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Interleukin-17 Orchestrates the Granulocyte Influx into Airways after Allergen Inhalation in a Mouse Model of Allergic Asthma

Laboratory of Experimental Immunology, Department of Otorhinolaryngology-Head and Neck Surgery, Laboratory of Hematology, and Laboratory of Molecular Immunology, Rega Institute, Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Department of Internal Medicine, University Hospitals, Faculty of Medicine, University of Leuven, Leuven, Belgium

Address correspondence to: Jan Ceuppens, Laboratory of Experimental Immunology, Onderwijs en Navorsing, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Jan.Ceuppens{at}med.kuleuven.ac.be

	Abstract

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Interleukin (IL)-17 is produced by activated memory CD4⁺ cells and induces cytokines and chemokines that stimulate neutrophil generation and recruitment. Here, we investigated the involvement of IL-17 in the bronchial influx of neutrophils in experimental allergic asthma. Inhalation of nebulized ovalbumin (OVA) by sensitized mice with bronchial eosinophilic inflammation resulting from chronic OVA exposure induced early IL-17 mRNA expression in inflamed lung tissue, concomitant with a prominent bronchial neutrophilic influx. Anti–IL-17 monoclonal antibodies (mAb) injected before allergen inhalation strongly reduced bronchial neutrophilic influx, in a manner equally as potent as the anti-inflammatory dexamethasone. Remarkably, anti–IL-17 mAb significantly enhanced IL-5 levels in both BAL fluid and serum, and aggravated allergen-induced bronchial eosinophilia. In another series of experiments, anti–IL-17 mAb were given repeatedly during the inhalatory challenge phase with OVA of sensitized mice. This treatment regimen abated bronchial neutrophilia in parallel with reduction of bone marrow and blood neutrophilia. In addition, anti–IL-17 mAb treatment elevated eosinophil counts in the bone marrow and bronchial IL-5 production, without alteration of allergen-induced bronchial hyperresponsiveness. In summary, our results demonstrate that IL-17 expression in airways is upregulated upon allergen inhalation, and constitutes the link between allergen-induced T cell activation and neutrophilic influx. Because neutrophils may be important in airway remodeling in chronic severe asthma, targeting IL-17 may hold therapeutic potential in human asthma.

Abbreviations: bronchoalveolar lavage, BAL • bronchial hyperresponsiveness, BHR • bone marrow, BM • enzyme-linked immunosorbent assay, ELISA • eosinophil peroxidase, EPO • experimental units, EU • granulocyte macrophage colony-stimulating factor, G-CSF • hematoxylin and eosin, H&E • interferon-, IFN- • interleukin, IL • monoclonal antibodies, mAb • methacholine, MCh • May-Grünwald-Giemsa, MGG • myeloperoxidase, MPO • ovalbumin, OVA • enhanced pause, Penh • T helper 2, Th2 • tumor necrosis factor, TNF

	Introduction

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In allergic asthma, inhalation of allergens leads to an inflammatory cascade in which T lymphocytes of the CD4⁺ subset play a central role. Activated CD4⁺ cells release so-called T helper 2 (Th2) cytokines that contribute substantially to the classic triad of allergic asthma, i.e., allergen-specific IgE production, bronchial hyperresponsiveness (BHR), and eosinophilic inflammation (1). Besides eosinophils, recent evidence is emerging for neutrophil participation in different features of human asthma: airway gland hypersecretion, BHR, and airway wall remodeling (2). Indeed, bronchial neutrophilia has been reported in severe asthma attacks (3–6), and after bronchial allergen challenge in patients with asthma (7). Experimental data in a rat model of allergic asthma suggest that T lymphocytes are essential for the induction of bronchial neutrophilia (8). However, factors responsible for T lymphocyte–mediated bronchial neutrophilic influx remain to be identified. We here hypothesized that interleukin (IL)-17, a T cell–derived cytokine whose biologic functions are beginning to be elucidated (9–11), may represent the link between activated CD4⁺ cells and bronchial neutrophilic influx in allergic asthma.

