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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The temporal association between airway inflammation and airway hyperresponsiveness (AHR) has been analyzed in BALB/c mice sensitized to, and subsequently exposed to, a single intranasal challenge of ovalbumin (OVA). In OVA-sensitized/challenged animals only a small increase in responsiveness to methacholine (MCh) was seen at 8 h, peaked at 24 to 48 h, and resolved by 96 h. An early bronchoalveolar lavage fluid (BALF) neutrophil infiltrate (peaking at 8 h postchallenge; ~ 72% total cells was observed) that returned to baseline by 48 h. BALF eosinophil numbers did not increase until 48 h (~ 32% of total cells), peaked at 96 h (~ 38%
total cells), and remained elevated at 8 d (~ 27% total cells). Airway tissue eosinophilia preceded changes in BALF. Eosinophil peroxidase (EPO) levels in BALF were elevated in OVA-sensitized/challenged mice at 48 h only. BALF TNF-
levels peaked at 8 h, whereas
IL-5 and IL-4 levels peaked at 24 h. IL-13 levels were increased at both 24 and 48 h. Mucus-positive cells were not observed in the airway epithelium until 48 h. Administration of IL-5 or VLA-4 antibody prior to OVA challenge prevented the development of AHR in sensitized mice as well as BALF and tissue eosinophilia. These data
identify a temporal association between Th2 cytokine production, tissue eosinophil infiltration and activation, and, importantly, both
the development and resolution kinetics of AHR. Moreover, the
antibody studies further support the association of eosinophilia with
the pathogenesis of AHR.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Airway hyperresponsiveness (AHR) is a well-established characteristic of allergic asthma and is believed to be the result of airway mucosal inflammation associated with goblet cell hyperplasia, as well as epithelial damage and smooth muscle hypertrophy. Clinical investigations have suggested a relationship between the presence of activated airway inflammatory cells (including T cells, mast cells, monocytes, eosinophils, and neutrophils), morphologic changes in airway tissues, and the development and severity of AHR (1).
The eosinophil is thought to be a major effector cell in the pathogenesis of allergen-mediated AHR. In particular, the release of cytotoxic granule proteins, which can damage the airway epithelium, are thought to contribute to this phenomenon (4). Although many clinical studies have demonstrated an association between the number of eosinophils in bronchoalveolar lavage fluid (BALF) and biopsy samples with disease severity/AHR, a number of studies have failed to demonstrate such a relationship (5). Indeed, it has been suggested that the sole presence of eosinophils is not sufficient for AHR and that eosinophil activation correlates better with AHR than with eosinophil number per se (6). Correspondingly, a number of murine studies support the hypothesis that increased levels of eosinophils and, more importantly, their cytotoxic products in the airways underlies the pathogenesis of AHR (7). However, as in the clinical studies, certain discrepancies exist. Several murine studies have dissociated airway eosinophilia from AHR (11). In the absence of reliable markers of eosinophil activation, the controversy remains unresolved.
Despite the fact that the eosinophil is usually the predominant infiltrating leukocyte in allergic asthma, other cell types may play a significant effector role in the development of AHR. For example neutrophils, like eosinophils, are capable of releasing proteases, reactive oxygen species, and lipid mediators
that may contribute to tissue damage and AHR (14, 15). Similarly, activation of airway mucosal mast cells may lead to the
release of proinflammatory mediators and the cytokines, IL-4,
IL-5, and tumor necrosis factor (TNF)-, all of which may contribute to AHR (16), although, mast cells may not be essential
under some conditions (17).
It is now increasingly clear that T cells, through the release of specific cytokines, which regulate effector cell recruitment and function, orchestrate the inflammatory response leading to AHR. Increased numbers of CD4+ T cells have been identified in the bronchial mucosa of allergic asthmatics expressing elevated levels of IL-3, IL-4, IL-5, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) consistent with the T helper (Th) 2 phenotype (18, 19). A number of murine studies have now demonstrated the critical role of CD4+ and CD8+ T cells of the type 2 phenotype in the development of allergic airway eosinophilia and subsequent AHR (12, 20).
Murine models of allergic asthma have provided further insight into the major mechanisms that may result in AHR. Importantly, discrepancies between studies, as discussed above, serve to further highlight the complex nature of the response. Among the issues that may contribute to some of the inconsistency is the fact that in many animal studies the relationship between airway inflammation and AHR has only been evaluated at a single time point when AHR is established. This is without consideration of the preceding events leading to AHR or the continuing events that result in resolution of AHR. Thus, studies investigating the sequence of inflammatory events after allergen challenge and the temporal association with AHR may be critical to further our understanding of the pathogenesis of AHR. Only a few studies have evaluated the sequence of inflammatory events taking place after allergen challenge (24). Even fewer have evaluated the temporal relationship between the kinetics of inflammatory responses and development/resolution of AHR (24, 26). These studies were limited by the use of multiple-challenge protocols in which inflammatory mediator release and cell recruitment may occur and accumulate between challenges, and the evaluation of limited time points. In the present study, we used an approach in which mice were first sensitized to ovalbumin and subsequently given a single intranasal challenge (SIN)3 of ovalbumin. This allowed us to evaluate the temporal association between airway tissue and (BALF) inflammation, and the development and resolution of AHR.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animals
Female BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were maintained on ovalbumin (OVA)-free diets. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.
Sensitization and Airway Challenge
Groups of mice 10 to 12 wk of age were sensitized intraperitoneally by injection of 20 µg OVA (Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg aluminum hydroxide (AlumImuject; Pierce Chemical, Rockford, IL) in a total volume of 100 µl on Days 1 and 14. Mice received a SIN with OVA (20 µl, 5 mg/ml in saline) on Day 28. At different time points (8, 24, 48, and 96 h and 8 d) after OVA challenge, using different groups of mice, airway hyperresponsiveness (AHR) was assessed and tissues were obtained for further analysis. Control mice groups received OVA SIN alone.
Administration of IL-5 and VLA-4 Monoclonal Antibodies
Rat antimouse IL-5 mAb (TRFK5) and VLA-4 mAb (PS/2) were purified from their respective hybridoma (American Type Culture Collection, Manassas, VA) using a Protein G-Sepharose affinity column (Pharmacia, Uppsala, Sweden). Anti-IL-5, anti-VLA-4 or control rat IgG (50 µg in 200 µl saline) was administered intravenously 1 h prior to intranasal OVA challenge.
