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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 170-178, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4811

Pan-Neurotrophin Receptor p75 Contributes to Neuronal Hyperreactivity and Airway Inflammation in a Murine Model of Experimental Asthma

Sebastian Kerzel, Günter Päth, Wolfgang A. Nockher, David Quarcoo, Ulrike Raap, David A. Groneberg, Q. Thai Dinh, Axel Fischer, Armin Braun* and Harald Renz*

Department of Clinical Chemistry and Molecular Diagnostics, Philipps-University Marburg, Marburg; Fraunhofer Institute of Toxicology and Aerosol Research, Drug Research and Clinical Inhalation, Hannover; and Clinical Research Unit of Allergy, Charité School of Medicine, Humboldt-University Berlin, Germany

Address correspondence to: Harald Renz, MD, Philipps-University Marburg, Department of Clinical Chemistry and Molecular Diagnostics; Central Laboratory Baldinger Strasse 35033 Marburg, Germany e-mail: renzh{at}post.med.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bronchial asthma represents a severe chronic inflammatory disease with increasing prevalence. The pathogenesis is characterized by complex neuroimmune dysregulation. Although the immunopathogenesis of the disease has been extensively studied, the nature of neuronal dysfunction still remains poorly understood. Recent data indicate that neurotrophins contribute to airway inflammation, broncho-obstruction and airway hyperresponsiveness. Using an established murine model of allergic bronchial asthma, the contribution of the pan-neurotrophin receptor p75NTR was defined. This receptor is expressed both in normal and asthmatic lungs and airways. Analysis of p75NTR-/- mice, as well as in vivo blocking of p75NTR, revealed that airway inflammation is to a large extent dependent upon functional receptor expression. Furthermore, neuronal hyperreactivity depends entirely on this receptor. Based on these data, a novel molecular pathway in the neuroimmune pathogenesis of bronchial asthma could be defined.

Abbreviations: airway hyperresponsiveness, AHR • fetal calf serum, FCS • interleukin, IL • interferon, IFN • immunoglobulin, IgG • nerve growth factor, NGF • ovalbumin, OVA • phosphate-buffered saline, PBS • substance P, SP • time of break, TB • Tris-buffered saline, TBS • tyrosine kinase receptors, trk


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic bronchial asthma is characterized by chronic airway inflammation, recurrent reversible broncho-obstruction, and development of airway hyperreactivity (AHR) to a broad range of stimuli, including methacholine, histamine, hypertonic saline, cold air, and cigarette smoke (1, 2). Although great advancement has been recently achieved in unraveling the immunopathogenesis of bronchial asthma, the development and regulation of nonspecific (allergen-independent) hyperresponsiveness remains still incompletely understood. One possible mechanism is an inflammation-induced hypersensitivity of sensory airway neurons which augments neuronal reflex circuits controlling lung function (3). A similar mechanism has been discussed for the development of inflammation-induced hyperalgesia. In this state of pathologic neuronal plasticity, the hypersensitivity of sensory neurons is induced by mediators derived from invading inflammatory cells. A number of these mediators has been implied to act in this manner, including biogenic amines (e. g. histamine and serotonin), lipid mediators (e.g., prostaglandin E2), and neurotrophic factors, including nerve growth factor (NGF) (4, 5).

NGF is a member of the neurotrophin family. These structurally related proteins exert their effects primarily as target-derived paracrine and autocrine factors, and were originally noted for their ability to promote survival, growth, and differentiation of neurons. In this context, the role of neurotrophins is well defined (6, 7). Initial evidence for an important role of neurotrophins in allergic bronchial asthma came from the finding of increased NGF levels in serum (8) and bronchoalveolar lavage fluids (BALF) from individuals with asthma. Further experiments demonstrated that allergen challenge induced neurotrophin content in BALF, and that a number of immune cells, including macrophages, T- and B-cells, and mast cells represent major sources of neurotrophins in this condition (9, 10). In a series of experiments in animal models of allergic bronchial asthma, the functional role of NGF could be further elucidated. NGF was shown to induce neuronal changes (increased excitability, hypersensitivity, and induction of neuropeptide production) during allergic immune response. Through these neuronal modifications, NGF may induce AHR to electrical field stimulation and histamine (11, 12). This concept received further support by experiments in which blocking of NGF prevented AHR and decreased airway inflammation (13).

