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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 25-32, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4532

Involvement of the Cysteinyl–Leukotrienes in Allergen-Induced Airway Eosinophilia and Hyperresponsiveness in the Mouse

Seok-Yong Eum, Karim Maghni, Qutayba Hamid, Holly Campbell, David H. Eidelman and James G. Martin

Meakins-Christie Laboratories and Respiratory Division, Department of Medicine, McGill University, Montreal, Quebec, Canada

Address correspondence to: Dr. James G. Martin, Meakins-Christie Laboratories, McGill University, 3626, Rue St-Urbain, Montreal, QC, Canada H2X 2P2. E-mail: jmartin{at}meakins.lan.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The leukotriene modifiers are a novel generation of therapeutic agents in the treatment of allergic asthma. However, the mechanisms by which the cysteinyl (cys) leukotrienes (LTs) participate in allergen-induced airway eosinophilia and airway hyperresponsiveness (AHR) are still unclear. In the present study, we have investigated the role of cys-LTs in ovalbumin (OVA)-induced airway responses in a murine model of asthma. Montelukast (3 or 10 mg/kg), a selective cys-LT1 receptor antagonist, reduced airway eosinophilia and AHR after OVA challenge. The levels of interleukin (IL)-5 and eotaxin in the bronchoalveolar lavage fluid (BALF) from montelukast-treated (3 mg/kg) mice were unaffected, although a decrease in IL-5 was observed with a dose of 10 mg/kg. LTD4 (50 ng) instilled intranasally to immunized mice augmented macrophages in the BALF, but in conjunction with OVA challenge it caused BALF eosinophilia and neutrophilia when given before challenge and BALF neutrophilia but not eosinophilia when given 2 h after challenge. However, there were no increases of IL-5 or eotaxin in BALF following LTD4 treatment. Repeated instillations of LTD4 to immunized mice, mimicking allergen challenge, did not induce AHR but in conjunction with OVA challenge LTD4 enhanced AHR. These results indicate that allergen-induced eosinophilia and AHR are in part mediated by the cys-LT1 receptor, and that, although LTD4 alone has no effect on airway eosinophilia, in conjunction with antigenic stimulation it potentiates the degree of airway inflammation and AHR.

Abbreviations: airway hyperresponsiveness, AHR • bronchoalveolar lavage, BAL • BAL fluid, BALF • cysteinyl, cys • enzyme-linked immunosorbent assay, ELISA • interleukin, IL • leukotrienes, LTs • methacholine, Mch • ovalbumin, OVA • respiratory system resistance, Rrs • regulated on activation, normal T cells expressed and secreted, RANTES


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is a chronic airway inflammatory disease characterized by an infiltration of the airways by inflammatory cells, including eosinophils, mast cells, and T lymphocytes (1). Even though a causal relationship is not proven, airway inflammation after allergen challenge is the likely cause of airway hyperresponsiveness (AHR). Leukotrienes (LTs), lipid mediators generated from arachidonic acid by the action of 5-lipoxygenase, play an important role in the pathogenesis of asthma (2, 3). Several airway cell types, including mast cells, eosinophils, macrophages, neutrophils, and epithelial cells, can synthesize LTs in response to a variety of stimuli (2). Increased amounts of LTs are found in the bronchoalveolar lavage (BAL) fluid (BALF) of asthmatics compared with normal subjects (4) and allergen challenge induces the release of LTs into the airways in several animal models of asthma (5–7).

