EDITORIAL FOCUS
Interleukin-1beta -induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway

Z.-X. Wu1, B. E. Satterfield1, J. S. Fedan2, and R. D. Dey1

1 Department of Neurobiology and Anatomy, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506; and 2 Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin (IL)-1beta causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1beta . Ferrets were instilled intratracheally with IL-1beta (0.3 µg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1beta . The IL-1beta -induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1beta -induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1beta . These results show that IL-1beta -enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons.

airway inflammation; airway smooth muscle contraction; muscarinic agonists; neurokinin receptor; airway innervation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTERLEUKIN-1 (IL-1), an inflammatory cytokine, has been reported to increase substance P (SP) release and gene expression in neurons of sensory and sympathetic ganglia (27, 31, 45). The IL-1beta level in bronchoalveolar lavage fluid is increased in asthmatic patients (6, 7), and IL-1beta induces airway hyperresponsiveness (AHR) in rats (50) and isolated human bronchi (4, 23). However, the mechanism of IL-1beta -induced AHR is still not clear. Recent studies show that IL-1beta can stimulate sensory afferent fibers (20, 27) and elicit the local release of SP. In enteric neurons of the gastrointestinal tract, IL-1beta causes SP release in a time- and concentration-dependent manner (26). Therefore, we hypothesized that the actions of IL-1beta in the airway may be mediated by altering SP expression of intrinsic airway neurons.

Neurons that comprise intrinsic airway ganglia are heterogeneous with regard to both function and neurotransmitter expression. Some intrinsic neurons represent classical postganglionic, cholinergic parasympathetic pathways, receiving input from vagal preganglionic neurons (46) and regulating smooth muscle, secretory glands, and blood vessels in the airway walls by releasing acetylcholine (ACh) (42). However, other neurons in the plexus contribute to inhibitory nonadrenergic noncholinergic innervation present in the airways (11, 56). Airway ganglia act as signal filters limiting electrical transmission of signals between presynaptic and postsynaptic neurons (8, 38, 39) and may be involved in the integration and control of airway function (2, 3, 10, 14, 17). Nerve cell bodies are located in large ganglia of the longitudinal trunk and in smaller ganglia of the superficial muscular plexus. Essentially all nerve cell bodies in the longitudinal trunk ganglia are cholinergic and do not normally contain detectable levels of nitric oxide (NO) synthase, SP, or vasoactive intestinal peptide (VIP) (14). Cell bodies in the superficial muscular plexus contain predominantly VIP and NO with a small population containing SP. Recently, the enzyme heme oxygenase-2, an enzyme that synthesizes carbon dioxide from heme, was identified in neurons of human and guinea pig airway ganglia (9). Neurons in both longitudinal trunk and superficial muscular plexus ganglia project to structures in the airway wall, including airway smooth muscle, and communications exist between airway neurons as well (57). Airway neurons may be capable of modulating neural activity within the airways and could provide local neural reflexes through their connections with the epithelium (18).

SP is a member of the tachykinin family and has potent effects on airway smooth muscle tone, vascular permeability to protein, and mucus secretion (5, 33, 35). Immunocytochemical studies have demonstrated that SP localized in the peripheral endings of nerves innervating the lung and airways originates in nerve cell bodies located both in sensory (15, 25) and intrinsic airway (13, 14, 16, 18, 33) ganglia. Furthermore, peptidergic innervation of airway smooth muscle, glands, and blood vessels originates from neurons with cell bodies located in intrinsic airway ganglia (13, 16). Inflammation and smooth muscle hyperresponsiveness are closely associated with increased SP release in the airway (34, 54). Although there is evidence suggesting that IL-1beta releases SP from sensory nerves, IL-1beta may also induce SP release from intrinsic airway neurons and result in airway inflammation or AHR. Therefore, the purpose of this study was to evaluate the effect of IL-1beta on airway responsiveness and to determine whether these effects are mediated through enhanced synthesis and release of SP from the intrinsic neurons of airway ganglia.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Female nonalbino ferrets (Marshall Farms, North Rose, NY) weighing 250-500 g were housed two to four per cage with access to food and water ad libitum in an American Association for Accreditation of Laboratory Animal Care-accredited facility. All procedures were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, and were also approved by the West Virginia University Animal Care and Use Committee.

