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INTRODUCTION
METHODS
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The effect of zafirlukast (Z) to alter the inflammatory response to segmental antigen challenge (SAC) was assessed by bronchoalveolar lavage (BAL) in this double-blind, placebo-controlled, two-period, crossover trial in 11 allergic asthmatic patients. Patients with asthma and positive skin tests to antigen received 7 d of treatment with Z (20 mg twice daily) or placebo (P) during two trial periods 14 to 21 d apart. At steady state (Day 5), patients underwent SAC followed by BAL immediately after challenge and 48 h later. Purified alveolar macrophages were analyzed ex vivo for phorbol myristate acetate (PMA)-driven superoxide release. Results were analyzed by analysis of variance (ANOVA). Forty-eight hours after SAC, Z therapy was associated with significantly reduced BAL lymphocytes and alcian blue-positive cells (presumably basophils) compared with P (p < 0.01), with a trend toward reduced numbers of alveolar macrophages (p = 0.06). PMA-driven superoxide release by purified alveolar macrophages was significantly reduced 48 h after SAC in the Z versus P arms (p < 0.05). Reduction of basophil influx, mediator release, and cellular activation may be important in attenuating the late phase of asthma. Collectively, the data suggest that zafirlukast therapy alters cellular infiltration and activation associated with antigen challenge.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Allergen exposure causes bronchoconstriction in patients with asthma and can lead to leukotriene production and increased bronchial hyperresponsiveness. The cysteinyl leukotrienes, LTC4, LTD4, and LTE4, are known mediators of bronchoconstriction, airway wall edema, and mucous hypersecretion in patients with asthma (1). Recent studies have shown that the leukotriene receptor antagonists, MK-571 and zafirlukast, decrease both early and late asthmatic responses to inhaled allergen (2, 3) and that zafirlukast attenuates the increased bronchial hyperresponsiveness that follows an inhaled allergen challenge (3).
Although a causal relationship has not been established, increased bronchial hyperresponsiveness develops simultaneously with airway inflammation after inhaled allergen challenges (4). Furthermore, stable airway hyperresponsiveness correlates with the number of mast cells, eosinophils, and neutrophils in asthmatic airways (4). Leukotrienes may play a role in eosinophil recruitment. In guinea pigs, LTD4 applied topically to the ocular surface or airway caused a pronounced infiltration of eosinophils into the conjunctival epithelium and airway, respectively (5). Infiltration was abolished by pretreatment with a leukotriene receptor antagonist (SKF104353 [5] or zafirlukast [7]). Similarly, antagonism of the LTD4 receptor blocked the development of both hyperresponsiveness and bronchoalveolar lavage (BAL) eosinophilia in antigen-challenged primates (8). In humans, mucosal biopsies of the lamina propria after LTE4 challenges showed increased numbers of eosinophils and neutrophils (9). Collectively, the data suggest that cysteinyl leukotrienes may have a causal but indirect role in facilitating eosinophil recruitment.
Bronchial challenge studies have been conducted to investigate cellular and molecular regulation of inflammation in asthma (10). Segmental antigen challenge (SAC) is used frequently because it provides a useful model to study airway inflammation and can be done safely in patients with asthma. BAL fluid obtained 48 h after SAC contains increased numbers of eosinophils, neutrophils, macrophages, and lymphocytes, and increased concentrations of albumin, LTC4, major basic protein, and eosinophil-derived neurotoxin (EDN) (14, 15). Additionally, purified alveolar macrophages are phenotypically distinct and are functionally activated, as evidenced by increased PMA-driven superoxide release 48 h after an allergen challenge (16). Monocytic cells express functional cysteinyl leukotriene 1 (cys-LT1) receptors (17). These cells could be activated by cysteinyl leukotrienes in the microenvironment of the asthmatic airway. Thus, secretion of cysteinyl LT could amplify monocyte-driven inflammation, and could contribute to the altered, and functionally activated phenotype observed in macrophages of asthmatic patients.
