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ABSTRACT |
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Airway inflammation and remodeling in chronic asthma are characterized by airway eosinophilia, hyperplasia of goblet cells and smooth muscle, and subepithelial fibrosis. We examined the role of leukotrienes in a mouse model of allergen-induced chronic lung inflammation and fibrosis. BALB/c mice, after intraperitoneal ovalbumin (OVA) sensitization on Days 0 and 14, received intranasal OVA periodically Days 14-75. The OVA-treated mice developed an extensive eosinophil and mononuclear cell inflammatory response, goblet cell hyperplasia, and mucus occlusion of the airways. A striking feature of this inflammatory response was the widespread deposition of collagen beneath the airway epithelial cell layer and also in the lung interstitium in the sites of leukocytic infiltration that was not observed in the saline-treated controls. The cysteinyl leukotriene1 (CysLT1) receptor antagonist montelukast significantly reduced the airway eosinophil infiltration, mucus plugging, smooth muscle hyperplasia, and subepithelial fibrosis in the OVA-sensitized/challenged mice. The presence of Charcot-Leyden-like crystals in airway macrophages and the increased interleukin (IL)-4 and IL-13 mRNA expression in lung tissue and protein in BAL fluid seen in OVA-treated mice were also inhibited by CysLT1 receptor blockade. These data suggest an important role for cysteinyl leukotrienes in the pathogenesis of chronic allergic airway inflammation with fibrosis.
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Keywords: eosinophil; fibrosis; interleukin-4; interleukin-13; mucus
Airway structural changes that occur in patients with asthma in response to persistent inflammation are termed airway remodeling and include airway wall thickening, subepithelial fibrosis, and hyperplasia of mucus glands, myofibroblasts, smooth muscle, and vasculature (1). By endobronchial biopsy, an increase in activated eosinophils is seen in the epithelium and submucosa in patients with asthma compared with normal individuals. Morphometric analyses of autopsied lungs from patients with asthma demonstrate a marked increase in the number of goblet cells as a percentage of total airway cells and the amount of mucus in the lumen of the airways compared with control subjects without asthma (2). Further, a 30-fold increase in goblet cells and 3-fold increase in the intraluminal amount of mucus are found in the peripheral airways of patients dying of an acute attack of asthma compared with the peripheral airways of patients with asthma not dying of an acute attack (2). Of particular concern, bronchial biopsies demonstrate thickening of the subepithelial lamina reticularis with excess collagen deposition in the lung interstitium of patients with asthma compared with normal control subjects (3).
We have previously demonstrated in an acute murine model of human asthma that specific inhibitors of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) that prevent leukotriene formation block airway mucus release and infiltration by eosinophils, indicating the importance of leukotrienes in these features of allergic pulmonary inflammation (4). In this study, using a chronic model of allergic airway inflammation with subepithelial fibrosis, we found an important role for cysteinyl leukotrienes in the pathogenesis of key features of airway remodeling.
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All animal use procedures were approved by the University of Washington Animal Care Committee. Female BALB/c mice (aged 6-8 wk; The Jackson Laboratory, Bar Harbor, ME) received an intraperitoneal injection of 100 µg of ovalbumin (OVA) complexed with alum on Days 0 and 14 (4). Mice received an intranasal dose of 500 µg OVA on Days 14, 27, 28, 29, 47, 61, 73, 74, and 75. The control group received normal saline with alum intraperitoneally on Days 0 and 14 and saline without alum intranasally on Days 14, 27, 28, 29, 47, 61, 73, 74, and 75. A group of OVA-treated mice was administered the cysteinyl leukotriene1 (CysLT1) receptor antagonist montelukast sodium (MK-0476; Merck & Co., Inc.) that was dissolved in distilled water containing 10% Na2CO3 (5). Then 200-µl Alzet Model 2004 miniosmotic pumps (6 µl/d delivery rate; Alza Corporation, Palo Alto, CA) containing montelukast (1 mg/kg) or vehicle control were placed subcutaneously on Day 26 and replaced on Day 54.
