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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 818-826, (2002)
© 2002 American Thoracic Society

Original Articles

Augmentation of Allergic Early-Phase Reaction by Nerve Growth Factor

Günter Päth, Armin Braun, Nina Meents, Sebastian Kerzel, David Quarcoo, Ulrike Raap, Gary W. Hoyle, Wolfgang A. Nockher and Harald Renz

Department of Clinical Chemistry and Molecular Diagnostic, University Hospital of Marburg, Marburg; Fraunhofer Institute of Toxicology and Aerosol Research, Hannover; Charité-Campus Virchow, Berlin, Germany; and Section of Pulmonary Disease, Critical Care and Environmental Medicine, Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana

Correspondence and requests for reprints should be addressed to Harald Renz, M.D., University Hospital of Marburg, Department of Clinical Chemistry and Molecular Diagnostic, Baldingerstrasse, 35033 Marburg, Germany. E-mail: renzh{at}post.med.uni-marburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The allergic early-phase reaction, a hallmark of allergic bronchial asthma, is caused by allergen and immunoglobulin E-dependent mediator release from mast cells. It was previously shown that nerve growth factor (NGF) contributes to acute airway inflammation. This study further investigates the role of NGF in the allergic early-phase reaction using a well-established mouse model of ovalbumin-induced allergic airway inflammation. Treatment of sensitized and aerosol challenged BALB/c mice with blocking anti-NGF antibodies inhibited allergen-induced early-phase reaction and suppressed airway inflammation. Transgenic mice constitutively overexpressing NGF in the airways (Clara-cell secretory protein promoter [CCSP]-NGF-tg) were employed and compared with wild-type animals. In sensitized and challenged CCSP-NGF-tg mice, early-phase reaction, airway inflammation, as well as percental relative increases in serotonin levels were augmented compared with wild-type mice. These effects were paralleled by increased serotonin levels in the airways, whereas immunoglobulin E levels remained unaffected. Furthermore, CCSP-NGF-tg mice developed an increased reactivity of sensory neurons in response to inhaled capsaicin demonstrating NGF-mediated neuronal plasticity. These data provide evidence for the functional role of NGF in the development of allergic early phase responses in the airways and the lung.

Key Words: nerve growth factor • allergy type I • airway inflammation • neurogenic inflammation • bronchial asthma • mouse model


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allergic bronchial asthma is characterized by allergen-specific immunoglobulin (Ig) E antibody levels, development of airway inflammation, reversible broncho-obstruction, and airway remodeling (1–3). Furthermore, the disease is accompanied by neuronal dysregulation (4). Increased levels of neuropeptides, in particular substance P (SP), have been detected in the lungs of patients with asthma, as well as in murine models of allergic airway inflammation (5, 6). SP has been described to trigger characteristic symptoms of bronchial asthma, including airway smooth muscle constriction and acute inflammation ("neurogenic inflammation") (7, 8). Furthermore, the production of SP by neurons is enhanced by nerve growth factor (NGF), a member of the neurotrophin family (9–12).

Growing evidence indicates that neurotrophins, in particular NGF, appear as mediators in the interactions between both immune and nerve cells (13). Neurotrophins are produced in nerve-associated cells and neurons (14, 15). In addition to these traditionally known sources, NGF is also produced by a wide range of immune cells, including mast cells (16), macrophages (17), T cells (18), and B cells (19), as well as lung fibroblasts and epithelial cells (20–22). Compared with healthy control subjects, patients with allergic rhinitis have elevated levels of NGF in nasal lavage fluids, which were further increased in response to allergens (23). Similar observations were made in patients with asthma. In these subjects, NGF levels were elevated in serum (24) and bronchoalveolear lavage fluids (BALF) after segmental allergen provocation (25). Recently, using a well-defined murine model of bronchial asthma, we have detected upregulation of local NGF production in allergic airway inflammation, and NGF was identified as an amplifier of the Th2 immune response and to contribute to the development of airway hyperreactivity (17).