Cytokines coordinate many types of inflammation. Interferon-
(IFN-) strongly upregulates monocyte and macrophage functions (18) and, importantly, is also a potent eosinophil-activating cytokine (19). Tumor necrosis factor-
(TNF-)
facilitates the development of inflammation by several mechanisms, including upregulation of intercellular adhesion molecule 1 (ICAM-1) expression (20) and potentiation of cysteinyl
LT release by eosinophils (21).
This evidence provides a compelling rationale for testing
the effect of leukotriene receptor antagonists on cellular inflammation after allergen exposure. Because mononuclear
cells express functional cys-LT1 receptor, we hypothesized
that a leukotriene receptor antagonist would block leukotriene-induced monocyte and macrophage activation and influx. In this trial, we evaluated the effect of the leukotriene
receptor antagonist zafirlukast on mediators of airway inflammation and macrophage function, specifically superoxide release and TNF- after SAC in patients with mild asthma.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Patient Population
Nonsmoking men and women between the ages of 18 and 45 yr with
mild asthma were enrolled in this trial. Each patient demonstrated reversible airway disease by a 15% or greater increase in 1-s forced expiratory volume (FEV1) after inhaled 
2-agonist or a positive response to methacholine challenge. All patients had an FEV1 at least
80% of predicted at screening (at least 6 h after 2-agonist use) and a
positive skin test (3+) to ragweed, cat dander, house dust mite, or
June grass. No patient was receiving, nor had received, oral or inhaled
corticosteroids within the preceding 6 mo. Women of childbearing potential used a nonhormonal birth control method during the trial.
Key exclusion criteria included the following: acute illness or disease; negative skin tests; respiratory tract infection or asthma exacerbation within 8 wk of screening; history of drug or alcohol abuse; theophylline, oral or inhaled corticosteroid, nedocromil sodium, or cromolyn sodium use within 4 wk of screening; astemizole use within 3 mo of screening; influenza virus or hepatitis B vaccination within 6 wk of screening; or participation in another clinical trial within 4 wk of randomization in this trial. In summary, patients with atopic asthma who were controlled on as needed inhaled beta-agonists alone were enrolled in the trial.
Overall Trial Design
This trial was designed as a randomized, double-blind, placebo-controlled, two-period, crossover trial. Each patient served as his or her own control and underwent both saline and allergen challenges. Thus, variation due to individual responses could be minimized and controlled. Because of this experimental design, and because the purpose of the trial was to determine the effect of zafirlukast on airway inflammation in asthmatic individuals, the inclusion of nonasthmatic control subjects was deemed not to be essential. Furthermore, we have shown previously that segmental allergen challenge of normal subjects does not evoke eosinophil influx (22).
The trial began with a screening visit, followed by a 14- to 21-d run-in period and two 7-d randomized treatment periods 14 to 21 d apart. Patients received twice-daily oral doses of zafirlukast (20 mg) or placebo in a crossover manner for 7 d during each treatment period. On Day 5 of each treatment period, patients underwent bronchoscopy, SAC, and BAL 4 h after zafirlukast or placebo administration. BAL was repeated 48 h later on Day 7 of drug or placebo treatment. Albuterol (Ventolin; Allen & Hanburys, Research Triangle Park, NC) was used as rescue medication. The protocol was approved by the Institutional Review Board for Biomedical Research of the University of Pittsburgh, and all patients gave informed consent before participating in the trial.
Screening
During the screening period, patients provided complete medical histories and had a routine physical examination and skin testing by the
prick-puncture method. FEV1 was measured at screening for each patient at least 6 h after the last use of 2-agonist. After the screening
FEV1 measurement, patients inhaled two puffs of albuterol; FEV1 was
measured 15 to 30 min later to determine airway reversibility (at least
15%). Patients who did not meet the reversibility criterion were excluded from the trial. Patients with positive skin tests underwent aerosol allergen challenge with standardized ragweed, cat dander, house
dust mite, or June grass extract as described previously (10) to demonstrate antigen responsiveness and to determine the provocative dose
that caused a 20% reduction in FEV1 (i.e., antigen PD20).