In vivo IL-1beta treatment. Ferrets were anesthetized with ketamine (25 mg/kg) and xylazine (2 mg/kg) in a single intraperitoneal injection. An 18-gauge steel tube 15 cm in length, marked to indicate when the tip reached the carina, was connected to a 1-ml tuberculin syringe filled with IL-1beta (1 µg/ml) or saline and inserted through the oral cavity and pharynx into the trachea. Once per day for 5 days 0.3 ml of IL-1beta or saline was instilled into the trachea and deposited at four equal intervals along the trachea from immediately superior to the carina to immediately inferior to the larynx. One hour after the last IL-1 or saline treatment, ferrets were killed and tracheas were removed and cut into several segments to measure smooth muscle contractions and for immunocytochemistry.

In vitro tracheal segments cultured for 24 h. Organotypic cultures of tracheas from normal ferrets were used following a modification of our previously described technique (18). Under sterile conditions, tracheas were removed and washed with cold culture medium (described below). The tissue was then placed in a petri dish with culture medium and cut into 10-mm-long segments beginning at the carina. After a second wash, the segments were placed directly on the bottom of petri dishes containing fresh culture medium with IL-1beta (final concentration 10 ng/ml) or saline. In some experiments, CP-99994 (3 × 10-6 M) was added to the culture media 30 min before IL-1beta and maintained throughout the experiment to determine the role of SP in intrinsic airway neurons. The antagonist concentration was based on previous studies (28, 51, 55). The petri dishes were then placed in a controlled atmosphere culture chamber and gassed with a mixture of 95% O2 and 5% CO2. The chamber was placed on a rocker and incubated at 37°C for 24 h. After culture, smooth muscle responses were measured in the segments. The culture medium consisted of CMRL 1066 containing 0.1 µg/ml hydrocortisone hemisuccinate, 1 µg/ml recrystallized bovine insulin, 60 µg/ml penicillin G (100 units/ml), 10 µg/ml amphotericin B, 100 µg/ml streptomycin, and 5% heat-inactivated fetal calf serum.

Verification of SP depletion during 24-h culture. Previous studies using fluorescence microscopy suggest that sensory fibers are depleted from the ferret trachea during culture periods of 3 days or longer (18). However, functional evidence supporting selective SP depletion in sensory nerves during culture is not available. Therefore, to verify for this study that sensory nerves were nonfunctional after 24 h in culture, tracheal smooth muscle contractility to electric field stimulation (EFS) was determined before and 20 min after application of capsaicin (10-7 M) in three separate experimental groups: 1) fresh, noncultured tracheal segments, 2) segments cultured for 24 h in capsaicin (10-5 M), and 3) segments cultured for 24 h in vehicle. The capsaicin doses were based on previous reports that 10-7 M capsaicin enhances smooth muscle contractility by releasing SP (47, 48) and that 10-5 M capsaicin is effective in depleting SP from sensory nerve terminals (21, 36, 53). Prior studies have shown that capsaicin depletes SP selectively from sensory nerves without affecting SP content in nonsensory nerves (29). The hypothesis that the 24-h culture functionally depletes SP from sensory nerves would be supported if smooth muscle responses to EFS in both cultured groups were not different before and after capsaicin application. The capsaicin response in the fresh trachea is included only to demonstrate the effectiveness of 10-7 M capsaicin on smooth muscle contractility.

