Pulmonary distribution, regulation, and functional role of Trk receptors in a murine model of asthma

Transcription

1 Pulmonary distribution, regulation, and functional role of Trk receptors in a murine model of asthma Christina Nassenstein, MD, PhD, a * David Dawbarn, PhD, b * Kenneth Pollock, PhD, c Shelley Jane Allen, PhD, b Veit Johannes Erpenbeck, MD, PhD, a Emma Spies, a Norbert Krug, MD, a and Armin Braun, PhD a Hannover, Germany, and Bristol and Surrey, United Kingdom Background: Neurotrophins have been implicated in the pathogenesis of asthma because of their ability to promote hyperreactivity of sensory neurons and to induce airway inflammation. Hyperreactivity of sensory nerves is one key mechanism of airway hyperreactivity that is defined as an abnormal reactivity of the airways to unspecific stimuli, such as cold air and cigarette smoke. Neurotrophins use a dualreceptor system consisting of Trk receptor tyrosine kinases and the structurally unrelated p75 neurotrophin receptor. Objective: The aim of this study was to characterize the distribution, allergen-dependent regulation, and functional relevance of the Trk receptors in allergic asthma. Methods: BALB/c mice were sensitized to ovalbumin. After provocation with ovalbumin or vehicle aerosol, respectively, Trk receptor expression was analyzed in lung tissue by means of fluorescence microscopy and quantitative RT-PCR. To assess the functional relevance of Trk receptors in asthma, we tested the effects of the intranasally administered pan-trk receptor decoy REN1826. Allergic airway inflammation was quantified and lung function was measured by using head-out body plethysmography. Results: Trk receptors were expressed in neurons, airway smooth muscle cells, and cells of the inflammatory infiltrate surrounding the bronchi and upregulated after allergen challenge. Local application of REN1826 reduced IL-4 and IL-5 cytokine levels but had no effect on IL-13 levels or the cellular composition of bronchoalveolar lavage fluid cells. Furthermore, REN1826 decreased broncho-obstruction in response to sensory stimuli, indicating a diminished hyperreactivity of From a the Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover; b the Henry Wellcome Laboratories, University of Bristol; and c ReNeuron Ltd, Guildford, Surrey. *These authors contributed equally to this study. Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 587, B4) and by direct financial support from ReNeuron Ltd and the Fraunhofer Society. Disclosure of potential conflict of interest: C. Nassenstein has received grant support from the German Research Foundation. D. Dawbarn has received grant support from Wellcome Trust and ReNeuron PLC. S. J. Allen has received grant support from Wellcome Trust and ReNeuron PLC. K. Pollock is employed by ReNeuron Ltd. The rest of the authors have declared that they have no conflict of interest. Received for publication January 25, 2006; revised March 22, 2006; accepted for publication April 24, Available online July 5, Reprint requests: Armin Braun, PhD, Immunology and Allergology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, Hannover, Germany. braun@item.fraunhofer.de /$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi: /j.jaci sensory nerves, but did not influence airway smooth muscle hyperreactivity in response to methacholine. Conclusion: These results emphasize the important role of Trk receptor signaling in the development of asthma. Clinical implications: Our data indicate that blocking of Trk receptor signaling might reduce asthma symptoms. (J Allergy Clin Immunol 2006;118: ) Key words: Sensory nerves, airway hyperreactivity, airway inflammation, reflex bronchospasm, capsaicin Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. In susceptible individuals this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and coughing. The inflammation also causes an associated increase in the existing bronchial hyperreactivity to a variety of stimuli. 1 On the basis of this definition, research activities of recent years were focused on discovering the immunologic events leading to allergic airway inflammation. However, it becomes clear that mediators of induce dysfunction in sensory nerves characterized by a long-lasting change of activity that becomes independent from inflammation during chronification. Growing evidence suggests that neurotrophins (NTs), such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, and NT-4, might be important mediators for plasticity of sensory nerves in several disorders associated with inflammation, including allergic asthma. 