In allergic bronchial asthma, inhalation of allergens results in acute broncho-obstruction, defined as the allergic early-phase reaction that occurs within minutes after allergen challenge and is short lasting (26, 27). The early-phase reaction is IgE dependent and mainly caused by activation of lung mast cells via allergen cross-linking of IgE antibodies bound to the high-affinity IgE receptor Fc{epsilon}RI (28). Activated cells rapidly degranulate and release proinflammatory mediators such as histamine, eicosanoids, cytokines, and reactive oxygen species (29–33). Once secreted, these substances induce airway smooth muscle constriction, mucus secretion, and vasodilatation. In this sense, a current study reported that NGF increased airway reactions during intravenous administration of histamine to allergic guinea pigs (34). Furthermore, a recent study performed in guinea pigs reported that anti-NGF antibodies reduced allergen-induced bronchoconstriction (35). The acute asthmatic reaction may be followed by a late-phase reaction that commences several hours later (36). The late-phase reaction is dependent on the recruitment and activation of immune cells, particularly eosinophils and T cells (2). In respect thereof, it has been demonstrated in a mouse model that treatment with anti-interleukin (IL)-5 antibodies prevented the influx of eosinophils into the airways and abolished the late-phase but not the early-phase reaction (27).

The role of NGF in the development of airway inflammation, airway hyperresponsiveness, and airflow limitation has been examined to some extent. To analyze further the relationship between NGF and allergic early-phase reaction and to assess further the role of NGF in the pathogenesis of bronchial asthma, the following study has been designed.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
BALB/c and C57BL/6 mice were obtained from Harlan Winkelmann GmbH (Borchen, Germany). Transgenic mice encoding NGF under the control of the lung-specific Clara-cell secretory protein promoter (CCSP) (37) were originally generated from a mixed background of C57BL/6 and SJL. Before experiments, these animals were mated with female C57BL/6 wild-type mice for three generations. Offsprings display either the wild-type (CCSP-NGF-wt) or the transgenic (CCSP-NGF-tg) phenotype. Mice were maintained under pathogen-free conditions. Mice taken for experiments were 6 to 8 weeks old.

Genotyping
Six-week-old CCSP-NGF offsprings were slightly anesthetized with 2.6 mg ketaminhydrochloride (Ketanest; Parke Davis, Berlin, Germany) and 0.18 mg xylazinhydrochloride (Rompun; Bayer AG, Leverkusen, Germany) per mouse by intraperitoneal injection. The tip of the tail was removed to isolate DNA using the QIAamp DNA MiniKit (Qiagen, Hilden, Germany). The existence of the promoter construct was determined by polymerase chain reaction using the following primer sequences: 5'-CATACCCACACATACCCACA-3' (upstream) and 5'-ACATTACGCTATGCACCTGG-3' (downstream). Polymerase chain reaction conditions were as follows: initial denaturing, 94°C for 5 minutes; 30 cycles with 94°C, 60°C, and 72°C for 30 seconds each step; final elongation, 72°C for 5 minutes. Polymerase chain reaction products were size fractionated in 3% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. CCSP-NGF-tg mice were recognized by the existence of a 311-bp fragment, whereas DNA from CCSP-NGF-wt animals was void of amplicons.

Allergic Sensitization and Allergen Challenge
Mice, 6 to 8 weeks of age, were sensitized with 10 µg of ovalbumin (OVA) (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) adsorbed to 1.5 mg Al(OH)3 (Alum; Pierce, Rockford, IL) diluted in phosphate-buffered saline (PBS) by intraperitoneal injection on Days 1, 14, and 21. Sensitization was followed by two local allergen challenges with 1% OVA (wt/vol) diluted in PBS delivered for 20 minutes by aerosolization on Days 27 and 28 (Protocol 1 and 2) and a further intranasal challenge on Day 35 (Protocol 1) (Figure 1) . Nonsensitized control animals received injections with 1.5 mg Al(OH)3 alone diluted in PBS. As a further control, untreated animals were recruited to determine the effect of intranasal fluid application (PBS alone) on airflow limitation. Experimental groups corresponding to the general Protocols 1 and 2 are depicted in Tables 1 and 2 .