Measurement of tracheal smooth muscle contraction in vitro. Tracheal smooth muscle reactivity was evaluated by measuring contractile responses to ACh, methacholine (MCh), or EFS. ACh and MCh responses measure smooth muscle responses to the applied agonist, whereas EFS evaluates cholinergic responses resulting from the release of ACh from airway nerves. Tracheal segments from ferrets 1 h after the last treatment with IL-1 or saline, from the 24-h cultures and capsaicin study were cut into 3-mm-wide strips, mounted in holders, and maintained in gassed (95% O2-5% CO2) modified Krebs-Henseleit (MKH) solution at 37°C with a composition (in mM) of 113 NaCl, 4.8 KCl, 2.5 CaCl, 1.2 MgSO4, 24 NaHCO3, 1.2 KH2PO4, and 5.7 glucose, pH 7.4. The strips were tied at each end with 4-0 silk and positioned between the rings of platinum electrodes attached to tissue holders. Each holder was anchored in a 10-ml water-jacketed organ bath, and the top string was attached to a force-displacement transducer connected to a recorder (Gould Instruments, Valley View, OH). Strips were equilibrated for 60 min at a resting tension of 1.0 g, determined in preliminary studies to be optimal for contraction, during which time the MKH solution in the baths was changed every 15 min. After equilibration, we constructed cumulative concentration-response curves for ACh and MCh for separate strips by adding a series of concentrations of ACh or MCh to the bath in half-log increment concentrations ranging from 10-9 to 10-3 M. The next concentration was not added until the previous response reached a plateau. After concentration response curves were completed, EFS-induced responses were obtained with a Grass S48 stimulator (Grass Instruments, West Warwick, RI). We constructed frequency-response curves by increasing the frequency from 0.3 to 30 Hz using a submaximum voltage of 120 V, 0.2-ms pulse duration, and 10-s train duration. Between each stimulation period, 10 min were allowed for the previous response to return to baseline. EFS-induced contractions were normalized as a percentage of the response to 10-3 M ACh (%ACh response). In some experiments, atropine (10-6 M) was added to the Krebs solution to verify that the responses elicited by EFS were mediated by the release of ACh from cholinergic neurons.

Immunocytochemistry. Tracheal segments from IL-1beta - or saline-treated ferrets were fixed in picric acid-formaldehyde fixative for 3 h and rinsed three times with a 0.1 M phosphate-buffered saline containing 0.3% Triton X-100 (PBS-Tx). The airways were frozen in isopentane, cooled with liquid nitrogen, and stored in airtight bags at -80°C. The tracheas were frozen on cock supports and oriented with the dorsal surface uppermost so the tracheal muscle would be sectioned in a coronal plane.

Cryostat sections (12 µm thickness) were collected on gelatin-coated coverslips and dried briefly at room temperature. Immunocytochemical procedures for localizing neuropeptides in neurons and nerve fibers are identical to those described previously (14, 15). Briefly, cryostat sections were covered with SP antibody diluted 1:200, incubated in a humid chamber at 37°C for 30 min, rinsed with a 1% bovine serum albumin (BSA)-phosphate saline buffer containing Triton X-100 solution (PBS-Tx plus BSA) three times allowing 5 min for each rinse. The sections were then covered with fluorescein isothiocyanate-labeled goat anti-rabbit antibody diluted 1:100, incubated at 37°C for 30 min, and rinsed. Then, the sections were processed for VIP immunoreactivity using mouse anti-VIP (1:100) and goat anti-mouse labeled with rhodamine (1:100). VIP labeling was done to allow efficient identification of superficial muscular plexus neurons, which are typically difficult to locate but can be easily visualized immunocytochemically because 90% are VIP immunoreactive (14). After all immunocytochemical procedures were conducted, the coverslips were mounted with fluoromount and observed under a fluorescence microscope equipped with fluorescein (excitation wavelengths from 455 to 500 nm and emission wavelengths >510 nm) and rhodamine (excitation 540-504 nm, emission >580) filters. Controls consisted of testing the specificity of primary antiserum by absorption with 1 µg/ml of the specific antigen. Nonspecific background labeling was determined by omission of primary antiserum.

