INTRODUCTION

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The neurokinins (NKs) are a family of structurally related neuropeptides. Substance P (SP) and neurokinin A (NKA) are encoded by the preprotachykinin-A gene and they are colocalized in secretory granules in the afferent C-fiber nerves (1). Two receptor types, NK-1 and NK-2 for SP and NKA, respectively, initially identified in biological assays, have been confirmed by molecular cloning and sequence analysis (2). Pharmacological studies have demonstrated that SP and NKA induce many of the proinflammatory processes involved in asthma, such as bronchoconstriction, nonspecific bronchial hyperresponsiveness, vasodilation and microvascular leakage, and airway mucous secretion (3). Higher levels of SP in the bronchoalveolar (BAL) and nasal (NAL) lavage fluids as well as an increase in levels after provocation of allergic subjects but not of nonallergic controls have been reported (4). An elevation of SP content in induced sputum from patients with asthma has been observed also and is correlated with eosinophil cell count in the sputum and with the degree of airflow obstruction (5). In a guinea pig model of allergic inflammation, the period corresponding to late-onset airway obstruction (6) is closely associated with an induction of neurokinin biosynthesis in the nodose ganglion and increased transport to the peripheral nerve endings (7). These and other studies (8) argue that NKs are proinflammatory mediators and that they may contribute to the inflammatory response of asthma.

Allergic asthma is a complex pulmonary syndrome in which chronic inflammation seems to be critical for the development of reversible airway obstruction, nonspecific bronchial hyperresponsiveness, and airway remodeling. Although various inflammatory cells are present in allergic airway inflammation, CD4⁺ T cells, in particular the helper T cell type 2 (Th2) subset, are believed to promote the allergic response in the airways of subjects with symptomatic asthma by releasing factors responsible for inflammatory cell recruitment into the airways (9, 10). Several animal models of allergic inflammation have been developed to investigate the physiopathology of bronchial asthma. We have previously demonstrated that allergen-induced dual airway responses in the atopic Brown Norway (BN) rat (11) are associated with eosinophilia and a predominance of Th2 cytokine expression in the airways (12, 13). More recently, we have shown that the injection of CD4⁺ T cells, isolated from cervical lymph nodes of ovalbumin (OA)-sensitized BN rats, into a naive recipient animal transfers the late allergic airway response (13). These studies indicate that the BN rat sensitized and exposed to OA develops allergic airway responses that are associated, at least in part, with CD4⁺ T cells and the predominant expression of Th2 cytokines.

NKs, in particular SP, modulate a number of important immunologic functions. SP enhances mitogen-induced T cell proliferation in vitro (14), and increases lymphocyte traffic from lymph nodes to the periphery (15) by acting preferentially on CD4⁺ T cells. SP also increases interleukin 4 (IL-4)-induced immunoglobulin synthesis by B cells (16) and stimulates human peripheral blood monocytes and bone marrow cells in vitro to produce cytokines, including IL-1, IL-3, IL-6, IL-10, IL-12, and tumor necrosis factor  (TNF-) (17, 18). These findings (14) support the concept that neuropeptides such as SP are important regulators of immune responses. In contrast to the extensive studies of NKs as proinflammatory mediators in the lungs, few investigations, if any, have been published on their potential role as neuroimmunoregulators in vivo, and particularly in modulating cytokine expression in the airways. To establish the suitability of our animal model for investigating the potential contribution of NKs in airway allergic responses, we have shown first that NK-1 and NK-2 receptors are expressed in BN rat lungs and that NKs are released after exposure to the allergen. Subsequently, to determine the respective contributions of the NK-1 and the NK-2 receptors in the potential modulation by NKs of allergen-induced allergic airway responses, two potent and selective NK antagonists were used. We show that NKs can regulate allergen-induced cytokine expression in the lungs, and that they are involved in the late airway response (LAR). However, our results suggest a dichotomy between the NK-1 and the NK-2 receptors in modulating the airway inflammatory responses, which may have important implications for potential therapy with NK antagonists in asthma.

