INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway inflammation is an important characteristic in patients with current symptomatic asthma (1). Sputum analysis has shown to be a valid and reliable means of measuring airway inflammation (2). With this technique, airway secretions can be collected noninvasively and repeatedly (3). Although repeated sputum inductions have been shown to cause a change in the composition of sputum, eosinophil counts remain unaffected (4).

Eosinophils and mast cells have been the most consistent types of cells found to be present in increased numbers in asthmatic airways (3, 5), and appear to participate in symptomatic asthma (6). Allergen inhalation can result in bronchoconstriction and airway hyperresponsiveness (AHR) in sensitized asthmatic individuals, as well as causing an increase in airway eosinophil numbers (7, 8). In contrast, inhalation of nonsensitizing stimuli, such as methacholine, does not induce an inflammatory response in asthmatic airways (9).

Both eosinophils and mast cells are sources of the cysteinyl leukotrienes (leukotriene C₄ [LTC₄], LTD₄, and LTE₄) (12), which are lipid mediators known to cause bronchoconstriction in human airways (13), and which play an important role in the pathogenesis of asthma (16). Inhaled LTD₄ causes maximal bronchoconstriction within 2 to 3 min, which usually resolves over a period of 1 h (14). Cysteinyl leukotrienes also cause mucus secretion (17), vasoconstriction, and enhancement of vasopermeability (18). More recently, inhaled LTD₄ has been shown to increase airway eosinophil counts in asthmatic subjects, suggesting that it is an eosinophil chemoattractant in human airways (10). However, in the study in which this was found, inhaled methacholine also increased airway eosinophil numbers, an action not previously believed to be mediated by muscarinic agonists.

Inhaled allergen increases urinary levels of LTE₄, a metabolite of LTD₄ (19). Inhibitors of leukotriene synthesis or leukotriene receptor antagonists have produced almost complete inhibition of allergen-induced early responses and partial inhibition of the late response (20). These findings indicate that leukotrienes are produced after allergen inhalation and are the main cause of allergen-induced bronchoconstriction. Recently, an LTD₄-receptor antagonist was shown to reduce inflammatory cell numbers in bronchoalveolar lavage fluid (BALF) after segmental allergen challenge in human airways, suggesting that interruption of the cysteinyl leukotriene pathway may regulate the allergen-induced inflammatory response (23).

To date, there is no conclusive evidence that leukotrienes play an important role in the recruitment of eosinophils to asthmatic airways in vivo. Bronchial biopsy specimens obtained after challenge with LTE₄ have shown increased numbers of eosinophils (11), suggesting that the cysteinyl leukotrienes may attract eosinophils into the airway. However, bronchial biopsies obtained from mildly to moderately asthmatic subjects after 4 wk of treatment with an LTD₄-receptor antagonist demonstrated a reduction in EG2 but not EG1-positive eosinophils (24). Calhoun and coworkers were unable to show an effect of LTD₄-receptor antagonist on allergen-induced eosinophil accumulation in BALF (23), possibly because of the dose of drug they used, since a subsequent study, using an 8-fold higher dose of the same LTD₄-receptor antagonist, demonstrated attenuation of allergen-induced airway eosinophil numbers (25). These studies provide some evidence that cysteinyl leukotrienes may have a role in eosinophil recruitment to the airway. To further evaluate the ability of inhaled LTD₄ to cause eosinophilic recruitment into asthmatic airways, we conducted a study in which we compared inhaled LTD₄ with inhaled methacholine and inhaled allergen on numbers of airway inflammatory cells measured in induced sputum and on AHR in a group of subjects with mild, stable, allergic asthma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Thirteen nonsmoking subjects with mild atopic asthma (Table 1) were recruited for the study. These subjects were selected because they had previously demonstrated allergen-induced airway responses and allergen-induced sputum eosinophilia. The subjects had stable asthma with an FEV₁ exceeding 70% of the predicted normal value on all study days before allergen inhalation. Subjects used no medication other than inhaled and/or oral ₂-agonists in the last 4 wk before and during the study. Medication was withheld for at least 8 h before each visit, and subjects were instructed to refrain from rigorous exercise or consumption of tea or coffee in the morning before visits to the laboratory. Subjects did not have asthma exacerbations or respiratory tract infections for at least 4 wk before entering the study. One subject was excluded from the study because of bronchoconstriction > 15% after challenges with diluent, and two subjects did not complete the study because of deterioration of their asthma. Comparisons of LTD₄ with methacholine were done with the remaining 10 subjects, which would give a statistical power of 85% to observe a doubling of the baseline percentage of sputum eosinophils. This number of mildly atopic asthmatic subjects has previously shown significant increases in sputum eosinophil counts after allergen inhalation (8). One subject was not available for allergen challenge, and one subject was unable to produce adequate sputum after allergen challenge. The study was approved by the ethics committee of the McMaster University Health Sciences Centre, and subjects gave their signed consent to participate in the study.