To measure fluorescence intensity in longitudinal trunk neurons, we recorded images using an AX 70 microscope (Olympus America, Melville, NY) with the SPOT 2 digital camera (Diagnostics Instruments, Sterling Heights, MI). Fluorescence intensity of SP was measured using commercial image processing software (Optimas 6.5; Media Cybernetics, Silver Spring, MD). The intensity recordings were calibrated with the InSpeck Green (505/515) microscope image intensity calibration kit (Molecular Probes, Eugene, OR). The longitudinal trunk neurons were identified by drawing the perimeter of the cell, and the fluorescence intensity was reported as gray level for each neuron. Neurons with a gray level <50 were considered negative because they were at or below the general background. Fluorescence intensities of >= 50 were counted as labeled neurons. To measure SP innervation of superficial muscular plexus neurons, we scored all identifiable VIP-positive neurons as either innervated or not innervated on the basis of the location of SP in varicosities in apparent direct contact with cell bodies. All identifiable longitudinal trunk and superficial muscular plexus neurons were evaluated in every 5th section collected from serial sections, usually amounting to a total of 10-15 sections analyzed.

For measuring nerve fiber density in tracheal smooth muscle, we collected images of SP-containing nerve fibers in series under the Zeiss LSM 510 confocal microscope. A series of images representing all of the tracheal smooth muscle in a section was collected in digital files and saved to an internal database and measured using Optimas software. We selected regions of smooth muscle using the rhodamine channel to avoid possible bias created by the presence or absence of nerve fibers. The smooth muscle regions were outlined to measure total cross-sectional area of smooth muscle. The microscope was then switched to reveal nerve fibers in the fluorescein channel, and the image was digitally captured. The proportion of nerve fibers was determined by segmentation using threshold gray levels with the Optimas software. Then percent nerve fiber density was calculated as a percentage of total cross-sectional area of smooth muscle occupied by SP-immunoreactive nerve fibers. At least 10 measurements were made for each section, and 15 sections were measured in each animal.

Data analysis. Unless otherwise stated, results are expressed as means ± SE. Contractions elicited by EFS were expressed as a percentage of the maximal contraction elicited by ACh. Contractions to ACh and MCh were normalized as a percentage of the respective maximal responses for each agonist. The half-maximal concentrations (EC50) for ACh and MCh were calculated using a four-parameter logistic curve fit (Sigmoidal, SigmaPlot 2000) and are presented with a 95% confidence interval in parentheses. Force development was expressed by normalizing force (g) divided by the wet weight of the tissue. Longitudinal trunk neurons were expressed as percent SP-positive cell bodies, and superficial muscular plexus neurons were expressed as percent SP-innervated cell bodies. Nerve fiber density was expressed as percent area of SP-immunoreactive nerve fibers in the total area of the smooth muscle. Statistical analyses of immunocytochemistry, EC50, and EFS were performed using Student's t-test or two-way repeated-measures analysis of variance. A P value <0.05 was considered significant, and n represents the number of animals studied.

Materials. ACh chloride, MCh chloride, atropine sulfate, IL-1beta human recombinant, hydrocortisone hemisuccinate, amphotericin B, and recrystallized bovine insulin were obtained from Sigma (St. Louis, MO). Penicillin G, streptomycin, fetal calf serum, and CMRL 1066 were obtained from GIBCO (Grand Island, NY). CP-99994 was obtained from Pfizer (Groton, CT). SP antibody was obtained from Peninsula (Belmont, CA). VIP was a gift from John Pocter (University of Texas, Health Science Center, Dallas, TX). Fluorescein isothiocyanate-labeled goat anti-rabbit antibody was obtained from ICN Immunobiologicals (Costa Mesa, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of IL-1beta on airway responsiveness in noncultured and cultured tracheas. The initial experiments examined the effect of IL-1beta on tracheal smooth muscle sensitivity in noncultured tracheas. Cumulative concentration-response curves for ACh and MCh were markedly shifted to the left (Fig. 1, A and B), and the EC50 values (Table 1) were decreased by 59 and 61%, respectively, in the IL-1beta treatment group (P <=  0.001). IL-1beta also increased contractile responses to EFS. A leftward shift in the frequency-response curve was observed in IL-1beta -treated animals (Fig. 1C), and contractions produced by EFS at 10 and 30 Hz were significantly increased by 19 and 16%, respectively, after treatment with IL-1beta (P <=  0.05).


