INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the airways of mammals, a network of capsaicin-sensitive sensory nerve fibers is present, containing various neuropeptide transmitters, including the tachykinins substance P (SP) and neurokinin A (NKA) (1, 2). Stimulation of the sensory nerves evokes the release of these tachykinins (3), thereby eliciting several airway responses collectively referred to as "neurogenic inflammation" (4). These responses include airway smooth muscle contraction, mucus secretion, microvascular leakage, vasodilation and recruitment and activation of inflammatory cells, and are mediated predominantly by tachykinin NK₁ and NK₂ receptors (for recent reviews see References 5 and 6). Thus, NK₂ receptors, which are preferentially activated by NKA, appear to be mainly involved in bronchoconstriction, whereas NK₁ receptors, preferentially activated by SP, are mainly involved in mucus secretion, microvascular leakage, vasodilation, and inflammatory cell responses (5, 6).

Because all of these responses are cardinal features of allergic asthma, mechanisms underlying neurogenic inflammation may be involved in the pathophysiology of allergic asthma (4). This hypothesis is supported by various observations in asthmatic patients. Thus, immunohistochemistry revealed evidence of an increase in SP-containing nerves in these patients (7), whereas enhanced levels of SP were observed in bronchoalveolar lavage (BAL) fluid (8) and in induced sputum (9). In addition, an enhanced expression of mRNA for the NK₁ receptor (10) as well as the NK₂ receptor (11) has been observed in lung tissue from asthmatic patients. As a possible consequence, both SP and NKA were shown to cause enhanced bronchoconstriction in asthmatics (12). However, SP, but not NKA, induced airway hyperreactivity (AHR) to methacholine in these patients (13, 14). The latter observation indicates that NK₁ receptor stimulation by SP may be important in the development of AHR, presumably by its potent inflammatory effects.

Direct evidence for the involvement of endogenous tachykinins in human allergic airway disease has thus far not been obtained. In guinea pigs, some controversy exists about the contribution of tachykinins to allergen-induced bronchoconstriction. Thus, sensory nerve depletion by chronic capsaicin treatment or administration of the selective nonpeptide NK₂ receptor antagonist SR48968 did not prevent the acute allergic asthmatic reaction in some studies (15), whereas positive results were reported by others with sensory nerve depletion, and with SR48968 or the nonpeptide dual NK₁/NK₂ receptor antagonist MDL105,212 (18). In the same species, several lines of evidence indicate that endogenous tachykinins released from sensory nerves may be involved in the development of AHR. Thus, it was demonstrated that capsaicin-induced release of endogenous sensory neuropeptides or exogenous SP enhanced airway responsiveness to various contractile agonists (22). Conversely, chronic treatment with high doses of capsaicin eliminated airway hyperreactivity to methacholine or histamine induced by acute capsaicin (22), platelet activating factor (27), and ovalbumin (OA) (18). In addition, the dual NK₁/NK₂ receptor antagonist MDL105,212 (21) as well as the NK₂ antagonist SR48968 (28) reduced the allergen- induced AHR at 24 h and 48 h after allergen challenge, respectively; however, the selective nonpeptide NK₁ receptor antagonist SR140333 appeared to be without effect at 48 h after the challenge (28).