LTB4 induces leukocyte chemotaxis through an action on BLT receptors, whereas the cysteinyl (cys)-LTs (LTC4, LTD4 and LTE4) principally cause contraction of airway muscle, increase microvascular permeability, stimulate mucus secretion, and induce smooth-muscle cell proliferation (2, 5, 8). Cys-LTs have also been shown to participate in the induction of AHR in patients with asthma; the inhalation of LTC4, LTD4, and LTE4 increases the airway responsiveness to histamine (9). Recent studies have shown a reduction in airway eosinophilia in asthmatic subjects treated with cys-LT1 receptor antagonists (10, 11), suggesting chemotactic effects of the cys-LTs in vivo. Indeed, cys-LTs have chemotactic activity in vitro on human eosinophils (12, 13). Similar effects have been observed in vivo, where inhaled LTD4 or LTE4 causes an increase of eosinophil numbers in the airways in human subjects with asthma (14, 15) and in the guinea-pig (16, 17). Other in vivo studies have failed to demonstrate the induction of eosinophilia by LTD4 (18, 19), suggesting that the effect of LTD4 on eosinophilia may depend on the characteristics of the subjects, on the species, or possibly the necessity for concomitant stimuli. The mechanisms by which cys-LTs induce airway eosinophilia and AHR are unclear, and whether the effect of cys-LTs on eosinophilia contributes to AHR or is independent of such effects is also still unknown.

The migration of eosinophils into the airways involves several steps, including eosinophilopoiesis and release from the bone marrow, upregulation of adhesion molecules on eosinophils and on the vascular endothelium, and the release of chemoattractant molecules within the airway tissues (20). The eosinophilopoietic cytokines, including interleukin (IL)-3, IL-5, granulocyte–macrophage colony-stimulating factor, and chemokines, including regulated on activation, normal T cells expressed and secreted (RANTES) and eotaxin, are thought to play a major role in eosinophil differentiation and trafficking (20, 21). It is plausible that cys-LTs are released after allergen challenge and promote airway eosinophilia by modulating the expression of cytokines (22) or chemokines as well as by inducing AHR. In the present study, we evaluated the effect of montelukast, a potent, specific cys-LT1 receptor antagonist on allergen-induced airway responses in a murine model. In addition, we tested the effect of LTD4 alone and in combination with allergen exposure by examining the effects of its instillation into the trachea on airway inflammation and AHR. We measured the levels of IL-5 and eotaxin in BALF, arguably the most important eosinophil-specific cytokine and chemokine, respectively, to examine their roles in mediating the effects of cys-LTs on allergen-induced airway eosinophilia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization and Antigen Challenge of Mice
Male Balb/c mice of 8–10 wk (24–25 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and immunized twice at 7 d intervals with injections of 100 µg ovalbumin (OVA) subcutaneously in 0.4 ml of a 4 mg/ml suspension of Al(OH)3. One week after the second injection, mice were challenged intranasally under light anesthesia, once per day for 3 consecutive days with 10 µg OVA in 50 µL of sterile saline. The protocols were approved by an institutional animal care committee.

Evaluation of Airway Responsiveness
Airway responsiveness was measured 48 h after the last challenge. Mice were sedated (xylazine, 8 mg/kg intraperitoneally), anaesthetized (pentobarbital, 70 mg/kg i.p.), tracheostomized, paralyzed (doxacurium, 0.5 mg/kg intraperitoneally) and placed on a small animal ventilator (Flexivent, SCIREQ, Montreal, PQ, Canada). Animals were ventilated quasisinusoidally (150 breaths/min, 6 ml/kg, positive end-expiratory pressure 1.5 kPa). Following a standard volume history, small amplitude volume oscillations at frequencies of 0.9, 4.8, and 10.4 Hz were applied at constant lung volume to the airway opening for 16 s and respiratory system resistance (Rrs) was calculated. Preliminary experiments indicated that responses at 0.9 Hz exceed those at other frequencies; thus, only results for this frequency of oscillation are reported. Methacholine (Mch) was injected through the jugular vein every 5 min at doses of 10, 33, 100, 330, and 1,000 µg /kg. At 1,000 µg /kg Mch, the model fit for the calculation of Rrs was frequently poor; these data are not reported.

The data acquisition process was modified for measurements of responsiveness to Mch in animals treated with OVA and concomitantly exposed to LTD4. For the measurement of Rrs a 0.9 Hz oscillation of 150 µL was applied at constant lung volume for consecutive 4 s periods after the bolus injection of Mch. The highest value of Rrs was taken for the analysis of responsiveness. The ventilatory parameters were otherwise identical to the above.