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Fig. 1.   Cumulative concentration-response curves for acetylcholine (ACh, A) and methacholine (MCh, B) and frequency-response curves for electrical field stimulation (EFS, C) in tracheal strips after repeated saline (open circle ) and IL-1beta () treatment for 5 days. Responses to ACh and MCh are plotted as a percentage of the maximum response. Responses to EFS are plotted as a percentage of the maximum response to ACh. black-triangle, Airway responses to the same challenge after administration of 10-6 M atropine. Values are means ± SE; n = 6. * Significant difference between saline- and IL-1beta -treated animals, P <=  0.05.


                              
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Table 1.   Effect of IL-1beta on cumulative concentration-response curves for ACh and MCh in trachea smooth muscle

The next studies examined whether SP-containing sensory neurons degenerated after 24 h in culture. Previous studies have shown that pretreatment with capsaicin depletes SP in sensory neurons (21, 36, 53) and releases SP, which enhances EFS-induced airway smooth muscle contraction (44, 49). In this study, smooth muscle responses to EFS were not different before and after application of capsaicin (10-7 M) in tracheal segments cultured with 10-5 M capsaicin and tracheal segments cultured with vehicle (Fig. 2, B and C) and were significantly enhanced in fresh tracheas after treatment with the same dose of capsaicin (Fig. 2A, P <=  0.05). These findings indicate that SP-containing sensory neurons are functionally depleted after 24 h in culture.


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Fig. 2.   Effect of capsaicin (10-7 M) on frequency-response curves for EFS in fresh tracheal (no culture) strips (A), strip cultured 24 h with capsaicin (B), and strips cultured with vehicle (C). open circle , Before the presence of capsaicin; , after the presence of capsaicin. Values are means ± SE; n = 5. * Significant difference between before and after the presence of capsaicin, P <=  0.05.

We performed the next studies using cultured tracheas to examine the contribution of intrinsic neurons on IL-1beta -enhanced airway responsiveness. Tracheal segments were maintained in organotypic culture and treated with IL-1beta or saline for 24 h. Cumulative concentration-response curves for ACh and MCh (Fig. 3, A and B) were shifted to the left, and EC50 values (Table 2) for ACh and MCh were decreased by 66 and 72%, respectively, in tracheal strips cultured with IL-1beta . Contractions produced by EFS at 10 and 30 Hz were significantly increased by 24 and 18%, respectively, in tracheal strips cultured with IL-1beta (Fig. 3C). The contractions to ACh, MCh, and EFS in both noncultured and cultured tracheal segments were totally abolished after treatment with 10-6 M atropine (Figs. 1 and 3).


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Fig. 3.   Cumulative concentration-response curves for ACh (A), MCh (B), and EFS (C) in tracheal strips cultured for 24 h with saline (open circle ) and IL-1beta (). black-triangle, Airway responses to the same challenge after administration of 10-6 M atropine. Values are means ± SE; n = 6. * Significant difference between culture with saline and culture with IL-1beta , P <=  0.05.