	METHODS

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Animals

Highly inbred BN rats, 7 to 9 wk old, ranging in weight from 180 to 210 g, were purchased from Harlan Sprague-Dawley UK (Blackthorn, UK). Rats were housed in a conventional animal facility at McGill University (Montreal, PQ, Canada). All rats were actively sensitized to ovalbumin (OA) on Day 0 with a 1-ml subcutaneous injection of a solution containing 1 mg of OA, 4.28 of Al(OH)₃ and isotonic sterile saline. In addition, 0.5 ml of Bordetella pertussis vaccine containing 10⁹ heat-killed bacilli was injected.

Extraction of the Total RNA from Lung Tissues

The expression of neurokinin receptors was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), using RNA extracted from the lungs of naive and OA-sensitized BN rats. Animals were killed with a lethal dose of Somnotol (pentobarbital) and the left major lobe of the lungs was removed. Lung tissue was flash-frozen in liquid nitrogen and stored at 80° C until RNA extraction. For RNA extraction, frozen tissue was ground to a fine powder under liquid nitrogen and 4 ml of TRIzol (Total RNA Isolation) reagent was added. Total RNA was extracted from lung tissues according to the manufacturers instructions.

RT-PCR Analysis

Single strand cDNA was made in a 20-µl reaction, using 2 µg of total RNA as a template, oligo(dT)_12-18 primer and Superscript II enzyme in the presence of acetylated bovine serum albumin (BSA) and RNAguard ribonuclease inhibitor. NK-1 and NK-2 receptors and cyclophilin (the housekeeping gene) mRNA were amplified from the resulting cDNA with the appropriate oligonucleotide primers by PCR. Thirty-five cycles were performed for the amplification of the NK-1 receptor (1 min of denaturation at 92° C, 2 min of annealing at 56° C, and 3 min of extension at 72° C) and 30 cycles were performed for the NK-2 receptor and cyclophilin (1 min of denaturation at 92° C, 2 min of annealing at 60° C, and 3 min of extension at 72° C), respectively, in a PTC-100 programmable thermal controller (MJ Research, Watertown, MA). Two set of primers were used to determine the expression of each NK receptor. The sequences used for NK-1 and NK-2 receptors and cyclophilin were based on the published sequences for rat NK-1 and NK-2 receptors and cyclophilin and were as follows: NK-1 receptor 5' sense primer (5' primer): 5'-TGCATGGCTGCATTCAATACGGT-3' (nucleotides 793-815); NK-1 receptor 3' antisense primer (3' primer): 5'-AGATGATGGGGTTGAACATGGTA-3' (nucleotides 1468-1446); NK-1 receptor inner 3' antisense primer (3'int. primer): 5'-TCCTGTTGGGATGCTCCGGCCACT-3' (nucleotides 1126-1103); NK-2 receptor 5' sense primer (5' primer I): 5'-GACCCGTGCCATTGTTTCTGAC-3' (nucleotides 454-475); NK-2 receptor 5' sense primer (5' primer II): 5'-AGGATGCGCACAGTCACCAACTA-3' (nucleotides 635-657); NK-2 receptor 3' antisense primer (3' primer): 5'-CATCTTGCCTCCGTTGTCATTGG-3' (nucleotides 1027-1005); cyclophilin sense primer: 5'-GGTCAACCCCACCGTGTTCTTCG-3' (nucleotides 45-67); cyclophilin antisense primer: 5'-GTGCTCTCCTGAGCTACAGAAGG-3' (nucleotides 598-576). These primers were designed to amplify cDNA fragments of 675 and 333 bp for the NK-1 receptor, 573 and 392 bp for the NK-2 receptor, and 556 bp for cyclophilin. The amplified products were visualized by ethidium bromide staining after gel-agarose electrophoresis.