The specific role of NK₁ receptors in allergen-induced early (EAR) and late (LAR) asthmatic reactions and the AHR observed immediately after these reactions is presently unclear. In addition, detailed studies on the role of NK₁ receptors in allergen-induced infiltration of eosinophils, neutrophils, and lymphocytes into the lung have not been reported thus far. Therefore, in a conscious and unrestrained guinea pig model of allergic asthma (29), we investigated the involvement of NK₁ receptors in these parameters, using the selective non-peptide NK₁ receptor antagonist SR140333 (30).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Specified pathogen-free Dunkin Hartley guinea pigs of either sex (Harlan, Heathfield, UK) were used in this study. All animals were sensitized to OA at 4 wk of age as described previously (29). To obtain a shift to IgE class antibodies, an allergen solution containing 100 µg OA and 100 mg Al(OH)₃ per ml saline was used. The mixture of allergen solution and Al(OH)₃ was gently rotated for 60 min to obtain an alu-gel, and 0.5 ml was injected intraperitoneally, and another 0.5 ml was divided over seven intracutaneous injection sites in the proximity of lymph nodes in the paws, lumbar regions, and neck. Animals were operated on 3 wk after sensitization and used experimentally 4 to 8 wk after sensitization. The animals were housed in individual cages in climate-controlled animal quarters and were given water and food ad libitum. All protocols described were approved by the University of Groningen Animal Health Committee, which is responsible for the care and proper use of experimental animals.

Measurement of Airway Function

Airway function was assessed by measurement of pleural pressure (Ppl) as described previously (31). In short, a small saline-filled latex balloon, connected to a saline-filled cannula, was surgically implanted inside the thoracic cavity. The free end of the cannula was driven subcutaneously to the neck of the animal, where it was exposed and attached permanently. Via an external saline-filled cannula the pleural balloon was connected to a pressure transducer (Ohmeda DTX; SpectraMed, Bilthoven, The Netherlands). Ppl (in cm H₂O) was continuously measured using an on-line computer system. Ppl data were sampled for 10 s every minute. Breath-by-breath variation was normally less than 10%; incidental large variations caused by sudden movements or deep sights were rejected from the calculation. We have previously shown that changes in Ppl are linearly correlated to changes in airway resistance and hence can be used as a sensitive index for allergic and nonallergic bronchoconstriction (31).

During the experimental protocol (1 to 5 wk after surgery) baseline Ppl measurements remained stable, and no signs of inflammation were observed at the sites of surgery. Airway function can be monitored repeatedly and continuously for periods of at least 24 h while the animals are unaware of the measurements being taken.

Provocation Procedures

OA and histamine provocations were performed by inhalation of aerosolized solutions. These provocations were carried out in a specially designed perspex cage of 9 L, in which the guinea pigs could move freely. A DeVilbiss nebulizer (type 646; DeVilbiss, Somerset, PA) driven by an airflow of 8 L/min, provided the aerosol with an output of 0.33 ml/min.

The animals were habituated to the experimental conditions and the provocation procedures on two sequential days at least 1 wk after surgery, when preoperative weight was restored. On the first day, the animals were placed in the provocation cage unconnected to the pressure transducer. After an adaptation period of at least 30 min, three consecutive provocations with saline were performed, each provocation lasting 3 min and separated by a 7-min interval. The next day, this procedure was repeated with the animals connected to the measurement system.

On the experimental days after the habituation procedure, allergen and histamine provocations were performed as indicated below. All provocations were preceded by an adaptation period of at least 30 min, followed by two consecutive control provocations with saline as described above. A baseline Ppl value was calculated by averaging the Ppl values from the last 20 min of the adaptation period.

In order to assess the airway reactivity for histamine, provocations with the agonist were performed starting with a 25-µg/ml solution in saline, followed by increasing dosage steps of 25 µg/ml. Histamine provocations lasted 3 min and were separated by 7-min intervals. Animals were challenged until Ppl was increased by more than 100% above baseline for at least 3 consecutive minutes. The provocation concentration, causing a 100% increase of Ppl (PC₁₀₀) was derived by linear interpolation of the concentration-Ppl response curve and was used as an index for airway reactivity towards histamine. Ppl returned to baseline within 15 min after the last histamine provocation.

Allergen provocations were performed by inhalation of increasing concentrations of 1.0, 3.0, and 5.0 mg/ml OA in saline for 3 min each, separated by 7-min intervals. Allergen inhalations were discontinued when an increase in Ppl of more than 100% was observed. Using these conditions, none of the animals developed anaphylactic shock after allergen provocation.

