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Published ahead of print on December 12, 2003, doi:10.1165/rcmb.2003-0303OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 793-800, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0303OC

Nerve Growth Factor and Substance P Regulation in Nasal Sensory Neurons after Toluene Diisocyanate Exposure

Erin R. Wilfong and Richard D. Dey

Department of Neurobiology and Anatomy, Department of Pharmacology and Toxicology, Robert C. Byrd Health Sciences Center, School of Medicine, West Virginia University, Morgantown, West Virginia

Address correspondence to: Dr. Richard D. Dey, Department of Neurobiology and Anatomy, Robert C. Byrd Health Sciences Center, P.O. Box 9128, Morgantown, WV 26506-9128. E-mail: rdey{at}hsc.wvu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Toluene diisocyanate (TDI) exposure produces rhinitis and nasal irritation, and increases the synthesis and release of substance P (SP) from airway sensory nerves. The mechanism leading to enhanced SP production following irritant inhalation remains unclear, but may involve actions of nerve growth factor (NGF). NGF binds trkA receptors located on sensory nerve terminals. Activation of trkA receptors initiates kinase-signaling cascades, which ultimately may increase SP. However, the effects of inhaled irritants on NGF release are not known. In this study, NGF levels in nasal lavages were examined following instillation of 10% TDI into both nasal cavities. NGF was significantly increased 2, 6, 12, and 24 h after TDI exposure compared with controls. The increase in NGF preceded the neuronal and mucosal increases in SP. Pretreatment with K252a, a nonselective tyrosine-kinase inhibitor, prevented the increase in SP-immunoreactivity in TG neurons and epithelial nerve fibers and the inflammatory response to TDI exposure. Because NGF binds to trkA tyrosine-kinase receptors, the NGF released during TDI exposure may mediate SP upregulation in airway sensory neurons, innervating the nasal cavity.

Abbreviations: bovine serum albumin, BSA • dimethyl sulfoxide, DMSO • ethyl acetate, EA • immunoreactivity, IR • mean gray value, MGV • nerve fiber density, NFD • nerve growth factor, NGF • phosphate-buffered saline, PBS • substance P, SP • toluene diisocyanate, TDI • trigeminal ganglia, TG


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhaled gases, vapors, or particles found in various occupational and environmental settings can act as irritants to the upper respiratory system (1). Toluene diisocyanate (TDI) is commonly used in the production and manufacture of polymer-containing products such as plastics, foams, adhesives, and surface coatings. Workers exposed to TDI vapor may develop inflammatory conditions including asthma, rhinitis, and nasal irritation (2). At the cellular level, TDI causes the release of neuropeptides, including substance P (SP), from activated sensory nerves in the nasal cavity and bronchi (3). Activation of C-fibers located in the airway mucosa produces neurogenic inflammation characterized by vasodilation, plasma extravasation, mucous secretion, and neutrophil chemotaxis resulting from the release of neuropeptides, especially SP and other tachykinins (4). Neurogenic inflammation has also been demonstrated in human airways and has been implicated in the pathology of chemical sensitivity syndromes (5).

There is substantial evidence that neurogenic inflammation in the airways is mediated by the release of the sensory neuropeptide SP from the peripheral endings of C-fibers distributed throughout the airway walls. SP is synthesized in nerve cell bodies through post-translational splicing of preprotachykinin mRNA (6). SP-containing sensory nerve endings in the nasal mucosa are found near blood vessels, mucous glands, and epithelium (7). The nerve cell bodies supplying sensory C-fibers to the nasal cavity are located in the trigeminal ganglia (TG) (8). Transient increases in SP protein and message have been demonstrated in sensory cell bodies innervating respiratory epithelium of the nose or lung following antigen challenge (9), TDI exposure (8), viral infection (10), and asphalt fume exposure (11).

Although the contribution of SP to a wide range of airway inflammatory conditions has been recognized, the mechanisms regulating irritant-enhanced SP expression in sensory neurons have not been established. Recent studies suggest that nerve growth factor (NGF), a neurotrophin released from inflamed tissues, may be a key mediator in the upregulation of SP levels in sensory neurons. NGF regulates neuronal growth and controls neuropeptide levels in mature sensory neurons (12). NGF is expressed in non-neuronal cells associated with the process of inflammation including airway epithelial cell lines (13), mast cells (14), and lymphocytes (15).