Measurement of Substance P Release

The release of the endogenous NKs after allergen challenge was determined by the measurement of SP in the bronchoalveolar lavage (BAL) of three different groups of rats. Group 1 (n = 4) corresponded to sham control animals exposed to an aerosol of 5% OA for 5 min. Groups 2 (n = 4) and 3 (n = 4) corresponded to OA-sensitized animals exposed to an aerosol of saline or 5 % OA for 5 min, respectively. Animals were killed 60 min after the saline or OA challenge and BAL was performed. Briefly, 5 ml of isotonic sterile saline at 37° C was infused into the lung, aspirated and placed in a tube containing peptidase inhibitors, phenylmethylsulfonyl fluoride (PMSF, 1 mM) and DL-thiorphan (5 µg/ml). After centrifugation of the lavage, the supernatant was divided into aliquots (500 µl) and immediately frozen at 20° C for further analysis. Before SP quantitation, peptides were extracted from a 2-ml aliquot of the lavage on Sep-Pak Cartridge Plus C₁₈ (Waters Millipore, Mississauga, ON, Canada) and concentrated under vacuum according to the manufacturer instructions. The determination of SP levels in BAL was performed with an enzyme immunoassay (EIA) kit for substance P, according to the manufacturer's instructions. The range of determination is 0 to 1,000 ng/ml, with a minimum detectable concentration of 35 pg/ml. The percentage of cross-reactivity is 100% for SP and fragments SP 2-11 to SP 5-11. There is no cross-reactivity of the assay for NKs A, B, and K and other neuropeptides.

Measurement of Pulmonary Resistance

Rats (180-220 g) were anesthetized by an intraperitoneal injection of urethane (1.5 g/kg). They were intubated with 6 cm of PE-240 polyethylene tubing (Becton Dickinson Diagnostics, Sparks, MD), using translumenal illumination (19). Animals were placed on a heating pad and rectal temperature was continuously monitored with an electric thermometer (Telethermometer; Yellow Springs Instrument, Yellow Springs, OH). Animals were kept in lateral recumbency. The tip of the tracheal tube was connected to a plastic box (volume, 175 ml; Commercial Plastics, Montreal, PQ, Canada). A Fleisch pneumotachograph (Fleisch 00; Bionetics, PQ, Canada) coupled to a piezoresistive differential transducer (Micro-Switch 163PC01D36; Honeywell, Scarborough, ON, Canada) was attached to the other side of the box to measure airflow (). Transpulmonary pressure (Ptp) was measured with a water-filled polyethylene catheter (PE-160; Becton Dickinson Diagnostics) placed in the lower third of the esophagus and connected to a port of a differential pressure transducer (Transpac II disposable transducer, Sorenson, Salt Lake City, UT); the other port of the transducer was connected to the plastic box. The pressure and flow signals were amplified and recorded with a 12-bit analog-to-digital converter at a rate of 200 Hz. All signals were recorded in 10-s segments, stored on a computer, and analyzed with a commercially available software package (RHT Infodat, Montreal, PQ, Canada). Pulmonary resistance (RL) was calculated by applying multiple linear regression to solve the equation of motion of the lung as follows: ELV + RL + K, where V is the volume obtained by the integration of , RL is resistance, EL is elastance, and K is a constant.

Experimental Protocol

Fourteen days after sensitization, rats were randomly divided into three groups and drug vehicle or neurokinin antagonists were injected 30 min before the allergen challenge. Group 1 (n = 8) received an intravenous injection of the drug vehicle corresponding to sterile isotonic saline and 0.01% dimethylsulfoxide (200 µl per 100 g body weight), Group 2 (n = 7) received an intravenous injection of the selective nonpeptide NK-1 antagonist CP-99,994 ((2s-methoxy-benzyl)- (2-phenyl-piperidin-3s-yl)-amine) (200 nmol/kg), and Group 3 (n = 7) received an intravenous injection of the selective NK-2 antagonist SR-48968 ((S)-N-methyl-N-[4-(4-acetylamino-4-phenyl piperidino)-2- (3,4-dichlorophenyl)butyl]benzamide) (200 nmol/kg). The NK-1 antagonist was obtained from E. Pagani (Pfizer, Groton, CT), and X. Edmonds-Alt (Sanofi Research, Montpellier, France) kindly provided the NK-2 antagonist. Baseline resistance in each animal group was measured before and 30 min after the administration of drug vehicle or NK antagonists (T = 0, starting baseline). Rats were challenged with OA (5% [w/v] in saline), using a nebulizer (model 1880; Hudson, Temecula, CA) with an airflow of 10 L/min for 5 min. RL was measured at 5-min intervals for 30 min after the allergen exposure and at 15-min intervals for a total of 8 h.