These observations form the basis of the concept that allergic bronchial asthma is a model disease of complex neuroimmune dysregulation. The biological effects of neurotrophins are mediated by binding either to the specific high affinity (Kd 10-11) tyrosine kinase receptors (trk) A (for NGF), trkB (for brain-derived neurotrophic factor), and trkC (for neurotrophin-3) or the low affinity (Kd 10-9) pan-neurotrophin receptor p75NTR. Whether the trks and the p75NTR act alone or in concert is subject to controversial discussion (14, 15). Recent observations suggest that p75NTR as well as trkA bind independently to NGF with predominantly low affinity, but produce high-affinity binding sites upon coexpression (16).

It was the aim of this study to examine the expression patterns of p75NTR in the normal and inflamed lung and to assess the role of p75NTR in a murine model of allergic airway inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
p75NTR-/- mice were obtained from Jackson Laboratory (Bar Harbor, ME). They were generated through targeted mutation of the gene encoding the p75NTR (17). Mice are bred on the background of C57BL/6J and maintained homozygously. C57BL/6J wild-type animals were purchased from Harlan Winkelmann (Borchen, Germany). Mice were held pathogen-free in single ventilated cages, fed an ovalbumin-free diet, and supplied with water ad libitum. The experiments were performed with the approval of the governmental authority (Regierungspraesidium Giessen).

Genotyping of Animals
Mice were genotyped for genetic quality control. According to the "neo PCR genotyping protocol" proposed by Jackson Laboratory (http://www.jax.org/resources/documents/imr/protocols/neo_generic_ko.html), the following neo primer sets were used: upstream primer, 5'-CTT GGG TGG AGA GGC TAT TC-3'; downstream primer, 5'-AGG TGA GAT GAC AGG AGA TC-3'.

Protocol of Allergic Sensitization
Mice were sensitized to ovalbumin (OVA) according to Braun and coworkers (13). Ten micrograms of OVA per injection (Sigma, Deisenhofen, Germany) were adsorbed to 1.5 mg Al(OH)3 and administered by intraperitoneal injections on Days 1, 14, and 21. Before analysis animals received two consecutive allergen challenges via the airways delivered by nebulization of 1% (wt/vol) OVA diluted in PBS for 20 min on Days 27 and 28. Nonimmunized control mice received intraperitoneal Al(OH)3 alone and were challenged with OVA on Days 27 and 28.

Immunohistochemistry of p75NTR
Lungs were preserved, distended with TissueTek (Sakura; Zoeterwonde, Netherlands), and cryo-fixed in liquid nitrogen. Cryostat sections (8 µm) were mounted in 3-aminopropyltriethoxysilane–coated slides, air-dried, fixed in acetone (1 min; -20°C), and placed in Tris-buffered saline (TBS; 0.05 M Tris in 0.15 M NaCl; pH 7.6). The sections were then subjected to the following staining methods:

To suppress endogenous activity of peroxidase, sections were incubated for 30 min with a peroxidase-blocking reagent (DAKO, Carpinteria, CA). After repeated washing in TBS (3 x 5 min), they were preabsorbed with 4% fetal calf serum (FCS) (Biochrom; Berlin, Germany) and 2% normal goat serum (DAKO) in TBS for 30 min. Then, specimens were incubated for another 30 min with rabbit anti-mouse p75NTR polyclonal antibody (Chemicon, Temecula, CA), diluted 1:300 in TBS (containing 4% FCS and 2% normal goat serum). Subsequent to washing, the secondary horseradish-peroxidase labeled antibody (EnVision; DAKO) was applied. After incubation with diaminobenzidine (DAKO) as a chromogen and H2O2 as the substrate, slides were counterstained with hematoxylin (Merck, Darmstadt, Germany).