Several studies suggest that NGF may play a role in sensory-neural responses during airway inflammation. Superficial nasal mucosal cells obtained from the inferior turbinate bones of healthy human subjects constitutively express mRNA for NGF, and NGF protein is present in nasal lavage fluid (16), suggesting that NGF may be important in maintaining normal levels of sensory innervation. However, airway inflammatory conditions may lead to increased NGF production. Significantly higher levels of NGF have been detected in serum of individuals with asthma compared with individuals without asthma (17) and patients with allergic rhinitis have significantly higher NGF concentrations in nasal lavage fluid compared with control subjects (16). The involvement of NGF in regulating neuropeptide expression in sensory neurons innervating the airways is also supported by recent studies in animal models. Transgenic mice overexpressing NGF in the airways have higher SP levels in the airway wall and exhibit enhanced sensitivity to capsaicin-induced airway contraction (18), a response mediated by sensory C-fibers, and also have enhanced inflammatory responses to ozone exposure (19). Tracheal instillations of NGF produces increased SP expression in neurons of the nodose and jugular ganglia innervating the guinea pig airways (20) and produces airway hyperresponsiveness through activation of SP-selective NK-1 receptors in rats (21). NGF levels are increased during viral infections in rats, and NGF antibodies attenuate the enhanced NK-1 receptor expression observed during viral infections (22). Recent studies demonstrate that transgenic mice deficient in the NGF receptor exhibit reduced inflammatory responses to antigen challenge (23).

The aim of the present study was to determine if NGF production and release in the nasal cavity increases during irritant exposures, and to determine if NGF mediates increased SP expression in airway sensory neurons. We first correlated the time course for NGF production in the nasal cavity, the arrival of NGF in the cell bodies of sensory neurons, SP production in sensory neurons, and nasal inflammatory responses. Then we showed that neuronal SP levels and inflammatory responses were attenuated using the nonselective tyrosine-kinase inhibitor, K252a. The findings support the hypothesis that TDI exposure causes the release of NGF in the nasal cavity and that NGF-bound tyrosine kinase receptors may regulate SP expression in sensory neurons innervating the nasal mucosa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
Adult male Sprague-Dawley rats (Hla:[SD]CVF) weighing 200–250 g purchased from Hilltop Lab Animals (Scottsdale, PA) were used for all the experiments. For the time-course studies, either 10% TDI or ethyl acetate (vehicle) was instilled into the nasal cavity. Tissues were removed and lavages were done 2, 6, 12, 24, 48, or 96 h later (n = 6/time point).

For the protein kinase inhibitor studies, either K252a or dimethyl sulfoxide (DMSO) (vehicle) was instilled into the nasal cavity 2 h before either TDI or ethyl acetate. Tissues were removed and nasal lavages were performed 24 h after irritant instillation (n = 6/group).

Rhodamine Latex Microsphere Instillation
Ten days before TDI or K252a/TDI instillations, rats were anaesthetized with an intraperitoneal injection of sodium brevitol (50 mg/kg body weight; Eli Lilly, Indianapolis, IN). Neurons in the trigeminal ganglion projecting to the nasal cavity were identified using a retrograde neural tracing procedure described previously (8). Briefly, the anterior and posterior regions of the right and left nasal cavities were each instilled with 4 µl of rhodamine-labeled latex microspheres using a 10-µl Hamilton syringe with plastic tubing covering the tip. The tubing was marked at 0.8 and 1.3 cm lengths to allow correct positioning into the anterior and posterior nasal regions. Even distribution over the entire nasal mucosa was achieved by rotating the rats in a circular pattern around the anterior-posterior axis five times after microsphere instillation. The microspheres are endocytosed by sensory nerve endings in the nasal epithelium and then retrogradely transported to the corresponding cell bodies in the TG.

TDI Instillation
Ten days after instillation of rhodamine-labeled latex microspheres, the rats were again anaesthetized with an intraperitoneal injection of sodium brevitol (50 mg/kg weight dose). Both nasal cavities were instilled with 5 µl of 10% TDI (Aldrich Chemical Co., Milwaukee, WI) or ethyl acetate (vehicle; Sigma Chemical Co., St. Louis, MO) by placing the tip of a 10-µl pipette at the entrance of the nasal cavity.