Procedures for BAL
After the measurement of airway responses, BAL was performed using phosphate-buffered saline instilled through the tracheostomy tube. A total volume of 4.5 ml was instilled in nine successive aliquots of 0.5 ml. The return from the first 0.5 ml of lavage fluid was centrifuged and the supernatant was used for the determination of IL-5 and eotaxin by enzyme-linked immunosorbent assay (ELISA). The pelleted cells were recovered and reconstituted in buffer and were pooled with the cells recovered by centrifugation of the subsequent eight aliquots of lavage fluid. Total cell numbers were counted with a hemacytometer. The cytospin slides of BAL cells were prepared using a cytocentrifuge (Cytospin model II; Shandon, Pittsburg, PA) and stained with May-Grünwald-Giemsa stain. Differential cell counts were determined by light microscopy from a count of at least 200 cells. Absolute cell numbers were also calculated.

Administration of Montelukast Sodium and LTD4
Montelukast sodium in 1% methyl cellulose was administered by gavage to immunized and fasted mice at 3 or 10 mg/kg one day before the first of three antigen challenges and 4 h before each antigen challenge. The same volume of vehicle was given to control mice. BAL was performed at 24 h and 48 h after the last challenge, whereas airway responsiveness was evaluated only at 48 h after the last challenge.

To test the effect of LTD4 administered alone on responsiveness to Mch, we instilled LTD4 (50 ng; Sigma-Aldrich, Milwaukee, WI) intranasally three times in a similar manner to the protocol employed for allergen-induced AHR. The same volume of vehicle was given to control mice. At 48 h after the last instillation of LTD4, BAL and responsiveness to Mch were evaluated.

To examine the possibility that an LTD4/allergen exposure interaction occurs and modifies the degree of airway inflammation and airway responsiveness, LTD4 (50 ng) was instilled intranasally to immunized mice in association with OVA challenge. We tested the effect of LTD4, administered once either 5 min before or 2 h after OVA challenge, on BAL leukocyte numbers. BAL was performed 24 h after LTD4 or OVA instillation. Using separate animals, we tested the effects of LTD4 and allergen challenge on airway responsiveness to Mch but measurements were made at 48 h after OVA challenge. This time point was chosen based on the time at which AHR was present after OVA challenge alone.

IL-5 and Eotaxin Levels in BALF
The levels of IL-5 and eotaxin in BALF were measured by ELISA. Briefly, 96-well ELISA plates were coated overnight with 50 µL of an anti-mouse IL-5 monoclonal antibody (TRFK-5; R&D Systems, Inc., Minneapolis, MN) at 2 µg/ml or with 100 µL of an anti-mouse eotaxin polyclonal antibody (R&D Systems, Inc.) at 0.2 µg/ml in 0.1 M Na2HPO4 (pH 9.0) at 4°C. The plates were blocked with PBS containing 10% fetal bovine serum (200 µl/well) for 1 h and then were washed with PBS containing 0.05% Tween 20 prior to incubation with the BAL supernatants (100 µl/well) for 2 h at room temperature. Following a further wash with PBS/Tween 20, the plates were incubated with biotinylated anti-mouse IL-5 monoclonal antibody (TRFK-4, Pharmingen, San Diego, CA) at 1 µg/ml, or with biotinylated anti-mouse eotaxin polyclonal antibody (R&D Systems, Inc.) at 25 ng/ml for 1 h at room temperature. After washing, the plates were incubated with 100 µL of streptavidin-horseradish peroxidase (Pharmingen) for 30 min and washed again with PBS/Tween 20. ABTS substrate solution containing H2O2 was added to the wells and the absorbance was read at a wavelength of 405 nm. Under these conditions, the assays were sensitive to < 10 pg/ml of murine IL-5 or eotaxin.