Determination of Airway Responsiveness
Airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine (MCh) using two different techniques.
Technique 1. Measurement of airway responsiveness in conscious, spontaneously breathing animals. Airway responsiveness to MCh in conscious, spontaneously breathing animals was measured by barometric plethysmography (Buxco, Troy, NY) as described (28). Pressure differences were measured between the main chamber of the plethysmograph, containing the animal and a reference chamber (box pressure signal). Mice were challenged with aerosolized saline (for the baseline measurement) or MCh (6 to 100 mg/ml) for 3 min and readings were taken and averaged for 3 min after each nebulization. Data are expressed as the fold increase above saline challenge baseline values using the dimensionless parameter Penh3 as described (28).
Technique 2. Measurement of airway responsiveness in anesthetized ventilated animals. Anesthetized (pentobarbital sodium, 70 to 90 mg/kg, given intraperitoneally), tracheostomized (stainless steel cannula, 18G) mice were mechanically ventilated and lung function was assessed using methods described by Takeda and colleagues (17). Mice were placed in a whole-body plethysmograph and ventilated (Model 683; Harvard Apparatus, South Natick, MA) via the tracheostomy tube at 160 breaths/min and a tidal volume of 150 µl with a positive end-expiratory pressure of 2 to 4 cm H2O. Transpulmonary pressure, lung volume, and flow were determined. Lung resistance (RL) was continuously computed (Labview, National Instruments, TX) by fitting flow, volume, and pressure to an equation of motion. MCh aerosol was administered for 10 s (60 breaths/min, 500 µl tidal volume) in increasing concentrations (1.56, 3.12, 6.25, and 12.5 mg/ml). Maximum values of RL were taken and expressed as a percentage change from baseline after saline aerosol.
Bronchoalveolar Lavage
Immediately after assessment of AHR, lungs were lavaged via a tracheal tube with Hanks' balanced salt solution (HBSS, 1 × 1 ml). Total leukocyte numbers were measured (Coulter Counter; Coulter Corporation, Hialeah, FL). Differential cell counts were performed by counting at least 200 cells on cytocentrifuged preparations (Cytospin 2; Cytospin, Shandon Ltd., Runcorn, Cheshire, UK), stained with Leukostat (Fisher Diagnostics, Fair Lawn, NJ), and differentiated by standard hematologic procedures.
Determination of Eosinophil Peroxidase (EPO) Activity
In a separate series of studies, levels of EPO in BALF supernatant were determined as previously described (29). Briefly, 100 µl of the substrate solution (0.1 mM o-phenylene-diamine-dihydrochloride, 0.1% Triton X-100, 1 mM hydrogen peroxide in 0.05 M TRIS-HCl; all reagents from Sigma) were added to 100 µl of supernatant in microtiter plates and incubated for 30 min at 37° C. The reaction was stopped by adding 50 µl of 4 M sulfuric acid, and the optical densities were read in a microtiter autoreader at 492 nm. This method has been shown to be specific for the peroxidase activity of eosinophils (29).
Histochemistry
Lungs were fixed by inflation (1 ml) and immersion in 10% formalin. Cells containing eosinophilic major basic protein (MBP) were identified by immunohistochemical staining as previously described using rabbit-antimouse MBP (7). The slides were examined in a blinded fashion with a Nikon microscope equipped with a fluorescein filter system. Numbers of eosinophils in the perivascular and peribronchial tissues were evaluated using the IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh counting six to eight different fields per animal. For detection of mucus-containing cells in formalin-fixed airway tissue, sections (6 µm) were cut and stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. The number of mucus-containing cells per millimeter of basement membrane was determined. In addition, epithelial cells were scored according to their mucus content (less than 75% of the cytoplasm PAS positive, 0; greater than 75% of the cytoplasm PAS positive, 1).
Measurement of Bronchoalveolar Lavage Fluid Cytokines
Cytokine levels in the BALF supernatants were measured by ELISA.
Briefly, 96-well plates (Immulon 2; Dynatech, Chantilly, VA) were
coated with either anti-TNF- (MP6-XT22), anti-IFN-
(R4-6A2), anti-IL-4 (11B11), or anti-IL-5 (TRFK-5) (all PharMingen, San Diego, CA)
and blocked with PBS/10% fetal calf serum overnight. Samples were
added: biotinylated anti-TNF- (MP6-XT3) anti-IFN- (XMG 1.2), anti-IL-4 (BVD6-24G2), or anti-IL-5 (TRFK-4) were used as detection antibodies (all PharMingen); and the reaction was amplified with avidin-horseradish peroxidase (Sigma). Cytokine levels were determined by
comparison with the known cytokine standards (PharMingen). The
limit of detection was 10 pg/ml. Similarly, IL-13 levels were determined by the use of a quantitative colorimetric sandwich ELISA kit
(Quantikine M mouse IL-13 ELISA, #M1300C; R&D Systems, Oxon,
UK). The limit of detection was 1.5 pg/ml.
Statistical Analysis
Analysis of variance (ANOVA) was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by Tukey-Kramer HSD test; p values for significance were set to 0.05. Values for all measurements are expressed as the mean ± standard error of the mean (SEM). Where possible, the correlation between airway responsiveness (measured at 50 mg/ml MCh for Penh or 12 mg/ml MCh for RL) and the different inflammatory parameters measured were performed. Paired data for challenged alone and sensitized/challenged mice were used in the analysis for each time point. Data are presented as the correlation coefficient, r2, and the p value for significance.