Study Design

The study was done with a double blind, diluent-controlled, randomized, crossover design. Subjects went through a screening period consisting of methacholine challenge, LTD₄ challenge, skin testing, and allergen challenge. The provocative concentration of methacholine causing a 20% decrease in FEV₁ (PC₂₀) was measured before and 24 h after allergen challenge, and samples of sputum were taken before, and at 7 h and 24 h after allergen challenge. Each subject then completed four treatment periods with inhalation of either methacholine, saline diluent, LTD₄, or saline/ethanol diluent. Each treatment period consisted of three visits to the laboratory. The PC₂₀ for methacholine, and induced sputum differential and total cell counts, were determined 1 d before inhalation challenge. Inhalation challenges were done the following morning. Spirometry was done until 4 h after challenge, and sputum samples were obtained at 4 h and 7 h after challenge. The PC₂₀ for methacholine and sputum samples were obtained 24 h after challenge. Each inhalation challenge was separated by a washout period of at least 7 d.

Spirometry

Spirometry was done with a Collins water-sealed spirometer and kymograph (Warren Collins, Braintree, MA). FEV₁ was measured in triplicate at baseline, and single FEV₁ measurements were made after inhalation of methacholine, LTD₄, and diluents. Volumes were recorded at body temperature-prevailing atmospheric pressure-water vapor saturation (BTPS).

Inhalation Challenges

Methacholine, LTD₄, and diluent inhalation. Methacholine was purchased from the McMaster University Hospital Pharmacy in a stock concentration of 128 mg/ml. This was diluted in physiologic saline into doubling concentrations ranging from 64 mg/ml to 0.016 mg/ml. These working concentrations were stored at 4° C for up to 3 mo. The diluent control solution for methacholine challenge was physiologic saline. LTD₄ was purchased at a concentration of 100 µg/ml ethanol (Cayman Chemical Co., Ann Arbor, MI), and stored in aliquots of 0.25 ml at 70° C. Immediately before use, a stock solution of 12.5 µg/ ml LTD₄ was prepared by adding a 0.25 ml aliquot of LTD₄ to 1.75 ml physiologic saline. This was further diluted in 2-fold concentrations ranging from 6.25 µg/ml to 0.006 µg/ml (12.5 mM to 12.2 nM). The diluent control solution for LTD₄ was prepared in the same manner, but with 0.25 ml of 100% ethanol instead of LTD₄ in ethanol.