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  		Figure 1. Experimental design. Protocols 1 and 2 are summarizing general animal treatment. A detailed description of experimental groups and substances used is presented in Tables 1 and 2.

 

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  		TABLE 1. Experimental groups and substances used for sensitization, pretreatment, and challenge: protocol 1

 

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  		TABLE 2. Experimental groups and substances used for sensitization, pretreatment, and challenge: protocol 2

 
Intranasal Anti-NGF Treatment of BALB/c Mice
Slightly anesthetized mice (see GENOTYPING) received intranasally 50 µl of polyclonal rabbit anti-mouse NGF antibody (Sigma) diluted 1:50 in PBS or isotype control antibody (Sigma). Intranasal treatment was performed 3 hours before allergen aerosol challenge on Days 27 and 28.

Head-Out Body Plethysmography
This method allows the continuous online recording of tidal airflow patterns in conscious mice. Experimental procedure and equipment used are described in detail by Neuhaus-Steinmetz and colleagues (38). The software used in this study has been described previously (39, 40). Data were collected over 14 seconds with a 1-second interval for computation of midtidal volume (EF50) and breathing pauses before expiration (time of break, TB). For experiments, mice were placed in the body plethysmograph chambers and allowed to adapt for approximately 15 minutes. After this period, the respiratory variables for each breath were obtained over a period of 15 minutes while the animals respired room air. Average values ± SD for each respiratory variable were calculated. These values were used as the baseline values from which deviations during the challenge period were calculated. Tidal volumes of mice tested in this study were similar in coeval animals tested.

Determination of Allergic Early-Phase Reaction and Airway Reactivity
Allergen- and methacholine-induced airflow limitations were evaluated by determining the respiratory EF50. Lung functions were continuously recorded using head-out body plethysmography as described previously here (38, 41, 42). Allergic early-phase reaction was induced by intranasal allergen challenge on Day 35. After assessment of baseline values for 15 minutes, animals were intranasally challenged with allergen for 5 minutes. Then the allergic early-phase reaction was determined during the next 30 minutes. Airway reactivity was assessed by aerosolization of methacholine on Day 29. After the evaluation of baseline values, animals were challenged with aerosolized methacholine, diluted in PBS, and administered in increasing concentrations of 25, 50, 75, and 100 mg/ml each delivered for 1 minute.

Collection of Blood and BALF Samples
Blood and BALF samples for evaluation of immunoglobulin and cytokine levels were taken 24 hours after the last challenge on Day 36. To verify that OVA-specific IgE baseline levels were not detectable in untreated animals, blood was collected on Day -1. Blood was obtained from the tail vein. After sacrifice of animals, the trachea was cannulated, and airways were lavaged with 1.6 ml (two times 0.8 ml) ice-cold PBS. Only BALF samples with a recovery volume of 1.4 ± 0.2 ml were further processed. One portion of the BALF samples was centrifuged at 250 x g, and the supernatant was aliquoted for measurement of cytokines. The other portion of the BALF samples was used to count the total cell number and to perform cytospins as previously described (43). BALF samples for determination of serotonin levels were taken 15 minutes post last allergen challenge on Day 35 by using 0.8 ml of ice-cold PBS for lavage. Only supernatants from samples with a recovery volume of 0.7 ± 0.1 ml were measured by enzyme-linked immunosorbent assay.

Assessment of Immunoglobulins and Cytokines by Enzyme-linked Immunosorbent Assay
Total and OVA-specific IgE levels as well as IL-4, IL-5, and interferon-{gamma} production were measured by enzyme-linked immunosorbent assay as described before (43). Serotonin was measured with a commercial enzyme-linked immunosorbent assay system (ICN Biomedicals GmbH, Eschwege, Germany) according to the manufacturer's protocol. Limits of detection were as follows: IL-4 and IL-5, 30 pg/ml; interferon-{gamma}, 20 pg/ml; total and OVA-specific IgE antibodies, 20 ng/ml and 20 arbitrary units, respectively; and serotonin, 0.05 ng/ml.