BAL Cell Counts

BAL was performed 8 h after OA challenge, lavage fluid was centrifuged, and the pellet was reconstituted in 5 ml of RPMI 1640 culture medium. Cells were counted with a hemacytometer and viability was assessed by the trypan blue dye exclusion test. The differential cell counts were determined on a cytospin slide that was prepared with a Cytospin model II (Shandon, Pittsburg, PA).

Immunocytochemistry and In Situ Hybridization

Cytospin slides were prepared on poly-L-lysine-coated glass slides, fixed in 4% paraformaldehyde for 30 min and washed with phosphate-buffered saline (PBS) (twice for 5 min each time) before processing. BAL cells were immunostained with monoclonal antibody (mAb) BMK13 (kindly provided by R. Moqbel, University of Alberta, Edmonton, AB, Canada), a mouse anti-major basic protein (MBP) MAb, using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. MBP-positive cells were quantified by microscopy by an investigator blinded to group status. A minimum of 500 BAL cells was counted and the percentage of cells expressing MBP immunoreactivity was evaluated.

Cytokine expression in BAL cells was assessed by in situ hybridization as previously described (12, 13). Antisense and sense riboprobes were prepared from cDNAs encoding rat IL-4, IL-5, and interferon  (IFN-) mRNA. cDNAs were first inserted into a pGEM vector and linearized with appropriate enzymes. In vitro transcription was carried out in the presence of ³⁵S-labeled UTP and the T7 or SP6 RNA polymerases. For detection of cytokine mRNAs, cytospin preparations obtained from BAL fluid were permeabilized with Triton X-100 and proteinase K (1 mg/ml) in 0.1 M Tris containing 50 mM EDTA for 20 min at 37° C. To prevent nonspecific binding of ³⁵S-labeled RNA probes, the preparations were incubated with 10 mM N-ethylmaleimide and 10 mM iodoacetamide for 30 min at 37° C, followed by incubation in 0.5% acetic anhydride and 0.1 M triethanolamine for 10 min at 37° C. Prehybridization was performed with 50% formamide and 2× standard saline citrate (SSC) for 15 min at 37° C. For hybridization, antisense or sense probes (10⁶ CPM/section) were diluted in hybridization buffer. Dithiothreitol (100 mM) was present in the hybridization mixture to ensure blocking of any nonspecific binding of the ³5S-labeled probes. Posthybridization washing was performed in decreasing concentrations of standard saline citrate (4× SSC to 0.5× SSC) at 40° C. Unhybridized single-strand RNA was removed by RNase A (20 mg/ml). After dehydration, the slides were immersed in NBT2 emulsion and exposed for 10 d. The autoradiographs were developed in Kodak D-19, fixed and counterstained with hematoxylin. Slides were coded, and positive cells were counted blindly at ×40 magnification with an eyepiece reticule. The results were expressed as the mean number of positive cells per 1,000 cells. For negative controls cytospins were hybridized with sense probes or pretreated with RNase before the application of probes.

Data Analysis

The early airway response (EAR) was derived from the highest RL measurements recorded < 30 min after OA challenge, and expressed as a percentage of baseline. The LAR was measured as the area under the curve for RL versus time above the baseline RL, between 3 h and 8 h after challenge. Data are represented as means ± SEM. Statistical comparison was performed by ANOVA followed by Fisher LSD test. A difference was considered to be statistically significant when the p value was less than 0.05.

Reagents

TRIzol, Superscript II, acetylated BSA, Platinum Taq polymerase, oligo(dT)_12-18, the dNTP set, and 1-kb DNA ladder were purchased from GIBCO-BRL (Burlington, ON, Canada). RNAguard ribonuclease inhibitor was purchased from Pharmacia Biotech (Quebec, PQ, Canada). PCR primers were synthesized and purified by fast protein liquid chromatography (FPLC) in the Sheldon Biotechnology Centre (Montreal, QC, Canada). The EIA kit for SP was purchased from Peninsula Laboratories (San Carlos, CA). Other chemical products and enzymes were purchased from Sigma (St. Louis, MO).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Neurokinin Receptor Expression in BN Rat Lung Tissues