K252a Instillation
Two hours before TDI instillation the rats were anaesthetized with an intraperitoneal injection of sodium brevitol as described. The anterior and posterior regions of the right and left nasal cavities were each instilled with 8 µl of K252a (100 µg/ml; Alexis Biochemicals, San Diego, CA) or dimethyl sulphoxide (vehicle, DMSO; Sigma). The K252a and DMSO were delivered using a 10-µl Hamilton syringe with plastic tubing covering the tip. The tubing was marked at 0.8 and 1.3 cm lengths to allow correct positioning into the anterior and posterior nasal regions. NGF action is inhibited by K252a, a carbazole alkaloid, which has been shown to inhibit trkA, trkB and trkC phosphorylation (24). K252a binds with high affinity to one site on the cytoplasmic kinase domain of the trkA receptor and inhibits NGF-stimulated phosphorylation of tyrosine residues on trkA receptors (25). Other limitations regarding the specificity of K252a as a tyrosine-kinase inhibitor are considered in the discussion.

Nasal Lavage
The rats were overdosed with 1.5 ml of 50 mg/ml sodium brevitol and the lower jaw was removed. A syringe with plastic tubing covering the needle was inserted into the posterior nares and sealed by finger pressure. Both sides of the nasal cavity were simultaneously lavaged with 15 ml of phosphate-buffered saline (PBS). The first 3 ml of lavage fluid was separated from the final 12 ml. Both aliquots for the nasal lavage fluid were centrifuged at 1,500 rpm (352 rgf) for 10 min. The supernatant from the initial 3 ml of nasal lavage was aliquoted and frozen at –80°C for subsequent assays. The two resulting cell pellets from each nasal sample were resuspended in a total of 1 ml cold PBS and pooled, plated on glass slides at a density of 1.5 x 105 cells/ml using a cytospin (Shandon Scientific, Ltd., Cheshire, UK) at 400 rpm (18.06 rgf) for 4 min, and stained with Wright-Giemsa on a Hema-Tek 2000 automated slide stainer (Bayer, Inc., Tarrytown, NY). A total of 100 cells were classified as neutrophils or nucleated cells (primarily epithelial cells) using light microscope (Olympus AX70) with a x40 magnification objective. The percentage of neutrophils was recorded for each slide.

Tissue Removal and Preparation
The right and left TG were removed by cutting distal to the division of the ophthalmic, maxillary, and mandibular trigeminal branches and at the junction of the trigeminal nerve with the ganglion. The nasal mucosa was also removed from the anterior and posterior regions of the nasal cavity. All tissue was immediately fixed in picric-acid formaldehyde fixative consisting of 2% paraformaldehyde, 15% saturated picric acid, and 0.15 M phosphate buffer at 4°C (26). After 3 h, the tissue was rinsed twice in 0.1 M PBS containing 0.3% (vol/vol) Triton X-100 (PBS-Tx, pH 7.8). After the second rinse, the tissues remained in PBS-Tx overnight at 4°C. The next day, TG were oriented on corks so the first section would be taken from the ventral surface. The nasal mucosa was laid flat and then rolled into a cylinder shape and stood upright on the cork. The tissues were covered with Tissue Tek O.C.T. compound (Sakura, Torrance, CA), frozen in isopentane cooled by liquid nitrogen and stored in airtight plastic bags at –80°C.

Continuous serial cryostat sections (12 µm thickness) of the entire TG were made as previously described (8). The first and second sections were collected on two separate gelatin-coated coverslips. The third, fourth, and fifth sections were discarded. This was repeated until the entire TG was sectioned. The first coverslip was used for SP immunocytochemistry and the second for NGF immunocytochemistry. The nasal epithelium was sectioned at 12 µm and used to evaluate SP nerve fiber density. A separate coverslip containing 16 sections randomly taken throughout the nasal mucosa was used for SP immunocytochemistry.

Immunocytochemistry
Procedures for immunocytochemistry were previously described (27). Cryostat sections on gelatin-coated coverslips were covered with either rabbit anti-SP (1:200; Peninsula, Belmont, CA) or rabbit anti-NGF (1:100; Chemicon International, Inc., Temecula, CA) primary antiserum diluted in PBS-Tx + 1% bovine serum albumin (BSA) (PBS-Tx-BSA, pH 7.8). The coverslips were incubated in a humid chamber at 37°C for 30 min, rinsed three times with PBS-Tx-BSA, allowing 5 min per rinse and then covered with fluorescein isothiocyanate–labeled goat anti-rabbit immunoglobulin IgG (ICN Pharmaceuticals, Inc, Costa Mesa, CA) diluted 1:100 in PBS-Tx-BSA and incubated at 37°C for 30 min. The coverslips were rinsed three times for 5-min increments in PBS-Tx-BSA and mounted on glass slides in Fluoromount (Southern Biotechnology, Birmingham, AL). The sections were observed using an Olympus AX70 fluorescence microscope equipped with fluorescein (excitation 495 nm and emission 520 nm, for antibodies) and rhodamine (excitation 540 nm and emission 580 nm for microspheres) filters.