2,3 Thus local application of NGF and BDNF induces airway hyperreactivity by contributing to an altered neuronal control of airway smooth muscle cells. 3,4 Antagonism of NGF inhibited allergeninduced allergic early-phase reactions and suppressed allergic inflammation in a murine model of asthma, whereas local treatment with NGF increased the influx of eosinophils into the lung. 5 NTs influence either directly by promoting survival and activation 6 and augmenting cytokine synthesis 2 or indirectly through amplification of proinflammatory neuropeptide synthesis in sensory neurons. 7 NTs exert individual cellular effects by means of interaction with 2 structurally unrelated receptors, the p75 NT receptor (p75ntr), which belongs to the TNF receptor superfamily, and the Trk receptor tyrosine kinases. Although p75ntr is capable of binding to all 597

2 598 Nassenstein et al J ALLERGY CLIN IMMUNOL SEPTEMBER 2006 Abbreviations used BALF: Bronchoalveolar lavage fluid BDNF: Brain-derived neurotrophic factor EF 50 : Tidal midexpiratory flow HBP: Head-out body plethysmography MCh: Methacholine NGF: Nerve growth factor NT: Neurotrophin OVA: Ovalbumin P75NTR: p75 neurotrophin receptor PC: Provocative concentration PD: Provocative dose REN1826: Pan-Trk receptor decoy (soluble TrkAd5 of a TrkA splice variant) Tb: Time of braking TBS: Tris-buffered saline TRPV1: Transient receptor potential vanilloid 1 mature NTs with equivalent affinity but unique kinetics, the Trk receptors exhibit ligand selectivity. Thus TrkA has been identified as the preferred receptor for NGF, TrkB for both BDNF and NT-4, and TrkC for NT-3. p75ntr and Trk receptors can form heteromeric complexes when both receptors are coexpressed, 8 resulting in an altered affinity and specificity of the Trk-NT interaction through conformational changes in Trk receptor proteins. 9 This is functionally associated with a prolonged survival after binding of NTs to neurons, 10 as well as to immune cells. 11 Under conditions in which concomitant Trk signaling is impaired, p75ntr can promote cell death in a variety of cell types, 12 whereas survival predominantly reflects Trk activation. 13 Because mechanisms involved in the regulation of sensory nerve and immune cell function play a central role in the pathogenesis of allergic airway diseases, we hypothesized that Trk receptors might play an important role in asthma. To address this question, we analyzed Trk receptor expression and its allergen-dependent regulation in murine lungs, constructed a novel soluble Trk receptor fragment (pan-trk receptor decoy [REN1826]) capable of binding all NTs with a high affinity, and investigated its effects after local application in a well-established mouse model of allergic asthma. REN1826 vector construction, protein expression, and purification To investigate the functional role of all Trk receptors, we generated REN1826. Details are provided in the Online Repository at Treatment protocol All animals were OVA sensitized, as previously described. 5 Aerosol challenges with either PBS (negative controls) or 1% OVA-PBS (positive controls and treatment groups) were performed on days 26, 27, and 35. Intranasal treatment with vehicle (negative and positive control groups) or 50, 200, or 800 ng of REN1826 per animal (treatment groups), respectively, was performed before each allergen challenge and consecutively every 2 days until day Lung function was assessed by using head-out body plethysmography (HBP) in response to capsaicin at days 28 and 36 and in response to methacholine (MCh) at day 29. Bronchoalveolar lavage was performed at day 36, as previously described (see Fig E1 in the Online Repository at 2 Quantification and localization of Trk receptor expression Trk receptor mrna expression was assessed by using real-time RT-PCR in murine lung tissue after exsanguination. To localize Trk receptor expression in the murine lung, we performed immunohistochemistry. Detailed protocols including primer sequences (Table E1) are provided in the Online Repository at Assessment of lung function Airway hyperreactivity was measured in response to inhaled capsaicin and MCh by using HBP in conscious and spontaneously breathing animals, as previously described. 