Provocation Protocol

On two different occasions, separated by a 1-wk interval, the guinea pigs inhaled either vehicle (saline) or SR140333 at a NK₁ receptor-selective concentration (100 nM) for 3 min, at 30 min before and at 5.5 h after OA provocation (i.e., between the EAR and LAR). Saline and SR140333 inhalations were alternated using a random crossover design.

In a control group of animals, saline was inhaled both in the first and in the second week at the time points indicated above.

Subsequent allergen provocations performed at Week 1 and Week 2 were identical with respect to the OA dose. Twenty-four hours before each allergen provocation, the basal histamine PC₁₀₀ was determined. Thirty minutes later, either saline (3 min) or SR140333 (100 nM, 3 min) was inhaled and a subsequent histamine PC₁₀₀ measurement was performed after another 30 min to assess the effects of saline and SR140333 on baseline histamine reactivity. In separate experiments, the effect of saline and SR140333 on basal histamine reactivity were also assessed at 5 h and 23 h after administration.

During the next day, histamine PC₁₀₀ values were assessed at 5 h (after the EAR) and 23 h (after the LAR) after OA provocation in the saline or SR140333 treated animals, to assess allergen-induced AHR at these time points. For the quantitative assessment of the EAR (between 0 h and 5 h after allergen provocation) and LAR (between 8 h and 23 h after allergen provocation), airway function was continuously measured during the whole procedure. Between the measurements of histamine PC₁₀₀ values at 5 h and 23 h, the animals were placed in their home cage (0.16 m²), in which water and food were freely accessible and where they could move around freely. During this transfer the animals remained connected to the measurement system.

Bronchoalveolar Lavage

BAL was performed at 25 h after the second OA provocation in the group of animals receiving saline at 30 min before and at 5.5 h after OA provocation in both weeks 1 and 2, and in the group receiving saline in Week 1 and SR140333 in Week 2. In a control group, OA-sensitized animals were lavaged 25 h after a provocation with saline instead of OA.

BAL was performed as described previously (29). In short, the animals were anaesthetized with 20 mg/kg Brietal, 35 mg/kg Ketalar, and 6 mg/kg Rompun, administered intraperitoneally. The trachea was exposed and cannulated, and the lungs were lavaged gently using 5 ml of sterile saline at 37° C followed by three subsequent aliquots of 8 ml of saline. The recovered lavage samples were cooled on ice, and centrifuged at 200 × g for 10 min at 4° C. The pellets were combined and resuspended to a final volume of 1.0 ml in phosphate-buffered saline and total cell numbers were counted using a Coulter Counter (Coulter, Hialeah, FL). For cytologic examination, cytospin-preparations were stained with May-Grünwald and Giemsa stain. A cell differentiation was performed by counting at least 400 cells in duplicate.

Data Analysis

The magnitudes of the allergen-induced EAR and LAR were expressed as the area under the Ppl time-response curve (AUC) between 0 and 5 h after allergen provocation for the EAR, and between 8 and 23 h after provocation for the LAR. Ppl was expressed as percentage change from baseline and AUC was calculated by trapezoid integration over discrete (5-min) time periods. Based on saline control provocations, threshold values of AUC (mean + 2 × SD; 99% confidence interval) were defined as 1,185% × 5 min for a positive EAR and 2,790% × 5 min for a positive LAR, respectively (29). Using these criteria, animals were characterized as single early responders and as dual responders (i.e., animals expressing an EAR as well as a LAR). Inherent to the research question, only dual-responding animals (67% of the animals) were included in this study.

Changes in airway reactivity towards histamine, allergen-induced asthmatic reactions and allergen-induced cell infiltration were analyzed by Student's t test for paired or unpaired data as indicated. Differences were considered to be statistically significant at p < 0.05. All data are presented as mean ± SEM.