For confirmation, staining was repeated with a second monoclonal rat anti-murine p75NTR antibody (Chemicon). Sections were preabsorbed for 20 min with 10% FCS in TBS, and then incubated with the antibody (10 µg/ml in TBS containing 10% FCS). After washing three times for 5 min each in TBS, specimens were incubated for 40 min with rabbit anti-rat immunoglobulins (DAKO), diluted 1:70 in TBS (containing 2% normal rabbit serum and 4% normal mouse serum [both from DAKO]). Slides were then incubated for 40 min with alkaline phosphatase rat anti-alkaline phosphatase complex (DAKO), diluted 1:70 in TBS (with 2% RNS, 4% MNS). For intensification, the last two steps were repeated. After washing, the chromogen and substrate solution (containing naphthol-AS-MX-phosphate, fast red TR, and levamisol; DAKO) was applied. Developed specimens were counterstained as described above.

Double Immunohistochemistry of Sensory Airway Neurons
Fluorescence–double immunohistochemistry was performed to assess the expression of the neuronal marker protein gene product 9.5 (PGP 9.5) and substance P (SP). Four groups were examined: (i) OVA-sensitized and -challenged p75NTR-/- mice; (ii) nonimmunized, OVA-challenged p75NTR-/- mice; (iii) OVA-sensitized and -challenged wild-type mice; and (iv) nonimmunized, OVA-challenged wild-type mice. Cryostat sections (8 µm) were washed in 1x phosphate-buffered saline (PBS), and preincubated for 1 h at room temperature with 2% lowfat milk powder in PBS pH 7.4. The sections were then incubated with a monoclonal rat anti-SP antibody (dilution 1:400; Boehringer Ingelheim, Heidelberg, Germany) and a polyclonal rabbit antiserum to PGP 9.5 (dilution 1:400; Biotrend, Cologne, Germany) in the preincubation solution overnight. After several washes in PBS, a biotinylated sheep anti-rat immunoglobulin (IgG) (dilution 1:100; Amersham, Braunschweig, Germany) was applied for 1 h. After several washing steps, all slices were incubated with a mixture of a streptavidin–Texas –Red conjugate (dilution 1:200; Amersham) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Cappel; ICN, Aurora, OH) for 1 h. Control experiments were performed in parallel sections which were incubated only with the secondary antibodies. Slides were coverslipped in carbonate-buffered glycerol (pH 8.6) and viewed using epifluorescence microscopy.

Determination of Antibody Titers
Serum concentrations of total IgE and allergen-specific IgE, IgG1, and IgG2a were measured by ELISA as previously described (18).

Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) was performed as previously described (13, 18). Briefly, 48 h after the last aerosol challenge (Day 27), animals were killed by cervical dislocation. The trachea was cannulated, and airways were lavaged two times with 0.8 ml ice-cold PBS containing proteinase inhibitor Complete (Boehringer, Mannheim, Germany). BAL fluids of each mouse were pooled, and the recovered volume and total cell number determined. Cells were centrifuged onto slides and differentially stained with Diff-Quik (Baxter Dade, Duedingen, Switzerland). They were classified by light microscopy according to common morphologic criteria. Two times 100 cells were assessed. Cell-free supernatants were stored at –20°C until measurements of cytokine content.

Determination of Cytokines in BAL Fluids
The concentrations of interleukin (IL)-4, IL-5, and interferon (IFN)-{gamma} were measured by ELISA as previously described (18) with the following minor modifications. The primary rat anti-mouse IFN-{gamma} (3 µg/ml) was purchased from R&D (Wiesbaden, Germany), diluted in PBS, and incubated at room temperature. Cytokine standards were purchased from PharMingen (Hamburg, Germany). Secondary biotinylated antibodies (PharMingen) were used in the following concentrations: IL-4 (0.25 µg/ml), IL-5 (1 µg/ml), and IFN-{gamma} (0.1 µg/ml). Sensitivities were 23 pg/ml for IL-4 and IL-5, and 100 pg/ml for IFN-{gamma}, respectively.