On Day 76, 24 h after the last intranasal administration of either normal saline or OVA, noninvasive pulmonary mechanics were determined after aerosolization of methacholine in conscious, freely moving, spontaneously breathing mice using whole body plethysmography (Model PLY 3211; Buxco Electronics Inc., Sharon, CT) as described by Hamelmann and coworkers (6). The degree of bronchoconstriction was expressed as enhanced pause (Penh), a calculated dimensionless value, that correlates with measurement of airway resistance, impedance, and intrapleural pressure. On Day 77, invasive pulmonary mechanics were measured in the mice in response to an intravenous infusion of methacholine. Resistance (R), lung conductance (GL=1/R), and dynamic compliance (Cdyn) were determined for both the control period and during the peak response to intravenous challenge with methacholine (120 µg/kg) (7). Each mouse was then exsanguinated by cardiac puncture, bronchoalveolar lavage (BAL) performed on the right lung, and left lung tissue obtained (4).
For light microscopy and morphometry, the lung sections were stained with hematoxylin and eosin to assess the inflammatory cell infiltrate (0-4+ scale) (4), 0.05% aqueous eosin with methylene blue counterstaining to identify eosinophils per unit airway (2,200 µm2) (4), Masson's trichrome to determine collagen deposition in the lungs (8), and alcian blue, pH 2.5, with nuclear fast red counterstaining to identify airway goblet cells (as percent of total airway cells) and the degree of mucus plugging of the airways (0.5 mm to 0.8 mm in diameter) with the percent occlusion of airway diameter by mucus classified on a 0-4+ scale (4). Airway smooth muscle thickness was determined in hematoxylin and eosin-stained lung sections by measuring the thickness of the smooth muscle cell layer beneath the airway epithelial cell basement membrane at three sites tangential to each airway cross section examined. Morphometry was performed by individuals blinded to the protocol design (4). Cell counts were determined using the Point Counting Stereology System II software (9). A minimum of 10 fields throughout the upper and lower left lung tissue were randomly examined for the morphometric analyses. The lung sections were also examined on an electron microscope (Model 1200EX; JEOL Ltd., Tokyo, Japan) at 60 kV (10).
Total RNA was isolated from the right lung of each mouse, and
mRNA levels for interleukin (IL)-4, IL-5, IL-10, IL-13, and eotaxin
were determined using BD RiboQuant Multi-Probe ribonuclease protection assay kits (BD PharMingen, San Diego, CA). BAL fluid levels
of IL-4 (
2 pg/ml), IL-5 ( 7 pg/ml), IL-10 ( 4 pg/ml), IL-13 ( 1.5 pg/ml), and eotaxin ( 3 pg/ml) were determined by ELISA (R&D
Systems, Inc., Minneapolis, MN).
Statistical Analyses
The data are reported as the mean ± SE of the combined experiments. Differences were analyzed for significance (p < 0.05) by either Student's two-tailed t test for independent means or analysis of variance (ANOVA) using the protected least significant difference method as indicated.
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Effect of CysLT1 Receptor Blockade on Allergen-induced Airway Inflammation and Remodeling
On Day 77, 48 h after the final intranasal OVA or saline treatment in mice from each experimental group, BAL was performed (Figure 1), and lung tissue was obtained to assess inflammatory cell infiltration and mucus release (Figure 2), and collagen deposition (Figure 3). The effect of the CysLT1 receptor antagonist montelukast on airway inflammation and remodeling (Figures 4-9) was determined. montelukast (1 mg/kg) was administered by miniosmotic pump beginning on Day 26 of the protocol, 24 h prior to the first intranasal dose of OVA or saline, and continuing until BAL was performed and lung tissue obtained on Day 77.
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BAL fluid cells. OVA-treated mice had a 3.0-fold increase in total cells recovered from BAL fluid compared with the saline group (Figure 1A; p < 0.0001, OVA versus saline). Of the BAL fluid cells 27.2% were eosinophils in the OVA-sensitized/challenged mice compared with 1.0% of total cells in controls (Figure 1B; p < 0.0001, OVA versus saline). The mean number of eosinophils in the BAL fluid in the saline-treated controls was 0.01 ± 0.001 × 105 cells (Figure 1C). The OVA-sensitized/challenged mice had a 100-fold increase in eosinophils recovered in the BAL fluid to 1.0 ± 0.07 × 105 cells (Figure 1C; p < 0.0001, OVA versus saline). Treatment with montelukast decreased the influx of eosinophils into the BAL fluid by 68.7% (Figure 1C; p = 0.0002, montelukast/ OVA versus OVA), respectively.