Methacholine, LTD₄, and diluent inhalation challenges were done by having the subject inhale 1 ml of solution, nebulized by a Pari LL jet nebulizer with an output of 0.6 ml/min, operated with a Pari Master compressor (Pari Respiratory Equip., Richmond, VA) and generating droplets with a mass median aerodynamic diameter (MMAD) of 3.1 µm. Solutions were nebulized by depressing a button on the nebulizer, and subjects were instructed to depress the button during the inhalation phase of tidal breathing. Inhalation continued until all solution was nebulized. The concentrations of methacholine and LTD₄ required to elicit a 30% decrease in FEV₁ were calculated from data gathered during screening methacholine and LTD₄ challenges. Challenges during the study periods were started at concentrations of methacholine and LTD₄ that were two doubling doses below the dose predicted to achieve a 30% decrease in FEV₁, and were increased in doubling concentrations until at least a 30% decrease in FEV₁ was achieved. The maximum bronchoconstrictor response was taken to be the largest decrease in FEV₁ measured within 1 h after inhalation.

Allergen inhalation challenge. Allergen extracts were stored at 70° C. Allergen for skin tests was diluted in phosphate-buffered saline (PBS) with 1.5% benzyl alcohol and was stored at 4° C, and allergen for inhalation was diluted in physiologic saline on the day of use. The allergen extract selected for inhalation produced the largest skin test wheal, and was diluted for inhalation at the concentration determined from a formula described by Cockcroft and coworkers (26), using the results of the skin test and the methacholine PC₂₀. Allergen inhalation challenges were done with a Wright nebulizer pressurized with oxygen at 50 psi and at a flow rate that gave an output of 0.13 ml/ min and produced droplets having an MMAD of 1.0 to 1.5 µm, using the method described by O'Byrne and coworkers (27). Doubling concentrations of allergen were inhaled by tidal breathing for 2 min, with FEV₁ measured 10 min after each inhalation. Inhalations were stopped when the FEV₁ had fallen by at least 15% from baseline, and was subsequently measured at 20, 30, 45, 60, 90, and 120 min and at hourly intervals thereafter up to 7 h after allergen inhalation. The early airway response, consisting of at least a 15% decrease in FEV₁, was taken to be the largest decline in FEV₁ within 2 h after challenge, and the late response was taken to be the largest decline in FEV₁ between 3 and 7 h after allergen inhalation. Subjects were considered to be dual airway responders if the late decline in FEV₁ exceeded 15%.

Methacholine PC₂₀. Methacholine inhalation challenge was performed as described by Cockcroft (28). Subjects inhaled normal saline followed by doubling concentrations of methacholine phosphate from a Wright nebulizer, pressurized with oxygen at 50 psi and at a flow rate that gave an output of 0.13 ml/min. Inhalation at each concentration of methacholine phosphate lasted for 2 min. FEV₁ was measured at 30, 90, 180, and 300 s after each inhalation. The test was terminated when the FEV₁ declined by 20% of the baseline value, at which point PC₂₀ was calculated.

Sputum Analysis

Sputum was induced and processed according to the method described by Pizzichini and coworkers (2). Subjects inhaled 3%, 4%, and then 5% saline for 7 min each. The induction was stopped when an adequate sample was obtained, or if the FEV₁ dropped by 20% from baseline. Cell plugs with little or no squamous epithelial cells were selected from the sample, using an inverted microscope, and were separated from saliva and weighed. Samples were aspirated in four times their volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Inc., San Diego, CA) and four times their volume of Dulbecco's PBS (DPBS; Gibco BRL, Life Technologies, Grand Island, NY). The cell suspension was filtered through a 52-µm nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, and was then centrifuged at 1,500 rpm for 10 min. The total cell count was made with a hemocytometer (Neubauer Chamber; Hausser Scientific, Blue Bell, PA) and expressed as the number of cells per milliliter of sputum. Cells were resuspended in DPBS at 0.75 to 1.0 × 10⁶/ml. Cytospins were prepared on glass slides, using 50 µl of cell suspension and a Shandon III Cytocentrifuge (Shandon Southern Instruments, Sewickly, PA), at 300 rpm for 5 min. Differential cell counts were obtained from the mean of two slides, with 400 cells counted per Diff-Quik (American Scientific Products, McGaw Park, IL)-stained slide. Metachromatic-cell (mast cell and basophil) counts were obtained from the mean of two slides stained with toluidine blue, with 1,500 cells observed on each slide.