Statistical analysis
The data are presented as mean ± SEM. Statistical comparisons were performed using one-way analysis of variance followed by Fisher's LSD test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Montelukast on Airway Eosinophilia and AHR after Antigen Challenge in Immunized Mice
After three consecutive OVA challenges at 1 d intervals, total cell and eosinophil numbers in BALF were increased in OVA-challenged mice (41.6 ± 5.0 and 19.9 ± 4.4 x 104 cells, respectively) compared with saline-challenged animals (11.6 ± 1.5 and 0.1 ± 0.04 x 104 cells, P < 0.001) (Figure 1). Montelukast (3 mg/kg) reduced total cells to 21.3 ± 2.9 and eosinophils to 6.3 ± 1.3 x 104 cells (P < 0.01). Similar results were observed with the 10 mg/kg dose of montelukast (Figure 1). There were no statistically significant differences in macrophage, neutrophil, or lymphocyte numbers (data not shown).



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  		Figure 1. Effect of montelukast (Mk, 3 mg and 10 mg/kg) on the recruitment of eosinophils into the airways after three OVA challenges. BAL was performed 48 h after the last of three challenges and montelukast was administered by gavage before each OVA challenge. Each bar represents the mean (± SEM) of six mice. OVA challenge increased total cell and eosinophil numbers (P < 0.001) in BALF compared with saline-challenged mice and significant differences between vehicle-treated OVA-challenged mice and Mk-treated OVA-challenged animals are indicated.

 
The baselines of respiratory resistance among vehicle-treated saline-challenged, vehicle-treated OVA-challenged, and montelukast-treated OVA-challenged group were not statistically different (0.33 ± 0.01, 0.34 ± 0.04, 0.28 ± 0.01 [3 mg, montelukast] and 0.34 ± 0.02 [10 mg] kPa · s/ml, respectively). OVA-challenged mice showed AHR as measured by responses to intravenous Mch. Respiratory resistance reached a value of 18.5 ± 6.1 kPa · s/ml at 330 µg/kg Mch (P < 0.001) compared with saline-challenged mice (2.4 ± 0.3 kPa · s/ml) (Figure 2). At both doses, treatment with montelukast reduced responses to 330 µg/kg Mch (6.8 ± 1.7 kPa · s/ml for 3 mg/kg and 4.8 ± 1.0 kPa · s/ml for 10 mg/kg; P < 0.01) compared with vehicle-treated OVA-challenged mice (Figure 2). The lack of significant dose-dependency indicates that a maximal effect was achieved at 3 mg/kg of montelukast.



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  		Figure 2. Effect of montelukast (Mk) on airway responsiveness to Mch in immunized Balb/c mice following three OVA challenges. Montelukast, 3 and 10 mg/kg, attenuates OVA-induced AHR to comparable degrees. Each bar represents the mean (± SEM) of six mice. OVA challenge increased airway responsiveness (P < 0.001) compared with that in saline-challenged mice and significant differences between vehicle-treated OVA-challenged mice and Mk-treated OVA-challenged animals are indicated.

 
The effects of montelukast treatment on airway responsiveness to Mch were tested. Animals were treated with montelukast (3 mg/kg intravenous) and measurements of responsiveness to Mch were made 15 min later. There was no significant difference between montelukast-treated and control animals in baseline Rrs or in responses to Mch at any of the doses tested (Table 1).


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  		TABLE 1 Effect of montelukast on respiratory system resistance after intravenous methacholine

 
Effect of Montelukast on IL-5 and Eotaxin Levels in BALF after Antigen Challenges in Immunized Mice
At the moment that AHR was present, 48 h after the last antigen challenge, the IL-5 levels in the BALF of antigen-challenged mice (72.9 ± 6.9 pg/ml) were significantly higher than saline-challenged animals (38.3 ± 4.1 pg/ml, P < 0.01) (Figure 3). Although 3 and 10 mg/kg of montelukast reduced eosinophilia and AHR, only at 10 mg/kg of montelukast was there a reduction in IL-5 (50.9 ± 3.9 pg/ml; P < 0.05) (Figure 3). No differences in eotaxin levels were observed among groups, either after OVA challenge or after montelukast treatment (Figure 3).



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  		Figure 3. Effect of montelukast (Mk) on the production of IL-5 and eotaxin in the BALF after OVA challenges. BAL was performed at 48 h after last OVA challenge. Montelukast reduces IL-5 levels at 10 mg/kg, but no differences are observed in eotaxin levels among groups. Each bar represents the mean (± SEM) of 6 mice. Significant differences are indicated.