Cellular infiltration of lung interstitium. By light microscopy, an extensive eosinophil and mononuclear cell infiltrate around the pulmonary blood vessels and airways was seen in the lung interstitium of OVA-sensitized/challenged mice compared with saline control animals (Figure 2A versus 2C). By morphometry, a 27.3-fold increase in total inflammatory cells infiltrating the lung interstitium was observed in OVA-sensitized/ challenged mice compared with saline control animals (Figure 5A; p < 0.0001, OVA versus saline). Airway eosinophil infiltration increased 41.3-fold from 0.53 ± 0.28 eosinophils/2,200 µm2 lung tissue in control animals to 21.8 ± 0.85 eosinophils/ 2,200 µm2 lung tissue in the OVA-treated mice (p < 0.0001, OVA versus saline) as determined by morphometry (Figure 5B). CysLT1 receptor antagonism by montelukast inhibited the total cellular and eosinophil infiltration of the lung tissue in OVA-sensitized/challenged mice (Figure 4A versus Figure 2A, and Figure 5A [p = 0.0002, montelukast/OVA versus OVA for total cells] and Figure 5B [p < 0.0001, montelukast/ OVA versus OVA for eosinophils]).
Airway goblet cell hyperplasia and mucus occlusion. Goblet cell hyperplasia was observed in the OVA-treated mice but not in the saline-treated control animals (Figure 2B versus 2D and Figure 5C). In the OVA-treated mice, goblet cells increased to 51.8 ± 2.0% of airway cells compared to 2.2 ± 1.0% of airway cells in controls (Figure 5C; p < 0.0001, OVA versus saline). montelukast (Figure 4B versus Figure 2B) significantly decreased airway goblet cell hyperplasia by 26.0% (Figure 5C; p = 0.0068, montelukast/OVA versus OVA).
A marked increase in airway mucus secretion was observed in the OVA-treated mice compared with control animals (Figure 2B versus 2D and Figure 5D). In the OVA-sensitized/challenged animals, a mean mucus occlusion score of 2.92 ± 0.14 was seen (Figure 5D; p < 0.0001, OVA versus saline) representing > 60% occlusion by mucus in 71.1% of the airways. With CysLT1 receptor antagonist treatment, the airway mucus occlusion score was significantly reduced by 38.4% to 1.86 ± 0.14 (Figure 5D; p = 0.0002, montelukast/OVA versus OVA). Of the airways in the montelukast/OVA treatment group 88.6% had < 30% occlusion of airway diameter by mucus.Airway smooth muscle hyperplasia. By morphometric analysis, the thickness of the smooth muscle layer surrounding the airways increased 2.1-fold to 29.4 µm in the OVA-treated mice compared with saline control animals (Figure 5E; p < 0.001, OVA versus saline). Airway smooth muscle thickness in the OVA-sensitized/challenged mice was reduced 80.1% by montelukast treatment (p < 0.001, montelukast/OVA versus OVA).
Airway collagen deposition/fibrosis. By Masson's trichrome staining, compared with saline control animals, dense collagen deposition/fibrosis was seen throughout the lung interstitium surrounding the airways and blood vessels and within the mixed inflammatory cell infiltrates in OVA-sensitized/challenged mice (Figure 3B-3E, versus 3A). CysLT1 receptor blockade by montelukast (Figure 4C versus Figure 3C-E) markedly reduced collagen deposition in the lung interstitium of OVA-treated mice, including the perivascular collagen deposition.
On a 0-4+ scale by morphometry, the mean airway fibrosis score was 2.2 ± 0.3 in the OVA-treated mice compared with 0.55 ± 0.07 in the saline-treated control animals (Figure 5F; p < 0.0001, OVA versus saline). Of note, the CysLT1 receptor antagonist montelukast reduced the airway fibrosis score in OVA-treated animals by 98.8% to 0.57 ± 0.09 (Figure 5F; p = 0.0005, montelukast/OVA versus OVA), which was not significantly different from the mean fibrosis score for the saline-treated controls (p = 0.8201, montelukast/OVA versus saline). By electron microscopy, montelukast was observed to markedly reduce the number and thickness of the collagen bundles in the lung interstitium of the OVA-treated mice (Figure 6A versus 6B).Eosinophil degranulation and Charcot-Leyden-like crystals. By morphometric analysis of the electron micrographs of eosinophils in the lung interstitium (Figure 7), 57.6% fewer cytoplasmic granules per eosinophil were observed in the OVA-treated mice (14.5 ± 1.2 granules/eosinophil cross section) compared with saline control animals (34.2 ± 4.1 granules/ eosinophil cross section; p < 0.0001, OVA versus saline). A significantly greater number of eosinophil granules/cell were seen in the OVA-sensitized/challenged mice treated with montelukast (22.5 ± 2.1 granules/eosinophil cross section; p = 0.0011, montelukast/OVA versus OVA).