Assessment of Lung Function
To assess airway function in vivo, head-out body plethysmography was used as described (19). Briefly, this well-established computer assisted system allows one to evaluate simultaneously the breathing pattern of four nonanesthetized, spontaneously breathing mice in response to different stimuli. For determination of bronchoconstriction, the midexpiratory airflow (EF50), i. e., the expiratory airflow (in ml/s) at 50% tidal volume, was measured. Sensory irritation was assessed by gauging the length of the pause prior to expiration (time of break, TB [s]) according to Vijayaraghavan and coworkers (20). Methacholine (diluted in PBS; Sigma, St. Louis, MO) or capsaicin (diluted in 10% ethanol; Sigma) were delivered by a jet nebulizer (Pari-Boy; Pari-Werke, Starnberg, Germany). As vehicle control the diluent itself was nebulized.

Treatment with Anti-p75NTR Antibodies
Intranasal application of 2 x 25 µl polyclonal rabbit anti-mouse p75NTR antibody (diluted 1:50 in sterile PBS) (Chemicon) or isotype control (rabbit IgG; Sigma) was performed according to Braun and colleagues (13) before each allergen challenge. For application procedure, mice were slightly anesthetized with 2.6 mg ketaminhydrochloride (Ketanest; Parke Davis, Berlin, Germany) and 0.18 mg xylazinhydrochloride (Rompun; Bayer, Leverkusen, Germany).

Statistical Analysis
Results are presented as mean values ± SD. Normality distribution was assessed by Kolmogorov-Smirnov test. Two-way ANOVA was used to detect differences among the groups presented in Figures 6 and 7. For determination of level of difference, Student's t test for unpaired observations was used. Values of P < 0.05 were considered significant.



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  		Figure 6. Methacholine responsiveness of wild-type (open diamonds) and p75NTR-/- (filled squares) mice. Dose–response curve of midexpiratory flow (EF50) in response to inhaled methacholine. All mice demonstrated a dose-dependent fall in EF50, which was significantly enhanced in OVA-sensitized mice (*P < 0.05), whereas the genotype had no effect on methacholine response pattern (n. s.). VC: vehicle control (aerosolic application of the diluent PBS alone). Baseline of 100% represents -4.13 ml/s. Error bars indicate SD.

 


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  		Figure 7. Capsaicin responsiveness. The sensory irritation to inhaled capsaicin was assessed by measuring time of break (TB). (A) Dose–response curves of nonsensitized but OVA-challenged wild-type (open diamonds; n = 10) and p75NTR-/- mice (filled squares; n = 9). Wild-type controls demonstrated a small increase in TB, which was abolished in p75NTR-/- mice. 100% baseline represents 31.4 ± 7.7 ms (mean ± SD). (B) In OVA-sensitized wild-type animals (open diamonds; n = 9) a marked increase in TB was found, which was almost completely prevented in p75NTR-/- mice (filled squares; n = 7). 100% baseline represents 33.6 ± 11.3 ms (mean ± SD). (C) OVA–sensitized wild-type mice were treated with anti-p75NTR antibodies (filled squares; n = 22) before each allergen challenge and showed a remarkably lower rise in TB following capsaicin provocation than isotype-treated control mice (open diamonds; n = 21). 100% baseline represents 30.2 ± 5.1 ms (mean ± SD). VC: vehicle control (aerosol application of the diluent alone); filled diamond: significant difference (P < 0.05) between nonsensitized and OVA-sensitized wild-type mice at doses of 500 and 1,000 µg/ml. Student's t test: *P < 0.05, **P < 0.01, ***P < 0.001. The error bars represent standard deviation (SD).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pattern of p75NTR Expression in Airways and Lungs
The expression pattern of p75NTR in normal lungs was assessed by immunohistochemistry, using two different antibodies and staining methods. Immunoreactivity was detected on large bundles of nerve fibers (Figure 1A) as well as on terminal subepithelial varicosities, most likely reflecting sensory nerves adjacent to the airways (Figures 1C and 1E). Figure 1F shows another example for p75NTR-positive nerve fibers. The expression pattern of p75NTR on these neuronal structures did not differ between OVA-sensitized and nonsensitized animals. In OVA-sensitized and challenged mice, however, additional p75NTR immunoreactivity was found within the inflammatory infiltrate surrounding larger airways (Figure 1G), a phenomenon absent in nonsensitized mice. The specificity of antibodies was verified by isotype-matched negative-control antibodies (Figures 1B, 1D, and 1H). This observation was further confirmed with sections obtained from p75NTR-/- mice, demonstrating no immunoreactivity for p75NTR (data not shown).