14 Baseline measurements were followed by aerosol provocation with vehicle (10% EtOH in PBS for capsaicin and PBS for MCh, respectively) and increasing doses of capsaicin or MCh generated by a Pari Master aerosol generator (Pari-Werke, Starnberg, Germany). Changes of the time of braking (Tb) in response to capsaicin were used as a surrogate marker to assess a hyperreactivity of afferent sensory nerves of the airways. 3,14 The tidal midexpiratory airflow (EF 50 ; in milliliters per second) was analyzed to assess broncho-obstruction after capsaicin or MCh challenge, respectively, to evaluate the role of Trk receptors for reflex bronchospasm or smooth muscle constriction, respectively. The provocative concentration (PC; capsaicin [in micrograms per milliliter]) or the provocative dose (PD; MCh [in micrograms]) causing a decrease in EF 50 to 25% (capsaicin) or 50% (MCh) of the vehicle value was calculated after extrapolation of the dose-response curves and is expressed as PC 25 EF 50 (capsaicin) or PD 50 EF 50 (MCh). Details are provided in the Online Repository at www. jacionline.org. METHODS Animals Six- to 8-week-old female BALB/c mice were obtained from Charles River, Sulzfeld, Germany. They were fed with ovalbumin (OVA) free laboratory food and tap water ad libitum and held in a regular 12-hour dark-light cycle at a temperature of 22 C. All animal care practices and experimental procedures were performed in concordance with the German animal protection law under a protocol approved by the appropriate governmental authority (Bezirksregierung Hannover). Differential cell count Cytospin preparations from bronchoalveolar lavage fluid (BALF) cells were stained according to the Pappenheim standard protocol. Details are provided in the Online Repository at Assessment of cytokine levels in BALF The measurement of cytokines in BALF was performed in duplicate according to the manufacturer s recommendation (DuoSet ELISA Development Kit; R&D Systems, Wiesbaden, Germany). The detection limit was 12.5 pg/ml for all cytokines.

3 J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3 Nassenstein et al 599 FIG 1. Trk receptor mrna expression in murine lung tissue 24 hours after allergen challenge. A, End-point analysis revealed the expected product lengths. B, Quantification of Trk mrna expression is shown (n 5 8 per group). PBGD, Porphobilinogen deaminase (housekeeping gene); RNA, RNA control; POS, positive control group; NEG, negative control group. **P <.01. Statistical analysis Statistical analysis was performed with GraphPad Prism Version 4.0 (GraphPad Software, San Diego, Calif). Details are provided in the Online Repository at RESULTS Allergen-dependent regulation of Trk mrna expression in the murine lung To investigate Trk receptor expression in lung tissue and to test its potential allergen-dependent regulation in the lung, we performed quantitative RT-PCR in lung tissue obtained from the positive control group (OVA-sensitized and OVA-challenged animals, intranasal treatment with vehicle) or negative control group (OVAsensitized and PBS-challenged animals, intranasal treatment with vehicle), respectively. No difference in TrkA mrna expression levels was observed, whereas TrkB and TrkC mrna expression levels in the lung tissue were upregulated in the positive control group (Fig 1). Localization of Trk receptor expression in the murine lung Cellular distribution of Trk receptors in the murine lung was investigated by means of immunohistochemistry. Immunoreactivity for all Trk receptors was detected in large bundles of nerve fibers, as well as on terminal subepithelial varicosities, most likely reflecting sensory nerves adjacent to the airways. Furthermore, TrkA and TrkC expression was observed in airway smooth muscle cells. The Trk expression pattern in neuronal cells and airway smooth muscle cells did not differ between the animals of the positive and negative control groups (data not shown). In addition, expression of all Trks was detected in inflammatory infiltrates surrounding the bronchi and blood vessels. Trk expression in immune cells was detected exclusively in the lungs of the positive control group (Fig 2). Further analysis revealed that TrkA 1, TrkB 1, and TrkC 1 cells of the inflammatory infiltrate belonged to different immune cell subtypes, including T cells, B cells, and antigen-presenting cells. In contrast, none of these receptors was expressed by eosinophils (Table I and Fig E2 in the Online Repository at REN1826 protein expression To study the functional relevance of Trk receptors, we generated REN1826. The molecular weight of purified recombinant REN1826 was tested by means of matrixassisted laser desorption/ionization time-of-flight and showed the correct predicted molecular weight, including the initiating methionine (data not shown). Endotoxin testing revealed that the preparation was endotoxin free. Hyperreactivity of afferent sensory nerves in response to capsaicin Tb was determined by means of HBP on days 28 and 36 in response to capsaicin to analyze the functional consequences of Trk receptors on afferent sensory nerves. Tb baseline values did not differ among the different groups and days of assessment. As described before, 14 the aerosol