In OVA-sensitized/challenged mice, electron-dense Charcot-Leyden-like crystals were noted in macrophages in the lung interstitium and alveolar macrophages (Figure 8A) and were membrane bound (Figure 8B). The Charcot-Leyden-like crystals were not seen in alveolar macrophages from OVA-sensitized/challenged mice treated with montelukast (Figure 8C) or saline-treated controls (Figure 8D).Cytokine production. Induction of significant levels of mRNA for IL-4, IL-5, IL-10, and IL-13 (Figure 9A) and eotaxin (not shown) was observed in the lungs of the OVA-treated mice compared with saline control animals. Montelukast reduced the mRNA levels of IL-4, IL-5, IL-10, and IL-13 (Figure 9A) but not eotaxin in the lung tissue of the OVA-treated mice. Significant levels of IL-4 and IL-13 were found in the BAL fluid of the OVA-treated mice compared with the saline control group (Figure 9B). Reductions of 63.7% and 58.1% in BAL fluid levels of IL-4 and IL-13, respectively, were seen in the montelukast/OVA group compared with OVA treatment alone (Figure 9B). IL-5, IL-10, and eotaxin were not detected in the BAL fluid of any of the study groups.
Effect of Leukotriene Blockers on Allergen-induced Airway Hyperreactivity to Methacholine
Noninvasive in vivo plethysmography. Airway reactivity was evaluated on Day 76, which was 24 h after the last intranasal challenge with OVA, by noninvasive in vivo plethysmography. In the OVA group, airway hyperreactivity was seen after aerosolized methacholine challenge (10 mg/ml), with a significant increase in Penh (% of air) compared with the saline group (Figure 10; p = 0.199, OVA versus saline). Montelukast did not significantly reduce the airway hyperresponsiveness to aerosolized methacholine in OVA-treated mice compared with the saline control animals (Figure 10; p < 0.05, montelukast/ OVA versus saline).
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Invasive in vivo plethysmography. Pulmonary mechanics were also assessed in response to intravenous methacholine on Day 77, which was 48 h after the last intranasal challenge with OVA, by invasive in vivo plethysmography. A significant decrease in both GL and Cdyn was seen in the OVA-sensitized/ challenged mice compared with the saline-treated control animals after intravenous methacholine (120 µg/kg) to indicate airway hyperreactivity in the OVA-treated group (Table 1; OVA versus saline, p < 0.05 for both GL and Cdyn). Treatment with montelukast did not reduce the airway hyperreactivity to intravenous methacholine in the OVA-treated mice (Table 1).
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METHODS
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We developed a mouse model with the following characteristic features of chronic airway inflammation and remodeling observed in patients with asthma: eosinophil infiltration into the lung interstitium and BAL fluid and release of Charcot-Leyden-like crystals, goblet cell hyperplasia with airway occlusion by mucus, airway smooth muscle hyperplasia, and increased collagen deposition around airways and blood vessels in the lungs. The airway remodeling changes observed in OVA-treated mice were significantly reduced by montelukast, which blocks the actions of cysteinyl leukotrienes (LT) C4, D4, and E4 mediated by the CysLT1 receptor. These data indicate that cysteinyl leukotrienes may be important in the pathogenesis of allergen-induced airway remodeling.
Cysteinyl leukotrienes have potent effects on leukocyte trafficking, airway mucus secretion, and collagen synthesis. In endothelial cells treated with LTC4 or LTD4, P-selectin (CD62) translocates to the luminal surface from subcellular sites in Weibel-Palade bodies (11). CysLT1 receptors are expressed in human eosinophils (12), and their activation by cysteinyl leukotrienes may lead to eosinophil recruitment to inflammatory sites. Inhaled LTD4 and LTE4 increase the number of eosinophils in induced sputum and bronchial lamina propria of patients with asthma (13, 14). CysLT1 receptor antagonists reduce peripheral blood (15), sputum (16), and BAL fluid (17) eosinophils, and decrease infiltration of activated eosinophils in bronchial tissue (18) in patients with asthma. The cysteinyl leukotrienes C4, D4, and E4 are also potent mucus secretagogues with greater activity in causing mucus secretion from isolated human bronchial tissue than LTB4 or cyclooxygenase products of arachidonic acid metabolism (19). Our results indicate that cysteinyl leukotrienes are also involved in airway goblet cell hyperplasia as CysLT1 receptor blockade significantly reduced the allergen-induced increase in the number of goblet cells in the airways observed in OVA-sensitized/challenged mice.