                              
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Table 2.   Effect of IL-1beta on cumulative concentration-response curves for ACh and MCh in organotypic cultured trachea smooth muscle

Role of SP in IL-1beta -induced AHR in cultured tracheas. The next experiments examined the involvement of SP in IL-1beta -enhanced airway responsiveness by blocking the neurokinin (NK)-1 receptor. Cumulative concentration-response curves for ACh and MCh (Fig. 4, A and C; Table 3) and the EFS-stimulated contractions at 10 and 30 Hz (Fig. 4E) demonstrated expected changes in organotypic cultured tracheal segments after IL-1beta treatment. EC50 values (Table 3) for ACh and MCh were decreased by 67 and 64% respectively, and contractions produced by EFS at 10 and 30 Hz were significantly increased in tracheal strips cultured with IL-1beta . However, administration of the NK-1 antagonist CP-99994 attenuated the IL-1beta -enhanced contractile responses to ACh, MCh (Fig. 4, B and D; Table 3), and EFS (Fig. 4F). EC50 values for ACh and MCh decreased by only 32 and 36%, respectively, after pretreatment with CP-99994 in tracheal strips cultured with IL-1beta . There was no significant difference in responses to the same frequency of EFS between tracheal strips cultured with IL-1beta and strips cultured with saline after treatment with CP-99994.


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Fig. 4.   Effects of saline (A, C, and E) or CP-99994 (B, D, and F) on cumulative concentration-response curves for ACh (A and B) and MCh (C and D) and frequency-response curves for EFS (E and F) in tracheal strips cultured for 24 h with saline (open circle ) and IL-1beta (). Values are means ± SE; n = 5. * Significant difference between culture with saline and culture with IL-1beta , P <=  0.05.


                              
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Table 3.   Effect of CP-99994 on cumulative concentration-response curves for ACh and MCh in organotypic cultured tracheal smooth muscle

Changes in immunoreactive SP-containing intrinsic airway neurons. SP-containing cell bodies were present within longitudinal trunk and superficial muscular plexus in control animals and after repeated IL-1beta treatment for 5 days (Fig. 5). About 70% of the longitudinal trunk cell bodies labeled for SP (Figs. 5A and 6 A) and ~66% of the superficial muscular plexus neurons were innervated by SP-containing nerve fibers in control ferrets (Figs. 5C and 6B). After repeated treatment with IL-1beta , >92% of the cell bodies in the longitudinal trunk contained SP (Figs. 5B and 6A), and nearly 84% of the cell bodies in the superficial muscular plexus were innervated by SP-containing nerve fibers (Figs. 5D and 6B). Also, SP nerve fiber density from in the tracheal smooth muscle was significantly increased by 38% from 0.39 (control) to 0.54 after repeated IL-1beta treatment (Figs. 5, E and F, and 6C; P <=  0.05).


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Fig. 5.   Fluorescence photomicrographs of substance P (SP)-immunoreactive nerve cell bodies and fibers in longitudinal trunk (A and B) and superficial muscular plexus (C and D) and SP-immunoreactive nerve fiber density in tracheal smooth muscle (E and F) after repeat saline and IL-1beta treatment for 5 days. A: negative SP-immunoreactive longitudinal trunk neurons are seen in the control ganglia. B: after IL-1beta treatment, most of the longitudinal trunk neurons contain SP immunoreactivity. C: negative SP-immunoreactive cell bodies (green) and positive vasoactive intestinal peptide (VIP)-immunoreactive cell bodies (red) in the superficial muscular plexus of control ferret. D: after IL-1beta treatment, SP-immunoreactivity around VIP-immunoreactive cell bodies in the superficial muscular plexus is increased. E: few SP-immunoreactive nerve fibers in tracheal smooth muscle of control (nerve fiber density of this micrograph is 0.40). F: increased SP-immunoreactive nerve fibers in tracheal smooth muscle after IL-1beta treatment (nerve fiber density of this micrograph is 0.55). Magnification: ×285.



