C-fiber afferents and triggers tachykinin release. An exaggerated response to capsaicin in CCSP-NGF mice would indicate that these mice have an increased ability to respond to irritant stimuli via sensory-nerve pathways. Baseline Rrs was not different in CCSP-NGF and normal mice. Inhaled capsaicin induced a dose-dependent increase in Rrs in both CCSP-NGF and normal mice. CCSP-NGF mice were more sensitive to the constrictive effect of capsaicin, as indicated by a lower dose of capsaicin required to induce a 50% increase in Rrs (Figure 8). This result indicated that the altered innervation in CCSP-NGF mice rendered them more susceptible to an irritant stimulus of the respiratory tract.
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Discussion |
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Neurogenic inflammation is a potentially important mechanism by which lung injury induced by irritants and allergens may be initiated and maintained. Enhanced accumulation of tachykinins in asthmatic and in normal subjects exposed to respiratory irritants highlights the need for further research into the neuronal control of airway inflammation (5, 6, 17, 18). A significant value of the mouse in airways-disease research lies in the ability to use it for performing genetic manipulations to test the role of specific mediators in disease processes. We have developed a novel approach to manipulating tachykinin levels by altering the growth of nerve fibers that contain these neuropeptides. In the transgenic mouse model presented here, lung-specific expression of NGF was used to alter the innervation of the airways. The advantages of this approach are that tachykinins will be released by stimuli that normally cause neuropeptide release, and that multiple peptides will be concurrently released, as happens in normal animals. In addition, since the alteration is confined to the airways, there is a specific increase in tachykinin levels at the anatomic site of interest.
The present model depends on the action of NGF on specific populations of peripheral neurons, and on its multiple effects on these responsive neurons. The actions of NGF on neuronal cells are mediated primarily by the high-affinity NGF receptor encoded by the trk protooncogene (45). Expression of trk in the peripheral nervous system has been detected exclusively in sympathetic neurons and in a subset of neural-crest-derived sensory neurons (46). Tachykinin-containing sensory C fibers appear to be among the neural-crest-derived sensory neurons most responsive to the effects of NGF (47). Other types of peripheral neurons, such as parasympathetic neurons and placode-derived sensory neurons, lack NGF receptors and cannot respond to NGF (48). These neurons rely on other neurotrophic factors for survival, growth, and differentiation during development.
NGF exerts multiple effects on developing neurons that possess the trk receptor. NGF is required for the survival of responsive neurons and controls neuronal gene expression (28). These effects are produced by binding and internalization of NGF, followed by retrograde axonal transport to the cell body (49, 50). NGF also promotes axonal branching and the directional growth of NGF-responsive axons to sites of NGF production (29, 51). These effects of NGF appear to be direct, local effects that do not require transport of NGF to the soma (51).
We have previously shown that overexpression of NGF in transgenic mice resulted in local hyperinnervation in tissues that expressed the transgene (29). In the present study, we used DNA regulatory sequences from the CCSP gene to direct expression specifically to the lung. This tissue-specific expression resulted in anatomic alterations that were confined to the lung and resembled the changes in innervation that were observed previously in other tissues. Because NGF is expressed in many organs and in epithelial cells (52, 53), it is likely that Clara cells normally synthesize and have the abililty to process the NGF polypeptide. However, the levels at which NGF is produced in the lungs of CCSP-NGF mice are much higher than would normally be the case, thereby accounting for the change in innervation. In the present study, we identified significant changes in both tachykinin-containing sensory fibers and sympathetic fibers in the airways of CCSP-NGF mice. Substance P was used as a marker for tachykinin-containing sensory nerve fibers. CCSP-NGF mice were found to have an increased density of innervation by substance P-containing fibers, which was demonstrated both by immunostaining and by enzyme immunoassay. As discussed earlier, these changes in innervation are consistent with what is known about the action of NGF during development and the distribution of NGF receptors in the peripheral nervous system.
Tachykinins, including substance P, have been documented to mediate inflammation and bronchoconstriction in the lung (3). Although the lungs of CCSP-NGF transgenic mice in the present study had a 5-fold increase in the amount of substance P over those of normal mice, the transgenic mice had a normal baseline Rrs and did not exhibit any evidence of inflammation. The lack of inflammation was not due to the presence of excess sympathetic nerve fibers, since no inflammation was observed in CCSP-NGF mice treated with 6-OHDA. Rather, the lack of inflammation was most likely due to sequestering of the tachykinins in sensory nerve endings and their lack of release in appreciable amounts until an irritant stimulus is encountered. In accordance with this concept, we have found that the level of substance P in lung lavage fluid from both CCSP-NGF and normal mice is below the limits of detection as measured with an enzyme immunoassay (< 7 pg/ml; not shown).
CCSP-NGF mice were more sensitive than normal mice to airway constriction induced by inhaled capsaicin. Capsaicin acts on sensory C fibers to stimulate both central reflex pathways and peripheral tachykinin release. Stimulation of central reflex pathways leads to airway constriction (54), whereas tachykinins act on mouse and rat airways to produce bronchodilation (16, 55). Our experiments showed that capsaicin inhalation produced an increase in Rrs in conscious mice, suggesting that the effects of the central pathway predominate in the intact mouse. CCSP-NGF mice required a lower dose of capsaicin than normal mice to produce the same increase in Rrs. This observation is consistent with the histologic and biochemical observations of an increase in tachykinin-containing nerve fibers in the lungs of CCSP-NGF mice. The capsaicin inhalation experiments showed that the additional tachykinin-containing sensory fibers in CCSP-NGF mice are functional, since they mediate a process in which C-fiber afferents are known to be involved (i.e., capsaicin-induced airway constriction). CCSP-NGF mice may be used to test the hypothesis that increased innervation by tachykinin-containing sensory neurons results in increased sensitivity to irritant-induced airway inflammation and hyperreactivity. CCSP-NGF mice therefore represent a novel model for examining neuronal mechanisms of irritant-induced lung injury.
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Footnotes |
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Address correspondence to: Gary W. Hoyle, Ph.D., Section of Pulmonary Diseases, Critical Care and Environmental Medicine, Department of Medicine, SL-9, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail: ghoyle{at}tmc.tulane.edu
(Received in original form October 8, 1996 and in revised form April 29, 1997).
Acknowledgments: The authors thank Dr. Richard Palmiter for providing NGF and insulin DNA clones, and Dr. Carol Phelps for assistance with fluorescence microscopy. This work was supported by NIH Grant AI39023 and by a grant from the Department of Defense to the Tulane/Xavier Center for Bioenvironmental Research. Regina Graham was supported by an award from the Tulane/ Xavier Center for Bioenvironmental Research.
Abbreviations CCSP, Clara-cell secretory protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NGF, nerve growth factor; 6-OHDA, 6-hydroxydopamine; Rrs, respiratory system resistance.
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References |
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Materials & Methods
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References
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