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  		Figure 1. Immunohistochemical localization of p75NTR in murine lungs. (A) Staining with polyclonal rabbit anti-p75NTR antibody revealed expression on large bundles of nerve fibers. (B) The isotype control showed no immunoreactivity. Magnification: both x200. (C) Immunoreactive small nerve fibers adjacent to alveolar tissue stained with a monoclonal rat anti-p75NTR antibody. (D) Corresponding isotype control. Magnification: both x200. (E) Immunopositive small subepithelial nerve indicating that the intranasally applicated polyclonal anti-p75NTR antibody was successful in reaching its target. Magnification: x600. (F) Large nerve bundle and smaller intramural fibers surrounding the esophagus, stained with the monoclonal anti-p75NTR antibody. Magnification: x200. (G) Cells of the inflammatory infiltrate surrounding bronchi immunopositive for p75NTR. (H) Isotype control. Magnification: both x600. Brown color indicates positive immunoreactivity in A, E, and G, whereas pink is positive in C and F.

 
Immunohistochemistry for PGP 9.5 and SP
Immunohistochemistry for PGP 9.5 reveals labeling of airway neurons in all study groups (Figure 2) . Nerve fibers were present in all airway compartments with decreasing frequency as the airways become smaller. Only few fibers were detected in bronchioli. To assess tachykinergic innervation, the sections were incubated with an SP antibody (Figure 2), and a population of tachykinergic nerves was identified within the PGP 9.5-positive neurons. These SP-positive nerve fibers projected to the lamina propria and the respiratory epithelium, and were also present around submucosal arteries and veins in both the OVA-sensitized/nonsensitized p75NTR-/- mice and the OVA-sensitized/nonsensitized wild-type mice. Qualitative assessment did not reveal any major differences between study groups.



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  		Figure 2. Expression of PGP 9.5 and substance P in airway nerve fibers. PGP 9.5 antibody stain for total airway innervation of nonimmunized OVA-challenged wild-type (A), OVA-sensitized and -challenged wild-type (C), nonimmunized OVA-challenged p75NTR-/- (E), and OVA-sensitized and -challenged p75NTR-/- mice (G). Double immunohistochemistry using a second antibody for substance P (B, D, F, H) revealed a subpopulation of substance P–positive (arrows) and a majority of substance P–nonreactive fibers (arrowheads) Original magnification: x400. veh/OA: non-sensitized mice received the vehicle alone intraperitoneally, but were challenged with allergen; OA/OA: OVA-sensitized and -challenged mice; PGP 9.5: neuronal marker protein gene product 9.5; SP: substance P.

 
Immune Responses in p75NTR-/- Mice
To assess the participation of p75NTR in the process of allergic immune response, wild-type and p75NTR-/- mice were sensitized and challenged with OVA. Serum levels of total IgE and OVA-specific IgE, IgG1, and IgG2a antibodies did not significantly differ between sensitized wild-type and p75NTR-/- mice (Table 1).


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  		TABLE 1 Serum immunoglobulin titres in C57BL/6 wt and p75NTR-/- mice (both sensitized to and challenged with ovalbumin, mean ± SD)

 
However, the local inflammatory response in the lung and airways showed marked differences. OVA-sensitized wt animals developed the typical allergic inflammation, characterized by the influx of eosinophils and lymphocytes into the airways. In p75NTR-/- mice, numbers of eosinophils and lymphocytes recruited into the airways were significantly lower (P < 0.0001 and P < 0.05, respectively) (Figures 3A and 3C) . In contrast, a slightly higher number of macrophages was detected in p75NTR-/- mice as compared with wild-type animals (P < 0.05) (Figure 3D). In nonsensitized but challenged mice, neither IL-4, IL-5, nor IFN-{gamma} were detectable in any study group (data not shown). In OVA-sensitized and -challenged animals, IL-5 concentrations were elevated. However, sensitized p75NTR-/- mice showed a tendency to lower levels (P = 0.067) compared with sensitized wild-type controls (Figure 4A) .