The expression of NK receptors in rat lungs has been suggested to be strain dependent (2, 20), so that we also wished to confirm their presence in the lungs of the highly inbred BN rat. RT-PCR analysis using two different sets of primers to selectively amplify rat NK-1 and NK-2 receptors demonstrated transcripts for both receptors in the lung tissues of OA-sensitized BN rats (Figure 1). Messenger RNAs encoding these two NK receptors were detected by RT-PCR assay in the lung tissues of naive BN rats also, and there was no evidence of a difference in their levels of expression in comparison with OA-sensitized animals (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1.   Determination of neurokinin receptor expression in the lungs of BN rats. Rats were sensitized to OA and were exposed to the allergen 2 wk later (n = 4). Thirty minutes after challenge, the lungs were removed and lung tissue was harvested for RNA extraction. The expression of both NK-1 and NK-2 receptors was determined by RT-PCR, using two different sets of primers. Cyclophilin was used as housekeeping gene.

Allergen Challenge Induces Release of the SP in the Airways of OA-sensitized BN Rat

The detection of NKs in a biological milieu is difficult to achieve because of their rapid proteolytic degradation into nonimmunoreactive fragments. To reduce their metabolism in the BAL sample collected, a neutral endopeptidase inhibitor (thi-orphan) and a peptidase inhibitor (PMSF) were added to the BAL. The release of NKs in the airways was assessed by the measurement of SP in BAL fluids. Using a highly selective and sensitive ELISA for SP and some of its related metabolites, no differences in the levels of this neuropeptide were detected between sham control animals challenged with OA and OA-sensitized animals exposed to a saline aerosol (Figure 2). However, allergen challenge in OA-sensitized rats increased by 2.4-fold the release of SP into the airways (Figure 2).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Determination of SP levels in BAL fluids by competitive ELISA. Three groups were investigated: (1) sham control rats exposed to OA (open squares; n = 4); (2) rats sensitized to OA and exposed to an aerosol of saline (open circles; n = 4); and (3) rats sensitized to OA and exposed to an aerosol of OA (open triangles; n = 4). Significant differences are **p < 0.01 from values compared with control, as assessed with an ANOVA following by the Fisher LSD test.

Effect of NK Antagonists on Allergen-induced Airway Responses

The expression of lung NK receptors and the subsequent release of NKs after allergen challenge in OA-sensitized BN rats suggest that NKs might play a role in the modulation of the allergic airway responses. To determine the potential role of endogenous NKs and their receptors in our animal model, highly potent and selective NK antagonists were used (21, 22). A dose of 200 nmol/kg (approximately 0.1 mg/kg) of each NK antagonist was administered to rats. This dose was selected according to their efficacy in reducing airway inflammation induced in guinea pigs by the intravenous injection of high doses of NKs (100 to 300 µg of SP or NKA per kilogram) (23). Since in our study the levels of NKs released in the airways in response to allergen challenge were low (50-210 pg/ml), we deduced that a sufficient dose of the respective NK antagonist was administered to the animals. The injection of the drug vehicle or the neurokinin antagonists before allergen challenge did not affect the baseline pulmonary resistance in anesthetized BN rats (vehicle: 0.157 ± 0.011 versus 0.161 ± 0.016; CP-99,994: 0.161 ± 0.015 versus 0.152 ± 0.014; SR-48968: 0.152 ± 0.014 versus 0.154 ± 0.015 cm H₂O ml¹ s), which suggests minimal if any role for endogenous NKs in the control of the airway basal tone. As previously published (10), OA-sensitized rats responded to allergen challenge with both EAR and LAR (Figure 3). The administration of the NK-1 or the NK-2 antagonist failed to reduce EAR (Figures 3 and 4). In contrast, both the NK-1 and NK-2 antagonists reduced the LAR in OA-sensitized rats (Figures 3 and 5).