IL-17 is a 20- to 30-kD protein that is primarily secreted by memory CD4⁺ T cells and that shares homology with a protein encoded by the open reading frame 13 of Herpesvirus saimiri (12). Neither IL-17 nor its receptor have sequence similarity with any known cytokine or cytokine receptor (11). Due to the ubiquitous distribution of its receptor (11), IL-17 exerts pleotropic biologic activities. Ligation of its receptor initiates the transcription of nuclear factor-B (NF-B) (13) and c-Jun NH₂-terminal kinase (14) via tumor necrosis factor (TNF) receptor-associated factor 6 (15). Recent reports illustrate that IL 17 plays a pivotal proinflammatory role in several inflammatory responses like experimental autoimmune neuritis (16), organ allograft rejection (17), tumorigenicity of cervical tumors (18), and bacterial pneumonia (19). In patients, expression of IL-17 is found in inflamed sites of rheumatoid arthritis (20, 21), multiple sclerosis (22), psoriasis (23), allergic contact dermatitis (24), and allergic asthma (2).

Increasing evidence suggests that IL-17, acting either directly or indirectly, significantly stimulates neutrophil maturation, migration, and function. Overexpression of IL-17 results in massive peripheral neutrophilia associated with increased levels of granulocyte colony-stimulating factor (G-CSF) and enhanced granulopoiesis (25). Alternatively, impaired IL-17 receptor signaling leads to delayed bronchial recruitment of neutrophils and impaired clearance of Klebsiella pneumoniae infection (19). In several cell systems, IL-17 induces the release of C-X-C chemokines (10, 24, 26) and upregulates interferon (IFN)-–mediated ICAM-1 expression (23, 24). These proinflammatory effects of IL-17 are further potentiated by the induction of TNF- and IL-1 (27), which are involved in upregulating cellular adhesion molecules and hence migration of inflammatory cells (28). In addition to favoring the recruitment of neutrophils, IL-17 has been reported to activate neutrophils within airways (29). Whether increased immunoreactivity for IL-17 in asthmatic bronchi (2) reflects allergen-induced stimulation of IL-17 secretion, and/or the release of biologically active IL-17, remains hypothetical. Therefore, we studied the relevance of IL-17 in a mouse model of allergic asthma. The mouse model we use here is characterized by production of allergen-specific IgE and induction of nonspecific BHR and bronchial eosinophilic inflammation (30, 31). To mimic allergen exposure of patients with asthma and chronically inflamed airways, we extended the routinely used biphasic protocol for induction of experimental allergic asthma, i.e., allergen sensitization and airway exposure, with an acute provocation phase. This provocation is performed by exposure of BALB/c mice to nebulized allergen and induces bronchial IL-17 expression and an early neutrophilic influx in airways. In the first part of this study, we investigated the effects of neutralization of IL-17 by administration of anti–IL-17 monoclonal antibodies (mAb), before provocation, on the inflammatory response following allergen provocation. Second, we investigated the effects of prolonged neutralization of IL-17 on bronchial inflammation, cytokine production, and bone marrow (BM) biology by administration of anti–IL-17 mAb during the inhalatory challenge phase of sensitized mice.

	Materials and Methods

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mAbs and Reagents
Ovalbumin (OVA; grade V), bovine serum albumin (BSA), o-dianisidine dihydrochloride, o-phenylenediamine, and metacholine (Mch) were purchased from Sigma (St Louis, MO). Neutralizing mAb against IL-17 (50,104.11, rat IgG) and recombinant mouse IL-17 were purchased from R&D Systems (Minneapolis, MN). Control rat IgG was purchased from Rockland (Gilbertsville, PA). Türk's solution was purchased from Merck Diagnostica (Darmstadt, Germany) and RPMI 1640 was purchased from BioWhittaker (Walkersville, MD). Enzyme-linked immunosorbent assay (ELISA) reagents for mouse IL-5 and IFN- quantification were purchased from PharMingen (San Diego, CA). An ELISA kit from Biosource International (Fleurus, Belgium) was used for measurement of IL-4 levels.