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  		Figure 3. Distribution of leukocyte subpopulations in BAL fluids from wild-type and p75NTR-/- mice. BAL was performed 48 h after last aerosol allergen challenge. In nonsensitized but challenged mice, mainly macrophages were detected (D). OVA-sensitized animals developed an allergic inflammation with a marked influx of eosinophils and lymphocytes (A, C). In p75NTR-/- mice this influx was significantly reduced as compared with wild-type (P < 0.0001 for eosinophils and P < 0.05 for lymphocytes). Number of macrophages was slightly increased in p75NTR-/- mice. Bars indicate the mean values.

 


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  		Figure 4. Levels of IL-5 in BAL fluids. (A) OVA-sensitized p75NTR-/- mice showed a tendency to lower levels of IL-5 in BAL fluids (P = 0.067). (B) Similarly, wild-type animals treated with an anti-p75NTR antibody demonstrated a tendency to diminished IL-5 concentrations compared with isotype-treated controls (P = 0.080).

 
To further substantiate these results, wild-type mice were treated with anti-p75NTR blocking antibodies. To ensure that antibodies were effective in reaching their targets, namely neuronal structures, immunohistochemistry was performed on lungs of intranasally treated animals without use of primary antibodies. Lungs of mice that were treated with the anti-p75NTR antibodies showed immunoreactivity of small subepithelial nerve fibers (Figure 1E), whereas isotype-control–treated mice showed no immunoreactivity (not shown), indicating that the applied antibodies succeeded in reaching their targets. Blockade of p75NTR in sensitized and challenged wild-type mice reduced the influx of eosinophils by ~ 50% as compared with controls (P < 0.05) (Figure 5A) . A similar effect was seen on the number of lymphocytes in BAL fluids (Figure 5C). As in p75NTR-/- mice, the number of macrophages increased (Figure 5D), whereas the number of neutrophils remained unaffected (Figure 5B). As observed in p75 NTR-/- mice, there was a tendency toward lower levels of IL-5 in BAL fluids from anti-p75NTR–treated mice (P = 0.08) (Figure 4B).



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  		Figure 5. Distribution of leukocyte subpopulations in BAL fluids from isotype-control and anti-p75NTR antibody–treated mice. Both groups developed a characteristic allergic inflammatory response, defined by the domination of eosinophils. In anti-p75NTR antibody–treated mice, the amount of eosinophils (A) as well as the amount of lymphocytes (C) was markedly and significantly reduced as compared with isotype controls (P < 0.05). Numbers of macrophages were increased (D) (P < 0.01).

 
Airway Reactivity
To assess the contribution of p75NTR on the development of airway reactivity (AR), we used head-out body plethysmography, allowing evaluation of the breathing pattern of nonanesthetized, spontaneously breathing mice in response to different stimuli (19). As a marker of broncho-obstruction, the midexpiratory airflow (EF50) was recorded. Nonsensitized mice demonstrated a dose-dependent decrease of ~ 40% in EF50 to methacholine. Wild-type and p75NTR-/- mice did not differ in their responses (Figure 6A) . However, airway reactivity to methacholine was markedly enhanced in OVA-sensitized animals (P < 0.05), indicating a state of constrictor hyperresponsiveness (Figure 6B, asterisk). Although the p75NTR-/- mice appeared to exhibit a slightly diminished ED50, they did not significantly differ from the wild-type controls.

Whereas methacholine mainly takes effect via the muscarinic acetylcholine receptor type 3 (M3 receptor), expressed on airway smooth muscle cells (21), capsaicin acts specifically via vanniloid receptors almost exclusively expressed on sensory neurons (22). Differences in sensory nerve activities were measured using inhalation of capsaicin. A characteristic feature of the sensory irritation so caused is lengthening of the pause before expiration (time of break, TB) in the airflow curve (20, 23). All mice demonstrated a slight increase in TB following administration of vehicle control (VC) alone (Figure 7) , reflecting some unspecific stimulation of sensory nerves by the diluent itself. Following capsaicin challenges, wild-type mice showed a considerable dose-dependent rise in TB. At concentrations higher than 100 µg/ml, OVA-sensitized and -challenged mice reacted significantly stronger than nonsensitized animals (diamond, Figures 7A and 7B), indicating hyperreactivity to capsaicin. In contrast, the capsaicin response was almost completely abolished in p75NTR-/- mice. The difference to the wild-types was significant in the nonsensitized groups (P < 0.05) (Figure 7A) and highly significant in the OVA-sensitized groups (P < 0.001) (Figure 7B).