Analysis of Immunoreactivity
SP and NGF immunoreactivity was evaluated in TG cell bodies innervating the nasal epithelium. Without knowledge of experimental grouping, neurons containing rhodamine-labeled latex microspheres were identified. The presence of microspheres was used as criteria that axons of the identified cell bodies projected to the nasal epithelium. Once the neurons of interest were identified using the rhodamine filter, a black and white image was captured with a SPOT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) and the perimeters of cell bodies containing microspheres were traced using Optimus, version 6.5 image analysis software (Media Cybernetics, L.P., Silver Springs, MD). Using the same field, an identical black and white image was captured with the fluorescein filter. The cell body outline obtained using the rhodamine filter was superimposed on the fluorescein image. The intensity of immunocytochemical labeling for SP or NGF was determined by calculating the mean gray value (MGV) for each neuron using Optimus software. Neurons with an MGV< 50 were considered negative and neurons with a MGV>=50 were classified as immunoreactive for the protein of interest. The cut-off range from positive to negative was based on an initial survey of several neurons in the TG directly observed to be positive or negative with the naked eye and then digitally analyzed. The percentage of SP-immunoreactivity (IR) or NGF-IR neurons innervating the nasal epithelium was determined by dividing the total number of positive microsphere-containing neurons (MGV >= 50) by the total number of microsphere-labeled neurons.

SP Nerve Fiber Density
Following immunocytochemical processing for SP, sections of nasal mucosa were observed on a Zeiss LSM 510 confocal microscope equipped with an argon laser (Zeiss, Oberkochen, Germany). Eighteen random images of respiratory epithelium were recorded from each coverslip. Using Optimus, the entire perimeter of epithelium was traced on each image of nasal mucosa. The threshold for each image was optimized so that only SP-IR nerve fibers were visible. The SP nerve fiber density (NFD) was calculated by dividing the SP-IR nerve fiber area by the total area of epithelium outlined. This represents the proportional cross-sectional area occupied by SP-immunoreactive nerve fibers.

NGF Enzyme-Linked Immunosorbent Assay
The nasal lavage supernatant samples (initial 3 ml) were frozen at –80°C. The concentration of NGF (7.8–500 pg/ml) in each sample was assayed using the NGF Emax ImmunoAssay System (Promega, Madison, WI) according to manufacturer's instructions. NGF was detected using an antibody sandwich format in 96-well plates. Each well was initially coated with 100 µl of anti-NGF pAb and incubated overnight followed by a 1-h incubation with blocking buffer (200 µl/well) to prevent nonspecific binding. Either 100 µl of lavage supernatant of 100 µl of NGF standard (7.8–500 pg/ml) was added to each well. The plate was incubated for six hours followed by an overnight incubation with anti-NGF mAb (100 µl/well). For color development an anti-rat IgG-horseradish peroxidase–conjugated antibody was added to each well (100 µl) followed by a tetramethlybenzidine solution, which reacts with the peroxidase-labeled conjugates to develop a blue color. The absorbance of each well was measured at 450 nm on a Spectra Max 340pc plate reader (Molecular Devices, Sunnyvale, CA). The concentration of NGF in each lavage sample was extracted from an NGF standard curve. All samples were run in duplicate or triplicate, and as a negative control, a PBS sample was run with each assay. The specificity of the NGF assay was validated by adding known concentrations of commercially available (Sigma) NGF. The limit of sensitivity of the NGF enzyme-linked immunosorbent assay was tested by performing 1:2 serial dilutions of nasal lavage supernatant samples. Promega reports a sensitivity range of 7.8–500 pg/ml. Serial dilutions of our samples parallel the standard curve until 15 pg. In preliminary experiments signal reduction averaged 50% (range 25–75%) after 1:2 dilution.

Protein Assays
The nasal lavage supernatant samples were also assayed for total protein using the bicinchroninic acid (BCA) Protein Assay Kit (Pierce, Rockford, IL), a modified Lowry assay that measures the concentration of total protein (20–2,000 µg/ml). The assay was performed according to the manufacturers instructions using the microplate procedure. The assay was performed in 96-well plates and 20 µl of unknown or BSA standard was added to each well followed by 200 µL of a working reagent. The plate was incubated at 37°C for 30 min before reading the absorbance at 562 nm on a Spectra Max 340pc plate reader (Molecular Devices). The concentration of total protein in each lavage sample was extracted from a BSA standard curve. All samples were run in duplicate or triplicate.