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DISCUSSION
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Kinetics of AHR after SIN OVA Challenge
At different time points (8, 24, 48, and 96 h and 8 d) after a SIN OVA challenge, the development and resolution of AHR to MCh was measured in conscious, spontaneously breathing animals by barometric plethysmography. Baseline (after saline) Penh measurements in OVA-challenged alone and OVA-sensitized/challenged mice at 8 h were 0.83 ± 0.07 Penh and 0.76 ± 0.07, respectively (p > 0.05). No significant differences were seen in baseline Penh values at any of the other time points investigated. In OVA-sensitized/challenged animals only a small increase in airway responsiveness to MCh was seen at 8 h when compared with animals receiving OVA challenge alone (Figure 1a). However, AHR to MCh continued to increase significantly with time in the OVA-sensitized/ challenged mice, peaking at 24 h (12.0 ± 1.6-fold increase in Penh at 50 mg/ml MCh) (Figure 1c). This AHR to MCh in the OVA-sensitized/challenged mice was maintained at 48 h (9.0 ± 0.9-fold increase in Penh at 50 mg/ml MCh) (Figure 1e) but had almost completely resolved by 96 h and 8 d (Figures 1g and 1i). No significant changes in airway responsiveness to MCh with time were seen in the mice receiving SIN OVA challenge alone.
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To confirm that the changes seen in airway responsiveness to MCh were indeed caused by changes in the lung, and not simply the result of changes in the nasopharynx resulting from the route of OVA challenge, RL changes to MCh were measured in anesthetized, tracheostomized, and ventilated mice using whole-body plethysmography. Baseline RL measurements in OVA-challenged alone and sensitized OVA-challenged mice at 8 h were 0.77 ± 0.04 cm/H2O/ml/s and 0.66 ± 0.06 cm/H2O/ ml/s, respectively (p > 0.05), and did not differ significantly at any of the other time points measured. Consistent with our observations in conscious, spontaneously breathing animals, a small but nonsignificant AHR to MCh initially was detected at 8 h in the OVA-sensitized/challenged animals, continued to increase significantly with time, peaking at 48 h and resolving between 96 h and 8 d (Figures 1b, 1d, 1f, 1h, and 1j).
Kinetics of BALF Cellular Infiltrate after SIN OVA Challenge
Total cell numbers recovered in BALF were essentially the same in OVA-challenged alone and OVA-sensitized/challenged mice at all but the 24-h time point (OVA-challenge alone: 226 ± 15 × 103 cells/ml, versus OVA-sensitized/challenged: 371 ± 35 × 103 cells/ml, p < 0.05) (Figure 2a). No significant changes in total cell number were seen with time in either challenge groups, and no correlation with airway responsiveness (Penh or RL) was observed.
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Although the inflammatory cell type recovered in the BALF of the mice receiving OVA-challenge alone consisted almost entirely of macrophages at all time points analyzed (Figure 2b), significant changes in the composition of the inflammatory cell infiltrate was observed with time in the OVA-sensitized/challenged animals. Macrophage number did not differ significantly with time (Figure 2b), and lymphocyte numbers were increased only at 8 d (Figure 2c). However, an early predominant neutrophil infiltrate peaking at 8 h (~ 72% of total cells) was observed. This was still significantly elevated at 24 h (~ 50% of total cells) but had returned to baseline levels by 48 h (~ 7% of total cells) (Figure 2d). In contrast, in OVA-sensitized/challenged mice, BALF eosinophil numbers did not significantly increase until 48 h (~ 32% of total cells), which was 24 h after the development of significant AHR. Furthermore, the eosinophil infiltrate peaked at 96 h (~ 38% of total cells) and remained elevated at 8 d (~ 27% of total cells) (Figure 2e), times at which AHR had resolved. Correlation analysis revealed no correlation of macrophage or lymphocyte number with airway responsiveness measured as Penh or RL at any time point. In contrast, the early increase in neutrophil number at 8 and 24 h correlated significantly with airway responsiveness measured by Penh (8 h, r2 = 0.475; p = 0.006: 24 h, r2 = 0.369; p = 0.007) and more so with RL (8 h, r2 = 0.556; p = 0.002; 24 h, r2 = 0.448; p = 0.017). Moreover, the later increase in BALF eosinophils at 48 h demonstrated good correlation with airway responsiveness measured by Penh (r2 = 0.566, p = 0.003) and RL (r2 = 0.864, p < 0.0001). The correlation between eosinophil number and airway responsiveness was still observed at later times, particularly when eosinophils numbers were compared with RL (96 h, r2 = 0.801; p = 0.0002; 8 d, r2 = 0.699; p = 0.0007).
Kinetics of Airway Tissue Eosinophil Inflammation after SIN OVA Challenge
The kinetics of eosinophilic inflammation were assessed in airway tissues using an antibody directed against MBP (Figures 3 and 4). Mice receiving OVA-challenge alone demonstrated little perivascular (PV) or peribronchial (PB) tissue eosinophilia (< 25 eosinophils/mm2) at any time point analyzed (Figures 3 and 4). In contrast, a PV (181 ± 29 eosinophils/mm2) and PB (117 ± 23 eosinophils/mm2) eosinophilia was present in the airways of OVA-sensitized/challenged mice at 8 h (Figures 3 and 4); i.e., before their appearance in the BALF and before the development of AHR. Both PV and PB eosinophilia increased with time, peaking at 48 h (PV: 623 ± 100 eosinophils/ mm2; PB: 552 ± 88 eosinophils/mm2) (Figures 3 and 4). Although still significantly elevated at 96 h and 8 d, both PV (96 h: 452 ± 103 eosinophils/mm2; 8 d: 292 ± 30 eosinophils/mm2) and PB (96 h: 273 ± 58 eosinophils/mm2; 8 d: 161 ± 22 eosinophils/mm2) eosinophilia resolved progressively (Figures 3 and 4), whereas the BALF eosinophilia remained elevated during this time span. Correlation between PB eosinophilia and airway responsiveness measured by Penh was demonstrated at 24 h (r2 = 0.476, p = 0.019), 48 h (r2 = 0.424, p = 0.022), and 96 h (r2 = 0.365, p = 0.049). A less significant correlation of PV eosinophilia and airway responsiveness was seen at the same time points.
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Kinetics of Eosinophil Peroxidase (EPO) Activity in BALF Supernatant
EPO activity in BALF supernatants from mice receiving OVA challenge alone or sensitized OVA-challenged mice was determined at the different time points. Significant levels of EPO were restricted to OVA-sensitized/challenged mice and only at 48 h (Figure 5). The significant levels of EPO at 48 h coincided with the dramatic eosinophil infiltrate in the BALF. However, unlike the BALF eosinophil numbers, which were still significantly elevated at 8 d, EPO levels rapidly returned to baseline.