To further explore the potential role of p75NTR in hyperreactivity, wild-type mice treated with anti-p75NTR antibodies were examined. Sensitized and challenged animals treated with isotype control antibody showed a similar dose-dependent rise in TB as observed in OVA-sensitized and -challenged nontreated mice (Figures 7B and 7C). However, this increase was significantly reduced in mice treated with the anti-p75NTR antibody (Figure 7C), again indicating that airway neuronal hyperreactivity depends on expression of functional p75NTR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, the contribution of neurotrophins on allergic airway inflammation and airway hyperresponsiveness has been established. However, their mechanism of action is not completely understood. Here, we were able to demonstrate that p75NTR is required for the development of several characteristic features of allergic bronchial asthma. Deactivation of p75NTR, either by genetic targeting or by treatment of wild-type animals with a blocking antibody, revealed several major effects. First, there was a marked reduction of allergic airway inflammation, characterized by the lowered influx of eosinophils and lymphocytes. These findings get strong support from the observations by Tokuoka and coworkers (24), who also found the numbers of eosinophils and levels of IL-5 in BAL fluids diminished in p75NTR-/- mice. Second, the neuronal hyperreactivity to capsaicin, following allergic airway inflammation, was almost completely abolished. In contrast, airway hyperresponsiveness to methacholine remained unaffected. Immunohistochemistry revealed an expression of p75NTR by neuronal structures in the inflamed lung as well as in the noninflamed lung. The finding of an additional expression pattern of p75NTR within the peribronchial inflammatory infiltrate is in line with previous findings of p75NTR expression by several immune cells. Although mast cells were found being p75NTR negative (25), p75NTR was detected on B cells (26) and basophils (27). Whether eosinophils express p75NTR remains to be elucidated. Furthermore, by assessment of tachykinergic innervation, we were able to demonstrate that sensory airway nerves are present even in p75NTR-/- mice.

The role of NGF in the allergic inflammation is well characterized (for review see Refs. 10, 28). The proinflammatory properties of NGF include facilitation of mast cell degranulation (25, 29), activation of eosinophils (30), augmentation of TH2 cytokine production and IgE synthesis (13), and the induction of B-cell differentiation into Ig-secreting plasma cells (31). However, it has been shown that the high-affinity NGF-receptor trkA plays the central role in mediating NGF effects to B lymphocytes (32). Furthermore, our observations indicate that the production of serum immunoglobulins was not affected in p75NTR-/- mice. The participation of the pan-neurotrophin receptor p75NTR in these events still awaits further elucidation.

A key feature in the pathophysiology of allergic bronchial asthma is a qualitative and quantitative change in the functional activity of peripheral sensory neurons, summarized by the term "neuronal plasticity" (33). This comprises an increase in mechanosensitivity of sensory nerve endings, resulting in exaggerated neuronal excitability (34, 35). In addition to several inflammatory mediators (e.g., prostaglandins and bradykinin), neurotrophins also have the capacity to induce this modulation of the nociceptive system (4, 5). Polymodal nociceptor neurons (unmyelinated C-fibers and A-{delta}-fibers) are very sensitive to capsaicin (22, 36, 37), an ingredient of hot pepper, which acts specifically via the vanilloid receptor 1 (VR1) (22, 38). Reflex activity of polymodal nociceptive terminals of sensory nerves is not only evoked by capsaicin, but also via several physiologic mediators, like bradykinin, or environmental irritants, like cold air, noxious agents including SO2, NO2, and ozone, changes in osmolarity, and smoke (39, 40). The allergen-independent reaction to these unspecific stimuli is an important feature of chronic asthma.