Important in the mediation of chronic allergic airway inflammation are CD4+ Th and CD8+ T cytotoxic (Tc) cells that
are recruited to the airways of patients with asthma after allergen challenge. In patients with asthma, CD4+ and CD8+ T
cells secrete cytokines (IL-4, IL-5, and IL-13) with a type 2 cytokine phenotype (Th2 and Tc2, respectively). Administration of IL-4 in mice induces mucus accumulation in the airways
(20). In IL-4 transgenic mice, the increased levels of IL-4 upregulate MUC5AC gene expression, leading to mucus accumulation in nonciliated airway epithelial cells and a 5- to 10-fold increase in mucus protein in the BAL fluid of these mice
compared with transgene-negative controls (21). We found
that montelukast reduced the elevated levels of IL-4 and IL-13
found in the BAL fluid of OVA-treated mice, suggesting an
important antiinflammatory action of this compound. We
have recently reported that soluble IL-4 receptor (sIL-4R),
which contains only the extracellular domain of IL-4R and
blocks the biological actions of IL-4, significantly inhibits airway mucus hypersecretion in OVA-sensitized/challenged mice (22). sIL-4R treatment was instituted in these mice after the extensive replacement of airway epithelial cells by mucus-producing goblet cells indicating an important role for IL-4 in the
maintenance of mucus glycoprotein hypersecretion in allergic
airways. IL-13 is also likely important in mediating allergen-induced airway mucus release. Exogenously administered IL-13
induces airway mucus hypersecretion in mice (20, 23). IL-13
transgenic mice, with increased IL-13 gene expression in the
airways and IL-13 levels in the BAL fluid compared with
transgene (
) littermate control mice, have increased production of neutral and acidic mucus and goblet cell hyperplasia in
the airways (24). Inhibition of IL-4 and IL-13 production by
LT receptor antagonism may be an important mechanism of
reducing goblet cell hyperplasia and mucus hypersecretion in
allergic airways.
We found that eosinophil degranulation in the OVA-treated mice was inhibited by CysLT1 receptor blockade. In
patients with asthma, CysLT1 receptor antagonists decrease
the levels of eosinophil cationic protein and LTC4 in nasal
lavage fluid (25), an effect that may be secondary in part to
reduction in the number of eosinophils, major producers of
LTC4 infiltrating the allergic respiratory tract. A striking feature of the airway histopathology of the OVA-treated mice was
the presence of Charcot-Leyden-like crystals in lung macrophages. Charcot-Leyden crystal protein, found in the sputum of
patients with asthma, is a hydrophobic polypeptide present
in the granules, cytoplasm, and euchromatin of the nucleus of eosinophils (26). A lysolecithin acylhydrolase, Charcot-Leyden crystal protein has moderate amino acid sequence
homology and x-ray crystallographic characteristics similar
to the galectin family of 
-galactoside binding proteins; the
mouse Charcot-Leyden crystal -galactoside binding site is
79%/69% (nucleotide/amino acid) identical to that of humans (27). Charcot-Leyden-like crystals have been previously observed in the airways of mice in association with
eosinophilia. In C57BL6 mice with eosinophilia after Cryptococcus neoformans infection, Charcot-Leyden-like crystal deposition in the lungs is found both extracellularly in bronchioles and alveoli and intracellularly; fibrotic foci are also
observed in their airways (28). Treatment of these mice with
IL-5 neutralizing antibody blocks airway crystal formation
(28). IL-13 transgenic mice also have Charcot-Leyden-like
crystal release into the alveoli and focal areas of airway fibrosis (24). Increased levels of eotaxin but not IL-4, IL-5,
MCP-5, or GM-CSF are found in the BAL fluid and lungs of
the IL-13 transgenic mice compared with control mice (24).
Although significant levels of eotaxin and IL-5 mRNA were
found in lung tissue of OVA-treated mice, these proteins were
not detected in their BAL fluid. The role of Charcot-Leyden-like crystal deposition in the airways in the pathogenesis of the
airway fibrosis we observed in our model of allergen-induced
lung injury is unknown.