Segmental Antigen Challenge and Bronchoalveolar Lavage
Spirometry was measured before bronchoscopy. Only patients with an
FEV1 
70% of predicted underwent BAL and SAC. Segmental challenges and BAL were conducted as described previously by Calhoun and colleagues (10, 16, 22) 4 h after administration of zafirlukast or
placebo on Day 5 of each treatment period (placebo or drug). Patients
were premedicated intramuscularly with 0.6 mg atropine and 1 mg midazolam. The bronchoscope was then inserted, four bronchopulmonary segments were identified, and the instrument was wedged into
the first segment or subsegment. For segmental challenges, 10 ml of
saline or antigen solution was instilled into the segment. Antigen concentrations used in the challenge were 0% (saline control), 1%, 5%,
or 20% of the previously determined antigen PD20. Antigen was diluted in 0.9% NaCl to a volume of 10 ml. All antigen solutions were
negative for endotoxin contamination by the limulus lysate assay to
the limit of detectability (6 pg/ml). After delivery of the challenge volume, the bronchoscope channel was cleared by flushing the channel
with 5 ml of air. After a 5-min dwell time, BAL was performed by instilling and immediately withdrawing two 60-ml aliquots of 0.9% NaCl
at 37° C. The bronchoscope was moved to another segment for the
next challenge. Each patient was challenged in four separate segments
with saline, and low (1%), medium (5%), and high (20%) doses of antigen, in that order; BAL was performed 5 min later. Typically, the
segments used were left upper lobe, anterior segment (0% segmental
saline challenge); superior lingula (1% SAC); right upper lobe, anterior segment (5% SAC); and right middle lobe, medial segment (20%
SAC). Bronchoscopy and BAL were repeated 48 h later in each segment.
BAL Fluid Processing
BAL fluid recovered from each bronchopulmonary segment was processed individually. Samples were centrifuged at 400 × g for 15 min to
separate cellular components, which were washed and resuspended in
Hanks' balanced salt solution, and counted using a hemocytometer.
Separate cytofuge preparations were stained with Diff-Quick and alcian blue, and at least 300 cells were enumerated. The supernatant
was collected and frozen at 
70° C for analysis of mediators. Samples
for analysis of leukotrienes were diluted with 4 volumes of 80% ethanol, and stored at 70° C until analyzed. BAL fluid was not concentrated for these studies, but rather was analyzed neat.
Purification of Alveolar Macrophages
Alveolar macrophages were purified from other BAL cells (> 98%) by centrifugation over a single cushion of Percoll (Sigma Chemical Co., St. Louis, MO) with a density of 1,075 mg/ml (16). Purified alveolar macrophages were used for superoxide production assays.
BAL Fluid Analysis
The eosinophil granule protein EDN was analyzed by an enzyme-linked immunosorbent assay (ELISA) kit (23). Because EDN is undetectable in BAL samples 5 min after SAC, only 48-h samples were analyzed (15). Samples reserved for leukotriene determination were
analyzed using a commercial assay according to the manufacturer's directions (Amersham Life Science, Arlington Heights, IL). Briefly, supernatants were acidified and extracted with methanol. The samples were then applied to PH (phenyl) columns and washed sequentially with 1.5 ml water (pH 3.0), 2 ml hexane, and 1.5 ml ethanol/water/2 M
HCl (ratio of 10:82:8 by volume). Leukotrienes were eluted with 2 ml
methyl formate, dried, resuspended in radioimmunoassay (RIA) buffer,
and assayed using a 3H-RIA kit. Cross-reactivities of the antibody for
leukotrienes are: LTC4, 100%; LTA4, 64%; LTE4, 64%; 11-trans LTD4,
30% and 11-trans LTE4, 24%. Recovery of radiolabeled LT was assessed by adding a known quantity of standard (LTC4) to buffer, and
subjecting the standard to extraction and analysis procedures. Recovery averaged 86%. Histamine was determined in BAL fluids by a
commercial kit (Immunotech, Westbrook, ME). Albumin was measured by ELISA. TNF- and IFN- were measured by enhanced
ELISA as described previously (22).