To assess the participation of p75NTR in the context of neurotrophin-induced neuronal hyperreactivity of the airways, the breathing pattern in response to inhaled capsaicin was analyzed. A characteristic feature of activation of sensory neurons is reflex prolongation of the breathing pause before expiration (time of break, TB). Remmers and colleagues were able to show that this postinspiratory apneic state, termed "stage I of expiration," is caused by stimulation of sensory airway nerves (23). It is due to a central chemoreflex mediated via the nucleus of the solitary tract. Electrical or chemical stimulation of laryngeal, vagal, or carotid sinus nerves were shown to prolong the period of depolarization in postinspiratory neurons without changing the duration of stage II expiratory or inspiratory inhibition, indicating a fairly selective prolongation of the first stage of expiration (23). Validation of body plethysmographic measurement was performed in a series of studies (20, 41) and recently adapted for asthma research in the mouse (19, 42, 43).

A key finding of this study is that the inflammation-induced neuronal hyperreactivity to capsaicin was almost completely prevented in p75NTR-/- mice. This observation was confirmed by experiments with wild-type mice treated with a blocking anti-p75NTR antibody. These results for the first time suggest an important role for the pan-neurotrophin-receptor p75NTR in the development of the hyperalgesia-like state in neuronal hyperreactivity. Hoyle and colleagues showed that transgenic mice with Clara-cell specific (CCSP) overexpression of NGF (NGF-tg) are more sensitive to inhaled capsaicin compared with wild-type mice (44). Consistently, we recently demonstrated that NGF-tg mice show a remarkable higher rise in TB than wild-type controls following aerosolic capsaicin challenge (45).

Another aspect to be considered is the possible anatomic differences between animals, due to the genotype. Although lungs of p75NTR-/- mice are histologically grossly similar to wild-type animals (46), it might be possible that the diminished reflex activity of sensory airway neurons seen in knockout animals was caused by a reduced innervation. However, wild-type animals treated with anti-p75NTR antibodies showed a similar effect as the p75NTR-/- mice, making this explanation rather unlikely. Despite this fact, experiments were performed to assess the sensory innervation of the lung in both p75NTR-/- mice and wild-type controls, and revealed that sensory nerves are present even in p75NTR-/- mice.

Several allergic conditions are associated with increased production of neurotrophins (8, 9, 13, 47). Neurotrophins are known to act on sensory neurons, resulting in augmented production of proinflammatory neuropeptides (SP, neurokinin A, calcitonin gene-related peptide) (48) and to lower the firing threshold of sensory nerves (44, 49). Therefore, it is not surprising that elevated levels of neuropeptides are another characteristic feature of inflammation-induced neuronal plasticity seen in allergic bronchial asthma (50). Furthermore, these tachykinins have highly proinflammatory properties, and play an essential part in the process termed neurogenic inflammation (33, 51).

Our findings suggest that blocking neurotrophin signaling to the sensory neurons via p75NTR prevents the subsequent neuronal plasticity. This may be related to inhibition of tachykinin synthesis and release. This hypothesis gets strong support from the very recent observation by Hoyle and colleagues, who found that the amount of SP in the lungs of p75NTR-/- mice was reduced by ~ 50% compared with wild-type mice (46). SP and other tachykinins elicit a broad range of proinflammatory actions on immune cells (reviewed in Ref. 52). For example, SP has a degranulating effect on eosinophils and induces human eosinophil migration in vitro. In an in vivo study involving patients with allergic rhinitis, it was shown that SP administered after repeated allergen challenge enhanced the recruitment of eosinophils (52).

In conclusion, we were able to demonstrate for the first time that neuronal hyperreactivity and allergic airway inflammation are p75NTR-dependent to a large extent. Our data indicate that this hyperalgesia-like state could be prevented by blocking of p75NTR. The receptor distribution in wild-type animals and the diminished content of SP in the lungs of p75NTR-/- mice suggest that these effects are partly mediated via sensory neurons and related to neurogenic inflammation. Whether p75NTR acts alone or in concert with specific trk receptors remains to be elucidated. Taken together, our data suggest that p75NTR acts as an important player in inflammation-induced neuronal plasticity in allergic bronchial asthma.


   Acknowledgments
 
This project was funded by the Deutsche Forschungsgemeinschaft and the SFB 587 B4.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form January 3, 2002

Received in final form July 25, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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