Establishment of a Chronic Asthma Model and Anti–IL-17 mAb Treatment
Male BALB/c mice (Harlan, Horst, The Netherlands) were kept under conventional pathogenic-free conditions and were actively sensitized by seven intraperitoneal injections of 10 µg OVA in 0.5 ml of pyrogen-free saline on alternate days from Days 1–13 as described previously (30) (Figure 1) . Mice were then exposed daily for 5 min to nebulized OVA (10 mg/ml; PARI TurboBOY, Starnberg, Germany) or saline from Days 33–40. On Day 41, eosinophilic airway inflammation was found with typical features reminiscent of human allergic asthma (30). After an allergen-free interval, an acute allergen provocation was performed on Day 50 by inhalation of nebulized OVA (200 mg/ml) or saline for 10 min. To study the early influx of white blood cells after acute allergen exposure, groups of 12 mice were killed before and at 3, 6, and 12 h after allergen inhalation. As neutrophilic influx was maximal at 3 h after provocation, this time-point was chosen for further analyses. Neutralizing mAb against IL-17 or control rat IgG were injected intraperitoneally at a dosage of 50 µg 30 min before allergen provocation (n = 12 per group). To compare the effects of blocking the proinflammatory cytokine IL-17 with the effects of the anti-inflammatory dexamethasone, we injected dexamethasone (3 mg/kg) or an equal volume of saline intraperitoneally 30 min before the allergen inhalation on Day 50 in another group of mice (n = 6 per group).

View larger version (15K): [in this window] [in a new window]   Figure 1. Experimental protocol for induction of experimental asthma. First, all BALB/c mice were sensitized to OVA by repeated injections of OVA (10 µg/500 µl, intraperitoneally) on alternate days from Days 1–13. Secondly, sensitized mice were repeatedly exposed to nebulized OVA (10 mg/ml) or saline for 5 min daily from Days 33–40. Finally, an acute provocation was performed by exposure of mice to nebulized OVA (200 mg/ml) or saline for 10 min on Day 50. In addition to induction of allergic airway inflammation, mice (n = 12 per group) were treated with anti–IL-17 mAb or control Ig (50 µg, intraperitoneally) 30 min before provocation on Day 50 and killed at 3 h after provocation. In a second series of experiments, anti–IL-17 or control Ig were injected on alternate days from Days 32–40 (n = 6 per group), i.e., during the induction of allergic inflammation, and killed at 24 h after the eighth inhalatory OVA challenge.

Evaluation of Eosinophilia in Peripheral Blood, Bronchoalveolar Lavage Fluid, and Bone Marrow
Mice were anaesthetized with intraperitoneal injection of urethane (2.1 g/kg) at 3 h after allergen provocation (Day 50) or at 24 h after chronic allergen inhalation (Day 41). After retro-orbital bleed, blood smears were stained with the classic May-Grünwald-Giemsa (MGG) stain, and differential cell counts were performed. The lungs were lavaged five times with 1 ml of phosphate-buffered saline (PBS) supplemented with BSA 5% through a tracheal polyethylene catheter (inner diameter 0.85 mm). The first lavage was centrifuged at 1400 x g for 5 min and the supernatant stored at –20°C until measurement of cytokines. The pellet was added to the subsequent four lavages. After centrifugation (1400 x g, 5 min), cells were washed and resuspended in 200 µl of PBS. For cell counting, 10 µl of cell suspension were added to 90 µl of Türk's solution and the number of cells was calculated in a Bürker chamber. After dissection of one femur, BM was prelevated by flushing the femoral shaft with RPMI 1640, and smears were made. Differential cell counts were performed on MGG-stained smears of peripheral blood and BM, and cytospin preparations of bronchoalveolar lavage (BAL) fluid cells.

Histologic Analysis
After performing BAL, lungs were dissected and fixed overnight in buffered formalin (5%). After dehydration and embedding in paraffin, 5-µm sections were stained with the classic hematoxylin and eosin (H&E). On H&E-stained sections, eosinophils could be easily recognized by their polyglobular nucleus and bright cytoplasmic granules. For assessment of neutrophilic influx, a classic myeloperoxidase (MPO) stain was performed as described previously (32). The maximal thickness of the inflammatory infiltrates around two bronchioli and two arterioli in the lung were measured using an eyepiece graticule at a magnification of x25, and the average of these four values was calculated for each mouse.

Measurement of IL-5, IFN-, and IL-4 Levels
IL-5, IL-4, and IFN- levels in BAL fluid (1:2 diluted) and serum (undiluted) were measured by sandwich ELISA using paired matched Ab according to the manufacturer's instructions. The sensitivity of these assays was 4.5, 2.0, and 1.2 pg/ml for IL-5, IL-4, and IFN-, respectively.