View larger version (27K): [in this window] [in a new window]   Figure 3.   Effect of neurokinin antagonists on the time course of ovalbumin (OA)-induced airway responses in OA-sensitized BN rats. Three groups were investigated: (1) control rats injected with the drug vehicle (open circles; n = 8); (2) rats treated with the NK-1 antagonist CP-99,994 (open squares; n = 7); and (3) rats treated with the NK-2 antagonist SR-48968 (open triangles; n = 7). The vehicle and the neurokinin antagonists were injected 30 min before the allergen challenge. All values represent means ± SEM.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effect of neurokinin antagonists on OA-induced early airway responses (EAR) in sensitized BN rats. (For a description of the groups, see the legend to Figure 3). EAR was expressed as the percentage of the peak resistance after allergen challenge to the baseline resistance. No significant difference was observed between antagonist-treated groups and the control group.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Effect of neurokinin antagonists on OA-induced late airway response (LAR) of sensitized BN rats. (For a description of the groups, see the legend to Figure 3.) LAR was calculated by the area under the curve of the airway resistance measured between 180 and 480 min after allergen challenge. Significant differences are *p < 0.05 and **p < 0.01 from values compared with control, as assessed with an ANOVA following by the Fisher LSD test.

Modulation of Allergen-induced Airway Eosinophilia by NK Antagonists

Immunocytochemical analysis of BAL eosinophil content, using a monoclonal antibody against the eosinophil-derived granule protein, MBP, confirmed the presence of eosinophils in the BAL 8 h after the allergen exposure (Figure 6). Interestingly, the administration of the NK-2, but not the NK-1, antagonist reduced allergen-induced BAL eosinophilia (Figure 7). The OA-sensitized and OA-challenged animals had significantly higher eosinophil numbers than unsensitized and unchallenged animals (9.45 [± 2.67] × 10⁴ cells, n = 6, p = 0.03) and the NK-2 antagonist treatment reduced BAL eosinophilia by 77%.

View larger version (15K): [in this window] [in a new window]   Figure 6.   Effect of neurokinin antagonists on OA-induced BAL eosinophilia in sensitized BN rats. (For a description of the groups, see the legend to Figure 3.) MBP-positive cells were quantified by microscopy. A minimum of 1,000 BAL cells was counted and the percentage of cells expressing MBP immunoreactivity was evaluated. Significant differences are **p < 0.01 from values compared with control, as assessed with an ANOVA following by the Fisher LSD test.

View larger version (15K): [in this window] [in a new window]   Figure 7.   Effect of neurokinin antagonists on OA-induced cytokine expression of BAL cells from OA-sensitized BN rats. (For a description of the groups, see the legend to Figure 3.) Cytokine expression [(A) IL-4; (B) IL-5; and (C) IFN-] was determined by in situ hybridization and quantified by microscopy. A minimum of 1,000 BAL cells was counted. Significant differences are **p < 0.01 from values compared with control, as assessed with an ANOVA following by the Fisher LSD test. V = vehicle, CP = CP-99,994, SR = SR-48968.

In Vivo Modulation of Th1 and Th2 Cytokine Expression in BAL Cells by NK Antagonists

Allergen challenge of OA-sensitized BN rats increased preferentially the expression of the Th2 cytokines IL-4 and IL-5 in the airways, in comparison with OA-challenged sham control or BSA-challenged OA-sensitized rats (24). In contrast, the number of IFN--positive cells detected in the airways of allergen-challenged OA-sensitized rats was less than in OA-challenged sham control or BSA-challenged OA-sensitized rats (24), consistent with a role for Th2 cytokines in modulating the allergic airway responses in this animal model. In the present study, we confirmed the predominant expression of Th2 cytokines in the BAL cells of OA-challenged animals (Figures 7A-7C). The administration of the NK-1 antagonist before allergen challenge failed to reduce either Th1 or Th2 cytokine expression in BAL cells (Figures 7A-7C). In contrast, the administration of the NK-2 antagonist reduced the number of both Th1 and Th2 cytokine-positive cells in BAL in comparison with the vehicle-injected OA-challenged animals (Figures 7A-7C). Both of the NK antagonists failed to reduce the number of BAL lymphocytes (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this report we have shown that NK-1 and NK-2 receptors are expressed in the lungs of the atopic BN rat and that NKs are released after allergen challenge. Moreover, our results strongly support a role for endogenous NKs in allergen-induced LAR and for an immunomodulatory role of NKs in the expression of both Th1 and Th2 cytokines in BAL cells. However, the results of this study also indicate a potential dichotomy between NK receptor actions in modulating airway inflammatory responses.