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Fig. 6.   Effects of 5 days in vivo saline (open bars) and IL-1beta (solid bars) treatment on SP-containing nerve cell bodies in longitudinal trunk (LT, A), SP innervation of airway neurons in superficial muscular plexus (SMP, B), and SP-immunoreactive (SP-IR) nerve fiber density in tracheal smooth muscle (C). Values are means ± SE; n = 6. * Significant difference between saline- and IL-1beta -treated animals, P <=  0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that IL-1beta enhances cholinergic responsiveness in ferret airway smooth muscle, as shown by elevated atropine-sensitive contractility to ACh, MCh, and EFS in ferrets. The elevation of airway smooth muscle responses by IL-1beta was attenuated by treatment with a NK-1 receptor antagonist, indicating that endogenously released SP was involved. NK-1-dependent AHR induced by IL-1beta was elicited in tracheal segments cultured for 24 h, a procedure both shown in previous study (18, 53) and verified in the present experiment to cause a significant anatomical and functional loss of SP from airway projections of sensory nerves. Because the IL-1beta effect was maintained in sensory-denervated tracheal segments, the findings suggest that neurons in airway ganglia may be the main source for SP in these airways. The observations that IL-1beta treatment increased the level of SP in longitudinal trunk neurons, SP innervation of superficial muscular plexus neurons, and SP innervation of tracheal smooth muscle all support the conclusion that IL-1beta elevates endogenous SP levels in intrinsic airway neurons.

IL-1beta is produced by alveolar macrophages and tracheal epithelial cells, and levels are elevated in bronchoalveolar lavage fluid during airway injury (12, 19, 37). Furthermore, IL-1beta is elevated in the bronchoalveolar lavage fluid from asthmatic patients (6, 7). IL-1beta has been implicated in the development of AHR in animal models and isolated human bronchi (4, 23, 50). Recent studies further found that IL-1beta plays an important role not only in up- and downregulation of acute and chronic inflammation (1, 32), but also in regulation of neuronal development and plasticity (20, 27, 30). SP is generally considered a sensory neuropeptide in the airways and has been associated with inflammation mediated through sensory pathways, but we have demonstrated previously that SP is also synthesized in the airway neurons (16, 18). Furthermore, inhalation of irritants or antigens enhances neuronal levels of SP and preprotachykinin mRNA in both sensory neurons and airway neurons (22, 34, 54, 55). One of the significant findings in this study is that IL-1beta enhances SP expression in airway neurons. The present study provides evidence that airway neurons, like sensory neurons, are able to respond to inflammatory cytokines. IL-1beta is known to cause the release and synthesis of SP from myenteric neurons of the gastrointestinal tract (26, 27, 31), which are embryologically and functionally analogous to intrinsic airway neurons. In the present study, we hypothesized that IL-1beta in the airways may mediate SP expression in intrinsic airway neurons as well. The immunocytochemical data demonstrating that SP in longitudinal trunk neurons and SP innervation of superficial muscular plexus neurons in trachea were both increased after repeated IL-1beta treatment provide evidence that IL-1beta treatment enhances SP levels in intrinsic airway neurons. In separate studies of airways from severe asthmatics, SP nerve fiber density in airway smooth muscle (43) and IL-1beta in bronchoalveolar lavage fluid were increased (6, 7). Thus IL-1beta released during airway inflammation may influence SP expression in airway neurons.

The finding that the NK-1 antagonist CP-99994 significantly attenuates the effect of IL-1beta on cholinergic and EFS-stimulated contractile responses implicates the involvement of SP as the mediator of IL-1beta action on airway smooth muscle. Although SP is a known bronchoconstrictor (5, 35), direct action of SP on smooth muscle does not appear to be an important effect in this study, because all of the smooth muscle contractile effects of IL-1beta were atropine sensitive. Thus the logical explanation of the IL-1beta effect is that SP alters cholinergic responsiveness. Previous studies have shown that SP enhances cholinergic responsiveness either through a direct effect on airway smooth muscle (49) or by enhancing ACh release from parasympathetic nerve terminals (40, 44, 52). The data in the present study do not discriminate between these two mechanisms because cholinergic responsiveness was enhanced both by cholinergic agonists and by EFS. This would suggest that enhanced smooth muscle responsiveness accounts for at least part of the enhanced cholinergic sensitivity but that enhanced ACh release from prejunctional terminals is not ruled out.