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Kinetics of Cytokine Appearance in BALF after SIN OVA Challenge
The levels of IL-4, IL-13, IL-5, IFN-, and TNF- proteins in
BALF were measured. After OVA challenge alone, TNF-
levels in BALF were slightly but nonsignificantly elevated at
the early time points 8 and 24 h (Figure 6a). Moreover, levels
of IFN- and IL-5 were found in the BALF fluid at all time
points, but neither cytokine differed significantly with time
(Figures 6b and 6c). Levels of IL-4 and IL-13 were almost undetectable at all time points (Figures 6d and 6e).
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In contrast to mice receiving SIN OVA challenge alone, a
defined kinetic pattern of cytokine appearance in BALF was
observed in OVA-sensitized/challenged mice. A significant
early (8 h postchallenge) TNF- response (325 ± 141 pg/ml)
was observed, which quickly (i.e., within 24 h) returned to
control levels (Figure 6a). In addition, an increase in BALF
IL-5 (Figure 6c) and IL-4 (Figure 6d) levels was observed at
8 h (IL-4: 23 ± 8 pg/ml; IL-5: 61 ± 33 pg/ml), reaching a maximum at 24 h (IL-4: 119 ± 29 pg/ml; IL-5: 123 ± 23 pg/ml). Levels of both cytokines remained elevated at 48 h (IL-4: 33 ± 13 pg/ml; IL-5: 65 ± 18 pg/ml) but returned to baseline by 96 h.
BALF IL-13 levels were significantly increased at 24 h (24 ± 9 pg/ml) and 48 h (31 ± 10 pg/ml) and returned to baseline by
96 h. OVA-sensitization/challenge did not appear to effect Th1
cytokine expression in the lung. For example, levels of IFN found in the BALF were similar to those found in the BALF
of mice receiving OVA challenge alone (Figure 6b). Furthermore, no significant differences in IFN- levels were seen with
time or between groups of mice at each time point. No correlation between airway responsiveness and any of the cytokines
measured in the BALF was observed at any time point.
Kinetics of Mucus Hyperproduction after SIN OVA Challenge
Lung sections were stained in order to identify mucus-containing cells in the airway epithelium of mice receiving OVA challenge alone and OVA-sensitized/challenged mice. The number of epithelial cells/mm of basement membrane of the large airways of mice receiving OVA challenge alone was 140 ± 6 cells/ mm at 8 h postchallenge and did not differ significantly at any of the other time points investigated. The airway epithelium of mice receiving OVA challenge alone was devoid of cells staining positive (+ve) for mucus at 8, 24, and 48 h, and only a few cells were mucus-positive at 96 h (5 ± 2 +ve cells/mm; 0 cells were scored 1), and 8 d (4 ± 3 +ve cells/mm; 0 cells were scored 1) (Figure 7). The number of epithelial cells/mm of basement membrane of the large airways of OVA-sensitized/challenged mice was 137 ± 7 cells/mm at 8 h postchallenge. However, the number of epithelial cells increased with time to 184 ± 5 cells/ mm at 8 d postchallenge. Very few mucus-positive cells were observed in the airway epithelium of OVA-sensitized/challenged mice at either 8 h (0 +ve cells/mm) or 24 h (1 ± 1 +ve cells/mm). At 48 h a number of cells were mucus-positive (55 ± 8 cells/mm); however, of these only 17 ± 3 were scored 1. The number of mucus-positive cells increased dramatically with time at 96 h (140 ± 6 +ve cells/mm) and 8 d (154 ± 8 +ve cells/ mm). Moreover, of those cells staining positive for mucus at 96 h and 8 d, 103 ± 6 and 110 ± 11, respectively, were scored 1. These data suggest both mucus cell hyperplasia and mucus hyperproduction at these later times.
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Inhibition of AHR by IL-5 and VLA-4 Antibodies
Given the temporal association of tissue eosinophilia, their possible activation, and the development of AHR, mice were treated with antibodies directed against IL-5 and VLA-4. Administration of rat IgG prior to OVA challenge in OVA-sensitized/challenged mice had no significant effects on the development of AHR to MCh at 8, 24, and 48 h (Figures 8a-8c). However, administration of IL-5 or VLA-4 antibody prior to SIN OVA challenge inhibited the development of AHR in sensitized mice at 24 and 48 h (Figures 8b and 8c).
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Inhibition of Airway Eosinophilia by IL-5 and VLA-4 Antibodies
Administration of rat IgG control antibody prior to OVA challenge of sensitized mice had no significant effect on the development of BALF (66 ± 11 cells/ml, n = 11; ~ 21% of total cells at 48 h) or tissue eosinophilia. In contrast, administration of IL-5 or VLA-4 antibody significantly inhibited BALF eosinophilia (anti-IL-5: 19 ± 9 × 103 cells/ml, n = 13; anti-VLA-4: 5 ± 4 × 103 cells/ml, n = 9) at 48 h, and tissue eosinophilia at all time points (75 to 90% inhibition, data not shown) (Figure 9). Neither anti-IL-5 (8 h: 135 ± 47 × 103 cells/ml, n = 7; 24 h: 90 ± 17 × 103 cells/ml, n = 9; 48 h: 41 ± 10 × 103 cells/ml, n = 13) nor anti-VLA-4 (8 h: 127 ± 49 × 103 cells/ml, n = 11; 24 h: 99 ± 38 × 103 cells/ml, n = 12; 48 h: 15 ± 7 × 103 cells/ml, n = 9) significantly affected neutrophil numbers at any time point, when compared with OVA-sensitized/challenged mice pre-treated with rat IgG control antibody (8 h: 187 ± 48 × 103 cells/ml, n = 11; 24 h: 132 ± 25 × 103 cells/ml, n = 12; 48 h: 29 ± 10 × 103 cells/ml, n = 11).