Statistical Analysis

All summary statistics are expressed as mean ± SEM, with the exception of methacholine PC₂₀ measurements, which were made by linear interpolation of log dose-response curves, resulting in logarithmic values for PC₂₀, and which are expressed as geometric mean and geometric SEM (GSEM). The two-tailed Student's t test for paired observations was used to compare the maximal declines in FEV₁ after administration of the bronchoconstrictor agents. Inhalation challenge-induced changes in methacholine PC₂₀ and in blood and sputum inflammatory cell counts were analyzed with a two-factor repeated-measures analysis of variance (ANOVA) (29) to determine the effects of treatment and time. Statistical analyses were done with specialized computer software (Statistica 4.5, 1993; Stat Soft Inc., Tulsa, OK).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Methacholine and LTD₄ challenges were followed by immediate bronchoconstriction, which reached maximum levels within 2 min after inhalation of each substance and returned to within 10% of baseline by 1 h (Figure 1). The maximum decrease in FEV₁ after methacholine inhalation challenge, of 39.4 ± 2.8% (range: 25.7% to 52.9%), was significantly greater than the maximum decrease in FEV₁ after LTD₄ challenge, of 31.4 ± 1.8% (range: 23.5% to 41.2%) (p = 0.02). In contrast, bronchoconstriction after allergen challenge had a slower onset, which was maximal at 20 to 30 min, and was more prolonged, as the FEV₁ had not returned to 10% of baseline by 1 h (Figure 1). Allergen challenge elicited a maximum decrease in FEV₁ of 30.1 ± 3.4% (range: 15.5% to 53.8%), which was similar to that with LTD₄ and methacholine (p > 0.05) (Figure 1). The maximum decreases in FEV₁ after the diluent challenges were 5.3 ± 0.9% after saline and 6.3 ± 0.8% after ethanol.

View larger version (17K): [in this window] [in a new window]   Figure 1.   Percent change in FEV1 for 60 min after inhalation challenges with methacholine (solid circles), saline (open circles), LTD4 (solid squares), ethanol (open squares) and allergen (solid diamonds).

The baseline methacholine PC₂₀ values were not significantly different before the challenges, and there was no significant change in methacholine PC₂₀ measured 24 h after inhaled methacholine or in methacholine PC₂₀ measured before and after diluent challenges (p > 0.05) (Figure 2). There was a trend toward a decrease in methacholine PC₂₀ measured 24 h after challenge with inhaled LTD₄, which fell from 8.6 mg/ml (GSEM = 1.6) to 5.7 mg/ml (GSEM = 1.7), but this difference was not statistically significant (p = 0.11) (Figure 2). By contrast, the methacholine PC₂₀ fell significantly, from 4.9 mg/ml (GSEM 1.3) to 1.7 mg/ml (GSEM 1.5), by 24 h after challenge with inhaled allergen (p = 0.02) (Figure 2).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Methacholine PC20 before (open rectangles) and after (solid rectangles) inhalation challenges with methacholine, saline, LTD4, ethanol, and allergen (*p < 0.05 for allergen-induced reduction in methacholine PC20).

Sputum eosinophils expressed as a percent of total cells did not change from its baseline value at 4 h, 7 h, or 24 h after inhaled methacholine, LTD₄, or diluent controls (p = 0.70), nor was there any significant difference among the challenges (p = 0.58) in terms of this percent (Table 2, Figure 3). Similar results were obtained when the data were analyzed as absolute number of eosinophils/ml sputum, with no effects of time (p = 0.20) or challenge (p = 0.66) (Table 3). By contrast, an increase in the percent eosinophils (p = 0.003) and in the number of eosinophils/ml sputum (p = 0.08) occurred 7 h and 24 h after allergen inhalation as compared with baseline and compared with the other challenges (p < 0.0003) (Figure 3, Tables 2 and 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Percent sputum eosinophils before and after inhalation challenges with methacholine (M), saline (S), LTD4 (L), ethanol (E), and allergen (A) (*p < 0.05 for allergen-induced increase in sputum eosinophils).