The development of airway inflammation is a pivotal event in the allergic airway pathology associated with asthma. To dissect the different events leading to eosinophilic inflammation and its relationship with bronchial hyperresponsiveness, various animal models have been developed (25). The BN rat sensitized and challenged with OA is among the best characterized models and demonstrates several features of asthma including EAR and LAR (10), mast cell activation (26), airway smooth muscle cells hyperplasia (27), and airway and BAL eosinophilia (28, 29). The eosinophilic inflammation is associated with a predominance of Th2 cytokine expression in the airways (12, 13) after allergen exposure. The LAR, defined as bronchoconstriction occurring several hours after allergen exposure, is mediated by cysteinyl-leukotrienes (30), appears to be CD4⁺ T cell driven (12, 13, 31), and is sensitive to glucocorticoid inhibition (19). NKs have been shown to be released in asthmatic airways after allergen challenge and both NK-1 and NK-2 receptors are expressed in human lungs (32, 33), which indicates the plausibility of a participation by these neuropeptides in the inflammatory reaction present in the airways of subjects with asthma. In the present study, we have shown that, similar to what has been previously reported in human subjects (4, 5, 32, 33), BN rat lungs express both NK-1 and NK-2 receptors and NKs are released in the airways after allergen challenge. The release of SP after allergen challenge further validates the animal model in terms of its similarity to the allergic airway responses observed in subjects with symptomatic asthma and its utility for the study of the role and the mechanisms of action of NKs in airway allergic responses.

The link between bronchial hyperresponsiveness and airway inflammation has been extensively investigated (34). Although the LAR is believed to precede the development of bronchial hyperresponsiveness (35), the mechanisms leading to LAR after allergen exposure are still poorly understood. A growing body of evidence links neuropeptides such as NKs to inflammation (36) and to the development of bronchial hyperresponsiveness in asthma (3, 37). However, whether the NKs participate in the LAR is still not well established. Fischer and coworkers (7) have shown an increase in NK mRNA expression and peptide synthesis in nodose ganglia of OA-challenged guinea pigs. They have postulated that these findings suggest a role for NKs in the increase of airway hyperreactivity developing in response to allergen challenge. More recently, Schuiling and coworkers (40, 41) have shown that the OA-induced LAR in guinea pigs is inhibited by the administration of an NK-2, but not an NK-1, antagonist. In the present study, we report that both NK-1 and NK-2 antagonists inhibit allergen-induced LAR in BN rats, although they fail to reduce the EAR. The lack of effect of both NK-1 and NK-2 on the EAR is in agreement with some other studies (40, 41) and suggests a minor contribution, if any, of NKs in allergen-induced immediate bronchoconstriction in BN rats. The discrepancy in the ability of NK-1 antagonists to reduce the LAR between our animal model and Schuiling's guinea pig model may be attributable to several differences between the two studies. The difference in the chemical nature of the NK-1 antagonists and the species under study could lead to differences in pharmacodynamic and the pharmacokinetic properties of these two antagonists. Furthermore, differences in the sensitization procedure, and in the route and timing of drug administration, may also be of importance. Finally, it is plausible that the NK-1 receptors may play a minor role in the development of the LAR in guinea pigs.