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Effect of IL-5 and VLA-4 Antibodies on Appearance of Cytokines in BALF
Administration of rat IgG control antibody prior to OVA challenge of sensitized mice had no significant effect on the early 8-h
increase in levels of BALF TNF- (278 ± 41 pg/ml) or the
maximum increases in IL-4 (131 ± 21 pg/ml) and IL-5 (157 ± 32 pg/ml) seen at 24 h. IFN- levels were also unaffected. Administration of IL-5 antibody significantly inhibited BALF IL-5
(51 ± 12 pg/ml) levels at 24 h, whereas VLA-4 antibody significantly inhibited (p < 0.05) both IL-5 (61 ± 27 pg/ml) and IL-4
(43 ± 8 pg/ml) levels at 24 h (n = 9 to 12 per group). Neither
IL-5 nor VLA-4 antibody affected BALF TNF- or IFN- levels.
Effect of IL-5 and VLA-4 Antibodies on Mucus Hyperproduction
The effect of IL-5 and VLA-4 antibodies on mucus hyperproduction at 48 h was assessed. In antibody-treated animals, the number of epithelial cells/mm of basement membrane of the large airways was similar to that observed in untreated OVA-sensitized/challenged mice. IL-5 antibody had no significant effect on the number of mucus-positive cells (64 ± 6 versus 55 ± 8 cells/mm), and the number of mucus-positive cells scored 1 (16 ± 3 versus 17 ± 3 cells/mm). Although VLA-4 antibody treatment had no significant effect on the number of mucus-positive cells (40 ± 8 cells/mm), the number of mucus-positive cells scored 1 was significantly decreased (5 ± 2 cells/mm).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The use of a single intranasal OVA challenge protocol, as opposed to a multiple challenge model, allowed us to evaluate the sequence of inflammatory events that follow allergen challenge of previously sensitized mice. Importantly, it enabled us to establish a temporal association of inflammatory events with the development and subsequent resolution of AHR. Initial studies were performed in which AHR was measured in conscious, spontaneously breathing animals by barometric plethysmography (Penh). After OVA sensitization and challenge, mice developed a low-level AHR to inhaled MCh at 8 h that continued to increase, peaking at 24 h. This was maintained at 48 h, but by 96 h had resolved. The kinetics of AHR measured directly (lung resistance, RL) in anesthetized and ventilated mice were generally in accordance with those measured by Penh, although the resolution of this response appeared to be slower. This confirmed that the changes seen in airway responsiveness were indeed largely due to changes in the lung and not simply the result of changes in the nasopharynx region resulting from the intranasal route of challenge. The sustained changes in airway responsiveness (or delayed resolution of AHR) observed with lung resistance measurements may reflect differences in the sensitivity of the two measurement systems. Indeed, Penh measurements reflect box flow changes and reflect changes in the whole respiratory system, including the lower airways and nasopharynx regions, and are measured in spontaneously breathing animals. In contrast, lung resistance is measured in ventilated animals and reflects changes in lung function at a specific level of the airways.
The cellular content of the BALF comprised an initial, but transient, neutrophil influx followed by a sustained eosinophil influx. Neutrophils peaked in the BALF at 8 h and were still significantly elevated at 24 h, the peak of AHR. In fact, neutrophils were the predominant cell type in the BALF at these times and were significantly correlated with increased airway responsiveness at these times. However, their numbers had resolved by 48 h, even though AHR was still maintained. Although the presence of neutrophils have been documented in allergic asthma and animal models of AHR, the role neutrophils may play remains controversial (3, 5, 24, 25, 30). Whether neutrophils have a direct role in the development of AHR or are an important prerequisite for the subsequent eosinophilic inflammatory changes that may contribute more directly to the AHR remains to be determined.
The eosinophil is a characteristic feature of allergic asthma and the correlation with disease severity/AHR in a number of studies has highlighted the eosinophil as a major effector cell in allergic AHR (5). Remarkably, in the present study significant numbers of eosinophils did not appear in the BALF until 48 h after the SIN OVA challenge; that is, 24 h after the peak of AHR was established. At this time (48 h), eosinophil number was highly correlated with AHR. However, BALF eosinophil numbers did not peak until 96 h, and remained elevated at 8 d, times at which AHR had significantly resolved. Subsequent evaluation of tissue eosinophilia using an antibody directed against MBP revealed an early (8-h) infiltration of eosinophils in the perivascular and peribronchial regions prior to the development of significant AHR at 24 h. The perivascular and peribronchial eosinophilia increased significantly by 24 h and peaked at 48 h, times at which increased airway responsiveness to inhaled MCh was easily detected. At these times, a significant correlation between tissue eosinophil number and AHR was observed. The presence of eosinophils in the airways or airway tissues per se does not, however, necessarily confer AHR. Indeed in the present study, we demonstrated that although perivascular and peribronchial eosinophilia were slowly and progressively resolving at 96 h and 8 d, they were, nonetheless, still elevated to a level that was not significantly different from those at 24 h. Moreover, correlation of tissue eosinophilia with AHR at these later times was less evident. Airway function at these times (96 h, 8 d) was similar to baseline levels, suggesting some element of dissociation of (late) tissue eosinophilia from AHR.
The activated eosinophil is believed to release cytotoxic granule proteins such as major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO), which may cause AHR (4). In the present study, an antibody against murine eosinophilic MBP was used to quantify tissue eosinophil numbers. However, detectable levels of extracellular MBP were not observed. Our observations are consistent with a number of murine studies using MBP antibody (31). Evidence of eosinophil activation in models of allergic AHR and in human asthma is marred by the absence of a good marker of eosinophil activation. EPO levels have been measured and used to give some indication of possible eosinophil activation (24). In the present study, elevated levels of EPO were detectable in BALF supernate at 48 h but not at 96 h or 8 d, even though eosinophils were similarly elevated in the BALF at these times. From these observations one might speculate that the resolution of AHR is associated with loss of eosinophil activation rather than simply the reduction in numbers of eosinophils. However, in the absence of good markers, it is difficult to evaluate the importance of eosinophil activation in the development of AHR.
Clinical and experimental evidence implicates a number of
specific cytokines in allergic airway inflammation. TNF-, which is generated by a number of inflammatory cells (T cells, macrophages, and mast cells), is one of the key cytokines in the induction ICAM-1 and VCAM-1 on endothelial cells, increasing
the recruitment of neutrophils, eosinophils, and T cells (32). In
the present study, peak levels of TNF- were present in the
BALF 8 h after SIN, and rapidly returned to baseline (24 h).