A significant increase in airway smooth muscle cell number is observed in endobronchial biopsies from patients with asthma compared with normal subjects (29). LTD4 augments epidermal growth factor-induced human airway smooth muscle proliferation in vitro (30). Our findings in OVA-treated mice are consistent with prior work in allergen-sensitized/ challenged Brown Norway rats (31), demonstrating that allergen-induced increases in airway smooth muscle are significantly reduced by treatment with a CysLT1 receptor antagonist. These studies indicate that cysteinyl leukotrienes are important mediators of the increase in airway smooth muscle observed after repeated allergen challenge.
Airway thickening beneath the basement membrane occurs with deposition of collagen and other extracellular matrix proteins, including fibronectin, tenascin, and laminin (1). The air- blood barrier between the alveolar type I epithelial cells and the capillaries is increased by this thickening of the basement membrane and deposition of collagen fibers. This airway wall thickening correlates with clinically severe asthma (34) and is a prominent feature of lung tissue from patients dying with fatal asthma. Collagen deposition occurs in the connective tissue layer surrounding the blood vessels and alveolar interstitium. In situ hybridization studies demonstrate type I collagen gene expression in connective tissue fibroblasts (35). In OVA-sensitized/challenged mice treated with montelukast, we found that airway collagen deposition was not significantly different from saline-treated control subjects. These data indicate an important role of cysteinyl leukotrienes in collagen deposition/lung fibrosis in this model of chronic asthma. Prior work has demonstrated that LTC4 stimulates collagen synthesis in vitro in human lung fibroblast cell lines from both normal individuals and patients with interstitial pulmonary fibrosis (IPF) who have increased accumulation of extracellular matrix proteins (36). Homogenates of lung tissue from patients with IPF have significantly greater levels of leukotrienes than nonfibrotic lung tissue from control subjects (37). Alveolar macrophages from patients with IPF also spontaneously release more leukotrienes in vitro than control alveolar macrophages suggesting that leukotriene overproduction may contribute to the pathogenesis of IPF (37).
Our data also dissociate the airway inflammation and remodeling changes from the airway hyperreactivity observed in this chronic asthma model. Despite significant reduction by montelukast of airway smooth muscle hyperplasia, fibrosis, inflammatory cell infiltration, and expression of type 2 cytokines, including IL-10, which is important for expression of airway hyperresponsiveness (38), CysLT1 receptor blockade had no effect on airway hyperreactivity to either aerosolized methacholine or intravenous methacholine as determined by noninvasive and invasive plethysmography, respectively. These data are consistent with data from our acute murine model of asthma in which 5-lipoxygenase inhibition by zileuton and FLAP inhibition by MK-886 (given 30 min before OVA challenge on three successive days with airway hyperreactivity to methacholine measured 24 h after the last OVA dose) to prevent leukotriene synthesis failed to reduce airway hyperreactivity despite blocking airway eosinophilia. Similarly, in prior work with this acute asthma model, we found that intraperitoneal mAb blockade of CD49d on circulating leukocytes prior to allergen challenge inhibits BAL fluid eosinophilia, but does not alter airway hyperreactivity, IL-4 or IL-5 production, or mucus hypersecretion (7). Treatment of OVA-sensitized/challenged mice with sIL-4R also blocks the influx of eosinophils into the airways without reducing airway hyperreactivity after methacholine challenge. Further, several studies have not found a correlation between airway inflammation and hyperreactivity in patients with asthma (39, 40).
Our studies demonstrate that CysLT1 receptor antagonism has a significant antiinflammatory effect on allergen-induced lung inflammation and fibrosis in an animal model reflective of the airway remodeling changes observed in patients with persistent asthma. The potent antiinflammatory effects of CysLT1 receptor blockade may be beneficial in the long-term management of asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to William R. Henderson, Jr., Department of Medicine, Box 356523, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-6523. E-mail: joangb{at}u.washington.edu
(Received in original form May 11, 2001 and accepted in revised form September 13, 2001).
Acknowledgments: The authors thank Wayne J. Lamm, Benjamin Sun, and Ying-Tzang Tien for excellent technical assistance and Rachel Norris for typing this manuscript.
Supported by NIH grants AI42989, HL61756, and HL30542 and by a Medical School grant from Merck & Co., Inc.
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References |
|---|
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ABSTRACT
INTRODUCTION
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REFERENCES
|
|---|
1. Elias JA, Zhu Z, Chupp G, Homer RJ. Airway remodeling in asthma. J Clin Invest 1999; 104: 1001 [Medline].
2.
Aikawa T,
Shimura S,
Sasaki H,
Ebina M,
Takishima T.