Efficacy Assessments
The following cell counts were determined from BAL fluid 5 min
(early phase) and 48 h (late phase) after SAC: alveolar macrophages, lymphocytes, neutrophils, eosinophils, and alcian blue-positive cells
(mast cells plus basophils). BAL fluid was also analyzed for concentrations of albumin, IFN-, TNF-, EDN (48 h only), total cysteinyl
leukotrienes, and histamine. Alveolar macrophages were analyzed ex
vivo for PMA-driven superoxide release 5 min and 48 h after SAC as
previously described (16). Briefly, 105 cells were incubated at 37° C with
ferricytochrome C, with or without activators. Absorbance at 550 nm
was measured over 60 min, was corrected for absorbance in parallel
wells containing 25 µg/ml superoxide dismutase, and was converted to
equivalent superoxide production using the appropriate molar extinction coefficient (16).
Concentrations of Zafirlukast in BAL Fluid and Plasma
BAL Fluid from the saline-challenged segment was collected and frozen at 10° C for analysis of zafirlukast concentrations. Samples were
analyzed by a high-performance liquid chromatography (HPLC)
method using fluorescence detection with a lower quantitation limit of
2.0 ng/ml. Blood samples were collected at screening and during double-blind treatment to determine plasma levels of zafirlukast. Plasma
concentrations of zafirlukast were analyzed by HPLC, using fluorescence detection with a quantitation limit of 0.75 ng/ml.
Safety Assessments
The safety of trial medications was assessed from adverse event reports, patient interviews for subjective symptoms, and results of clinical laboratory tests, electrocardiographic and physical examinations, and measurements of vital signs.
Statistical Methods
No previous data existed regarding the effect of zafirlukast on cell mediators of inflammation; therefore, published results of total cell
counts after SAC and BAL recoveries were used to estimate the variation when comparing high and low doses of allergen (15). The study
was powered to identify alterations in total cell counts and the production of inflammogens by alveolar macrophages. A sample of 12 patients in a two-period, crossover design was deemed sufficient to detect a difference of 40 million cells in total cell counts from BAL, with
80% power at = 0.05 (95% confidence level), or to detect a difference of 10 nmol superoxide per million cells per hour. Sample size estimation for either of these parameters suggested a requirement for 10 to 12 patients for adequate power.
The following variables played an important role in testing our hypothesis a priori and are presented in detail: concentration of neutrophils, eosinophils, basophils, and lymphocytes; respiratory burst of purified alveolar macrophages; histamine; albumin; IFN-; TNF-; EDN;
and total cysteinyl leukotrienes.
An analysis of variance (ANOVA) model in the framework of a
two-period, crossover design was used to compare the effects of zafirlukast and placebo on the parameters of interest. Appropriate data transformations of cell counts were used to meet ANOVA assumptions, as many parameters were not normally distributed. ANOVA
was performed for each assessment in a model containing treatment
sequence, patient number within a treatment sequence, trial period,
allergen dose level, BAL time, and treatment as the factors. Factors
that were analyzed by nonparametric methods included albumin, total protein, histamine, TNF-, and IFN-. Determination of group differences was made by appropriate post hoc testing using Student-Newman-Keuls procedure. Descriptive statistics were used to summarize
the zafirlukast concentrations in plasma and BAL fluid. Additionally,
simple correlations were performed for cell concentrations and their
products.