Materials

Histamine hydrochloride, OA (grade III), May-Grünwald stain and Giemsa stain were obtained from Sigma Chemical Co. (St. Louis, MO). Al(OH)₃ was obtained from JT Baker Chemical Co. (Phillipsburg, NJ). Brietal (methohexital sodium) was purchased from Eli Lilly (Nieuwegein, The Netherlands), Ketalar (ketamine hydrochloride) from Parke-Davis (Hoofddorp, The Netherlands), Rompun (2-(2,6-xylidino)- 5,6-dihydro-4H-1,3-thiazine-hydrochloride, methylparaben) from Bayer (Leverkusen, Germany).

SR140333((S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2,2,2]octane chloride) was a kind gift of Sanofi Recherche (Montpellier, France).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of SR140333 on Allergen-induced Early and Late Asthmatic Reactions

Allergen provocation of OA-sensitized guinea pigs induced a pronounced EAR and LAR (Table 1). In addition, comparable early and late reactions were found after the two subsequent allergen challenges when the animals were treated with saline at both occasions (first and second weeks). Treatment with inhaled SR140333, either at Week 1 or at Week 2 of the protocol, did not significantly affect the EAR and LAR as compared with saline inhalations by the same animals (control week) (Table 1). In addition, SR140333 had no effect on basal Ppl (not shown).

Effect of SR140333 on Allergen-induced Airway Hyperreactivity to Histamine

Both saline and SR140333 inhalations had no significant effect on the basal histamine PC₁₀₀ at 30 min and at 5 and 23 h after inhalation (Figure 1). In saline-treated animals, exposure to OA aerosol induced a marked 2.50 ± 0.25-fold increase in airway reactivity to histamine after the EAR (at 5 h after allergen provocation), whereas a 1.98 ± 0.34-fold increase in airway reactivity was found after the LAR (at 23 h after allergen provocation) (Figure 2). Inhalation of SR140333 by these animals at 30 min before and at 5.5 h after allergen challenge significantly attenuated the observed AHR to histamine, both at 5 h after the EAR (1.77 ± 0.13-fold increase in airway reactivity; p < 0.01) and at 23 h after the LAR (1.15 ± 0.13-fold increase in airway reactivity; p < 0.05) (Figure 2). No changes in allergen-induced AHR were observed when animals were treated with saline at two subsequent occasions (Figure 3).

View larger version (43K): [in this window] [in a new window]   Figure 1.   Effect of inhalations (3 min) of saline and the selective NK1 receptor antagonist SR140333 (100 nM) on basal airway reactivity to histamine. Subsequent histamine PC100 measurements were performed 30 min before (open bars) and 30 min (hatched bars), 5 h (cross-hatched bars), and 23 h (closed bars) after saline or SR140333 inhalation. Data represent mean values ± SEM of four to seven animals.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Effect of inhalation of the selective NK1 receptor antagonist SR140333 (100 nM, 3 min) on ovalbumin (OA)-induced airway hyperreactivity to histamine at 5 h (i.e, after the allergen-induced EAR) and at 23 h (i.e., after the allergen-induced LAR) after OA provocation. SR140333 (closed bars) or saline (open bars) were inhaled at 30 min before and 5.5 h after OA provocation. Data represent mean values ± SEM of seven animals. Statistical analysis: *p < 0.05, p < 0.001, Student's t test for paired observations.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effect of saline inhalation (3 min) on ovalbumin (OA)- induced airway hyperreactivity to histamine at 5 h (i.e., after the allergen-induced EAR) and at 23 h (i.e., after the allergen-induced LAR) after OA provocation on two subsequent occasions with a 1-wk interval. Saline was inhaled at 30 min before and 5.5 h after OA provocation in both Week 1 (open bars) and Week 2 (closed bars). Data represent mean values ± SEM of five animals. Statistical analysis: *p < 0.05, p < 0.001, Student's t test for paired observations.