4 600 Nassenstein et al J ALLERGY CLIN IMMUNOL SEPTEMBER 2006 FIG 2. Immunohistochemical localization of Trk receptors in the murine lungs. Left row, Trk receptor expression in the trachea. Right row, Trk receptor expression in cells of the inflammatory infiltrate surrounding the bronchi. Inserts, Corresponding isotype controls. White bars represent 50 mm. The figure shows representative results from one of 4 independent experiments. N, Nerves; SM, smooth muscle; E, epithelium; L, airway lumen. TABLE I. Expression and distribution of Trk receptors in murine lung tissue TrkA TrkB TrkC Epithelium Nerves Airway smooth muscle CD45 1 immune cells 1* 1* 1* CD3 1 T lymphocytes 1* 2 1* CD19 1 B lymphocytes 1* 1* 1* MHC II 1 antigen-presenting 1* 1* 1* cells MBP 1 eosinophils MBP, Major basic protein. *Expression can be observed only after allergen provocation. of the vehicle control caused a slight prolongation of Tb in all investigated groups, which was further enhanced by increasing doses of capsaicin aerosol. Hyperreactivity in sensory nerves, as indicated by a markedly prolonged Tb in response to capsaicin aerosol, was observed in the positive control groups when compared with the negative control groups. Intranasal treatment with REN1826 in OVA-sensitized and OVA-challenged animals reduced hyperreactivity of sensory nerves in a dose-dependent manner. Contrary to day 28, hyperreactivity was also absent in those animals that received 50 ng of REN1826 when measurements were repeated on day 36 to evaluate a sustained effect of repeatedly administered REN1826 after iterated allergen challenge (Fig 3). Hyperreactivity of sensory nerves in response to capsaicin was measured

5 J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3 Nassenstein et al 601 FIG 3. Hyperreactivity of sensory nerves to capsaicin. A, Original monitor picture during capsaicin aerosol challenge. B, Dose-response curves at days 28 and 36. C, Exemplary statistical analysis for Tb during aerosol challenge with 100 mg/ml capsaicin (n 5 16 per group). *P <.05; **P <.01. neg, Negative control group; pos, positive control group. in an independent experiment after intratracheal treatment with vehicle (positive and negative control groups) or 200 ng of REN1826 to confirm that these results are related to pulmonary deposition of intranasally applied REN1826. Changes in Tb were similar to those obtained after intranasal treatment with REN1826 (data not shown). Broncho-obstruction in response to capsaicin The EF 50 was measured and the PC 25 EF 50 (in micrograms per milliliter) values were calculated to assess broncho-obstruction in response to selective activation of afferent sensory nerve fibers; low values correspond to an increased broncho-obstruction. No difference in EF 50 baseline values and EF 50 values after inhalation of the vehicle control was seen between the investigated groups. The PC 25 EF 50 values were significant lower in the positive control group compared with those in the negative control group. Intranasal treatment with REN1826 increased the PC 25 EF 50 in a dose-dependent manner both on day 28 and on day 36 (Fig 4). Similar results were observed after intratracheal application of REN1826 (data not shown). Broncho-obstruction in response to MCh MCh aerosol provocation was performed on day 29 by using HBP to investigate the contribution of Trk receptors to the development of airway smooth muscle cell hyperreactivity. No difference in EF 50 baseline values and EF 50 values after inhalation of vehicle control was seen among the investigated groups (data not shown). Hyperreactivity of airway smooth muscle cells to MCh was observed in the positive control group, indicated by a significantly lower PD 50 EF 50 compared with that of the negative control group. This response was not altered by intranasal treatment with REN1826 (Fig 5) or after intratracheal application (data not shown).