Adverse events were tabulated by treatment group and body system using COSTART (Coding Symbols for Thesaurus of Adverse Reaction Terms) terminology. The results of clinical laboratory tests, vital sign measurements, and physical examinations were summarized by treatment and protocol time. Changes from baseline were analyzed using ANOVA to determine if changes were treatment related. The ANOVA model used included treatment sequence, patient number within treatment sequence, challenge day, and treatment as factors.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Twelve patients with mild asthma between the ages of 19 and 26 yr (mean age 22.5 yr) were enrolled and 11 completed the study (five women and six men). One patient was withdrawn from the study because of an intercurrent syndrome consistent with a viral upper respiratory tract infection that developed in the interphase between randomized arms. The mean percent of predicted FEV1 for these patients at entry was 95.9% predicted (range 78% to 146%). Table 1 summarizes the baseline characteristics of the study volunteers who completed the study.
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Safety and Detectability of Zafirlukast
Safety. No serious adverse events were reported for any patient, and no patients withdrew from the trial. Adverse events were reported for seven (64%) patients during zafirlukast treatment and five (45%) patients during placebo treatment. Table 2 summarizes the number of patients reporting adverse events by the most recent treatment received. No statistically or clinically significant changes were observed in results of clinical laboratory tests, electrocardiographic or physical examinations, or vital signs.
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Immediate Response to SAC
Allergen Response
There was no significant effect of allergen dose on recovered
BAL volume. Within the placebo arm (but not the zafirlukast arm) we observed a significant effect of allergen challenge on the concentration of BAL eosinophils obtained 5 min after
SAC (Table 3). This effect was seen at all levels of allergen
challenge and by a posteriori testing was significant for the low
and high allergen doses. Moreover, 5 min after high-dose SAC,
there was a significant increase in the concentration of IFN-,
which was seen in both treatment arms. In addition, there was
a significant allergen dose-dependent increase in BAL histamine concentration immediately after SAC in both arms.
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Immediate Response to SACEffect of Zafirlukast
There was no significant effect of zafirlukast treatment on any
BAL measure, including volume recovery, 5 min after SAC.
No effect on total or differential cell counts, histamine, cysteinyl leukotrienes, albumin, TNF-, or IFN- was observed
(Table 3).
Late Response to SACAllergen Response
BAL fluid samples obtained 48 h after SAC had significantly higher concentrations of total cells, neutrophils, eosinophils, and basophils (placebo only) compared with samples obtained 5 min after SAC, confirming cellular influx as a late consequence of SAC in our model. There was no significant effect of allergen challenge on the volume or percent recovery of BAL fluid.
Forty-eight hours after SAC, there were significant (p < 0.05) allergen dose-dependent increases in the concentration
of total cells, neutrophils, eosinophils, basophils, albumin,
TNF-, EDN, superoxide release by purified alveolar macrophages, and total cysteinyl leukotrienes compared with saline challenge (Table 4, Figures 1-4).
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) denotes a significant (p < 0.05) effect of zafirlukast versus placebo for the indicated antigen dose. Basophils were undetectable (zero) in the zafirlukast groups; therefore, bars for this
measure are not visible. By two-way ANOVA with repeated measures, there was a significant effect of both antigen dose (p < 0.001) and zafirlukast (p < 0.01).
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) denotes a significant (p < 0.05) effect
of zafirlukast versus placebo for the indicated antigen dose. By
two-way ANOVA with repeated measures, there was a significant
effect of zafirlukast (p < 0.01).
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Late Response to SACEffect of Zafirlukast
Cell counts. Compared with placebo, zafirlukast treatment was associated with significantly blunted influx of basophils (p < 0.01) and lymphocytes (p < 0.01) 48 h after SAC (Figures 1 and 2); there also was a trend toward reduced accumulation of alveolar macrophages (p = 0.06) 48 h after SAC (Table 4). The number and proportion of eosinophils recruited to the airway 48 h after SAC was lower during zafirlukast treatment, although the difference was not statistically significant compared with placebo (Figure 3). No zafirlukast treatment differences were observed in neutrophil proportions or numbers 48 h after SAC.
