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Published ahead of print on January 23, 2004, doi:10.1165/rcmb.2003-0395OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 837-843, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0395OC

Inhibition of Early Airway Neutrophilia Does Not Affect Development of Airway Hyperresponsiveness

Christian Taube, Jerry A. Nick, Britta Siegmund, Catherine Duez, Katsuyuki Takeda, Yeong-Ho Rha, Jung-Won Park, Anthony Joetham, Katie Poch, Azzeddine Dakhama, Charles A. Dinarello and Erwin W. Gelfand

Division of Cell Biology, Department of Pediatrics, and Department of Medicine, National Jewish Medical and Research Center, Denver; Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and Charité Humanmedizin Berlin, Campus Benjamin Franklin, Medical Department I, Berlin, Germany

Address correspondence to: Erwin W. Gelfand, M.D., National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: gelfande{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of modifying early neutrophil-mediated inflammation on the development of airway hyperresponsiveness (AHR) was investigated using an interleukin (IL)-1 receptor antagonist (IL-1Ra), an anti–IL-18 antibody (anti–IL-18) or a p38 mitogen-activated protein kinase (MAPK) inhibitor (M39). Balb/c mice were sensitized to ovalbumin (OVA) and challenged with a single intranasal dose of OVA. Treatment with the IL-1Ra or anti–IL-18 was initiated 20 min before challenge, whereas M39 was administered 4 h before the challenge. Eight hours after challenge, sensitized mice showed significantly higher numbers of neutrophils in bronchoalveolar lavage (BAL) fluid; treatment with IL-1Ra, anti–IL-18, or M39 significantly decreased the influx of neutrophils. At 48 h, none of the treatments affected eosinophil inflammation in BAL fluid and lung tissue, goblet cell hyperplasia, or cytokine levels (IL-4, IL-5, IL-12, IL-13, interferon-{gamma}) in BAL fluid. Anti–IL-18 or IL-1Ra had no effect on the development of AHR, whereas M39-treated mice showed a decrease in methacholine responsiveness. These results demonstrate that early neutrophil influx following allergen challenge is mediated by IL-1, IL-18, and p38 MAPK. However, neutralization of IL-1 and IL-18 did not affect the later development of AHR and eosinophilic airway inflammation. The effects of inhibiting p38 MAPK in decreasing AHR indicate activities independent of its prevention of neutrophil accumulation.

Abbreviations: airway hyperresponsiveness, AHR • bronchoalveolar lavage, BAL • dynamic compliance, Cdyn • enzyme-linked immunosorbent assay, ELISA • immunoglobulin, Ig • interleukin, IL • lipopolysaccharide, LPS • mitogen-activated protein kinase, MAPK • major basic protein, MBP • methacholine, MCh • macrophage inflammatory protein, MIP • ovalbumin, OVA • periodic acid Schiff, PAS • lung resistance, RL • single intranasal challenge, sin • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is a syndrome characterized by airway obstruction, hyperreactivity (AHR), and inflammation, with eosinophils, lymphocytes, and mast cells increasing in the airway wall and in the airway lumen. The contribution of these cells to the development of AHR has been intensively studied, and a number of reports suggest a critical role for eosinophils (1), and CD4+ and CD8+ lymphocytes (1, 2). An increasing number of clinical investigations in patients with allergic and nonallergic asthma have described the accumulation of neutrophils in the airways (3), especially in patients with more severe disease (4), with nocturnal asthma (5), and with steroid-dependent asthma (6). In addition, a marked increase in numbers of neutrophils within the airways has been found after allergen challenge (7) and during asthma exacerbation (8). Neutrophils are capable of releasing a number of different substances, including proteases, reactive oxygen species, and mediators, which may contribute to the development of AHR (9, 10). Further, AHR has been described in patients with pulmonary diseases other than asthma, where neutrophils are the predominant inflammatory cells (e.g., cystic fibrosis, chronic obstructive pulmonary disease) (11–13).

Following allergen challenge, early neutrophil infiltration is transient, followed by an influx of eosinophils and lymphocytes (14). We have demonstrated recently that this early neutrophil influx is allergen-specific and dependent on allergen-specific antibodies and on the presence of Fc{gamma} receptor III (15), similar to models of immune complex disease. This early neutrophil influx was preceded by increased levels of the neutrophil chemokines macrophage inflammatory protein (MIP)-2 and cytokine-inducible neutrophil chemoattractant (KC), homologs of human interleukin (IL)-8. Models of immune complex–mediated inflammation have associated IL-1ß and IL-18 with neutrophil inflammation (16, 17). Both of these cytokines are known to mediate the release of neutrophil chemokines. Neutrophil chemotaxis toward these chemokines appears to be mediated through p38 mitogen-activated protein kinase (MAPK) (18) and inhibition of p38 MAPK decreased neutrophil influx into the lung following instillation of lipopolysaccharide (LPS) (19).

In the present study, we examined the role of the antigen-specific early transient influx of neutrophils into the lung following challenge of sensitized mice in the development of airway inflammation and AHR. We demonstrate that inhibition of neutrophil inflammation using three different approaches reduces the numbers of neutrophils recruited into the airways, but that later inflammatory changes and increase in cytokine levels are not affected. In addition, inhibition of IL-1 or IL-18 failed to affect the development of AHR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained on an ovalbumin (OVA)-free diet. 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
Mice were sensitized as previously described (14). Briefly, mice 8–12 wk of age received an intraperitoneal injection of 20 µg OVA (Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg aluminum hydroxide (AlumImuject; Pierce, Rockford, IL) in a total volume of 100 µl on Days 0 and 14. On Day 28, mice received a single intranasal (sin) challenge with OVA (40 µl, 2.5 mg/ml in normal saline). At 8 and 48 h after OVA challenge, using different groups of mice, AHR was assessed and tissue was obtained for further analysis. Control mice received injections of phosphate-buffered saline (PBS) on Days 0 and 14 and OVA sin. Application of OVA to nonsensitized mice resulted in a small but insignificant increase in neutrophils in bronchoalveolar lavage (BAL) fluid at 8 h following the administration.

In Vivo Inhibition of IL-1, IL-18, and p38 MAPK
The IL-1 receptor antagonist (IL-1Ra, 10 mg/kg) (provided by Amgen Inc., Thousand Oaks, CA) was administered 30 min before the challenge by intraperitoneal injection. IL-18 neutralizing antiserum was raised in rabbits against murine recombinant IL-18 (Peprotech, Rocky Hills, NJ) as previously reported (20), and 400 µl were administered 30 min before the allergen challenge by intraperitoneal injection. Normal rabbit serum was used as control. Systemic inhibition of p38 MAPK was achieved with M39 ([(S)-5-[2-(1-phenyl-ethylamino) pyrimidine-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4 piperidinyl) imidazole]) (provided by Merck, Rahway, NJ) (21). The mice were fasted for 6 h and then M39 was administered per oral gavage (22-gauge straight feeding needle with 2.25-mm ball) at 3 mg/kg (suspended in 100 µl hydroxypropylmethylcellulose; Abbott Laboratories, Abbott Park, IL) 4 h before sin.

Assessment of IL-1ß, IL-18, and Phosphorylated p38 MAPK Levels in Lung Homogenates
After killing, lungs including trachea and main bronchi were dissected from the animals, weighed, and completely homogenized in dilution buffer (PBS, 0.1% Triton X [Sigma] and Proteinase Inhibitor [PharMingen, San Diego, CA]) using a tissue homogenizer (Tissue Tearor, BioSpec, Bartlesville, OK) followed by centrifugation at 14,000 RPM for 30 min. Supernatants were collected and then frozen at –80°C until further analysis. Total protein content was measured by colorimetric assay (DC Protein Assay; Bio-Rad Laboratories, Hercules, CA), levels of IL-1ß were assessed by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN), and concentrations of IL-18 were assessed as previously described (22). Levels of phosphorylated p38 MAPK were assessed by ELISA (Biosource International, Camarillo, CA) following the manufacturer's instructions. Limits of detection for IL-1ß was 30 pg/ml, for IL-18 20 pg/ml and for phosphorylated p38 MAPK 0.8 U/ml.

Determination of Airway Resistance and Dynamic Compliance
Airway resistance (RL) and dynamic compliance (Cdyn) were determined as a change in airway function after aerosolized methacholine (MCh) challenge. Mice were anesthetized with sodium pentobarbital (90 mg/kg), tracheotomized, and mechanically ventilated at a rate of 160 breaths/min with a constant tidal volume of air (0.2 ml). Lung function was assessed as previously described (23). Aerosolized MCh was administered in increasing concentrations (1.56, 3.125, 6.25, and 12.5 mg/ml). After each MCh challenge, the data were continuously collected for 1–5 min and maximum values of RL and minimum values of Cdyn were taken to express changes in these functional parameters.

Determination of Cell Numbers and Cytokine Levels in BAL
Eight hours after the allergen challenge and immediately after the assessment of AHR, lungs were lavaged via the tracheal cannula with one milliliter of Hanks' balanced salt solution. Total leukocyte numbers were measured (Coulter Counter; Coulter Corporation, Hialeah, FL). Differential cell counts were made from cytocentrifuged preparations (Cytospin 2; Shandon Ltd., Runcorn, UK), stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA). Cells were identified, in a blinded fashion, as macrophages, eosinophils, neutrophils, and lymphocytes by standard hematological procedures and at least 200 cells counted under x400 magnification.

BAL supernatants were collected and kept frozen at –80°C until assayed. The levels of cytokine secreted into the supernatants of BAL fluid samples were determined by ELISA. IL-4, IL-5, IL-10, IL-12, interferon-{gamma} (all from PharMingen), as well as IL-13, KC, MIP-2, and tumor necrosis factor (TNF)-{alpha} (all from R&D Systems) were measured following the manufacturer's direction.

Measurement of Serum OVA-Specific Antibody and Total IgE
OVA-specific immunoglobulin (Ig)E and IgG1 levels were measured by ELISA as previously described (24). Total IgE levels were determined using the same method compared with a known mouse IgE standard (PharMingen). The limits of detection were 100 pg/ml for IgE.

Histologic and Immunohistochemistry Studies
After obtaining BAL fluid, lungs were inflated through the trachea with 2 ml of 10% formalin and then fixed in the same solution by immersion. Tissue sections, 5 µm thick, were affixed to microscope slides and deparaffinized. The sections were stained with periodic acid Schiff (PAS) for identification of mucus-containing cells, examined under light microscopy, and analyzed using NIH Scion Image software (version 1.62, developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Eosinophils were identified by immunohistochemistry using a rabbit anti-mouse eosinophilic major basic protein (MBP) antibody (generously provided by Dr. J. J. Lee, Mayo Clinic, Scottsdale, AZ) as previously described (25). Four different sections per animal were examined in a blinded fashion with a Nikon microscope equipped with a fluorescein filter system (Nikon, Garden City, NY) and analyzed using IPLAB2 software (Signal Analytics, Vienna, VA) for the Macintosh computer.

Statistical Analysis
Values of all measurements were expressed as the mean and SEM. ANOVA was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by Tukey-Kramer HSD test. Statistical significance was assumed for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Upregulation of IL-1ß, IL-18, and Phosphorylated p38 MAPK following Allergen Exposure of Sensitized Host
Levels of IL-1ß, IL-18, and phosphorylated p38 MAPK were assessed in lung homogenates 8 h after the allergen challenge. Sensitized and challenged mice showed a significant (P < 0.01) increase in levels of IL-1ß (3-fold), IL-18 (2-fold), and phosphorylated p38 MAPK (2.5-fold) compared with nonsensitized but challenged mice (Figure 1).



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  		Figure 1. Levels of IL-1ß, IL-18, and p38 MAPK in lung homogenates obtained 8 h after allergen challenge. Levels of IL-1ß, IL-18, and phosphorylated p38 MAPK were assessed in lung homogenates 8 h after the challenge from either challenged-only mice (open bars, n = 8) or sensitized and challenged mice (filled bars, n = 8) mice. Mean ± SEM. *P < 0.01.

 
Pretreatment with IL-1Ra, anti–IL-18, or M39 Decreases Early Neutrophil Influx
As described previously (15), sensitized mice showed significantly (P < 0.001) higher neutrophil numbers in BAL fluid 8 h after a single intranasal challenge compared with nonsensitized but challenged mice (Figure 2). Treatment with IL-1Ra, anti–IL-18, or M39 resulted in significantly (P < 0.05 for each) lower neutrophil numbers compared with sensitized mice, as well as sensitized mice treated with normal rabbit serum (Figure 2).



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  		Figure 2. Differential cell counts in BAL fluid 8 h after the single intranasal OVA challenge. Differential cell counts were assessed in challenged-only mice (challenged only), sensitized and challenged mice (sensitized/challenged), sensitized and challenged mice treated with either IL-1Ra (IL-1Ra), anti–IL-18 ({alpha}IL-18), rabbit serum (rabbit serum), or p38 MAPK inhibitor (M39) (n = 12 in each group). No eosinophils were detected at this time point. Mean ± SEM are given. *P < 0.01 compared with challenged only, IL-1Ra, {alpha}IL-18, and M39. #P < 0.05 to challenged only.

 
Following allergen challenge, sensitized mice showed significantly (P < 0.05) higher levels of TNF-{alpha} and the neutrophil chemokines MIP-2 and KC in BAL fluid compared with the nonsensitized but challenged mice (Figure 3). Pretreatment with either IL-1Ra or IL-18 significantly reduced levels of TNF-{alpha}, MIP-2, and KC in BAL fluid. In contrast, inhibition of p38 MAPK did not decrease BAL fluid concentrations of TNF-{alpha}, MIP-2, or KC (Figure 3).



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  		Figure 3. Levels of chemokines and cytokines in BAL fluid supernatant 8 h after the single intranasal OVA challenge. Levels of MIP-2 (A), KC (B), and TNF-{alpha} (C) in challenged-only mice (chall), sensitized and challenged mice (sens/chall), sensitized and challenged mice treated with either IL-1Ra (IL-1Ra), anti–IL-18 ({alpha}IL-18), rabbit serum (rabbit serum), or p38 MAPK inhibitor (M39) (n = 12 in each group). Results are expressed in mean ± SEM. *P < 0.05 compared with chall, IL-1Ra, and {alpha}IL-18. #P < 0.05 to chall.

 
Inhibition of Early Neutrophil Influx Does Not Alter Airway Inflammation and Cytokine Levels 48 h after OVA Challenge
At 48 h after the allergen challenge, inflammatory cell accumulation in BAL fluid was assessed. Sensitized and challenged mice showed significantly (P < 0.05) higher total cell numbers and eosinophil numbers compared with nonsensitized but challenged animals (Figure 4). Treatment with IL-1Ra, anti–IL-18, and M39 had no significant effect on total cell counts or eosinophil numbers in BAL fluid at this time point. In addition, the small but significant increase in BAL lymphocyte numbers was not affected by any of the treatments (Figure 4).



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  		Figure 4. BAL fluid cellular composition at 48 h following the single intranasal challenge. Cellular composition in BAL fluid 48 h after the single intranasal OVA challenge in challenged-only mice (challenged only), sensitized and challenged mice (sensitized/challenged), and sensitized and challenged mice treated with either IL-1Ra (IL-1Ra), anti-IL-18 ({alpha}IL-18), rabbit serum (rabbit serum), or p38 MAPK inhibitor (M39) (n = 12 in each group). Results are expressed in mean ± SEM. *P < 0.05 compared with all other groups.

 
Similar findings were observed when BAL cytokine levels were examined. Sensitized and challenged mice showed significantly (P < 0.05) higher levels of IL-4, IL-5, and IL-13 and decreased IL-10, IL-12, and IFN-{gamma} concentrations in BAL fluid when compared with the nonsensitized but challenged controls (Table 1). There were no significant differences in any of the cytokine levels between treated and nontreated sensitized and challenged mice (Table 1).


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  		TABLE 1. Inhibition of early neutrophilia does not affect cytokine levels in BAL fluid of sensitized and challenged mice

 
Inhibition of Early Neutrophil Influx Does Not Affect Lung Tissue Inflammation or Goblet Cell Hyperplasia at 48 h after OVA Challenge
Tissue inflammatory cell infiltration was assessed using hematoxylin/eosin staining of lung sections (Figure 5). After the challenge, sensitized mice showed increased numbers of inflammatory cells in lung tissue when compared with the challenged-only mice. Sensitized and challenged mice pretreated with IL-1Ra, anti–IL-18, or the p38 MAPK inhibitor demonstrated histologic evidence of inflammatory changes similar to that of the nontreated sensitized and challenged mice (Figure 5).



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  		Figure 5. Tissue inflammation and goblet cell hyperplasia at 48 h following the single intranasal challenge. Inflammation in tissue sections was detected using hematoxylin and eosin staining (H&E), and goblet cell hyperplasia was quantified in Periodic Acid Schiff (PAS)-stained sections, in challenged-only mice (chall), sensitized and challenged mice (sens/chall), and sensitized and challenged mice treated with IL-1Ra (IL-1Ra), anti–IL-18 ({alpha}IL-18), or p38 MAPK inhibitor (M39). Bar = 50 µm.

 
To evaluate tissue eosinophil inflammation, tissue sections were stained for MBP (Figure 6). Sensitized and challenged mice showed increased peribronchial numbers of MBP-positive cells (mean ± SEM: 86 ± 18 MBP-positive cells/mm basement membrane [BM]) compared with the challenged-only mice (3 ± 1) (Figure 6). Treatment of sensitized and challenged mice with IL-1Ra (92 ± 24), anti–IL-18 (80 ± 14), M39 (98 ± 29), and rabbit serum (88 ± 18) (n = 8 mice in each group) did not affect numbers of peribronchial MBP-positive cells (Figure 6).



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  		Figure 6. Eosinophil inflammation in lung tissue 48 h after the single intranasal challenge. Immunohistochemical (MBP-staining) localization of lung tissue eosinophils was determined 48 h after the OVA challenge in challenged-only mice (A), sensitized and challenged mice (B), and sensitized and challenged mice receiving IL-1Ra (C), anti–IL-18 (D), p38 MAPK inhibitor (E), or rabbit serum (F) (final magnification: x64).

 
To assess goblet cell hyperplasia, tissue sections were stained with PAS. Sensitized and challenged mice showed significantly (P < 0.001) higher numbers of PAS-positive cells (mean ± SEM: 76 ± 15 PAS-positive cells/mm BM) compared with the challenged-only mice, where virtually no PAS-positive cells were detectable (Figure 5). Treatment of sensitized and challenged mice with IL-1Ra (72 ± 12 PAS positive cells/mm BM), anti–IL-18 (68 ± 14), M39 (73 ± 14), and rabbit serum (79 ± 18) (n = 8 mice in each group) did not affect the numbers of PAS-positive cells (Figure 5).

Inhibition of Early Neutrophil Influx Does Not Affect Levels of OVA-Specific IgE and IgG1
Serum levels of OVA-specific and total Igs were measured 48 h after the challenge. Sensitized mice had significantly (P < 0.05) increased serum levels of OVA-specific IgE, IgG1, and total IgE compared with nonsensitized mice (Table 2). In sensitized and challenged mice treated with IL-1Ra, IL-18, or M39, serum levels of total IgE, and OVA-specific IgE and IgG1, were similar to the nontreated sensitized and challenged mice (Table 2).


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  		TABLE 2. Inhibition of early neutrophilia does not affect serum immunoglobulin levels in sensitized and challenged mice

 
Development of AHR after the Allergen Challenge Is Decreased by Treatment with p38 MAPK Inhibitor but Is Not Affected by IL-1Ra and Anti–IL-18
Airway responsiveness to MCh was measured in anesthetized, tracheotomized, and ventilated animals 48 h after the allergen challenge. Sensitized and challenged mice developed significant AHR to inhaled MCh compared with nonsensitized but challenged-only mice; RL values were significantly increased and Cdyn values significantly decreased in a MCh dose-dependent manner (Figure 7). Prior treatment with IL-1Ra or anti–IL-18 did not affect the development of AHR; increases in RL and decreases in Cdyn were not significantly different from levels in sensitized and challenged mice or sensitized and challenged mice treated with normal rabbit serum. In contrast, administration of the p38 MAPK inhibitor, 4 h before challenge, decreased the response to MCh throughout the dose–response curve to MCh (Figure 7).



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  		Figure 7. Airway responsiveness to MCh 48 h after the single intansasal challenge. RL and Cdyn in response to inhaled MCh was monitored in challenged-only mice (open squares), sensitized and challenged mice (open diamonds), and sensitized and challenged mice treated with either IL-1Ra (open circles), anti–IL-18 (triangles), rabbit serum (filled squares), or p38 MAPK inhibitor (filled diamonds) (n = 12 mice in each group). 48 h after the single intranasal OVA challenge. RL and Cdyn values were obtained in response to increasing concentrations of inhaled MCh as described in MATERIALS AND METHODS. Data represent the mean ± SEM. *P < 0.05 compared with M39 and chall. #P < 0.05 compared with chall.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that the early neutrophil response following allergen challenge of sensitized animals is regulated by the cytokines IL-1 and IL-18. Inhibition of either cytokine significantly decreased neutrophil numbers as well as chemokine levels in the BAL fluid 8 h after allergen challenge. Similarly, the number of neutrophils in the BAL fluid significantly decreased following oral administration of M39, a p38 MAPK inhibitor. This inhibitor decreases neutrophil accumulation, likely by direct inhibition of neutrophil chemotaxis (18), because neutrophil chemokine levels in BAL fluid were unaffected by this inhibitor treatment.

Previously, we demonstrated that the early neutrophil influx following allergen exposure of sensitized mice is dependent on allergen-specific antibodies and expression of Fc{gamma} receptor III (15), similar to findings in immune complex–induced lung inflammation (26). Immune complex–induced neutrophil infiltration into the lung and airways has been linked to increased expression of IL-1 (16) as well as IL-18 (17). In the present study, we detected increased levels of IL-1ß and IL-18 in lung homogenates of sensitized and challenged mice at 8 h after challenge, the time point when neutrophil numbers and levels of neutrophil chemokines peak in the BAL fluid (14, 27). These findings suggested that similar to the neutrophil influx (27), sensitization of the host is required to upregulate IL-1ß and IL-18 levels following allergen exposure. Inhibition of IL-1, using an IL-1Ra, or of IL-18, using a neutralizing antibody, effectively decreased levels of neutrophil chemokines as well as neutrophil numbers in BAL fluid following the allergen exposure, implying that both cytokines are involved in the development of this early neutrophil response. However, inhibition of IL-1 or IL-18, and the resultant early neutrophil influx, did not affect the development of later changes in this model. For example, at 48 h after the allergen exposure, eosinophil numbers in BAL fluid and lung tissue, goblet cell hyperplasia, levels of cytokines in the BAL fluid, and the development of AHR were unaffected by neutralization IL-1 and IL-18 and the early neutrophil response. These findings suggest that the early but transient neutrophil influx following allergen challenge (15), though allergen-specific, is not essential for the development of later events.

The present findings are consistent with inhibition of IL-17 to decrease neutrophil numbers following allergen exposure. Despite the decrease in neutrophil numbers, no effects were detected on the later development of AHR (28). These findings are also similar to those in humans, where a decrease in neutrophil numbers after allergen challenge, following treatment with a leukotriene B4 receptor antagonist, did not affect the early and late airway response to allergen challenge (29).

Both IL-1 and IL-18 have been directly linked to allergic disorders. IL-1 has been associated with eosinophil migration (30), late asthmatic responses (31), altered smooth muscle responsiveness (32), and development of AHR (33). The role of IL-18 in the development of allergic responses appears more complex. In the absence of IL-12, IL-18 has the capacity to induce IgE production in vivo (34). However, during airway challenge, co-administration of IL-12 and IL-18 can inhibit the development of allergic airway inflammation and AHR (35). Furthermore, overexpression of IL-18 in the lung either by vaccination with an allergen–IL-18 fusion DNA (36) or by IL-18 gene transfer (37) effectively reduced the development of AHR and reversed preexisting airway disease. In contrast to these findings, administration of IL-18 during the airway challenge phase enhanced eosinophil infiltration in the lung tissue, (38, 39). The inconsistencies may be due to the differences in the amounts of IL-18 administered exogenously or overexpressed. In the present study, we used specific neutralization of IL-1 or IL-18 after sensitization but before challenge, which nearly abolished the early neutrophil inflammation but did not affect the later development of eosinophilic airway inflammation or AHR, suggesting that the early neutrophil inflammatory response following allergen challenge is not essential for the development of AHR and eosinophilic airway inflammation. Although neutralizing IL-18 with anti–IL-18 likely persists for 48 h, a single dose of IL-1Ra blocks IL-1 receptors only transiently because the half-life is short (40).

Through in vitro studies of various cell lines and primary cells, p38 MAPK has been linked to a variety of inflammatory responses and p38 MAPK has been identified to play a critical role in neutrophil activation. Stimulation with LPS leads to an upregulation of the p38 MAPK pathway in neutrophils, and p38 MAPK regulates neutrophil chemotaxis and degranulation (18). In the present study, the levels of phosphorylated p38 MAPK were increased in the lungs of sensitized and challenged mice at 8 h after the allergen challenge. This suggests increased activity of p38 MAPK as the enzymatic activity of p38 MAPK is closely linked to its phosphorylation (41). Preventing p38 MAPK activation using M39 in an LPS-induced murine model of pulmonary inflammation resulted in a selective reduction of neutrophil accumulation in the airspaces (19). The activity of M39 as a p38 MAPK inhibitor seems to be fairly specific for neutrophils, because a 1,000-fold greater concentration of M39 was required to block the release of TNF-{alpha}, KC, and MIP-2 from alveolar macrophages (19). M39 has a good oral bioavailability, and following oral application M39 is detectable in the serum for more than 24 h (21). Treatment with M39 did not affect levels of MIP-2, KC, or TNF-{alpha} in the BAL fluid 8 h after the allergen challenge. Therefore, the decrease in neutrophil numbers observed may be attributed to a decrease in neutrophil chemotaxis, comparable to results obtained in LPS models (19).

Inhibition of p38 MAPK was effective in reducing neutrophil influx at 8 h after allergen challenge. Similar to inhibition of IL-1 and IL-18, administration of M39 had no effect on the numbers of other inflammatory cells at 48 h after the allergen exposure. Numbers of eosinophils were not significantly different in BAL fluid or lung tissue in sensitized/challenged and M39-treated mice. In addition, inhibition of p38 MAPK did not affect the levels of cytokines 48 h after the allergen challenge. As a result, it was somewhat surprising that mice treated with M39 actually showed a decrease in AHR when compared with the nontreated sensitized and challenged mice or the IL-1Ra– or anti–IL-18–treated mice.

The p38 MAPK pathway has been linked to airway smooth muscle hyporesponsiveness (42), cytokine gene expression in human airway myocytes (43), and smooth muscle cell migration (44). In eosinophils, signaling through the p38 MAPK pathway has been associated with the activation by eotaxin (45), whereas IL-5 has been found to activate both p38 and ERK (46). In one study, inhibition of p38 MAPK has been shown to effectively reduce eosinophil numbers in BAL fluid in a mouse model of allergic airway inflammation and to reduce AHR in a guinea pig model (47). In a rat model of allergic airway inflammation, a p38 MAPK inhibitor reduced early TNF-{alpha} levels in BAL fluid but did not reduce airway neutrophil or eosinophil numbers (48). In the present study, there was no effect of the p38 MAPK inhibitor on eosinophil numbers in the BAL fluid or in the lung tissue. This might be due to the fact that in other studies, a different p38 MAPK inhibitor was tested at higher doses and more frequent dosing intervals (47). At this time, the reduction in AHR with the p38 MAPK inhibitor remains unexplained. Possible mechanisms might be a direct effect on airway smooth muscle or a decrease in the activation of eosinophils.

Here, we studied a model of acute development of AHR following allergen exposure of sensitized hosts. The findings of the absence of neutrophil involvement in the development of AHR under these conditions may not simply translate to other aspects of allergic airway disease, for example, early and late phase airway responses or chronic airway disease. This potential heterogeneity in the requirement for neutrophils is likely similar to the role of eosinophils in the development of AHR, where eosinophil-dependent as well as eosinophil-independent pathways may be involved.

In summary, these findings demonstrate that the early neutrophil influx into the airways after allergen challenge is regulated by IL-1 and IL-18 and can be reduced by inhibition of either cytokine or by inhibition of p38 MAPK. This reduction in neutrophil number does not affect the development of later inflammatory changes in the airways or the development of AHR, suggesting that this early and transient neutrophil response does not play a direct role in the development of allergen-induced AHR.


   Acknowledgments
 
The authors thank Dr. J. J. Lee (Mayo Clinic, Scottsdale, AZ) for providing the anti-MBP antibody, and L.N. Cunningham and D. Nabighian (National Jewish Medical and Research Center, Denver, CO) for their assistance. This work was supported by NIH grants AI-42246, HL-36577, HL-61005 (to E.W.G.), AI-15614, HL-68743 (to C.A.D.), HL-68743 (to J.A.N.), and EPA grant R825702 (to E.W.G.). C.T. is supported by the Deutsche Forschungsgemeinschaft (Ta 275/2-1). B.S. is supported by the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (DFG Si 749/3-1).

Received in original form November 4, 2003

Received in final form December 22, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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