This coincided with the development of the BALF neutrophil
response and the initial recruitment of eosinophils to the airway tissue. It is now increasingly clear that Th2 cells, through
the release of specific cytokines, particularly IL-4, IL-13, and
IL-5, regulate effector cell, particularly eosinophil, recruitment
and function, orchestrating the inflammatory response and
leading to AHR (12, 18, 33). In the present study, measurement of BALF cytokines after sensitization and SIN OVA challenge revealed the presence of increased levels of
IL-4, IL-13, and IL-5, whereas IFN was unchanged. Elevated
levels of IL-4 and IL-5 were present in BALF as early as 8 h,
and peaked at 24 h, coincident with the development of tissue
eosinophilia and AHR. Although reduced, levels of both cytokines were still elevated at 48 h but had returned to baseline
levels by 96 h. IL-13 levels were not elevated in the BALF until 24 h but were still similarly increased at 48 h, although levels returned to baseline by 96 h. The disappearance of IL-5 a
potential activation and survival factor for eosinophils was
concordant with the loss of EPO activity in BALF supernatants and the resolution of AHR at 96 h and 8 d. Although we
could make a number of temporal associations between cytokines and the inflammatory events in the lung, no significant
correlation could be made between any one specific cytokine
and AHR. The lack of correlation observed between any of
the cytokines and AHR may reflect the role of these cytokines in events upstream of the effector response leading to AHR,
and highlights the role of multiple cytokines at different times.
Mucus hyperproduction and mucus cell hyperplasia was not observed in the present study until 48 h after the challenge. At this time, a number of cells stained positive; however, only few cells demonstrated significant staining. But, by 96 h a significant increase in mucus cell hyperplasia and mucus production was observed that persisted at 8 d. Our results are consistent with two recent reports identifying similar changes in the airway epithelium after OVA sensitization and challenge of mice (25, 27). The mucus hyperproduction observed in our study was preceded by an increase in BALF IL-4 and IL-13 levels; cytokines that have been implicated in mucus production (33, 34). Interestingly, the development of mucus cell hyperplasia was temporally related to the resolution of the BALF EPO response, and normalization of airway function. Further, we have shown that development of AHR is independent of mucus cell hyperplasia (10). Under physiologic conditions, normal production of mucus may be protective to the epithelium. In addition, a recent study suggests that mucin may inhibit degranulation of eosinophils (35).
Given the temporal association of lung tissue eosinophilia,
eosinophil activation, and development and resolution of AHR
in the present study, it is tempting to link the eosinophilia to the development of AHR. However, recruitment of eosinophils to the airways and development of AHR may represent
two parallel but independent phenomena. Although a number
of murine studies suggest that increased numbers of eosinophils and their cytotoxic products in the airways underlies the
pathogenesis of AHR, several studies have been able to dissociate eosinophilia from AHR (7). It is possible, given the
early recruitment of neutrophils to the airways that airway
function is differentially modulated at different time points, or
that neutrophils are prerequisite for the eosinophilic-driven alterations in airway function. In an attempt to more clearly link
these phenomena, we adopted two different strategies to inhibit airway eosinophilia. Antibodies directed against IL-5 or
VLA-4 were utilized. Interleukin-5, and the 4 integrin, VLA-4,
expressed on the surface of eosinophils (and T cells) are considered central to the development of allergic airway eosinophilia. IL-5 is critical for eosinophil proliferation, migration,
activation, and survival (36, 37). Alternatively, VLA-4 binds
to VCAM-1 on the surface of endothelial cells, facilitating the
transendothelial migration of blood eosinophils into the airway tissues (38). Administration of IL-5 or VLA-4 antibody prior to SIN OVA challenge significantly inhibited eosinophil recruitment to the airway tissues at 8, 24, and 48 h, and to the BALF
at 48 h. Moreover, this was associated with inhibition of AHR.
These observations highlight the importance of IL-5 and
VLA-4 in the development of allergic airway eosinophilia and
support the role of eosinophils in AHR. These observations
are consistent with a number of previous studies in BALB/c mice
using antibodies directed against IL-5 and VLA-4 (7, 9, 39).
Overexpression of IL-5 in the lung epithelium of C57BL/6
mice or airway administration of recombinant IL-5 to BALB/c
mice has been shown to produce eosinophilic inflammation and
AHR (40, 41). In addition, IL-5 deficient BALB/c mice and
IL-5 receptor chain-deficient 129/BALB/c mice do not develop airway eosinophilia after sensitization and challenge (8,
42). Nonetheless, the role of IL-5 and eosinophils in the development of AHR still remains controversial. Using the same
sensitization and challenge protocol, IL-5-deficient mice on a
C57BL/6 background were shown to develop AHR independent of eosinophils, suggesting possible strain differences (12). However, mouse strain does not explain all of the differences, as a number of other studies using anti-IL-5 in either BALB/c or C57BL/6 mice have implied a dissociation between eosinophilia and AHR (11, 43). Association and dissociation of eosinophils is not simply limited to studies in which IL-5 is manipulated. In the present study we have demonstrated the
effectiveness of anti-VLA-4 antibody administered intravenously in inhibiting airway eosinophilia and AHR. VLA-4 has
also been shown to affect T cell recruitment and possibly activation (38). Indeed, administration of VLA-4 antibody inhibited IL-4 and IL-5 in the BALF in this study without significantly affecting neutrophil influx, TNF, and IFN levels.
Previously, it was demonstrated that local blockade of CD49d,
a subunit of the adhesion molecule VLA-4, inhibited local eosinophilia, Th2 cytokine production, and AHR. In contrast, systemic blockade of CD49d inhibited airway eosinophilia without effect on AHR (39). Interestingly, one common feature of
many of the studies demonstrating a dissociation of AHR from
airway eosinophilia, is that the bronchoconstrictor used to evaluate AHR was administered intravenously (11, 39, 43).
Despite these differences in methods to measure AHR and in
mice strain, interpretation between studies is further complicated by the use of different sensitization and challenge protocols, and differences in the timing, route of administration, and
dose of antibody.
In summary, we have developed a murine model of allergic
airway inflammation and AHR in which BALB/c mice were
sensitized to OVA and subsequently given a single intranasal
challenge of OVA. The use of a single challenge protocol has
allowed us to identify the precise sequence of inflammatory
events that follow allergen challenge of sensitized mice. The
data identify a sequence in the production of the proinflammatory cytokines TNF-, IL-4, IL-5, and IL-13, the ordered
recruitment of neutrophils followed by eosinophils into the
tissue and airways, and their temporal association with the development and resolution of AHR. Moreover, the inhibitory
effect of IL-5 and VLA-4 antibodies on the development of
airway eosinophilia and AHR strongly suggest a causal link.
| |
Footnotes |
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Correspondence and requests for reprints should be addressed to Erwin W. Gelfand, M.D., Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org
(Received in original form May 2, 2000 and in revised form August 10, 2000).
Dr. Tomkinson is the recipent of an ILSI/AII Fellowship Award.Acknowledgments: The writers wish to thank Lynn M. Cunningham and Diana Nabighian for their technical assistance.
Supported by NIH grants HL-36577 and HL-61005 from the National Institutes of Health, by Grant R825702 from the Environmental Protection Agency, and by the Arnold & Sheila Aronsen Fellowship in Pediatric Pulmonary Medicine at the National Jewish Medical and Research Center.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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B. D. Medoff, A. M. Tager, R. Jackobek, T. K. Means, L. Wang, and A. D. Luster Antibody-antigen interaction in the airway drives early granulocyte recruitment through BLT1 Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L170 - L178. [Abstract] [Full Text] [PDF]
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A. L. Greene, M. S. Rutherford, R. R. Regal, G. H. Flickinger, J. A. Hendrickson, C. Giulivi, M. E. Mohrman, D. G. Fraser, and J. F. Regal Arginase Activity Differs with Allergen in the Effector Phase of Ovalbumin- versus Trimellitic Anhydride-Induced Asthma Toxicol. Sci., December 1, 2005; 88(2): 420 - 433. [Abstract] [Full Text] [PDF]
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K. Takeda, N. Miyahara, T. Kodama, C. Taube, A. Balhorn, A. Dakhama, K. Kitamura, A. Hirano, M. Tanimoto, and E. W. Gelfand S-carboxymethylcysteine normalises airway responsiveness in sensitised and challenged mice Eur. Respir. J., October 1, 2005; 26(4): 577 - 585. [Abstract] [Full Text] [PDF]
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N. Miyahara, K. Takeda, S. Miyahara, S. Matsubara, T. Koya, A. Joetham, E. Krishnan, A. Dakhama, B. Haribabu, and E. W. Gelfand Requirement for Leukotriene B4 Receptor 1 in Allergen-induced Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 161 - 167. [Abstract] [Full Text] [PDF]
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N. Miyahara, K. Takeda, S. Miyahara, C. Taube, A. Joetham, T. Koya, S. Matsubara, A. Dakhama, A. M. Tager, A. D. Luster, et al. Leukotriene B4 Receptor-1 Is Essential for Allergen-Mediated Recruitment of CD8+ T Cells and Airway Hyperresponsiveness J. Immunol., April 15, 2005; 174(8): 4979 - 4984. [Abstract] [Full Text] [PDF]
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M. Fonseca-Aten, A. M. Rios, A. Mejias, S. Chavez-Bueno, K. Katz, A. M. Gomez, G. H. McCracken Jr., and R. D. Hardy Mycoplasma pneumoniae Induces Host-Dependent Pulmonary Inflammation and Airway Obstruction in Mice Am. J. Respir. Cell Mol. Biol., March 1, 2005; 32(3): 201 - 210. [Abstract] [Full Text] [PDF]
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E. W. Gelfand, A. Joetham, Z.-H. Cui, A. Balhorn, K. Takeda, C. Taube, and A. Dakhama Induction and Maintenance of Airway Responsiveness to Allergen Challenge Are Determined at the Age of Initial Sensitization J. Immunol., July 15, 2004; 173(2): 1298 - 1306. [Abstract] [Full Text] [PDF]
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 R. A. Joachim, V. Sagach, D. Quarcoo, Q. T. Dinh, P. C. Arck, and B. F. Klapp Neurokinin-1 Receptor Mediates Stress-Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness in Mice Psychosom Med, July 1, 2004; 66(4): 564 - 571. [Abstract] [Full Text] [PDF]
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C. Taube, J. A. Nick, B. Siegmund, C. Duez, K. Takeda, Y.-H. Rha, J.-W. Park, A. Joetham, K. Poch, A. Dakhama, et al. Inhibition of Early Airway Neutrophilia Does Not Affect Development of Airway Hyperresponsiveness Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 837 - 843. [Abstract] [Full Text] [PDF]
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C.-C. Lee, J.-W. Liao, and J.-J. Kang Motorcycle Exhaust Particles Induce Airway Inflammation and Airway Hyperresponsiveness in BALB/C Mice Toxicol. Sci., June 1, 2004; 79(2): 326 - 334. [Abstract] [Full Text] [PDF]
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A. Pastva, K. Estell, T. R. Schoeb, T. P. Atkinson, and L. M. Schwiebert Aerobic Exercise Attenuates Airway Inflammatory Responses in a Mouse Model of Atopic Asthma J. Immunol., April 1, 2004; 172(7): 4520 - 4526. [Abstract] [Full Text] [PDF]
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 H. Wan, K. H. Kaestner, S.-L. Ang, M. Ikegami, F. D. Finkelman, M. T. Stahlman, P. C. Fulkerson, M. E. Rothenberg, and J. A. Whitsett Foxa2 regulates alveolarization and goblet cell hyperplasia Development, February 15, 2004; 131(4): 953 - 964. [Abstract] [Full Text] [PDF]