Superoxide production by purified alveolar macrophages. Antigen challenge was associated with an increase in PMA-driven superoxide release from purified alveolar macrophages during placebo (threefold increase versus saline) and zafirlukast treatment (less than 50% increase). During zafirlukast treatment, PMA-driven superoxide production was significantly lower 48 h after SAC compared with placebo (Figure 4), and a posteriori testing confirmed a significant effect of zafirlukast on superoxide responses after medium- and high-dose SAC. EDN. Zafirlukast had no significant effect on EDN concentrations in BAL fluid obtained 48 h after SAC compared with placebo (Table 4). However, a significant correlation (r = 0.78, p < 0.01) was observed between EDN concentration and the number of eosinophils recruited to the airway 48 h after SAC. Histamine. Persistent histamine release was observed 48 h after SAC compared with saline challenge. Histamine concentrations in BAL fluid 48 h after SAC were significantly lower during treatment with zafirlukast than with placebo, considering all allergen doses by ANOVA. Appropriate a posteriori comparisons failed to identify differences between zafirlukast and placebo for individual allergen doses, but in aggregate there was a significant treatment effect (p < 0.05, Figure 5). The largest drug effect was observed in those segments with the greatest histamine release. This observation suggests that there is an effect of zafirlukast on last histamine release, but that it is small in magnitude.
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, and TNF- concentrations were significantly (p < 0.05) higher 48 h after SAC
compared with 5 min after the challenge, and compared with
saline challenge (Table 4). IFN- and TNF- concentrations
in BAL fluid were also significantly (p < 0.05) greater 48 h after SAC with high-dose antigen than with saline. Furthermore,
this increase occurred in an antigen dose-response fashion.
No zafirlukast treatment differences were observed in albumin or IFN-concentrations in BAL fluids obtained 48 h after SAC (Table 4). Forty-eight hours after SAC, TNF- concentrations increased significantly compared with saline (p < 0.05) and the rise in TNF- concentrations was significantly
blunted in the zafirlukast arm compared with the placebo arm
(p < 0.04, Figure 6).
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INTRODUCTION
METHODS
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DISCUSSION
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This is the first study in humans to show that blockade of the
cys-LT1 receptor in vivo reduces indices associated with the development of airway inflammation after SAC. Treatment
with the leukotriene receptor antagonist zafirlukast was associated with reduction in several indices of the inflammatory allergic response. Histamine concentrations, PMA-driven superoxide release from purified alveolar macrophages, and
TNF- concentrations in BAL fluid were significantly lower
in the zafirlukast arm compared with placebo 48 h after SAC.
In addition, zafirlukast therapy decreased the number of lymphocytes and alcian blue-positive cells (presumably basophils) present in BAL fluid 48 h after SAC, compared with placebo, and produced a strong trend (p = 0.06) toward reduced alveolar macrophage influx. Additionally, late eosinophil recruitment in response to SAC was decreased by an average of 33% but did not reach statistical significance due to
large intersubject variation. Previous investigations have suggested that zafirlukast at this dose blocks late-phase physiologic responses and hyperresponsiveness to allergen challenge
(3). The results of the current study suggest that reduced infiltration and activation of relevant cells may be a contributing
mechanism for the effect of zafirlukast on late-phase physiology. In this trial, however, we did not measure bronchial hyperresponsiveness. Furthermore, the relationship between whole-lung bronchial responsiveness and segmental inflammation is conceptually weak. For these reasons, we cannot
draw direct parallels between the current study of airway inflammation and previous physiologic trials. Nonetheless, our
data showing reduced indices of airway inflammation are generally consistent with previous observations of reduced hyperresponsiveness and with current concepts of the relationship
between bronchial responsiveness and inflammation.