Statistical Analysis
The means and standard errors were calculated for each endpoint measured. For the time-course study, a two-way ANOVA with a Tukey post hoc test was run with treatment (TDI or EA) and time (2, 6, 12, 24, 48, or 96 h) as the variables. For the protein-kinase inhibitor studies, a two-way ANOVA with a Tukey post hoc test was run with pretreatment (K252a or DMSO) and treatment (TDI or EA) as the variables. Significance was set at P <= 0.05 for each endpoint measured.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NGF in the Nasal Lavage Fluid following TDI
The concentration of NGF in the nasal lavage fluid was significantly increased over controls at 2, 6, 12, and 24 h following instillation of 10% TDI into the nasal cavity (Figure 1). NGF increased as early as 2 h following TDI (104.74 ± 23.86 pg/ml) compared with control animals (42.00 ± 7.16) that received ethyl acetate. The amount of detectible NGF (pg/ml) continued to increase 6 h (184.38 ± 31.64), 12 h (194.65 ± 33.62) and 24 h (229.32 ± 28.54) after TDI exposure compared with controls (64.08 ± 5.71 at 6 h, 57.07 ± 6.36 at 12 h, and 54.60 ± 11.95 at 24 h). By 48 h and 96 h, there was no significant difference in NGF in the nasal lavages of TDI and control animals. The elevated concentration of NGF in the nasal lavage fluid as early as 2 h and continued increase until 24 h following TDI exposure demonstrates a time-dependent increase during the first 24 h followed by a return to baseline values. The findings demonstrate that TDI exposure promotes NGF release in the nasal cavity.



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  		Figure 1. Concentration of NGF (pg/ml) in the nasal lavage fluid of Sprague-Dawley rats at various time points following intranasal instillation of 10% TDI (filled bars) or ethyl acetate (control vehicle; open bars). Asterisk denotes significant TDI-induced change relative to controls (P <= 0.05). n = 6 for each group.

 
SP and NGF Immunoreactivity in TG Neurons Innervating the Nasal Epithelium following TDI
Trigeminal neurons innervating the nasal cavity were identified by the localization of rhodamine-labeled latex microspheres in the cell bodies (Figure 2A). Neurons projecting to the nasal cavity were identified by immunocytochemistry for SP (Figure 2B) or NGF (not shown) and captured as grayscale images for quantification (Figure 2C). The percentage of SP-IR in TG neurons innervating the nasal epithelium was significantly increased 24 h after TDI instillation (55.24 + 4.63%) compared with controls (10.50 ± 1.71%) (Figures 3A). After a peak at 24 h, the percentage of SP-IR neurons in TDI animals was decreased at 48 h but remained elevated over controls (30.68 ± 2.36 and 7.72 ± 1.28, respectively). By 96 h, there was no observable difference between the percentage of SP-IR TG neurons in control and TDI-exposed animals. These findings suggest that TDI exposure produced increased levels of SP in TG neurons projecting to the nasal cavity.



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  		Figure 2. Images of TG processed for SP using immunocytochemistry. Tissue was removed 24 h after exposure to TDI. Image in A was taken with a rhodamine filter to identify neurons containing rhodamine-labeled latex microspheres, which are known to innervate the nasal epithelium. Images in B and C were both taken using a fluorescein filter, except B was taken in color whereas C was taken in black and white. Asterisks indicate rhodamine-labeled cell bodies identified in A. In C, the three microsphere-containing neurons were outlined and the MGV was calculated to be 32.14, 100.17, and 59.46 (top to bottom). The first neuron was negative for SP-IR (MGV < 50) and the remaining two were SP-IR (MGV > 50).

 


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  		Figure 3. The percentage of SP-IR TG neurons (A) and the percentage of NGF-IR TG neurons (B) innervating the nasal epithelium of Sprague-Dawley rats at various time points following intranasal instillation of 10% TDI (filled bars) or ethyl acetate (control vehicle; open bars). Asterisk denotes significant TDI-induced change relative to controls (P <= 0.05). n = 6 for each group in A and B.

 
Changes in NGF-IR in TG neurons innervating the nasal epithelium were also time-dependent after TDI exposure (Figure 3B). The proportion of NGF-IR TG neurons was significantly increased 24 h after TDI exposure relative to controls (75.58 ± 9.72% and 11.67 ± 0.84%, respectively). A significant difference in the percentage of NGF-IR TG neurons, from 21.12 ± 5.41 in TDI-exposed rats to 7.50 ± 0.46 in control rats, was still detected at the 48 h time point. TDI-induced changes in NGF-IR TG cell bodies returned to pre-exposure levels 96 h after TDI instillation. The occurrence of NGF in the cells bodies of TG neurons probably reflects the uptake and axonal transport of NGF after being released in the nasal cavity.