6 602 Nassenstein et al J ALLERGY CLIN IMMUNOL SEPTEMBER 2006 FIG 4. Broncho-obstruction to inhaled capsaicin. The PC (in micrograms per milliliter) of capsaicin causing a 25% decrease in EF 50 of the appropriate vehicle control was calculated (PC 25 EF 50 [in micrograms per milliliter]; n 5 16 per group). *P <.05; **P <.01. neg, Negative control group; pos, positive control group. DISCUSSION FIG 5. Broncho-obstruction to inhaled MCh. The PD (in micrograms) of MCh causing a 50% decrease in EF 50 of the appropriate vehicle control was calculated (PD 50 EF 50 [in micrograms]; n 5 16 per group). *P <.05. neg, Negative control group; pos, positive control group. Airway inflammation To investigate the effect of Trk receptors on airway inflammation, we assessed cytokine levels, as well as differential cell counts, in the BALF. As expected, marked increases in T H 2 cytokine levels (IL-4, IL-5, and IL-13) and the numbers of eosinophils, lymphocytes, and neutrophils were observed in the positive control animals when compared with the negative control animals. IFN-g levels of all groups were under the detection limit of the appropriate ELISA (data not shown). IL-4 and IL-5 levels were dose-dependently reduced by intranasal treatment with REN1826, whereas IL-13 levels and the numbers of infiltrating inflammatory cells, including eosinophils and lymphocytes, remained unaffected (Fig 6 and Table II). There were no differences in BALF recovery between the different groups. In this study the expression, distribution, and function of Trk receptors in the murine airways were analyzed. Trk receptors were expressed in airway smooth muscle cells and nerve fibers of healthy control mice and additionally in cells of the inflammatory infiltrate surrounding the bronchi and blood vessels of allergen-challenged animals. These results were associated with a marked increase in mrna levels of TrkB and TrkC in the inflamed lung tissue. Our data are therefore consistent with the results that were observed in the human lung sections showing Trk receptor expression in various cells types in healthy individuals 15 and in infiltrating cells after allergen provocation in patients with asthma. 6 The functional relevance of Trk receptor expression in asthma development was demonstrated by inhibition of Trk receptor binding with REN1826 before repetitive allergen challenges in a mouse model of allergic asthma. Our results clearly indicate that Trk receptors are involved in hyperreactivity of afferent sensory nerve fibers, reflex broncho-obstruction to capsaicin, and airway inflammation. Responsiveness of bronchial and pulmonary sensory nerve fibers was measured on the basis of prolongation of Tb as a central reflex mechanism after stimulation with capsaicin. Capsaicin is an exogenous agonist of the transient receptor potential vanilloid 1 (TRPV1) that is expressed almost selectively in C and Ad nociceptive fibers (reviewed in Szallasi and Blumberg 16 ). Although capsaicin was used as a surrogate to assess functional changes in sensory nerve fibers, it is likely that responses to stimulation with capsaicin reflect the responses to endogenous TRPV1 ligands. Endogenous ligands of this receptor, which might also play a role in allergic asthma, are acids and lipid mediators, such as anandamide and lipoxygenase products. In addition, stimulation of G-coupled receptors, such as the bradykinin B2 receptor, exert their effects, probably through activation of TRPV1 (reviewed in Suh and Oh 17 ).

7 J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3 Nassenstein et al 603 NTs contribute to the onset of an enhanced sensitivity of afferent sensory nerve fibers to capsaicin, 3,5 for example by potentiating the response to TRPV1 agonists. 18 The results of our study indicate that Trk receptor signaling is important in the development of hyperreactive sensory nerves in allergic asthma. Therefore it might be likely that the pathways responsible for transducing the Trk-dependent sensitization of TRPV1 in asthma are comparable with those in thermal hyperalgesia. NGF-induced rapid sensitization of nociceptive sensory neurons to painful thermal stimuli depends on activation of second messengers of the Trk cascade, including phosphatidylinositol-39-kinase, protein kinase C, and calcium-calmodulin dependent protein kinase II. 19 However, because p75ntr is also involved in the increased excitability of sensory neurons, as has been demonstrated both in vitro 20 