Because monocytic cells express the cys-LT1 receptor, it is
plausible to suggest that the blunted upregulation of markers of macrophage activation, superoxide production, and TNF-
release may be a direct consequence of blockade of the receptor by zafirlukast. We observed that zafirlukast therapy significantly blunted the antigen-driven augmentation of superoxide release by alveolar macrophages observed 48 h after
challenge. Superoxide anion produces airway hyperresponsiveness, and bronchial obstruction when applied to or generated in the airway, suggesting a pathogenic role in asthma for
this reactive oxygen species (24, 25). Thus, the reduction in superoxide production may reflect an important anti-inflammatory effect of zafirlukast in the context of allergic inflammation. In addition, alveolar macrophages are likely to be an
important source of TNF- in the airway. Although we did not
specifically measure the production of this cytokine by alveolar macrophages, it is plausible to suggest that zafirlukast
treatment may downregulate several important macrophage
functions.
TNF- is an important inflammogenic cytokine that may
be crucial in the development of late-phase asthmatic reactions. This cytokine is prominently expressed at sites of allergic inflammation (26). Gosset and colleagues demonstrated
that production of TNF- by alveolar macrophages after antigen challenge was increased only in patients who developed a
late asthmatic response (27). Thus, modulation of the activity
of this cytokine could be an important therapeutic strategy.
The reduced production of TNF- in the zafirlukast arm suggests an important potential mechanism by which airway inflammation may be modulated by this compound.
The number of alveolar macrophages recovered 48 h after SAC was lower during treatment with zafirlukast than with placebo. Previous studies have demonstrated that SAC in allergen-sensitive subjects is associated with active recruitment of the more dense monocytic cells into the airway (16). Although a number of reasons are possible, the most likely explanation for reduced numbers of alveolar macrophages in the zafirlukast arm is less intense recruitment of monocytes to the airway following SAC.
Basophils appear to play a role in asthma, although they are not typically found in BAL fluid or bronchial biopsies of patients with stable asthma. Basophils are, however, found in the sputum of symptomatic patients and in large numbers in the BAL fluid of patients experiencing late asthmatic responses to inhaled allergen (28, 29). Additionally, blood basophil counts increase during pollen season (30). Drugs such as corticosteroids, which inhibit the late asthmatic response, downregulate basophil function (31) and also blunt basophil influx after SAC (32). In the current study, zafirlukast reduced the number of alcian blue-positive cells found in BAL fluid 48 h after SAC. These cells were very likely basophils, rather than mast cells, as previously reported (29). Our identification of markedly reduced alcian blue-positive cell influx (presumably basophils) after SAC suggests a possible causal relationship with reduced bronchial hyperresponsiveness. However, the specific cells directly responsible for the development of hyperresonsiveness have not been uniquely identified, and furthermore, the cellular source of either early or late histamine response was not established in this trial.
That our study did not identify a significant effect of zafirlukast treatment on eosinophil influx, despite animal evidence of such an effect, may be due to several factors. First, the stimulus of SAC is quite intense, and is considerably more so than aerosol challenge (10); thus, the stimulus may have overwhelmed any potential protective effect of zafirlukast. Second, and perhaps most importantly, our study was not powered to detect differences in eosinophil numbers or concentrations, and was demonstrably insufficiently powered to do so. Even with a cadre of 12 rather than 11 patients, the study would have achieved a power of less than 80% to detect a twofold change in eosinophil numbers.
The effect of zafirlukast on cellular recruitment in this trial,
coupled with existing animal data (7, 33), indicates that leukotriene receptor blockade might affect cellular recruitment through a mechanism common to several cell types. One such
mechanism is an increase in adhesion molecule expression.
Basophils and eosinophils adhere to the vascular cell adhesion
molecule (VCAM-1) expressed on endothelium via very-late
activation antigen 4 (VLA-4) (34). It is possible that zafirlukast
affects adhesion molecule expression perhaps by modulation
of TNF- or other cytokines; however, this process was not
evaluated in our trial.