SP Nerve Fiber Density in the Nasal Epithelium following TDI
The percent area of SP-IR nerve fibers in the nasal mucosa significantly changed over time following intranasal instillation of TDI (Figure 4). The SP NFD was significantly increased from 0.19 ± 0.02 in control animals to 0.56 ± 0.08 in the TDI group 12 h after irritant exposure. The percent area of SP-IR nasal mucosal nerve fibers remained elevated at 24 h in TDI-exposed animals compared with controls (0.53 ± 0.02 and 0.20 ± 0.03, respectively). By 48 h, the SP NFD in TDI-treated animals returned to control levels, where it remained at 96 h. These findings suggest that the levels of nerve fiber–derived SP in the nasal mucosa is increasing during the period 12 and 24 h after TDI exposure.



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  		Figure 4. The density of SP-IR nerve fibers in the nasal epithelium of Sprague-Dawley rats at various time points (n = 6/group) following intranasal instillation of 10% TDI (filled bars) or ethyl acetate (control vehicle; open bars). Asterisk denotes significant TDI-induced change relative to controls.

 
Inflammatory Markers in the Nasal Lavage Fluid following TDI
The percentage of neutrophils in the nasal lavage fluid was significantly increased above controls at 6, 12, 24, and 48 h following intranasal TDI instillation (Figure 5A). The proportion of neutrophils in the nasal lavage fluid steadily increased from 44.93 ± 2.06 at 6 h to 68.87 ± 9.00 at 12 h compared with controls (21.66 ± 2.18 and 12.68 ± 2.94, respectively). The highest neutrophil count, 79.45 ± 2.23%, was observed 24 h after TDI exposure compared with 14.70 ± 4.22% observed in control animals. Thereafter, the percentage of neutrophils began to decrease at 48 h (55.7 ± 6.14) and returned to control levels by 96 h (27.57 ± 4.55) compared with control values (13.17 ± 12.87 and 13.75 ± 3.97, respectively). Although not counted directly, the abundance of epithelial cells, the predominant cell type in nasal lavage, was not noticeably reduced in lavages from the TDI-treated rats, suggesting that changes in percentage of neutrophils represented an increased neutrophil infiltration into the nasal cavity. These findings suggest that TDI induces neutrophilic inflammation in the nasal cavity during the first 6 h lasting at least 2 d.



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  		Figure 5. The percentage of neutrophils (A) and the concentration of total protein (µg/ml) (B) in the nasal lavage fluid of Sprague-Dawley rats at various time points (n = 6/group) following intranasal instillation of 10% TDI (filled bars) or ethyl acetate (control vehicle; open bars). Asterisk denotes significant TDI-induced change relative to controls (P < 0.05).

 
The concentration of total protein (µg/ml) in the nasal lavage fluid was significantly increased above controls 12 and 24 h after intranasal TDI instillation (Figure 5B). Total protein was significantly increased (236.12 ± 39.00 µg/ml) 12 h after TDI treatment compared with control levels (38.30 ± 3.71 µg/ml). The largest amount of protein was detected in the nasal lavage fluid 24 h after TDI compared with controls (393.47 ± 61.00 and 54.11 ± 10.02 µg/ml, respectively). By 48 h, the concentration of protein (µg/ml) in the nasal cavity of TDI-exposed animals (41.49 ± 11.33) had returned to control levels (63.39 ± 22.47), where it remained at 96 h (147.94 ± 27.29 and 78.47 ± 34.79, respectively). The protein assay supports the conclusion that protein is leaking from the nasal vasculature 12 and 24 h after TDI exposure. Together with the neutrophil efflux into the nasal cavity during the same period, the findings support the occurrence of inflammation of the nasal cavity during the period between 12 and 24 h.