There was a significant increase in the percent neutrophils in sputum at 7 h (p = 0.000004), and a significant decrease in the percent and number of sputum macrophages/monocytes at 7 h (p = 0.00002 and p = 0.003, respectively) after inhalation of methacholine, LTD₄, and diluents (Tables 2 and 3). There were no significant effects of time or treatment on percent sputum metachromatic cells (p > 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have shown that inhalation of LTD₄ causing submaximal bronchoconstriction does not increase sputum eosinophils in the airways of mildly asthmatic subjects. When comparing inhalation of LTD₄ with that of methacholinea bronchoconstrictor agent that does not induce airway inflammationwe found no effect of LTD₄ on sputum eosinophils or on the activation state of eosinophils following challenge.

Our observations do not support the suggestion that inhaled LTD₄ increases sputum eosinophil numbers in asthmatic airways, as was reported by Diamant and coworkers (10). These investigators reported that inhalation of LTD₄ resulting in maximal bronchoconstriction induced a significant increase in the percentage of sputum eosinophils, as compared with inhalation of its diluent, whereas a similar trend with methacholine challenge failed to reach statistical significance. In their study, however, Diamant and coworkers found no significant difference in the percent sputum eosinophils after LTD₄ or methacholine challenge. These results could be interpreted to indicate that this sputum eosinophilia was not a consequence of inhalation of LTD₄, but was rather the result of an event associated with maximal bronchoconstriction. The current study was designed to achieve a submaximal level of bronchoconstriction, preventing this possible consequence of maximal bronchoconstriction. This study showed that LTD₄ does not induce airway eosinophilia in mildly asthmatic subjects after submaximal bronchoconstriction.

Allergen is a stimulus known to cause sputum eosinophilia in atopic asthmatic individuals. Allergen inhalation challenge was included in the current study to document the development and magnitude of sputum eosinophilia observed in subjects with mild atopic asthma after allergen-induced submaximal bronchoconstriction. Except for allergen, none of the inhalation challenges caused a significant eosinophilic response in the airways.

The airway response during the first hour after challenge was similar with LTD₄ and methacholine (rapid onset, rapid recovery), but was different with allergen (slow onset, prolonged recovery). This suggests that LTD₄-induced bronchoconstriction in the asthmatic subjects in our study operated through a mechanism similar to that of methacholine, through direct binding to specific receptors on airway smooth muscle. The mechanism of response to allergen challenge, in contrast, is cell-mediated and can result in a late airway bronchoconstrictor response, AHR, and airway inflammation (8, 30, 31). AHR, which has been shown to correlate with sputum eosinophil numbers in asthmatic individuals (32), was found only after allergen challenge in the current study, in association with an allergen-induced increase in sputum eosinophils. There was no significant AHR following challenge with LTD₄. Comparing the kinetics of the early airway response to allergen and to LTD₄, and the lack of AHR following challenge with LTD₄, supports our view that LTD₄ by itself does not cause eosinophil accumulation in the airways of asthmatic individuals.