and in vivo in a mouse model of allergic asthma, 14 our data support the hypothesis that NTs probably bind to heteromeric complexes consisting of Trk and p75ntr 8,9 on sensory nerves innervating the airways. This assumption has to be confirmed experimentally. Subsequent activation of sensitized neurons (eg, by TRPV1 agonists, 21 ozone, exercise, and a variety of inflammatory mediators [reviewed in Joos et al 22 ]) could increase broncho-obstruction by both central reflex pathways facilitating efferent parasympathetic nerve activity and by local or axon reflexes characterized by release of neuropeptides from sensory nerve endings We have shown in our study that capsaicin-induced broncho-obstruction is also inhibited by local application of a pan-trk receptor decoy, indicating that NT signaling through Trk receptors is involved in reflex bronchospasm. NTs might influence reflex bronchospasm through upregulation of neuropeptide synthesis in ganglion neurons 26 and increase of the number of neuropeptide-immunoreactive nodose ganglion neurons projecting to the airways during. 27,28 After antidromic transport, neuropeptides are then stored in peripheral endings. Stimulation of sensory nerve endings in the respiratory tract results in a local axon reflex and finally in neuropeptide release, 29 which can then act on a number of effector cells, including airway smooth muscle cells. 30 The functional role of neuropeptides in NT-induced airway hyperreactivity has been emphasized by inhibiting broncho-obstruction after application of a neurokinin 1 receptor antagonist. 31 These mechanisms leading to broncho-obstruction can be facilitated by a heightened parasympathetic vagal reflex activity on stimulation of afferent endings of sensory nerves. 32,33 Although this assumption is compatible with our observation that blocking of Trk receptor signaling reduces hyperreactivity of sensory neurons in the airways and in the following broncho-obstruction, it could not be ruled out that Trk receptors might also directly influence cholinergic neurons and airway smooth muscle cells, in which we could observe TrkA and TrkC receptor immunoreactivity. Lung function was assessed in response to MCh to investigate the role of Trk receptors on airway smooth muscle cells. We saw an enhanced responsiveness FIG 6. Cytokine levels in BALF. Cytokine levels were measured in BALF 24 hours after last aerosol challenge. Levels of IL-4 (A), IL-5 (B), and IL-13 (C) were significantly increased in the positive control group (pos) compared with those in the negative control group (neg). Treatment with REN1826 partly reduced IL-4 and IL-5 levels (n 5 16 per group). *P <.05; **P <.01. of OVA-sensitized and OVA aerosol challenged animals to MCh, but this effect was not altered by treatment with REN1826, indicating that Trk receptor signaling in airway smooth muscle cells does not contribute to reflex bronchoobstruction after stimulation of afferent sensory nerves.

8 604 Nassenstein et al J ALLERGY CLIN IMMUNOL SEPTEMBER 2006 TABLE II. Distribution of leukocyte subpopulations in BALF Recovery (ml) Macrophages (cells ) Lymphocytes (cells ) Eosinophils (cells ) Neutrophils (cells ) Negative control ** ** ** Positive control ng of REN ng of REN ng of REN Cell numbers are given in absolute numbers (means 6 SEM; n 5 16 per group). **P <.01 compared with positive control. Because of the fact that disruption of p75ntr signaling also had no effect on broncho-obstruction in response to MCh, 14 we propose that NTs and Trk receptors in vivo are not involved in the development of hyperreactivity of airway smooth muscle cells, as could be suggested by in vitro studies. 34 The influence of Trk receptors on cholinergic nerves remains to be investigated. Although it has been suggested that NTs exert proinflammatory properties contributing to allergic airway inflammation (reviewed in Nassenstein et al 35 ), the role of Trk receptors has not yet been investigated. In this study we could demonstrate Trk receptor immunoreactivity in immune cells of the airways exclusively after allergen challenge. This observation was accompanied by increased mrna levels for TrkB and TrkC in the inflamed lung, indicating that Trk receptors might be involved in mediating proinflammatory properties of NTs. NTs are known to promote lymphocyte development and to increase neuropeptide production. In addition, they modulate cytokine synthesis and might play a critical role in regulating the T H 1/T H 2 balance (reviewed in Nassenstein et al 35 ). The relevance of the latter hypothesis has been shown in a mouse model of allergic asthma. Mononuclear cells of OVA-sensitized animals responded to exogenously added NGF with a dose-dependent increase in T H 2 cytokine production, whereas synthesis of IFN-g remained unaffected. 