Protein Kinase Inhibitor Studies
Treatment with K252a, a nonspecific tyrosine kinase inhibitor, attenuated the TDI-induced changes in airway and sensory nerve responses. The expected increase in the percent of SP-IR neurons innervating the nasal epithelium 24 h after TDI exposure was significantly reduced from 52.88 ± 2.63% to 35.92 ± 4.49% (Figure 6A). DMSO, the vehicle for K252a, did not alter the TDI response. Similarly, the increase in intraepithelial SP-IR NFD (Figure 7) observed 24 h after TDI treatment was not observed in rats pretreated with the receptor tyrosine kinase inhibitor, K252a (0.22 ± 0.02, Figure 6B), but not affected in rats treated with K252a vehicle (0.52 ± 0.03). The increase in nasal lavage neutrophils observed 24 h after DMSO-TDI treatment (79.40 ± 2.94%) treatment did not occur in rats pretreated with K252a (20.00 ± 14.36%; Figure 6C). These findings support a role for NGF activation of the tyrosine kinase–linked trkA receptor. An unexpected finding was a mild (not significant) increase in SP-positive cell bodies of trigeminal ganglion after the K252a treatment alone. This may represent a mild irritation induced by K252a sufficient to activate SP synthesis in sensory neurons, but insufficient to cause inflammation in the nasal mucosa.



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  		Figure 6. The percentage of SP-IR TG neurons innervating the nasal epithelium (A), the density of SP-IR nerve fibers in the nasal epithelium (B), and the percentage of neutrophils in the nasal lavage fluid (C) of Sprague-Dawley rats pretreated with an intranasal instillation of K252a or DMSO (control vehicle) 2 h before intranasal instillation of 10% TDI or ethyl acetate (control vehicle) and killed 24 h later. Asterisk denotes significance (P < 0.05). n = 5 or 6 for each group in A, B, and C.

 


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  		Figure 7. Images of rat nasal mucosa showing SP-IR nerve fibers in the nasal epithelium. The rats were pretreated with either K252a or DMSO before either EA or TDI exposure and killed 24 h later. TDI treatment increases the number of SP-IR nerve fibers projecting between epithelial cells in the nasal mucosa (DMSO/TDI image). Pretreatment with K252a prevented the increase in SP-IR nerve fibers in the nasal epithelium (K252a/TDI image). Pretreatment with either K252a or DMSO before ethyl acetate did affect SP NFD in the epithelium.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The production and release of neuropeptides from sensory nerves in the airways is an important part of the inflammatory response to inhaled antigens and to environmental or occupational irritants. The present study was done to investigate the possibility that NGF generated during irritant exposure mediates the SP content of airway neurons. Previous studies have demonstrated SP upregulation in airway sensory neurons after instillation of NGF into guinea pig airways (20) and in transgenic mice overexpressing NGF in airway epithelial cells (18). The present study shows that NGF and SP levels in the nasal cavity and SP levels in sensory neurons innervating the nasal cavity are upregulated after TDI exposure, an occupational irritant and model of airway inflammation, and that inhibition of tyrosine kinase, an intracellular signaling molecule associated with NGF receptor binding to trkA, substantially reduces the TDI-induced upregulation of SP and the inflammatory response. These findings support the conclusion that NGF is produced during irritant exposures and influences SP production in sensory neurons of the nasal cavity. SP synthesis has also been reported in macrophages, lymphocytes, and eosinophils (28, 29). The effects of NGF on SP synthesis in these inflammatory/immune cells has not been determined.

The studies demonstrate a temporal relationship between NGF production in the nasal cavity, upregulation of SP in TG neurons, and the appearance of inflammatory markers such as increased neutrophil influx and protein levels in nasal lavage fluid. Increased levels of NGF in the nasal lavage fluid occurred before the observed increases in SP levels in epithelial nerve endings and TG neurons and the inflammatory markers. Previous studies have demonstrated that levels of NGF increased significantly in the nasal lavage fluid of allergic rhinitis patients following allergen challenge (16), and our findings demonstrate similar increases in NGF using an irritant model. In addition to increased NGF in the nasal cavity, the number of NGF-containing TG neurons innervating the nasal epithelium was increased. Further, the arrival of NGF in the TG cell bodies and the upregulation of SP are closely matched, both being increased at 24 and 48 h, supporting the possibility that NGF is transported to nerve cell bodies and induces SP upregulation. The apparent latency between increased NGF in the nasal cavity, which was apparent at 2 h, and the increase in SP-containing cell bodies in TG, observed at 24 h, is attributable to the length of time required for receptor binding and transport. Binding of NGF to trkA receptors located on sensory nerve terminals is well established (30). The role of NGF in supporting neurogenic inflammation in the airways suggests important implications in lung defense mechanisms. Other beneficial effects of NGF include the control of neuronal survival by reducing apoptosis. NGF also has benficial effects in wound healing promoting migration and survival of fibroblasts and white blood cells (31).