 
Effect of Instilled LTD4 on the Recruitment of Eosinophils into the Airways and the Levels of IL-5 and Eotaxin in BALF
To examine the effect of LTD4 on eosinophil recruitment in the absence of antigen challenge, we performed a BAL 24 h after LTD4 (50 ng) instillation. In mice that received LTD4 alone, the total cell numbers were increased to almost twice the vehicle-instilled control values (P < 0.05) (Figure 4 and Table 2) and more than 90% of these cells were macrophages. LTD4 (50 ng) was also administered in conjunction with antigen either 5 min before or 2 h after OVA challenge. When LTD4 was instilled just before OVA challenge, there were no changes in macrophage or lymphocyte numbers in BALF, but significant increases in eosinophils (P < 0.01) and neutrophils (P < 0.01) were observed compared with OVA-challenged mice (Figure 4 and Table 2). However, when LTD4 was instilled 2 h after OVA challenge, eosinophil numbers were unchanged but neutrophils were significantly increased (P < 0.001) (Figure 4 and Table 2).



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  		Figure 4. Effect of LTD4 instillation on the airway eosinophilia. LTD4 was instilled intranasally alone or in combination with OVA challenge. LTD4 was instilled either 5 min before (LTD4 + OVA) or 2 h after OVA challenge (OVA + LTD4) and BAL was performed 24 h after challenge. LTD4, when instilled before OVA challenge, promotes airway eosinophilia. Each bar represents the mean (± SEM) of five to eight mice. Significant differences are indicated.

 

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  		TABLE 2 Effect of instilled leukotriene D4 on bronchoalveolar lavage cells composition in immunized Balb/c mice

 
To test the possibility that the effect of LTD4 on the recruitment of eosinophils into the BALF was caused by increases in either IL-5 or eotaxin, we measured the levels of IL-5 and eotaxin in the BALF 24 h after LTD4 instillation, with and without concomitant OVA challenge. Similar levels of IL-5 were detected in vehicle- and LTD4-instilled mice (26.9 ± 10.6 and 33.5 ± 5.9 pg/ml, respectively). Significant increases in IL-5 levels were observed after OVA challenge (125.7 ± 8.5 pg/ml) compared with vehicle-challenged mice (P < 0.001). However, LTD4, instilled either before or after OVA challenge, effected no changes in IL-5 compared with OVA challenge alone (Figure 5). Detectable levels of eotaxin were found in BALF, but there were no differences among groups (Figure 5).



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  		Figure 5. Effect of LTD4 instillation on the production of IL-5 and eotaxin in the BALF. LTD4 was instilled as described in Figure 4 and BAL was performed at 24 h after challenge. OVA increases IL-5 levels but LTD4 does not affect it. No differences of eotaxin levels are observed among groups. Each bar represents the mean (± SEM) of five to eight mice. Significant differences are indicated.

 
Effects of Instilled LTD4 on the Induction of AHR
To evaluate if LTD4 alone could induce AHR, LTD4 was instilled three times to mimic OVA challenges and BAL and AHR were evaluated 48 h after the last instillation. Similar to the result after one instillation of LTD4, more than 90% of BAL cells were macrophages in both vehicle- and LTD4-instilled groups, but their number was significantly increased in LTD4-instilled mice compared with vehicle-instilled animals (13.1 ± 1.5 and 7.4 ± 0.6 x 104 cells, respectively; P < 0.05). No significant changes were observed in the other BAL leukocytes between the two groups (data not shown). The baseline Rrs was similar between vehicle- and LTD4-instilled mice (0.38 ± 0.02 and 0.36 ± 0.01 kPa · s/ml, respectively). The mice that received LTD4 showed no changes in responsiveness to intravenous Mch compared with vehicle-treated mice (Table 3).


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  		TABLE 3 Effect of intranasal leukotriene D4 on respiratory system resistance after methacholine

 
The three administrations of LTD4 in conjunction with OVA challenge caused substantial changes in baseline pulmonary function. The values of Rrs were significantly higher in the animals that were LTD4-treated and OVA-challenged than in the sham and OVA-challenged groups (P < 0.002; Figure 6). The LTD4-treated OVA-challenged animals were also significantly more hyperresponsive to Mch than were the OVA-challenged and sham-challenged animals. The augmentation of AHR by LTD4 was of comparable degree, irrespective of the timing of the administration of LTD4 in relationship to OVA challenges.



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  		Figure 6. Effect of LTD4 instillation on the induction of AHR by OVA challenge. LTD4 was instilled three times to OVA-immunized and -challenged mice either 5 min prior to OVA challenges or 2 h after challenges. Airway responsiveness to intravenous Mch was measured 48 h after the last challenge. Statistical analysis was performed for the baseline values of Rrs and for values of Rrs after 330 ug/kg Mch. Each bar represents the mean (± SEM) for seven to eight mice per group. Significant differences are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cys-LTs exert their biologic actions by binding to and activating specific receptors located on the cell membranes of target cells, and are important mediators of allergic airway responses (2). In this study, to evaluate the actions of cys-LTs in the airway responses in a murine model of allergic asthma we used montelukast, a selective antagonist of the cys-LT1 receptor (23). In the model, marked eosinophilia and AHR developed following three allergen challenges, and both eosinophilia and AHR were attenuated by montelukast. These results are consistent with prior observations of cys-LT antagonists in allergic airway responses of humans (24), sheep (25), monkeys (26), rats (27), and mice (28). Corroboration of the importance of 5-lipoxygenase products in allergic airway responses has also been obtained using knock-out mice in which airway eosinophilia and AHR were also reduced compared with wild-type mice (29). Curiously, Henderson and coworkers (30) failed to demonstrate an effect of 5-lipoxygenase inhibition on allergen-induced AHR, although marked effects on eosinophilia were shown. The reason for the discrepant results is not clear because the same strain of mouse was studied (Balb/C) and similar methods were used for challenge testing as in the current study.

Although clear evidence of an effect of a cys-LT1 receptor antagonist on eosinophilia has been obtained, the mechanism by which this effect is accomplished is obscure. LTD4 has been shown to promote the recruitment of eosinophils into the airways of guinea pigs (22) and of subjects with asthma (15). Chemotactic effects have also been shown in vivo following instillation of LTD4 into the conjunctiva of the guinea pig (13). However, other studies have failed to show recruitment of eosinophils into the airways of humans and rats with asthma after administration of LTD4 (18, 19), suggesting that the chemotactic effects of cys-LTs for the eosinophil found in vitro (12) may not pertain in vivo in all species. The current study may help to explain some of the discrepancies in the literature. Whereas LTD4 alone did not induce eosinophilia, LTD4 in conjunction with allergen challenge was associated with substantial augmentation of the degree of eosinophilia. Perhaps LTD4 acts in conjunction with other priming or chemotactic signals without which it may not recruit eosinophils from the blood stream or bone marrow. Curiously, the augmentation of eosinophilia by LTD4 and OVA challenge did not occur when the administration of LTD4 was delayed to two hours after allergen challenge, suggesting that the interaction of LTD4 with other signals occurs early after the initiation of the response to allergen. The effect of LTD4 on BALF cells was not limited to the eosinophil; there was also an augmentation of macrophage numbers. Functional cys-LT1 receptors have been identified on alveolar macrophages (31), but whether this receptor mediates chemoattractant effects does not appear to have been investigated. There was also a pronounced effect on neutrophil numbers, again by mechanisms that have not been explored.

We were particularly interested in the role that either IL-5 or eotaxin might play in mediating the effects of montelukast on eosinophilia. These cytokines were chosen for study because of the dominant role that they play in mediating allergen-induced eosinophilia in several species, including the mouse (32). The induction of airway eosinophilia by LTD4 in the guinea pig is inhibited by an anti–IL-5 antibody (22). This result has been interpreted as indicating that IL-5 may be involved in the mechanism of LTD4's actions. Two pieces of evidence suggest that this may not be the case in the mouse. LTD4 alone or in conjunction with OVA challenge did not affect either IL-5 levels in the BAL or induce an airway eosinophilia. A caveat is the fact that we made measurements of IL-5 at a single time point, 24 h after exposure. However, in support of our argument is the finding that the lower of the two doses of montelukast (3 mg/kg) that we used also did not reduce IL-5 at 48 h after challenge despite a significant reduction of eosinophilia. The higher dose (10 mg/kg) did reduce IL-5, indicating that the threshold for the effects of the LT modifier on allergen-induced AHR and eosinophilia is not the same as for the suppression of IL-5 in this model. In the BN rat montelukast also caused a reduction in IL-5 messenger RNA–expressing cells in the BALF in association with inhibition of allergen-induced late responses (27). The greater efficacy in inhibiting IL-5 in the rat may be attributable to the fact that the same dose (3 mg/kg) of montelukast was given by the intravenous route just prior to challenge. In addition, multiple allergen challenges were employed in the mouse, whereas a single challenge was used in the rat. Furthermore, the time points examined are quite different, 8 h after challenge in the rat and 48 h in the mouse.

Eotaxin, an eosinophil-specific chemokine, plays a major role in the eosinophil migration into the airways (33), potentially providing an alternative mechanism by which cys-LT receptor antagonists could reduce eosinophilia. An interaction between eotaxin and cys-LTs has been shown previously. The instillation of eotaxin induces airway eosinophilia and increases the LTC4 levels in the BALF of IL-5–transgenic mice (34). However, in the current study, there were no changes of eotaxin levels in BALF after treatment with montelukast or LTD4 instillation, either alone or in conjunction with antigen, arguing that eotaxin may not be involved in the promotion of airway eosinophilia by LTD4 in this model. Alternatively, the lack of an OVA-induced increase in eotaxin suggests that the compartment sampled by BAL may not be a good reflection of eotaxin levels in the airways.

The mechanism by which LTD4 modulates allergen-induced AHR is not yet established. There are contradictory reports about the effect of LTD4 on airway responsiveness in humans with asthma (9, 18). In the guinea pig, inhalation of LTD4 failed to induce AHR despite airway eosinophilia (22), indicating that LTD4-induced airway eosinophilia per se is not sufficient to cause AHR. To examine the role of LTD4 in induction of AHR in our mouse model, we instilled LTD4 three times and airway responsiveness was measured 48 h after the last instillation, similar to the antigen challenge protocol required to evoke AHR. Instillations of LTD4 in this manner failed to induce AHR. However, when given in conjunction with OVA challenge, LTD4 augmented the degree of OVA-induced AHR, indicating that the cys-LT1 receptor is involved in the mediation of AHR after allergen challenge by interaction with other factors. Interestingly, the augmentation of OVA-induced AHR was unaffected by the timing of administration of LTD4 although the pattern of inflammation was affected differently. When given before OVA challenge, LTD4 accentuated eosinophilia, and when given after challenge it accentuated neutrophilia. A dissociation of eosinophilia from AHR has been previously reported for a murine model of asthma (35).

In summary we have examined the role of the cys-LT1 receptor in a murine model of allergic asthma, more specifically in the induction of airway eosinophilia and AHR. By using montelukast, a specific cys-LT1 receptor antagonist, we have shown an important role for cys-LTs in OVA-induced AHR and eosinophilic inflammation. By examining the effects of administration of LTD4 alone and in conjunction with OVA challenge on airway responsiveness to Mch and BALF composition, we conclude that LTD4 by itself does not induce airway eosinophilia and AHR, but may amplify airway inflammation and AHR induced by antigen challenge. Furthermore, the role of the cys-LT1 receptor in the induction of eosinophilia and AHR appears unlikely to be mediated by IL-5 and eotaxin.


   Acknowledgments
 
Supported by a fellowship award and grant from Merck, Inc. and by MRC grant 10381.

Received in original form February 13, 2001

Received in final form July 22, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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