Effect of SR140333 on Allergen-induced Airway Inflammation

Total and differential BAL cell counts from saline (control) and OA-challenged guinea pigs are presented in Figures 4A and 4B.

View larger version (19K): [in this window] [in a new window]   Figure 4.   Bronchoalveolar lavage cell counts at 25 h after inhalation of saline (open bars) or ovalbumin (OA). The OA-challenged animals received saline (3 min) at 30 min before and 5.5 h after OA challenge in Week 1, and either saline (3 min) (hatched bars) or SR140333 (100 nM, 3 min) (closed bars) at 30 min and 5.5 h after OA challenge in Week 2. (A) Effects on total cells, eosinophils, and macrophages. (B) Effects on lymphocytes, neutrophils, and epithelial cells. Data represent mean values ± SEM of six to seven animals. Statistical analysis: *p = 0.10, p < 0.05, p < 0.01, §p < 0.001, Student's t test for unpaired observations.

In all groups measured, the recovery of the lavage fluid was high, with an overall average of 83.9 ± 0.3% (n = 19). Compared with a control challenge with saline, OA provocation caused a significant increase in total BAL cells (p < 0.01), eosinophils (p < 0.001), macrophages (p < 0.05), lymphocytes (p < 0.01), and neutrophils (p < 0.01) in animals that were treated with saline at 30 min before and 5.5 h after the second allergen challenge, whereas the total cell number (p < 0.05), as well as the numbers of infiltrated eosinophils (p < 0.05), lymphocytes (p < 0.01), and neutrophils (p < 0.05) were significantly reduced in the animals treated with SR140333 at these time points. In addition, the number of epithelial cells in the BAL fluid tended to be decreased in the SR140333-treated animals (p = 0.10) (Figure 4B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, we have demonstrated that tachykinin NK₁ receptors are involved in the development of airway hyperreactivity to histamine after both the EAR and the LAR, and that NK₁ receptor-mediated infiltration of inflammatory cells in the airways may contribute to the observed hyperreactivity after both reactions.

The selective and highly potent nonpeptide tachykinin NK₁ receptor antagonist SR140333 was used to investigate the involvement of NK₁ receptors in the development of allergen-induced EAR and LAR, the AHR after these reactions as well as airway inflammation after the LAR. In previous studies, SR140333 has been demonstrated to be highly selective towards the NK₁ receptor. Thus, in radioligand displacement experiments, SR140333 competitively inhibited the binding of SP in a variety of assays with inhibition constants (K_i) ranging from 0.019 to 0.7 nM, whereas the K_i values in binding assays for NK₂ and NK₃ receptors were 700 to 50,000 times higher (30). A potency in the subnanomolar range was also found in functional in vitro NK₁ receptor assays, whereas SR140333 had no effect on NK₂ or NK₃ receptor-mediated responses up to 1 µM (30). On the basis of these findings, it can be argued that the inhaled dose of SR140333 (100 nM solution, nebulized during 3 min in a 9-L animal provocation cage) is both NK₁-selective and effective.