Determination of Sensory Reactivity
Reactivity of sensory neurons was continuously measured in a head-out body plethysmograph by assessment of breathing pauses before expiration (TB) in response to local capsaicin provocations. Capsaicin, diluted in PBS with 10% ethanol, was administered in increasing concentrations of 10, 50, 100, 500, and 1,000 µg/ml delivered by 1-minute aerolization for each concentration.

Statistical Analysis
Results are presented as mean values ± SD or as single data points and median. Statistical significance of differences between experimental groups was determined by using a t test (Figures 2C, 3, 4 , and 7) or analysis of variance and Bonferroni's post test (all groups versus each other) (Figures 2B, 6, and 8). Curves in Figure 2A were smoothed using the average of 13 nearest neighbors.



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  		Figure 2. Allergic early-phase reaction in BALB/c and CCSP-NGF mice measured by head-out body plethysmography on Day 35 according to Protocol 1. (A) Mean airflow at EF50 in BALB/c mice before and after nasal application of either PBS alone (NIL) or OVA diluted in PBS (isotype control or anti-NGF pretreatment). The curve represents means of single data points taken every 0.25 minutes from each mouse per group. The gray area represents the period of challenge. Dashed line represents NIL. Thin solid line represents isotype control. Thick solid line represents anti-NGF. (B) Statistical analysis of curves from A. Columns represent means ± SD of single data points from the time periods indicated. White bars represent NIL. Light gray bars represent isotype control. Dark gray bars represent anti-NGF. (C) Statistical analysis of allergic early-phase reaction in CCSP-NGF-wt and CCSP-NGF-tg mice within 10 to 20 minutes post challenge with 50 µl OVA (PBS control and OVA) or BSA (BSA controls) diluted in PBS. White bars represent wt. Light gray bars represents tg. Columns represent means ± SD in percent baseline (-20 to -10 minutes) of single data points from the time period indicated. NIL, n = 12; isotype control, n = 16; anti-NGF, n = 16; CCSP-NGF-wt, n = 12; CCSP-NGF-tg, n = 12; ***p < 0.001.

 


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  		Figure 3. Numbers and types of cells in BALF taken from BALB/c (A) and CCSP-NGF-wt and CCSP-NGF-tg mice (B–D) at 24 hours post last challenge on Day 36 according to Protocol 1. Columns represent mean ± SD. Isotype control and anti-NGF, n = 16; CCSP-NGF-wt and CCSP-NGF-tg, n = 12; *p < 0.05; **p = 0.01; ***p < 0.001. eos, eosinophils; lym, lymphocytes; m{phi}, macrophages; neu, neutrophils.

 


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  		Figure 4. Levels of IL-4 (A), IL-5 (B), and interferon-{gamma} (C) in BALF taken from BALB/c mice pretreated with isotype control or anti-NGF antibodies on Day 36 according to Protocol 1. The scattergram represents single BALF samples and medians of data points from experimental groups. Dashed lines indicate limits of detection (DL). Open circles represents isotype control. Solid circles represents anti-NGF. Isotype control, n = 15; anti-NGF, n = 18; ***p < 0.001.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Allergic Early-Phase Reaction
To assess the contribution of NGF to the development of allergic early-phase reaction, BALB/c mice were locally pretreated with anti-NGF or isotype control antibody before allergen provocation. As a control, nonsensitized/nonchallenged (untreated animals) BALB/c mice were nasally treated with PBS to determine the effects of fluid application on EF50 values per se. Isotype control mice and anti-NGF–treated animals responded immediately to nasal application of OVA with a marked fall in EF50 values (p < 0.001). While in the isotype control group significant airflow limitation persisted for at least 25 minutes, the anti-NGF group recovered rapidly within 15 minutes (Figures 2A and 2B). Similar to BALB/c mice, allergic early-phase reaction was also inducible in sensitized and challenged CCSP-NGF-wt and CCSP-NGF-tg mice constitutively overexpressing NGF locally in the lungs (Figure 2C). In all experimental groups, CCSP-NGF-tg demonstrated a significantly higher degree of expiratory airflow limitation than CCSP-NGF-wt animals (p < 0.001). In both the PBS control groups and the bovine serum albumin (BSA) control groups, no allergic early-phase reaction was elicited.

Allergic Airway Inflammation
Allergic airway inflammation was assessed by measuring numbers and distribution of leukocyte subpopulations and levels of interferon-{gamma}, IL-4, and IL-5 in BALF 24 hours after nasal allergen challenge. In anti-NGF–treated BALB/c mice, the number of eosinophils recruited into the airways was significantly reduced (p < 0.01), whereas numbers of macrophages, lymphocytes, and neutrophils were not altered (Figure 3A). Furthermore, in the anti-NGF group, local production of IL-4 and IL-5 was significantly reduced (p < 0.001), whereas interferon-{gamma} was not affected (Figure 4).

Conversely to the effects of anti-NGF-treatment, OVA-sensitized and challenged CCSP-NGF-tg (OVA group) demonstrated a significantly higher influx of eosinophils, lymphocytes, and neutrophils into the airways than corresponding CCSP-NGF-wt animals (p < 0.01, p < 0.001, and p < 0.05) (Figure 3B). The OVA-sensitized and challenged mice of the BSA control group were nasally challenged with BSA as a control for unspecific responses to proteins per se. In this group, eosinophil numbers did not differ significantly between transgenic and wild-type animals (Figure 3C). Notably, allergic CCSP-NGF-tg mice of the OVA and the BSA group displayed an elevated level of lymphocyte recruitment (p < 0.001 and p < 0.01) (Figures 3B and 3C). As this enhancement was similar in both groups, lymphocyte recruitment seems to be rather the result of OVA aerosol challenges on Days 27 and 28 than the nasal challenge on Day 35. As a further control, in the nonsensitized PBS controls, eosinophil recruitment to the airways was not detected (Figure 3D).

IgE Production
One possible mechanism through which NGF influences allergic early-phase reaction may be by altering IgE production. Therefore, total and allergen-specific IgE antibody levels were measured in OVA-sensitized mice. In BALB/c mice, anti-NGF treatment had no effect on IgE levels. Although CCSP-NGF-wt and CCSP-NGF-tg mice had lower IgE levels than BALB/c mice, no differences were detectable between CCSP–NGF study groups (Figure 5) . As a control, it was verified that OVA-specific IgE baseline levels were not detectable 1 day before the start of experimental treatment (data not shown).



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  		Figure 5. Levels of total (A) and OVA-specific (B) IgE antibodies in serum samples taken from BALB/c mice pretreated with isotype control or anti-NGF antibodies and CCSP-NGF-wt and CCSP-NGF-tg mice on Day 36 according to Protocol 1. As a control, it was verified that OVA-specific IgE was not detectable on Day -1 (data not shown). The scattergram represents single serum samples and medians of data points from experimental groups. Dashed lines indicate limits of detection (DL). Isotype control, n = 34, anti-NGF, n = 35; CCSP-NGF-wt, n = 21; CCSP-NGF-tg, n = 17; AU, arbitrary units.

 
Airway Reactivity
The effects of anti-NGF and NGF treatment on airway reactivity were tested in BALB/c mice by aerosol challenge with methacholine (Figure 6) . Although methacholine-induced EF50 values did not differ between mice from all OVA-sensitized and -aerosol challenged groups (control IgG, anti-NGF, and NGF), significant airflow reduction between allergic and untreated animals was observed at methacholine concentrations of 75 and 100 mg/minute aerosolization (p < 0.01 and p < 0.001). This demonstrates that the development of methacholine-induced airway hyperreactivity in allergic animals was not affected by treatment with anti-NGF or NGF.



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  		Figure 6. Airway reactivity in BALB/c mice measured in response to inhaled methacholine by head-out body plethysmography on Day 29 according to Protocol 2. Curves represent means ± SD. Isotype control, anti-NGF, and NGF groups display no statistical differences if compared with each other. Short dashed line, asterisk represents NIL. Dashed line, open circle represents isotype control. Solid line, filled circle represents anti-NGF. Solid line, filled square represents NGF. NIL, n = 4; isotype control, n = 21; anti-NGF, n = 22; NGF, n = 20; NIL versus isotype control: ++p < 0.01; +++p < 0.001. NIL versus anti-NGF: ***p < 0.001. NIL versus NGF: °°°p < 0.001.

 
Serotonin Release and Early-Phase Reaction
To assess whether NGF modulates mast cell degranulation and subsequent mediator release, serotonin levels were measured in BALF immediately after allergen challenge on Day 35 according to Protocol 1. Baseline values, obtained from the PBS control groups after intranasal allergen challenge, were 1.45 ± 0.43 ng/ml (mean ± SD) for the CCSP-NGF-wt and 1.10 ± 0.42 ng/ml for the CCSP-NGF-tg groups, indicating higher baseline values in the wild-type mice (data not significant). After normalization of baseline values to 100%, sensitized CCSP-NGF-tg mice intranasally challenged with OVA displayed a significantly higher increase in serotonin release than respective wild-type groups (p < 0.05) (Figure 7) .



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  		Figure 7. Serotonin levels in bronchoalveolar lavage fluids (BALF) taken from CCSP-NGF-wt (open triangles) and CCSP-NGF-tg (filled triangles) mice immediately after last allergen challenge on Day 35 according to Protocol 1. Shown are values from individual mice. Bars represent the median. Serotonin values were expressed in percent of the mean baseline levels. Baseline levels, evaluated in nonsensitized mice, were as follows: wt, 1.45 ± 0.43 ng/ml; tg, 1.10 ± 0.42 ng/ml (not significant). All groups, n = 13; *p < 0.05.

 
Sensory Reactivity
Hyperreactivity caused by capsaicin-induced irritation of sensory neurons is characterized by prolongation of the breathing pause before expiration (TB) (44) and results in the secretion of tachykinins like SP. Therefore, functional alterations in sensory reactivity were measured during inhalation of capsaicin acting specifically via vanilloid receptors, which are in the lung predominantly expressed on sensory neurons. Treatment of OVA-sensitized CCSP-NGF-wt and CCSP-NGF-tg mice with capsaicin 24 hours after last OVA aerosol challenge resulted in a dose-dependent rise in TB, which was markedly increased in transgenic animals at all capsaicin concentrations given (Figure 8A) . To control that, under the conditions used, capsaicin does not influence airway smooth muscle cell constriction, alterations of EF50 in response to capsaicin were determined simultaneously. As expected, no changes in EF50 were inducible (Figure 8B).



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  		Figure 8. Sensory reactivity (A) and airflow limitation (B) in CCSP-NGF-wt (open triangles, dashed lines) and CCSP-NGF-tg (filled triangles, solid lines) mice, respectively, measured in response to inhaled capsaicin by head-out body plethysmography on Day 29 according to Protocol 2. Curves represent means ± SD in percent baseline. CCSP-NGF-wt and CCSP-NGF-tg, n = 24; *p < 0.05; **p < 0.01; ***p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented in this study demonstrate that NGF augments the allergic early-phase reaction in the lung. Furthermore, NGF appears as a promoter of allergic airway inflammation by increasing eosinophil and lymphocyte recruitment into the lungs as well as local IL-5 and IL-4 production after allergen challenge. In addition, neither anti-NGF nor NGF treatment altered methacholine-induced airway hyperreactivity, but constitutive overexpression of NGF in the lung stimulates sensory hyperresponsiveness to inhalation of the nonallergic irritant capsaicin.

The allergic early-phase reaction is directly related to the mediator release from mast cells. NGF exerts its biologic actions via binding to two distinct types of cell surface receptors: the low-affinity receptor p75NTR and the high-affinity receptor TrkA. p75NTR binds to all neurotrophins, whereas the high-affinity receptor TrkA binds specifically to NGF (45, 46). Both receptors were demonstrated to be expressed by mast cells. Mouse mast cells maintained in the presence of IL-3 express mRNA for both TrkA and p75NTR (47), whereas rat mast cells express only functionally TrkA, but not p75NTR (48, 49). In addition, functional TrkA was found to be expressed by human mast cells, including immature mast cells of the human mast cell line 1 (HMC-1) as well as umbilical and lung mast cells (20, 50, 51).

Several lines of evidence indicate that NGF influences rodent mast cell development and function. In neonatal rats, a subcutaneous injection of NGF induced mast cell hyperplasia and hypertrophy in many organs (13, 52, 53), and in patients with asthma, a current study reported that inhalation of allergen at low-dose increased both NGF mRNA production and mast cell numbers in the airways (20). Conversely, mast cell numbers were greatly decreased in rats by antibodies to NGF (53). In the presence of IL-3, NGF promotes colony formation and maturation of bone marrow-derived mouse mast cells in vitro (54). NGF also promotes the survival of rat peritoneal mast cells by suppressing apoptosis (48, 55). Moreover, it was demonstrated that rat, mouse, and human mast cells as well the HMC-1 are a source of NGF themselves (16, 50, 51, 56). Regarding its biologic actions, it is notable that NGF functions as a chemoattractant for rat peritoneal mast cells (57) and can induce mediator release by these cells (49, 58–60). This is in line with our finding that constitutive overexpression of NGF in OVA-sensitized CCSP-NGF-tg mice significantly enhances the release of serotonin in response to allergen provocation. It is also conceivable that NGF may directly or indirectly increase serotonin release by mast cells without changing their number.

Exposure of mouse mast cells to IgE results in the upregulation of high-affinity Fc{epsilon}RI expression and thus increases the ability to release serotonin, IL-6, and IL-4 (61). NGF could be also released via IgE by cultured murine bone marrow–derived mast cells (56). Therefore, enhancement of IgE levels appears as an important mechanism to increase mast cell effector functions. NGF may participate in these processes, as it induces differentiation of activated B cells into immunoglobulin-secreting plasma cells (62–64), and NGF serum levels are related to total IgE antibody titers and severeness of disease in patients with allergic asthma (24). However, we found no differences in serum IgE concentrations between anti-NGF–treated or CCSP-NGF-tg animals versus their corresponding controls, indicating that enhancement of allergic early-phase reaction by NGF is not caused by modulating IgE antibody production.

The actions of NGF on neurons are primarily mediated via the high-affinity TrkA receptor expressed on sympathetic neurons and a subset of neural crest-derived sensory neurons (65, 66). On responsive neurons, NGF promotes survival, axonal outgrowth, and branching; functions as a chemoattractant for growing axons (67, 68); and induces local hyperinnervation in transgenic mice overexpressing NGF by tissue-specific promoters (37, 68, 69). Furthermore, NGF upregulates SP production in sensory neurons (9–12), and as a consequence, Hoyle and colleagues (37) detected substantially elevated levels of SP in the BALF of CCSP-NGF-tg mice with lung-specific NGF overexpression. SP is produced by sensory neurons (70), and its release is induced by a variety of mechanical, thermal, chemical (i.e., capsaicin) and inflammatory stimuli (8). Interestingly, SP on itself is in turn capable of to augmenting bronchopulmonary sensory reflex output (71). Once secreted, SP also exerts a broad range of proinflammatory actions, including mast cell chemoattraction and degranulation, as well as activation of macrophages, lymphocytes, neutrophils, and eosinophils (72). The observed reduction of eosinophil influx as well as local IL-4 and IL-5 production in anti-NGF–treated BALB/c mice and, vice versa, increased recruitment of eosinophils and lymphocytes in CCSP-NGF-tg mice, may be related to NGF-mediated modulation of SP production and release. The late-phase asthmatic reaction is mainly under the control of eosinophils and lymphocytes (2). Therefore, these data could point also to a possible role for NGF in the development of the allergic late-phase reaction.