Several studies have demonstrated a relationship between activated CD4⁺ T cells, in particular the Th2 subset, and the pathogenesis of asthma (9, 42). Activated CD4⁺ T cells have been detected in the bronchial wall and BAL fluid of subjects with atopic asthma (9, 10). IL-4 and IL-5 produced by Th2 cells are believed to play a potential role in the onset and maintenance of allergic responses. IL-4 is believed to play a crucial role in the regulation of IgE production by B cells and for T cell commitment to the Th2 phenotype (34, 43). In contrast, IL-5 regulates eosinophil maturation, activation, and migration of this leukocyte to sites of allergic inflammation (34, 43). In the present study, IL-4; IL-5-, as well as IFN--producing BAL cells were detected in the lung lavage fluid of BN rats 8 h after the OA challenge, with a predominant expression in the number of Th2 cytokine-positive cells, as previously reported in this animal model (12, 13, 24). We have also reported in a previous study using double-labeling techniques that the majority of cytokine-positive cells detected in the BAL from OA-challenged BN rats were lymphocytes (44). The administration of SR-48968, but not CP-99994, in BN rats reduced the expression of all cytokines measured after allergen exposure. However, neither neurokinin antagonist affected the number of BAL lymphocytes. These results suggest that NK-2, but not NK-1, receptors modulate both Th1 and Th2 cytokine expression in BAL cells. The nonselective inhibition of Th1 and Th2 cytokine expression in BAL cells after the administration of the NK-2 antagonist is also interesting because these results suggest that a decrease in Th2 cytokine expression is not necessarily associated with an increase in the expression of Th1 cytokines, in particular IFN-. We postulate that in vivo other mechanisms implicating, at least in part, the NKs might regulate the predominance of one particular phenotype independent of the conventional model for a reciprocal control of cytokine expression by Th1 and Th2 cytokines. This hypothesis, and our data, are in agreement with the study by Levite (45) showing that neuropeptides such as SP directly induce a marked secretion of both Th1 and Th2 cytokines, and also drive distinct Th1 and Th2 populations to a "forbidden" cytokine secretion characterized by the secretion of Th2 cytokines from a Th1 T cell line and vice versa. Furthermore, the inhibition of both Th1 and Th2 cytokine expression in association with the decrease of airway eosinophilia in this animal model supports assertion that the two types of cytokines act in concert to regulate eosinophil recruitment in allergic airways (46). This concept accords with our results with the NK-1 antagonist, which failed to reduce both Th1 and Th2 cytokine expression and airway eosinophilia.

The potential interaction between NKs and cytokines in the airways and the role played by NK-2 receptors in this process have also been suggested by Kraneveld and coworkers (47), who showed that an NK-2, but not NK-1, antagonist abolished IL-5-induced bronchial hyperresponsiveness in guinea pigs. In this study the cellular targets for IL-5 action and the release and actions of NKs have also not been identified. Although the reduction in the LAR by the administration of an NK-2 antagonist is clearly associated with a modification of the inflammatory process within the airways, such is not the case for the NK-1 antagonist. These data indicate that NK involvement in the LAR is not uniquely linked to eosinophilia and cytokines of the Th2 variety. One possibility is that the NK-1 receptor, which is expressed, as evidenced by RT-PCR, on airway smooth muscle cells from the BN rat and that mediates intracellular calcium mobilization in response to substance P (48), may also allow a spasmogenic effect of NKs. We speculate that the reduction in the LAR by the NK-1 antagonist may therefore be a direct effect on airway smooth muscle.

Few studies have been performed on the potentially beneficial effects of NK antagonist administration in subjects with asthma. Joos and coworkers (49) have shown that the dual NK antagonist FK224 was not effective in reducing asthmatic symptoms, however, this drug was also ineffective in inhibiting NKA-induced bronchoconstriction in subjects with asthma. Interestingly, an NK-1 antagonist (FK-888) has been shown to improve exercise-induced airway narrowing in subjects with asthma (50). Furthermore, SP-induced mast cell-mediator release was not inhibited by pretreatment with hydrocortisone or disodium cromoglycate (DSCG) (51), which suggests that diseases involving SP modulation of mast cell activation may not be successfully treated with corticosteroids or DSCG. Indeed, it is still difficult to speculate on the potential clinical efficacy of NK antagonists in the treatment of asthma. However, our results suggest that in allergic conditions such as asthma, the administration of NK-2 receptor antagonists might be more efficacious than NK-1 antagonists as a treatment to reduce and control airway inflammatory responses.

In conclusion, we have shown that in the atopic BN rat both NK-1 and NK-2 receptors are expressed in lung tissues and that NKs are released after allergen challenge. Our results provide initial in vivo evidence linking neurokinins to the regulation of cytokine expression in cells without discrimination as to their phenotype. In the same context, the results also change the current paradigm for the inhibition of allergic airway inflammation through preferential downregulation of the Th2 cytokines in response to an upregulation of the Th1 cytokines such as is observed with corticosteroids (52). Our results also suggest that a dichotomy exists between NK receptors in regulating airway inflammatory responses. These findings have implications for both the pathophysiology and treatment of asthma.