Two other trials have studied the effect of interruption of the leukotriene cascade on airway inflammation in asthma. Kane and colleagues used the orally active 5-lipoxygenase inhibitor zileuton in a trial design similar to the current study (35). Urinary LTE4 was significantly reduced in the zileuton arm. There were no significant differences in BAL parameters of inflammation between the zileuton and placebo arms; however, significant eosinophil influx was observed 24 h after SAC during placebo and was not observed during active treatment. On that basis, the researchers suggested that zileuton has an anti-inflammatory effect on allergen-driven airway inflammation. Wenzel and colleagues used the endogenous stimulus of nocturnal asthma to evaluate the effects of the same compound (36). At 4:00 A.M., blood and BAL eosinophilia were both significantly reduced in the zileuton arm compared with the placebo arm, but no significant changes in lung physiology were seen. These data are also consistent with the hypothesis that leukotrienes (whether hydroxy or cysteinyl cannot be determined from the zileuton studies) play a role in the recruitment of eosinophils to the airway in humans.
Several caveats must be considered. First, it is possible that allergen could spill out of the intended segment to produce more generalized lung inflammation. We view this as a minor problem for several reasons. First, our previously reported experience with the technique using BAL immediately after SAC minimized eosinophil recruitment to the saline-challenged segment (22), in marked contrast to an earlier technique in which washout was not performed and "saline segment" eosinophilia was more marked after 48 h (15). In the current study, we observed only a threefold increase in eosinophil concentration 48 h after saline challenge, but a 50- to 200-fold increase in eosinophil concentrations in the allergen-challenged sites. This observation supports our suggestion that there was little generalized pulmonary inflammation as a consequence of unintentional allergen spillage. A second consideration is the effect of the 5-min BAL on cell, mediator, and cytokine recovery 48 h after challenge. Some effects on the later lavage are likely, but these would have been equivalent in both drug and placebo arms. Finally, the degree of spill would not likely have differed between the drug and placebo arms of the study.
The concentrations of cysteinyl leukotrienes that we measured at baseline (about 300 pg/ml) are roughly comparable to those observed by Wenzel and colleagues (about 65 pg/ml) (37), and are somewhat lower than those reported by Miadonna and coworkers (320 pg/ml) (38). Considerable technical differences exist among the published studies of BAL in asthma, and direct interstudy comparisons are therefore not always valid. Furthermore, there is striking heterogeneity in leukotriene production (eightfold to tenfold) (37) and response to leukotriene blockade among patients; thus, some of the observed differences may be attributed to variation in the specific characteristics of the patient populations studied by various investigators. What is consistent throughout is the observation that cysteinyl leukotrienes are increased to a variable degree in allergic patients after allergen challenge.
In summary, SAC induces antigen dose-dependent airway inflammation 48 h after a challenge in atopic patients with asthma. Treatment with zafirlukast for 7 d was associated with reduced BAL fluid indices of airway inflammation 48 h after SAC, which suggests that interruption of the cysteinyl leukotriene pathway has potential for modifying the inflammatory response that underlies asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. William J. Calhoun, 3350 Terrace St./440 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261.
(Received in original form September 4, 1996 and in revised form November 12, 1997).
Accolate is a trademark, the property of Zeneca Limited.Dr. Lavins is currently employed by McNeil Pharmaceuticals, Spring House, Pennsylvania.
Acknowledgments: The authors thank Jennifer J. Brick, B.A., and Tammy L. Kanuch, B.S., for help with recruiting and screening subjects for this trial; Kimberley L. Hinton, B.S., for technical assistance; and Mary Jo Psomas, M.S., and Gregg Truitt, B.S., for editorial assistance.
Supported by a grant from Zeneca Pharmaceuticals.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Chung, K. F., and
P. J. Barnes.
1992.
Role of inflammatory mediators in
asthma.
Br. Med. Bull.
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