Although our studies support NGF production in the nasal mucosa, other possible sources of NGF have been reported. A number of inflammatory and immune cell types, including mast cells (14), lymphocytes (32), eosinophils (33), and macrophages (34), are capable of synthesizing and releasing NGF. The lack of significant inflammatory cell influx at 2 h when NGF levels are already increasing does not support the concept that these migratory cells are the initial source of NGF, although mast cells present in normal airway mucosa could be responsible for NGF production. A more likely explanation is that epithelial cells produce NGF, an observation supported in studies using airway epithelial cell lines (13).

NGF has been shown to increase the synthesis of neuropeptides, including SP (35). Inhibition of NGF action by injections of NGF antibody reduces the SP content of dorsal root ganglion neurons further emphasizing the important role of NGF in maintaining SP levels in adult sensory neurons (36). We have provided evidence that NGF mediates the upregulation of SP in sensory neurons. Our findings show that treating the nasal cavity with K252a, a nonspecific tyrosine kinase inhibitor previously shown to inhibit or interfere with NGF activity, reduced the TDI-induced increases in SP innervation in the nasal mucosa, SP-positive cell bodies in the TG projecting to the nasal cavity, and inflammation in the nasal cavity. Previous studies showing that K252a inhibits NGF-dependent SP production in cultured sensory neurons (37) support an inhibitory effect on NGF action and demonstrate the possible involvement of NGF in maintaining SP levels in sensory neurons. However, limitations regarding the lack of specificity of K252a as a tyrosine kinase inhibitor must be considered. K252a inhibits the phosphorylation of the neurotrophin receptors trkB and trkC (24) in addition to trkA, and also inhibits protein kinase C and calmodulin (38). Tyrosine kinase is a signaling molecule in cascades activated by multiple growth factors, although some data suggest a relative selectivity of K252a for the trkA-associated tyrosine kinase but not for kinases mediated by EGF, insulin, and v-src (24). Because K252a inhibits phosphorylation of tyrosine kinase receptors and NGF binds to the tyrosine kinase–coupled trkA receptor, attenuation of the neural response by K252a further supports, and does not contradict, a role for NGF as a trophic modulator of neural activation after irritant exposure. However, the K252a data do not conclusively prove that NGF mediates the observed changes in SP levels in airway sensory neurons, and growth factors other than NGF could contribute to or be entirely responsible for the observed changes in sensory nerves after TDI exposure.

The attenuation by K252a of the TDI-induced neutrophil influx can be correlated with the reduction in SP innervation of the nasal mucosa. Upon binding to NK-1 receptors localized within epithelium (7), SP initiates a receptor mediated inflammatory response which includes neutrophil chemotaxis. Thus, the reduced neutrophil influx after K252a is possibly due to decreased amounts of SP released from nerve fibers of the nasal epithelium. However, other effects of K252a have been described in antigen-sensitized and -challenged rats where the neutrophil-chemotactic factor normally produced by leukocytes infiltrating inflamed tissue is decreased in a concentration-dependent manner when K252a is present (39). In addition to the inhibitory effects of K252a on tyrosine kinase, K252a also blocks the NGF-mediated release of inflammatory mediators from resident mast cells and leukocytes by inhibiting protein kinase C and calmodulin (38). Therefore, the effect of K252a on neutrophil influx cannot be unequivocally attributed to the NGF regulation of SP in sensory neurons. An inhibitor that solely targets the trkA receptor would clarify the involvement of NGF in the activation of sensory nerves and stimulation of SP synthesis.

Overall, our findings demonstrate the progress and resolution of NGF release into the nasal cavity following irritant exposure. Although the cellular source of the TDI-induced NGF remains unclear, the early rise in NGF levels in nasal lavage fluid suggest production in the nasal mucosa. Based on the time course of increased NGF in cell bodies of the TG ganglia and the inhibitory effects of the tyrosine kinase inhibitor, K252a, the findings supporting the concept that irritant-induced SP expression in sensory neurons is mediated by NGF acting through a receptor binding mechanism, probably involving the trkA receptors. Final assessment of NGF in airway neural changes will depend on the development of suitable inhibitors of NGF action. The findings still have important health implications because human subjects with allergic rhinitis, an inflammatory disease of the upper airways, have significantly higher baseline concentrations of NGF protein in nasal lavage fluids (16). Identifying not only the source of endogenous NGF but also the mechanisms controlling NGF synthesis and release will aid in the understanding and treatment of airway inflammation.


   Acknowledgments
 
This study was supported by NIH Grant HL 35812.

Received in original form August 14, 2003

Received in final form December 8, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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