Allergen provocation of sensitized guinea pigs results in the activation of pulmonary mast cells and subsequent release of various mediators, causing an immediate bronchoconstriction of the airways. Among the mediators that are released, histamine, leukotrienes, platelet-activating factor, lipoxins, prostanoids, and kinins are all capable of releasing neuropeptides from sensory nerves (32), which have been shown to enhance the airway responsiveness for contractile agonists (22). In the present study, the selective NK₁ receptor antagonist SR140333 had no effect on basal airway reactivity to histamine, but it clearly attenuated the development of allergen- induced AHR after both the EAR and LAR, indicating that NK₁ receptor activation is involved in this process. This observation appears to be in contrast with recent observations by other investigators, who found that in guinea pigs NK₂ rather than NK₁ receptors are involved in the development of allergen-induced AHR (28, 35). Thus, the selective NK₂ receptor antagonist SR48968, but not the selective NK₁ receptor antagonist SR140333, both being administered intraperitoneally 30 min before allergen provocation, inhibited AHR to inhaled acetylcholine 48 h after allergen provocation (28). In addition, the dual NK₁/NK₂ receptor antagonist FK224, but not the selective NK₁ receptor antagonist FK888, both administered intravenously 5 min before allergen provocation, inhibited the AHR to inhaled methacholine at 30 min after allergen provocation (35). These apparently conflicting results may reflect differences in conditions of lung function measurement (anesthetized versus conscious unrestrained guinea pigs in our study), routes of drug administration (systemically versus locally in our study), the time points of AHR measurements as well as the provoking agents used. With respect to the latter point, it is important to note that allergen-induced AHR in our study was assessed by histamine inhalation, whereas methacholine or acetylcholine were used in the other studies. Acetylcholine- and methacholine-induced bronchoconstriction results from direct stimulation of the airway smooth muscle, whereas histamine-induced bronchoconstriction is partially mediated by stimulation of afferent vagal nerve endings, leading to additional reflex bronchoconstriction (36, 37). Recently, we demonstrated that an enhanced cholinergic reflex mechanism importantly contributes to the allergen-induced AHR to histamine in conscious, unrestrained guinea pigs (37). Because tachykinins, predominantly via stimulation of NK₁ receptors, facilitate cholinergic neurotransmission in guinea pig airways (38), the enhanced cholinergic contribution to histamine-induced bronchoconstriction may involve exaggerated NK₁ receptor stimulation and be inhibited by SR140333. Interestingly, in our previous study it was found that the role of this enhanced cholinergic reflex in the AHR to histamine was more pronounced after the EAR than after the LAR (37), which was completely in parallel with the stronger dysfunction of the prejunctional inhibitory muscarinic M₂-autoceptors after the EAR than after the LAR (41). Consequently, after the LAR the NK₁ receptor-mediated facilitation of the reflex cholinergic outflow would be expected to play quantitatively a more important role than after the EAR. This is supported by the larger inhibition by SR140333 of the late AHR as compared with the early AHR to histamine, as found in the present study.

Another mechanism that could be important is an allergen-induced increase in afferent nerve excitability, possibly leading to enhanced vagal as well as eNANC activity in response to histamine (42, 43). In addition, the accessibility of these afferents for stimuli may also be increased, as a consequence of epithelial damage caused by cytotoxic mediators derived from infiltrated inflammatory cells such as eosinophils (44).

The present study also indicates that the SR140333-induced inhibition of allergen-induced AHR after the LAR was accompanied with a reduced number of eosinophils, neutrophils, and lymphocytes in the BAL fluid, whereas there was a tendency to reduced epithelial cells in the airway lumen, indicating that NK₁ receptor-mediated infiltration of proinflammatory cells and subsequent epithelial damage may be involved in the observed AHR after this reaction. Although the composition of BAL fluid was not determined after the EAR in the present study, we have previously shown that a pronounced infiltration of both eosinophils and neutrophils, as well as activation of eosinophils is already present after this reaction and may similarly be involved in the development of the early AHR (29, 45). Recently, neuropeptides have been shown to modulate human eosinophil (46) and rat neutrophil (47) chemotaxis in vitro. In vivo, inhaled SP elicits eosinophil influx, whereas inhaled NKA causes neutrophil recruitment in BAL fluid from guinea pigs 24 h after exposure of the aerosolized neuropeptides (48). In addition, by capsaicin inhalation it was demonstrated that influx of eosinophils and neutrophils can also be induced through endogenous tachykinins (48). Because in vitro studies have indicated that SP alone did not exert chemotatic activity to eosinophils, and SP only induced neutrophil infiltration at very high concentrations (47), the SP-mediated influx of these proinflammatory cells into the airways occurs probably through an indirect mechanism, presumably by priming the cells for enhanced chemotactic activity in response to LTB₄, PAF, and IL-5 (46, 49). In addition, SP-induced eosinophil and neutrophil infiltration may occur through activation of mast cells (50) and/or stimulation of alveolar macrophages (53, 54), via release of chemotactic factors from these cell types (55). Because SP has higher affinity for NK₁ than for NK₂ receptors, the role of SP in inflammatory cell infiltration may well be a NK₁ receptor-mediated process. Indeed, the potentiation of PAF-induced migration of eosinophils was prevented by the NK₁ receptor antagonist FK888 in vitro (49), and SP-induced neutrophil infiltration into murine air pouch was prevented by coadministration of the selective NK₁ receptor antagonists RP-67,580 and CP-96,345 in vivo (56), whereas the SP-mediated degranulation of mast cells was shown to be attenuated by NK₁ receptor antagonists (51, 52). The role of NK₁ receptors in SP-induced inflammatory cell infiltration was further strengthened by the observation that CP-96,345 reduced SP- and capsaicin-induced adhesion of eosinophils and neutrophils in the venules of rat trachea (57). Moreover, Kaltreider and colleagues (58) recently demonstrated inhibition of antigen-induced infiltration of lymphocytes and total granulocytes by CP-96,345 in mice, which is in line with the present study.