Another possible mechanism of SP action may be related to the complex circuitry of the ferret tracheal plexus. Nerve cell bodies are located in large ganglia of the longitudinal trunk and in ganglia of the superficial muscular plexus. Longitudinal trunk neurons are predominantly cholinergic, and cell bodies in the superficial muscular plexus contain predominantly VIP and NO with a small population containing SP (16, 18). Both longitudinal trunks and superficial muscular plexus project to airway smooth muscle and communicate freely between neurons in the plexus (57). The present study suggests that SP release may be enhanced at VIP/NO neurons in the superficial muscular plexus ganglia. We demonstrated recently that ozone inhalation causes enhanced SP content in longitudinal trunks neurons, increased SP innervation of superficial muscular plexus neurons, and increased SP innervation of airway smooth muscle (55). These findings suggest that SP innervation of VIP/NO neurons may be involved in modulating SP-mediated responses in the ferret tracheal plexus. Unfortunately, the effects of SP on these neurons are not known, and the presence of NK receptors has not been determined in these specific neurons. There is evidence that NK-3 receptors on presumably cholinergic neurons of guinea pig airway mediate membrane hyperpolarization in response to topical SP application (41). Thus it is likely that enhanced SP production in airway ganglia induced by IL-1beta increases neural activity within the airways through local neural reflexes that could involve modulation of cholinergic or VIP/NO pathways.

Although IL-1beta caused enhanced SP expression in airway neurons, the precise signaling pathways involved are still not clear. Treatment of pure neuronal cultures with IL-1beta fails to induce expression of preprotachykinins in sympathetic neurons (31), suggesting either that IL-1beta acts on nonneuronal cells, which, in turn, release another factor or that a nonneuronal cell cofactor is necessary for IL-1beta actions on neurons. A recent study has found that IL-1beta may regulate leukemia inhibitory factor (LIF) release from ganglion nonneuronal cells, that treatment of pure neuronal cultures with LIF induces SP expression, and that cocultures with LIF antibody prevent the SP increase caused by nonneuronal cells (31, 45). These studies provide further support for LIF as a signaling molecule regulating SP expression in airway ganglia. Another mechanism of IL-1-induced SP release may involve a prostaglandin intermediate pathway. Pretreatment with cyclooxygenase inhibitors attenuates the stimulation of sensory neurons by IL-1beta (20). Furthermore, cyclooxygenase inhibitors completely abolish SP release induced by IL-1beta in dorsal root ganglia. Also, IL-1beta increases cyclooxygenase-2 mRNA expression in the same ganglia (27). Prostaglandins directly evoke SP release from sensory neurons (24), but their effect on intrinsic airway neurons is not known.

In conclusion, our results show that repeated intratracheal treatment with IL-1beta increases SP levels in and around airway neurons and tracheal smooth muscle. At the same time, sensitivity of airway smooth muscle is increased. This effect is maintained in tracheal segments cultured for 24 h. Administration of CP-99994, an antagonist of the NK-1 receptor, attenuates the IL-1beta -induced airway responses in cultured tracheal segments. The findings indicate that IL-1beta induces AHR by enhancing SP expression in airway neurons.


    ACKNOWLEDGEMENTS

The authors are grateful to Dr. G. Hobbs (Department of Statistics, West Virginia University) for statistical analysis. The authors also thank Pfizer for the supply of CP-99994.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-35812.

Address for reprint requests and other correspondence: R. D. Dey, Dept. of Neurobiology and Anatomy, PO Box 9128, West Virginia Univ., Morgantown, WV 26506 (E-mail: rdey{at}hsc.wvu.edu).

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.

April 12, 2002;10.1152/ajplung.00363.2001

Received 12 September 2001; accepted in final form 4 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 283(5):L909-L917