In addition to these proinflammatory properties of NGF, there is also evidence for antiinflammatory activities of this cytokine. For example, NGF is observed to inhibit monocyte transendothelial migration in a model of experimental autoimmune encephalomyelitis (73) or to reduce calcitonin gene-related peptide (CGRP) gene expression in activated B cells (74). Moreover, a body of literature reported a role of NGF in wound healing. This effect of NGF seems to be predominantly mediated via induction of proliferation of keratinocytes (75) or endothelial cells (76) and induction of fibroblast migration and differentation (77). In this sense, NGF seems also to be involved in tumor growth (78–80).

Mast cells from a variety of distinct tissues are located in close proximity to neurons (81–85). The interactions between nerve and mast cells involve SP operating via its high-affinity neurokinin-1 receptor and inducing mast cell activation (86). Blocking of the neurokinin-1 receptors by the antagonist RP67580 was shown to abolish completely tracheal hyperreactivity in allergic mice (87). CCSP-NGF-tg mice from all experimental groups displayed a more intensive early-phase reaction than the corresponding wild-type groups. This was accompanied by an substantial increase in the reactivity of sensory neurons innervating the lung of CCSP-NGF-tg animals in response to inhaled capsaicin. Furthermore, the CCSP-NGF-tg mice display significantly elevated SP levels (37). Thus, it is likely that the sensory hyperreactivity in response to irritant stimuli enhances the release of SP, which in turn affects local mast cells. Although SP in high doses may cause mast cell degranulation by itself (88) and vanilloid receptors are present on murine mast cells (89), we did not observe airflow limitation (EF50 values) in response to capsaicin-induced sensory hyperreactivity. Vanilloid receptors are also expressed to some extend by some non-neuronal cells such as epithelial linings of both trachea and airways, serous cells of submucosal glands, and mononuclear cells (90, 91). As TB values changed immediately without any alterations of EF50 values, it could be concluded that the enhanced susceptibility of CCSP-NGF-tg animals to capsaicin is rather the result of sensory hyperinnervation of the airways observed in these mice (37) than by a possible indirect action via products secreted by non-neuronal cells after capsaicin activation. For this reason, a direct or SP-mediated capsaicin-induced activation of mast cell release seems to be not relevant in the in vivo model employed. Furthermore, neither anti-NGF nor NGF treatment altered methacholine-induced airway hyperreactivity (EF50 values), indicating that NGF has no effect on the susceptability of airway smooth muscle cells. However, SP in low doses can prime mast cells for activation by subthreshold concentrations of other stimuli (92). Therefore, a priming effect of sensory neuron-released SP on mast cells may be an additional indirect mechanism enhancing allergic early-phase reaction and may serve as a link between allergen-independent airway hyperreactivity and allergen-induced asthmatic reaction.

Most of the proposed pathways by which NGF may enhance allergic early-phase reaction rest on the paradigm that IgE-activated mast cells are the pivotal inducers of the allergic early-phase reaction and that we measure elevated serotonin release in CCSP-NGF–transgenic mice. In the mouse, the central role for the mast cells in airway inflammation was recently evaluated (93, 94). Alternatively, a current study demonstrated early-phase reaction in mast cell deficient mice (95). These conflicting observations suggest that, in mice, strain-specific differences may exist or that the asthma model employed could be a modulating factor. However, the contribution of mast cells to the development of allergic airway reaction remains to be further characterized.

In conclusion, our data demonstrate that NGF augments allergic early-phase reaction via increased release of mast cell mediators. This is strongly supported by the work of de Vries and colleagues (35) demonstrating reduction of allergen-induced bronchoconstriction in guinea pigs. Moreover, during acute allergic airway inflammation, NGF augments the local production of the Th2 cytokines IL-4 and IL-5 as well as the recruitment of eosinophils and lymphocytes into the inflamed tissue. Finally, chronic exposure to NGF results in hyperreactivity of sensory neurons. Therefore, concerning immune cells and neurons in the employed murine model of asthma, NGF appears as a mediator in the interactions between the immune and the nervous system in states of acute and chronic allergic airway inflammation.


   FOOTNOTES
 
Supported by the Volkswagen Foundation.

Received in original form February 21, 2002; accepted in final form June 24, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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