In addition to migration of inflammatory cells into the airway lumen, SP has also been shown to activate both eosinophils (59) and neutrophils (60), which may similarly be important in the reduction of allergen-induced AHR by SR140333 as observed in our study. Finally, because airway edema may contribute to allergen-induced AHR because of thickening of the airway wall (61), inhibition of SP-induced microvascular leakage after allergen challenge by NK₁ receptor antagonists such as CP-96,345 (62) and SR140333 may be important.

In the present study, the allergen-induced EAR was not altered by inhalation of the NK₁ receptor antagonist, indicating that NK₁ receptors are not importantly involved in the allergen-induced immediate bronchoconstriction. This is in line with previous observations in capsaicinized guinea pigs, indicating that reduced tachykinin levels in lung tissue did not prevent the allergen-induced immediate bronchoconstriction (15, 16). In addition, Bertrand and colleagues (20) have demonstrated that the selective NK₂ antagonist SR48968, but not the selective NK₁ receptor antagonist CP96,345, attenuates the allergen-induced immediate bronchoconstrictor response in the presence of the neutral endopeptidase (NEP) inhibitor phosphoramidon, whereas the inhibitory effect of SR48968 could be potentiated in the presence of CP96,345. This indicates that the involvement of NK₁ receptors in allergen-induced EAR is minor and may only be visualized when NK₂ receptors are blocked and the breakdown of tachykinins by NEP is inhibited. In line with the above results, the allergen-induced immediate bronchoconstriction in guinea pigs was inhibited by the dual NK₁/NK₂ receptor antagonist MDL105,212, which was paralleled by an inhibition of OA-induced histamine release from lung tissue in vitro, indicating that endogenous tachykinins facilitate allergen-induced histamine release from mast cells (21).

Although allergen-induced airway inflammation is generally considered to play an important role in the development of the LAR, we previously could not demonstrate a direct correlation between the severity of the LAR and eosinophil or neutrophil numbers in the BAL fluid collected after this reaction (29). Also in the present study, we found a dissociation between these parameters; although SR140333 reduced the numbers of eosinophils and neutrophils in the BAL fluid collected after the LAR, the magnitude of this reaction was unaffected. We therefore hypothesize that inhibition of NK₁ receptor-mediated eosinophilia and neutrophilia per se is not sufficient to suppress the development of the LAR.

In conclusion, it was demonstrated that NK₁ receptors are involved in the development of allergen-induced AHR to histamine after both the EAR and the LAR, and that NK₁ receptor-mediated inflammation of the airways may contribute to this process. Therefore, this study emphasizes the important role of SP in the development of AHR and indicates that tachykinin NK₁ receptor antagonists may be useful in the treatment of allergic asthma.