2 Our data indicate that Trk receptors, in particular TrkA and TrkC, are involved in regulation of cytokine synthesis in vivo, as indicated by decreased IL-4 and IL-5 levels but sustained IFN-g levels after local application of REN1826 in our mouse model of asthma. Because previous studies revealed that airway inflammation in asthma is also p75ntr dependent, 14 we suggest that NTs might bind to heteromeric complexes consisting of Trk and p75ntr in immune cells. 8,9 Although it has been demonstrated that NTs contribute to airway eosinophilia in a mouse model of allergic asthma, 2 probably through direct interaction with those cells, 6 antagonization of Trk-mediated signals had no influence on cell infiltration in our study. Therefore we suggest that eosinophilia in our mouse model is to a large extent dependent on p75ntr, but not on Trk, expression. 14 Nevertheless, there is an ongoing discussion about functional differences of eosinophils in mice and human subjects. 36 Therefore the functional role of Trk receptors in human eosinophils remains to be investigated. Because Trk receptors are expressed in immune cells and several in vitro studies indicate that NTs can directly affect immune cell function, can be complimentarily modulated by Trk-dependent activation of sensory nerves. Direct evidence for this so-called neurogenic inflammation in the airways characterized by enhanced neuropeptide release of sensory neurons came from Quarcoo et al. 37 In this study application of a dual neurokinin 1 neurokinin 2 antagonist in NGF-transgenic mice partly inhibited airway inflammation. However, the involvement of neurogenic inflammation in human subjects is still uncertain because initial clinical studies with strategies to block neurogenic inflammation have not been encouraging. 38 The observations of our study indicate that Trk receptors might play an important role in the development of hyperreactivity of sensory nerves, leading to reflex bronchospasm in patients with asthma. The latter is probably due to an increased local release of neuropeptides and facilitation of cholinergic neurotransmission. In contrast, Trk receptor signaling has no direct role on the hyperreactivity of airway smooth muscle cells but might play an important role in allergic airway inflammation. On the basis of these results, it becomes more and more evident that NT antagonism (eg, with a Trk receptor antagonist, such as REN1826) might be effective in inhibiting hyperreactivity of sensory nerves that could be responsible for many symptoms of asthma, including broncho-obstruction after inhalation of smoke or cold air and exercise. We thank Drs Nancy A. Lee and James J. Lee (Mayo Clinic Scottsdale) for providing the rat anti-mouse major basic protein antibody. We also gratefully acknowledge the statistical advice of Professor Hartmut Hecker, Institute Biometry, University Medical School, Hannover, Germany, and the excellent technical assistance of Sabine Schild and Olaf Macke. REFERENCES 1. National Institutes of Health. Global initiative for asthma. Natl Heart Lung Blood Inst Publ 1995; Publication no Braun A, Appel E, Baruch R, Herz U, Botchkarev V, Paus R, et al. Role of nerve growth factor in a mouse model of allergic airway inflammation and asthma. Eur J Immunol 1998;28: Braun A, Lommatzsch M, Neuhaus-Steinmetz U, Quarcoo D, Glaab T, McGregor GP, et al. Brain-derived neurotrophic factor (BDNF)

9 J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3 Nassenstein et al 605 contributes to neuronal dysfunction in a model of allergic airway inflammation. Br J Pharmacol 2004;141: Braun A, Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, Hoyle G, Renz H. Nerve growth factor induces airway hyperresponsiveness in mice. Int Arch Allergy Immunol 2001;124: Path G, Braun A, Meents N, Kerzel S, Quarcoo D, Raap U, et al. Augmentation of allergic early-phase reaction by nerve growth factor. Am J Respir Crit Care Med 2002;166: Nassenstein C, Braun A, Erpenbeck VJ, Lommatzsch M, Schmidt S, Krug N, et al. The neurotrophins nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 are survival and activation factors for eosinophils in patients with allergic bronchial asthma. J Exp Med 2003;198: Goedert M, Stoeckel K, Otten U. Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons. Proc Natl Acad Sci U S A 1981;78: Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors trk and p75ntr. EMBO J 1999; 18: Esposito D, Patel P, Stephens RM, Perez P, Chao MV, Kaplan DR, et al. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor. J Biol Chem 2001;276: Lee KF, Davies AM, Jaenisch R. p75-deficient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF. Development 1994;120: Caroleo MC, Costa N, Bracci-Laudiero L, Aloe L. Human monocyte/ macrophages activate by exposure to LPS overexpress NGF and NGF receptors. J Neuroimmunol 2001;113: Dechant G. Molecular interactions between neurotrophin receptors. Cell Tissue Res 2001;305: Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 2001;24: Kerzel S, Path G, Nockher WA, Quarcoo D, Raap U, Groneberg DA, et al. Pan-neurotrophin receptor p75 contributes to neuronal hyperreactivity and airway inflammation in a murine model of experimental asthma. Am J Respir Cell Mol Biol 2003;28: Ricci A, Felici L, Mariotta S, Mannino F, Schmid G, Terzano C, et al. Neurotrophin and neurotrophin receptor protein expression in the human lung. Am J Respir Cell Mol Biol 2004;30: Szallasi A, Blumberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 1999;51: Suh YG, Oh U. Activation and activators of TRPV1 and their pharmaceutical implication. Curr Pharm Des 2005;11: Shu X, Mendell LM. Nerve growth factor acutely sensitizes the response of adult rat sensory neurons to capsaicin. Neurosci Lett 1999;274: Bonnington JK, McNaughton PA. Signalling pathways involved in the sensitisation of mouse nociceptive neurones by nerve growth factor. J Physiol 2003;551(suppl):S Zhang YH, Nicol GD. NGF-mediated sensitization of the excitability of rat sensory neurons is prevented by a blocking antibody to the p75 neurotrophin receptor. Neurosci Lett 2004;366: Saria A, Theodorsson-Norheim E, Gamse R, Lundberg JM. Release of substance P- and substance K-like immunoreactivities from the isolated perfused guinea-pig lung. Eur J Pharmacol 1984;106: Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000;55: Szolcsanyi J, Bartho L. Capsaicin-sensitive non-cholinergic excitatory innervation of the guinea-pig tracheobronchial smooth muscle. Neurosci Lett 1982;34: Mazzone SB, Canning BJ. Synergistic interactions between airway afferent nerve subtypes mediating reflex bronchospasm in guinea pigs. Am J Physiol Regul Integr Comp Physiol 2002;283:R Fuller RW, Dixon CM, Barnes PJ. Bronchoconstrictor response to inhaled capsaicin in humans. J Appl Physiol 1985;58: Lindsay RM, Harmar AJ. Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature 1989;337: Hunter DD, Myers AC, Undem BJ. Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons. Am J Respir Crit Care Med 2000;161: Fischer A, McGregor GP, Saria A, Philippin B, Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation. J Clin Invest 1996;98: Barnes PJ. Asthma as an axon reflex. Lancet 1986;1: Lundberg JM, Martling CR, Saria A. Substance P and capsaicininduced contraction of human bronchi. Acta Physiol Scand 1983; 119: de Vries A, Dessing MC, Engels F, Henricks PA, Nijkamp FP. Nerve growth factor induces a neurokinin-1 receptor-mediated airway hyperresponsiveness in guinea pigs. Am J Respir Crit Care Med 1999;159: Barnes PJ. Neural mechanisms in asthma. Br Med Bull 1992;48: Hahn HL. Role of the parasympathetic nervous system and of cholinergic mechanisms in bronchial hyperreactivity. Bull Eur Physiopathol Respir 1986;22(suppl 7): Bachar O, Adner M, Uddman R, Cardell LO. Nerve growth factor enhances cholinergic innervation and contractile response to electric field stimulation in a murine in vitro model of chronic asthma. Clin Exp Allergy 2004;34: Nassenstein C, Kerzel S, Braun A. Neurotrophins and neurotrophin receptors in allergic asthma. Prog Brain Res 2004;146: Malm-Erjefalt M, Persson CG, Erjefalt JS. Degranulation status of airway tissue eosinophils in mouse models of allergic airway inflammation. Am J Respir Cell Mol Biol 2001;24: Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, Schierhorn K, Hoyle GW, Renz H, et al. Nerve growth factor induces increased airway inflammation via a neuropeptide-dependent mechanism in a transgenic animal model of allergic airway inflammation. Clin Exp Allergy 2004; 34: Barnes PJ. Neurogenic inflammation in the airways. Respir Physiol 2001; 125: