Published ahead of print on September 17, 2002, doi:10.1164/rccm.200205-387OC
© 2002 American Thoracic Society
Montelukast Prevents Antigen-induced Mucociliary Dysfunction in SheepDivision of Pulmonary and Critical Care Medicine, University of Miami at Mount Sinai Medical Center, Miami Beach, Florida Correspondence and requests for reprints should be addressed to William M. Abraham, Ph.D., Department of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140. E-mail: abraham{at}msmc.com
The cysteinyl leukotrienes are potent proinflammatory mediators that, in addition to their bronchospastic actions, can also contribute to mucociliary dysfunction, a central component of the pathophysiology of asthma. In this study, we determined whether montelukast, a cysteinyl leukotriene 1 receptor antagonist, could prevent and/or reverse antigen-induced mucociliary dysfunction in allergic sheep. We measured tracheal mucus velocity, a marker of mucociliary clearance, before and for 8 hours after antigen challenge in six animals treated with montelukast (0.15 mg/kg, intravenously) 30 minutes before, 1 hour after, or 4 hours after antigen challenge. In the control trial, the sheep received 0.9% saline intravenously at each of the previously mentioned time points. The maximum decrease in tracheal mucus velocity seen in the control trial was 56 ± 4% (mean ± SE) of baseline at 8 hours. Pretreatment with montelukast significantly protected against this reduction. However, treatment at 1 and 4 hours neither protected against nor reversed the allergen-induced fall in tracheal mucus velocity. We conclude that the early release of cysteinyl leukotrienes may contribute to the fall in tracheal mucus velocity that follows acute antigen challenge and that pretreatment with montelukast reduces this impairment.
Key Words: asthma • mucus • leukotrienes • animal model • therapy
The impairment of normal mucociliary function in the airways is an important consequence of asthma exacerbation. Clinically, there is evidence that suggests a relationship between mucociliary dysfunction and asthma severity. Messina and colleagues (1) found that during an asthma attack warranting hospital admission, mucociliary clearance was significantly impaired, with marked improvement after discharge, whereas O'Riordan and colleagues (2) found that patients with expiratory flow limitation during tidal breathing had reduced mucociliary clearance compared with patients without airflow limitation. Thus, although there is a tendency to focus on airflow obstruction in asthma, excessive secretion and clearing of airway luminal mucus are other important pathophysiologic features of the disease that contributes to its morbidity and, in the case of status asthmaticus, its mortality (3). Acute asthma exacerbations in patients can be reproduced in the laboratory by allergen provocation. Under these conditions, mucociliary clearance is reduced in both allergic patients with asthma (4–6) and allergic animal models of asthma (7–10), and thus, allergen challenge in the laboratory offers a mechanism through which to study possible pathways affecting mucociliary dysfunction. After allergen provocation, activated mast cells release a number of preformed and newly synthesized inflammatory mediators. A number of these mediators have been shown to increase mucus secretion and/or impair mucociliary clearance in vivo or in vitro, suggesting that they may contribute to allergen-induced mucociliary dysfunction (6). The cysteinyl leukotrienes (CysLTs) are one such class of newly formed mediators that are both potent bronchconstrictors (11) and mucus secretagogues (12, 13) and when inhaled can cause mucociliary dysfunction (14). Collectively, these data suggest that CysLTs may contribute to antigen-induced mucociliary dysfunction. Previous studies examining the effects of three different CysLT modifiers on antigen-induced mucociliary dysfunction in animals and humans have been equivocal, despite the fact that in some of the same studies the bronchospastic contributions of the CysLTs were blocked (10, 15). Montelukast is a new, orally active CysLT1 receptor antagonist that is currently in use for the treatment of asthma (16–22). Experimentally, the drug blocks CysLT-induced bronchoconstriction, antigen-induced early and late bronchoconstriction, and nonspecific bronchial responsiveness to a variety of challenges (19, 20, 23). Montelukast improves baseline pulmonary function and reduces symptoms (16, 18, 21). Although it is clear that the drug has positive effects on airway smooth muscle, part of its beneficial action in the treatment of asthma could be related to improvement in mucociliary function, especially during asthma exacerbations. This hypothesis, however, has not been tested. In this study, then, we used the model of antigen provocation in sheep with airway hypersensitivity to Ascaris sum antigen to determine whether montelukast could protect against and/or reverse the antigen-induced depression in tracheal mucus velocity (TMV), a marker of mucociliary clearance (24).
This study was approved by the Mount Sinai Medical Center Animal Research Committee, which is responsible for ensuring the humane care and use of laboratory animals. A total of nine adult ewes with natural Ascaris suum hypersensitivity and previously demonstrated Ascaris suum–induced depression of TMV were used for this study. Animals were nasally intubated with a modified endotracheal tube after topical anesthesia of the nasal passages with 2% lidocaine. All animals were conscious throughout the study. TMV was measured in vivo by a previously described roentgenographic technique (9, 24, 25). Between 8 and 10 radiopaque Teflon/bismuth trioxide disks were insufflated into the trachea. Individual disk velocities were calculated by measuring the cephalad–axial distance traveled by each disk during a 1-minute observation period. The mean value of all individual disk velocities was calculated for each run. Intravenous administration of saline or drug was performed via an external jugular vein. Once the vein was localized and the skin area shaved and sterilized with 70% isopropyl alcohol, a 22-gauge Surfler intravenous catheter was inserted into the vessel and taped into place. The catheter was then connected to a 3-way stopcock (Medex, Hilliard, OH), which was accessed throughout the study. Ascaris suum extract was diluted in phosphate-buffered saline to a concentration of 82,000 protein nitrogen U/ml and delivered as an aerosol (20 breaths/minute for 20 minutes) using a disposable medical nebulizer (Raindrop; Puritan Bennett, Lenexa, KS). A dosimeter system was used to control aerosol delivery as previously described (9).
Protocol The studies were done in two phases. In the first series of studies, we determined whether pretreatment with montelukast would affect the antigen-induced responses in TMV. The sheep received saline infusion at all three time points described previously here (control), or the sheep received montelukast 30 minutes before antigen challenge, with saline infusions at 1 and 4 hours after challenge. These experiments were randomized to each other. In the remaining two trials, which were done after the completion of the initial experiments, the sheep were administered montelukast at either 1 or 4 hours after antigen challenge, receiving saline at the other time points described previously here. These studies were randomized to each other. To ensure that the dose of montelukast used was effective against leukotriene D4(LTD4)-induced decreases in TMV, three sheep were challenged with aerosolized LTD4 (50 µg) with and without montelukast pretreatment. TMV was measured at baseline and then 30 minutes, 1 hour, 2 hours, and 3 hours after LTD4 challenge. Finally, to ensure that montelukast itself had no effect on TMV, we treated three sheep with montelukast alone and followed TMV for up to 8 hours.
Statistical Analysis
Baseline TMV values for the individual sheep for all experiments are given in Table 1. When the sheep were treated with saline 30 minutes before antigen challenge, the expected decrease in TMV was observed. Baseline TMV was 9.2 ± 0.4 mm/minute and then progressively decreased to 56 ± 4% of baseline by 8 hours after antigen challenge (Figure 1). When the sheep were treated with montelukast 30 minutes before antigen challenge, there was significant protection against the antigen-induced fall in TMV. At baseline, TMV was 8.9 ± 0.2 mm/minute, and at 8 hours, TMV remained at 91 ± 4% of baseline (p < 0.05 versus antigen alone). If, however, montelukast was given either 1 or 4 hours after antigen challenge, no protective effect was seen. In the 1-hour postchallenge treatment trial, baseline TMV was 8.4 ± 0.4 mm/minute and fell to 70 ± 4% of baseline by 8 hours, and in the 4-hour treatment trial, baseline TMV was 8.8 ± 0.3 mm/minute and fell to 59 ± 2% of baseline by 8 hours (Figure 1). Treatment with montelukast alone did not affect TMV (Figure 2).
Figure 3 illustrates that the dose of montelukast used in this study was effective against LTD4-induced slowing of TMV. The maximum fall in TMV occurred 1 hour after LTD4 challenge. LTD4 alone reduced TMV to 59 ± 4% of baseline (9.9 ± 0.4 mm/minute), whereas in the presence of montelukast, the TMV value at 1 hour was 92 ± 5% of baseline (9.0 ± 0.2 mm/minute) (p < 0.05 versus LTD4 alone).
The results of this study indicate that pretreatment with intravenous montelukast at a clinically relevant dose prevents antigen-induced slowing of TMV but that posttreatment with intravenous montelukast does not reverse the established antigen-induced impairment in TMV. Because the dose of montelukast used also blocked LTD4-induced decreases in TMV, these findings suggest that the CysLTs play an early role in the pathogenesis of allergen-induced mucociliary dysfunction. The failure of montelukast to reverse the allergen-induced decrease in TMV may be reflective of its inability to attenuate the mucociliary effects of subsequently recruited inflammatory cells (neutrophils)/mediators (9). The protocol design in this experiment is based on our experience with this animal model (7, 9). We have previously shown that in these animals the fall in TMV is dependent on challenge with the specific antigen to which they are sensitive and not a nonspecific allergen such as ragweed or after challenges with saline (7, 9). The time to recovery after allergen provocation is within 2 weeks, and based on the animals' starting TMV for the four trials, this appeared to be the case in this study. Likewise, the instrumentation used in these studies is designed to minimize nonspecific effects on TMV. Although the animals are intubated throughout the course of the study, the cuff of the tracheal tube is placed just below the vocal cords and is inflated only during the time of allergen provocation, which minimizes impairment of TMV caused by prolonged cuff inflation. Finally, between measurement periods, the animals' breathe warm humidified air to prevent artificial reductions in TMV due to drying of the airway mucosa.
The observation that antigen challenge induces an impairment in TMV, which becomes apparent 2 hours after challenge and reaches a maximum approximately 6 hours after challenge is consistent with previous studies from this laboratory (9, 26); however, the mechanism behind this impairment has yet to be fully elucidated. One of our more recent studies suggests an important role for neutrophil elastase in this event. Thus, the natural elastase inhibitors The difference in the results between these studies and the previous studies may be related to the difference in potency among the compounds (i.e., montelukast > pranlukast > LY171883). The difference in potency could also explain why allergen-induced early- and late-phase bronchoconstrictor responses in sheep are significantly inhibited to varying degrees with these CystLT1 antagonists including, LY171883 (15), zafirlukast (ICI 204,219) (27), pranlukast (ONO-1078) (10), but only montelukast was effective in protecting against both the antigen-induced bronchoconstrictor (19) responses as well as the antigen-induced fall in TMV. The results of the aforementioned studies in sheep appear to differ from those reported with the prototypic CysLT antagonist, FPL-55712, in dogs (28) and humans (4), in which FPL-55712 was reported to block the antigen-induced depression in TMV. However, in both the dog and human studies, the TMV measurements were only performed 2 hours after antigen challenge. Furthermore, in both studies, there was an initial increase in TMV immediately after antigen challenge that resolved slowly over this 2-hour period. Thus, if these earlier studies were performed for longer than 2 hours, TMV may have continued to decline even in the presence of the drug. Subsequent studies examining the effects of FPL-55712 on the LTD4-induced depression in TMV in sheep (14, 29) support this concept. In these studies, unlike this study when montelukast did protect against the LTD4-induced fall in TMV, FPL-55712 did not block the LTD4-induced depression in TMV. It is not surprising that inhibiting the actions of the CysLTs can affect mucociliary dysfunction. CysLTs have been shown to stimulate mucus secretion from in vitro preparations from a variety of species, including sheep (13), guinea pigs (30), and humans (12), and can themselves slow TMV (14). Although, two types of CysLT receptors, CysLT1 and CysLT2, have been identified (31), the protection afforded by montelukast, a CysLT1 antagonist, against LTD4- and antigen-induced induced depressions in TMV suggests that these actions are mediated through blockade of CysLT1 receptors in sheep. This conclusion is supported by our previous findings showing trends toward protection with the weaker CysLT1 antagonist, LY171883, and pranlukast (ONO-1078). These effects appear to be different from what has been reported in sheep airways in vitro, which suggests that CysLT2 receptors predominate (32). If this is the case, however, it is unclear why CysLT1 receptor antagonists block both antigen and LTD4-induced responses in this model in vivo (10, 15, 19, 27, 33). The effect of montelukast is limited to prophylaxis because giving the drug after antigen challenge did not reverse the fall in TMV. As stated, reversal of the antigen-induced fall in TMV has been observed with elastase inhibitors (9). Although not tested directly in this study, one possible mechanism that could explain such results would be that the early release of CysLTs serves as an important signal for mast cell–mediated cytokine/chemokine release. In particular, it is conceivable that the CysLTs could modulate interleukin-8 release, which is specific for neutrophil recruitment and activation and which in turn is important for the subsequent events leading to the prolonged depression in TMV. The fact that montelukast failed to reverse the antigen-induced fall in TMV would suggest that these signaling events occur within 1 hour of stimulation. In support of such a mechanism is the identification of CysLT1 receptors on mast cells (34), that mast cells secrete interleukin-8 (35), and the fact that sheep treated with CysLT receptor antagonists have a reduced airway neutrophil response after allergen challenge (8, 36). Thus, CysLT1 stimulation could be an important signaling mechanism in terms of recruiting inflammatory cells that have effects on TMV. In summary, the early administration of the CysLT1 receptor antagonist montelukast was effective in preventing LTD4- and allergen-induced depressions in TMV, a marker of mucociliary clearance. Thus, prophylactic use of this drug could be beneficial in reducing the mucociliary dysfunction that occurs during asthma exacerbations and in so doing lessen the overall severity of the attack.
Supported in part by an educational grant from Merck, Inc. This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form May 3, 2002; accepted in final form September 12, 2002
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
INTRODUCTION
1 protease inhibitor and secretory leukocyte protease inhibitor as well as chemical inhibitors of neutrophil elastase such as ICI 200,355 can prevent and reverse the antigen-induced fall in TMV (