Marked goblet
cell hyperplasia with mucus accumulation in the airways of patients
who died of severe acute asthma attack.
Chest
1992;
101:
916-921
3. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989; 1: 520-524 [Medline].
4.
Henderson WR Jr,,
Lewis DB,
Albert RK,
Zhang Y,
Lamm WJE,
Chiang GKS,
Jones F,
Eriksen P,
Tien Y,
Jonas M,
Chi EY.
The importance of leukotrienes in airway inflammation in a mouse model of
asthma.
J Exp Med
1996;
184:
1483-1494
5. Jones TR, Labelle M, Belley M, Champion E, Charette L, Evans J, Ford-Hutchinson AW, Gauthier J-Y, Lord A, Masson P, et al . . Pharmacology of montelukast sodium (SingulairTM), a potent and selective leukotriene D4 receptor antagonist. Can J Physiol Pharmacol 1995; 73: 191-201 [Medline].
6.
Hamelmann E,
Schwarze J,
Takeda K,
Oshiba A,
Larsen GL,
Irvin CG,
Gelfand EW.
Noninvasive measurement of airway responsiveness in
allergic mice using barometric plethysmography.
Am J Respir Crit
Care Med
1997;
156:
766-775
7.
Henderson WR Jr.,,
Chi EY,
Albert RK,
Chu S-J,
Lamm WJE,
Rochon Y,
Christie PE,
Harlan JM.
Blockade of CD49d (
4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.
J Clin Invest
1997;
100:
3083-3092
[Medline].
8. Sheehan DC, Hrapchak BB. Connective tissue and muscle fiber stains. In: Theory and practice of histotechnology. Columbus, OH: Battelle Press; 1980, p. 180-201.
9. Bertram JF, Bolender RP. Counting parenchymal cells in the goat lung with serial section reconstruction and stereology. Am Rev Respir Dis 1986; 133: 891-898 [Medline].
10. Henderson WR, Chi EY. Ultrastructural characterization and morphometric analysis of human eosinophil degranulation. J Cell Sci 1985; 73: 33-48 [Abstract].
11. McEver RP. Selectins: novel receptors that mediate leukocyte adhesion during inflammation. Thromb Haemost 1991; 65: 223-228 [Medline].
12.
Figueroa DJ,
Breyer RM,
Defoe SK,
Kargman S,
Daugherty BL,
Waldburger K,
Liu Q,
Clements M,
Zeng Z,
O'Neill GP, et al
.
. Expression
of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes.
Am J Respir Crit Care Med
2001;
163:
226-233
13. Diamant Z, Hiltermann JT, van Rensen EL, Callenbach PM, Veselic-Charvat M, van der Veen H, Sont JK, Sterk PJ. The effect of inhaled leukotriene D4 and methacholine on sputum cell differentials in asthma. Am J Respir Crit Care Med 1997; 155: 1247-1253 [Abstract].
14. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet 1993; 341: 989-990 [Medline].
15.
Knorr B,
Matz J,
Bernstein JA,
Nguyen H,
Seidenberg BC,
Reiss TF,
Becker A.
montelukast for chronic asthma in 6- to 14-year-old children: a randomized, double-blind trial.
JAMA
1998;
279:
1181-1186
16. Pizzichini E, Leff JA, Reiss TF, Hendeles L, Boulet LP, Wei LX, Efthimiadis AE, Zhang J, Hargreave FE. montelukast reduces airway eosinophilic inflammation in asthma: a randomized, controlled trial. Eur Respir J 1999; 14: 12-18 [Abstract].
17. Calhoun WJ, Williams KL, Simonson SG, Lavins BJ. Effect of zafirlukast (Accolate® ) on airway inflammation after segmental allergen challenge in patients with mild asthma [abstract]. Am J Respir Crit Care Med 1997; 155: A662 .
18.
Nakamura Y,
Hoshino M,
Sim JJ,
Ishii K,
Hosaka K,
Sakamoto T.
Effect of
the leukotriene receptor antagonist pranlukast on cellular infiltration in
the bronchial mucosa of patients with asthma.
Thorax
1998;
53:
835-841
19. Larivée P, Levine SJ, Rieves RD, Shelhamer JH. Airway inflammation and mucous hypersecretion. In: Takishima T, Shimura S, editors. Airway secretion. Physiological bases for the control of mucous hypersecretion. New York: Marcel Dekker, Inc.; 1994. p. 469-511.
20.