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J. A. J. Vanoirbeek, C. Mandervelt, A. R. Cunningham, P. H. M. Hoet, H. Xu, H. M. Vanhooren, and B. Nemery Validity of Methods to Predict the Respiratory Sensitizing Potential of Chemicals: A Study with a Piperidinyl Chlorotriazine Derivative That Caused an Outbreak of Occupational Asthma Toxicol. Sci., December 1, 2003; 76(2): 338 - 346. [Abstract] [Full Text] [PDF]
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C. Taube, Y.-H. Rha, K. Takeda, J.-W. Park, A. Joetham, A. Balhorn, A. Dakhama, P. C. Giclas, V. M. Holers, and E. W. Gelfand Inhibition of Complement Activation Decreases Airway Inflammation and Hyperresponsiveness Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1333 - 1341. [Abstract] [Full Text] [PDF]
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V. V. Jain, T. R. Businga, K. Kitagaki, C. L. George, P. T. O'Shaughnessy, and J. N. Kline Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L1137 - L1146. [Abstract] [Full Text] [PDF]
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L. S. van Rijt, N. Vos, D. Hijdra, V. C. de Vries, H. C. Hoogsteden, and B. N. Lambrecht Airway Eosinophils Accumulate in the Mediastinal Lymph Nodes but Lack Antigen-Presenting Potential for Naive T Cells J. Immunol., October 1, 2003; 171(7): 3372 - 3378. [Abstract] [Full Text] [PDF]
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K. Takeda, N. Miyahara, Y.-H. Rha, C. Taube, E.-S. Yang, A. Joetham, T. Kodama, A. M. Balhorn, A. Dakhama, C. Duez, et al. Surfactant Protein D Regulates Airway Function and Allergic Inflammation through Modulation of Macrophage Function Am. J. Respir. Crit. Care Med., October 1, 2003; 168(7): 783 - 789. [Abstract] [Full Text] [PDF]
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Y.-S. Hahn, C. Taube, N. Jin, K. Takeda, J.-W. Park, J. M. Wands, M. K. Aydintug, C. L. Roark, M. Lahn, R. L. O'Brien, et al. V{gamma}4+ {gamma}{delta} T Cells Regulate Airway Hyperreactivity to Methacholine in Ovalbumin-Sensitized and Challenged Mice J. Immunol., September 15, 2003; 171(6): 3170 - 3178. [Abstract] [Full Text] [PDF]
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G. C. Koo, K. Shah, G. J. F. Ding, J. Xiao, R. Wnek, G. Doherty, X. C. Tong, R. B. Pepinsky, K.-C. Lin, W. K. Hagmann, et al. A Small Molecule Very Late Antigen-4 Antagonist Can Inhibit Ovalbumin-induced Lung Inflammation Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1400 - 1409. [Abstract] [Full Text] [PDF]
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C. Taube, A. Dakhama, Y.-H. Rha, K. Takeda, A. Joetham, J.-W. Park, A. Balhorn, T. Takai, K. R. Poch, J. A. Nick, et al. Transient Neutrophil Infiltration After Allergen Challenge Is Dependent on Specific Antibodies and Fc{gamma}III Receptors J. Immunol., April 15, 2003; 170(8): 4301 - 4309. [Abstract] [Full Text] [PDF]
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H. H. Shen, S. I. Ochkur, M. P. McGarry, J. R. Crosby, E. M. Hines, M. T. Borchers, H. Wang, T. L. Biechelle, K. R. O'Neill, T. L. Ansay, et al. A Causative Relationship Exists Between Eosinophils and the Development of Allergic Pulmonary Pathologies in the Mouse J. Immunol., March 15, 2003; 170(6): 3296 - 3305. [Abstract] [Full Text] [PDF]
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A. Koarai, M. Ichinose, S. Ishigaki-Suzuki, S. Yamagata, H. Sugiura, E. Sakurai, Y. Makabe-Kobayashi, A. Kuramasu, T. Watanabe, K. Shirato, et al. Disruption of L-Histidine Decarboxylase Reduces Airway Eosinophilia but not Hyperresponsiveness Am. J. Respir. Crit. Care Med., March 1, 2003; 167(5): 758 - 763. [Abstract] [Full Text] [PDF]
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 C. Taube, A. Dakhama, K. Takeda, J. A. Nick, and E. W. Gelfand Allergen-Specific Early Neutrophil Infiltration After Allergen Challenge in a Murine Model Chest, March 1, 2003; 123(2007): 410S - 411S. [Full Text] [PDF]
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A. Kibe, H. Inoue, S. Fukuyama, K. Machida, K. Matsumoto, H. Koto, T. Ikegami, H. Aizawa, and N. Hara Differential Regulation by Glucocorticoid of Interleukin-13-induced Eosinophilia, Hyperresponsiveness, and Goblet Cell Hyperplasia in Mouse Airways Am. J. Respir. Crit. Care Med., January 1, 2003; 167(1): 50 - 56. [Abstract] [Full Text] [PDF]
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C. Taube, C. Duez, Z.-H. Cui, K. Takeda, Y.-H. Rha, J.-W. Park, A. Balhorn, D. D. Donaldson, A. Dakhama, and E. W. Gelfand The Role of IL-13 in Established Allergic Airway Disease J. Immunol., December 1, 2002; 169(11): 6482 - 6489. [Abstract] [Full Text] [PDF]
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J. N. Kline, K. Kitagaki, T. R. Businga, and V. V. Jain Treatment of established asthma in a murine model using CpG oligodeoxynucleotides Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L170 - L179. [Abstract] [Full Text] [PDF]
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M. J. TOBIN Asthma, Airway Biology, and Nasal Disorders in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 598 - 618. [Full Text] [PDF]
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J. R. Crosby, H. H. Shen, M. T. Borchers, J. P. Justice, T. Ansay, J. J. Lee, and N. A. Lee Ectopic expression of IL-5 identifies an additional CD4+ T cell mechanism of airway eosinophil recruitment Am J Physiol Lung Cell Mol Physiol, January 1, 2002; 282(1): L99 - L108. [Abstract] [Full Text] [PDF]
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D. M. Walter, J. J. McIntire, G. Berry, A. N. J. McKenzie, D. D. Donaldson, R. H. DeKruyff, and D. T. Umetsu Critical Role for IL-13 in the Development of Allergen-Induced Airway Hyperreactivity J. Immunol., October 15, 2001; 167(8): 4668 - 4675. [Abstract] [Full Text] [PDF]
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