Studies with allergic guinea pigs have demonstrated eosinophil influx into the airway following challenge with LTD₄ (33). Guinea pigs have been used because of their sensitivity to the bronchoconstrictor effects of leukotrienes; however, their relevance to human studies is unclear since there are species differences in the response to leukotrienes (34). Inhaled LTE₄ was shown to induce an influx of eosinophils and neutrophils into the bronchial mucosa of four asthmatic patients (11), but there is no evidence that LTD₄ would attract eosinophils into the airways of asthmatic or nonasthmatic subjects. In vitro, LTD₄ appears to be a potent chemoattractant for isolated human peripheral blood eosinophils (35), but this has not been clearly demonstrated in vivo. Smith and colleagues (36) did not find any change in BALF eosinophil numbers at 24 h after inhalation of LTD₄ by normal subjects, and inhalation of LTD₄ causing maximal bronchoconstriction in asthmatic subjects was shown to increase sputum eosinophil numbers, but not to a significantly greater extent than methacholine (10). Furthermore, no late response is seen after inhalation of LTD₄ (14), in contrast to the late airway response following allergen inhalation which occurs among 50% or more of atopic asthmatic individuals, which begins 3 to 4 h after allergen inhalation (37) and is accompanied by airway eosinophilia (8, 30).

The increase in percent sputum neutrophils observed 7 h after inhalation challenges is likely to be a result of the sputum induction procedure, since repeated induction of sputum has been shown to cause increases in the percentage of sputum neutrophils (4). The numbers of sputum macrophages at 7 h after inhalation challenges are most likely depressed as a direct result of the observed neutrophilia. Although a number of studies have shown that neither methacholine inhalation nor sputum induction is associated with changes in the level of airway eosinophilia, it is possible that these procedures mask small changes in inflammatory cell differential counts. However, analyses of the challenge-induced shift in percent sputum eosinophils also show no difference between challenges, supporting our conclusion that LTD₄ is not an eosinophil chemoattractant when delivered into the airway.

We have provided strong evidence that inhaled LTD₄ does not attract eosinophils into the airways; however, a number of limitations of our study must be considered. The subpopulation of mildly asthmatic subjects in the study clearly cannot exhibit the LTD₄-induced response of those with more severe asthma. Nonetheless, we have demonstrated tremendous differences in the recruitment of eosinophils to the airway after allergen as compared with LTD₄ challenge. A Pari jet nebulizer (MMAD = 3.1 µm) was utilized for methacholine and LTD₄ challenges, ensuring that these agents were deposited in the same area of the lung. However, a Wright nebulizer (MMAD = 1.0 to 1.5 µm) was used for allergen challenges, which may have introduced a bias into our comparisons with LTD₄ if, in fact, LTD₄ was not deposited optimally in the lung as compared with allergen. We could not measure the amounts of LTD₄ or methacholine actually delivered to the airway, but we achieved responses to these agents, since, as compared with the diluent challenges, they produced bronchoconstriction. In addition, it could be argued that use of LTC₄ rather than LTD₄ would have produced more of a biologically relevant challenge, but there is no evidence that we would have seen a difference in response.

In this study we compared effects of inhalation of methacholine and LTD₄ on airway eosinophil recruitment. Inhalation of LTD₄ alone did not result in an eosinophil response in this investigation; however, LTD₄ may still be a cofactor in allergen-induced bronchoconstriction and the resulting airway eosinophilia. Increased levels of the cysteinyl leukotrienes in airway samples after allergen challenge (38) support the role of these mediators in allergen-induced responses. Studies with LTD₄ receptor antagonists have been instrumental in demonstrating the involvement of LTD₄ in asthma. Blocking the LTD₄ receptor has been shown to reduce airway eosinophil numbers in mildly uncontrolled asthma (39), as well as to improve airway physiology (40) and inflammatory cell recruitment (25) after allergen challenge, and provides additional benefit to corticosteroid in treating chronic asthma (41), suggesting that LTD₄ may be a cofactor contributing to the accumulation of eosinophils in asthma. Further studies will be required to determine exactly how LTD₄ is involved in allergic asthma, and studies combining LTD₄-receptor blockade and corticosteroid treatment may clarify the role of LTD₄ in eosinophil accumulation in asthma. Although we did not observe a direct effect of LTD₄ on eosinophil recruitment, LTD₄ may still play a cooperative role in eosinophil recruitment in asthma.