Grünig G,
Warnock M,
Wakil AE,
Venkayya R,
Brombacher F,
Rennick DM,
Sheppard D,
Mohrs M,
Donaldson DD,
Locksley RM,
Corry DB.
Requirement for IL-13 independently of IL-4 in experimental asthma.
Science
1998;
282:
2261-2263
21. Temann U-A, Prasad B, Gallup MW, Basbaum C, Ho SB, Flavell RA, Rankin JA. A novel role for murine IL-4 in vivo: Induction of MUC5AC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol 1997; 16: 471-478 [Abstract].
22.
Henderson WR Jr.,,
Chi EY,
Maliszewski CR.
Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse
model of asthma.
J Immunol
2000;
164:
1086-1095
23.
Wills-Karp M,
Luyimbazi J,
Xu X,
Schofield B,
Neben TY,
Karp CL,
Donaldson DD.
Interleukin-13: central mediator of allergic asthma.
Science
1998;
282:
2258-2261
24. Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999; 103: 779-788 [Medline].
25. Volovitz B, Tabachnik E, Nussinovitch M, Shtaif B, Blau H, Gil-Ad I, Weizman A, Varsano I. montelukast, a leukotriene receptor antagonist, reduces the concentration of leukotrienes in the respiratory tract of children with persistent asthma. J Allergy Clin Immunol 1999; 104: 1162-1167 [Medline].
26. Calafat J, Janssen H, Knol EF, Weller PF, Egesten A. Ultrastructural localization of Charcot-Leyden crystal protein in human eosinophils and basophils. Eur J Haematol 1997; 58: 56-66 [Medline].
27. Dyer KD, Handen JS, Rosenberg HF. The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes. Genomics 1997; 40: 217-221 [Medline].
28.
Huffnagle GB,
Boyd MB,
Street NE,
Lipscomb MF.
IL-5 is required for
eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in
genetically susceptible mice (C57BL/6).
J Immunol
1998;
160:
2393-2400
29. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990; 3: 507-511 .
30.
Panettieri RA,
Tan EM,
Ciocca V,
Luttmann MA,
Leonard TB,
Hay DW.
Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction in vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists.
Am J Respir Cell Mol Biol
1998;
19:
453-461
31. Sapienza S, Du T, Eidelman DH, Wang NS, Martin JG. Structural changes in the airways of sensitized brown Norway rats after antigen challenge. Am Rev Respir Dis 1991; 144: 423-427 [Medline].
32. Wang CG, Du T, Xu LJ, Martin JG. Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in the rat. Am Rev Respir Dis 1993; 148: 413-417 [Medline].
33. Salmon M, Walsh DA, Huang TJ, Barnes PJ, Leonard TB, Hay DW, Chung KF. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats. Br J Pharmacol 1999; 127: 1151-1158 [Medline].
34.
Boulet LP,
Laviolette M,
Turcotte H,
Cartier A,
Dugas M,
Malo JL,
Boutet M.
Bronchial subepithelial fibrosis correlates with airway responsiveness to methacholine.
Chest
1997;
112:
45-52
35. Jonas M, Su ML, Henderson WR, Chi E. In situ hybridization and immunochemistry of type 1 collagen in idiopathic and asthmatic lung fibrosis. Am J Respir Crit Care Med 1995; 151: A704 .
36. Medina L, Perez-Ramos J, Ramirez R, Selman M, Pardo A. Leukotriene C4 upregulates collagenase expression and synthesis in human lung fibroblasts. Biochim Biophys Acta 1994; 1224: 168-174 [Medline].
37. Wilborn J, Bailie M, Coffey M, Burdick M, Strieter R, Peters-Golden M. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J Clin Invest 1996; 97: 1827-1836 [Medline].
38.
Makela MJ,
Kanehiro A,
Borish L,
Dakhama A,
Loader J,
Joetham A,
Xing Z,
Jordana M,
Larsen GL,
Gelfand EW.
IL-10 is necessary for
the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization.
Proc Natl Acad Sci USA
2000;
97:
6007-6012
39. Ollerenshaw SL, Woolcock AJ. Characteristics of the inflammation in biopsies from large airways of subjects with asthma and subjects with chronic airflow limitation. Am Rev Respir Dis 1992; 145: 922-927 [Medline].
40.
Crimi E,
Spanevello A,
Neri M,
Ind PW,
Rossi GA,
Brusasco V.
Dissociation between airway inflammation and airway hyperresponsiveness
in allergic asthma.
Am J Respir Crit Care Med
1998;
157:
4-9
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