MPO and Eosinophil Peroxidase Assay
MPO activity in BAL fluid was measured using a method described by Bradley and coworkers (33). In brief, 100 µL of undiluted BAL fluid were incubated for 15 min with 2.9 ml of PBS containing 0.0005% of H₂O₂ and 0.167 mg/ml of o-dianisidine dihydrochloride at pH 6.0. The reaction was stopped by adding 100 µL NaN₃ 1% and the optical density was measured at a wavelength of 460 nm. Eosinophil peroxidase (EPO) activity in BAL fluid was assessed by incubation of undiluted BAL fluid (100 µl) with Tris-HCl buffer (50 µl) for 10 min. Then, 100 µl of substrate solution, containing 2 mg/ml of o-phenylenediamine and 1.3 µl/ml of H₂O₂ in Tris-HCl buffer, were added. The reaction was stopped by adding H₂SO₄, and the optical density was measured at 492 nm.

RT-PCR for IL-17 and Other Cytokines
Part of the left lung was dissected at 3 h after acute allergen provocation, immediately frozen in liquid nitrogen, and stored at –80°C. Total RNA was extracted using TRIzol (Life Technologies, Gaithersburg, MD). A constant amount of 1 µg of target RNA was reverse transcribed using 100 U Superscript II RT (Life Technologies) at 42°C for 80 min in the presence of 5 µM Oligo(dT)₁₆. Real-time quantitative RT-PCR was performed for IL-17, IL-4, IL-5, IL-10, IL-13, IFN-, IL-12 p40, and IL-1ß mRNA in the ABI prism 7,700 Sequence detector (Applied Biosystems, Foster City, CA) as described before (34). The primer and probe for mouse IL-17 were designed based on the published sequence (35), and are as follows: IL-17 forward primer, 5' GCTCCAGAAGGCCCTCAGA 3'; IL-17 reverse primer, 5' AGCTTTCCCTCCGCATTGA 3'; IL-17 Taqman probe, 5'FAM-CTCTCCACCGCAATGAAGACCCTGA-TAMRA 3'. The amplified PCR-fragment is 142 bp in length, located from bp 229 to 370. The primer and probe sequences used for IL-4, IL-5, IL-10, IL-13, IFN-, IL-12, and IL-1ß were as previously published (34). Each PCR amplification was performed in triplicate wells, using the following conditions: 10 min at 94°C, followed by a total of 40 or 45 two-temperature cycles (15 s at 94°C and 1 min at 60°C).

Measurements of Nonspecific BHR
On Day 41, bronchial responsiveness to inhaled metacholine (Mch) was measured using whole body plethysmography (Buxco; EMKA Technologies, Paris, France) as described previously (30). In brief, mice that had been treated with either control Ig or anti–IL-17 mAb during the phase of challenges with nebulized OVA (Days 33–40), were exposed to incremental doses of nebulized Mch (0, 5, 10, 20, and 50 mg/ml) for 1 min, and mean Penh values were recorded during 3 min after each dose of Mch.

Data Analyses
Data are expressed as means ± SEM. Statistical analyses were performed using the Mann-Whitney test. A difference was considered to be significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Allergen Provocation of Mice with Eosinophilic Airway Inflammation Induces Bronchial Neutrophilic Influx and IL-17 Expression
All mice in this study were sensitized by i.p. injection of OVA (Days 1–13) and then exposed daily (Days 33–40) to aerosols containing OVA or saline as described previously (30) (Figure 1). In this way, peribronchial eosinophilic inflammation and airway hyperresponsiveness were induced in OVA-challenged mice (30). After an allergen-free interval of 10 d, during which (peri-)bronchial inflammation partially resided, an acute allergen provocation was performed by means of inhalation of nebulized OVA (200 mg/ml) or saline for 10 min. At 3 h after provocation with OVA, bronchial cellular influx was maximal and constituted primarily of neutrophils (51.2% of cells in BAL fluid; Figure 2A) . As neutrophils were hardly present before provocation (0.4 ± 0.1 x 10³/ml of BAL fluid), their prominent influx in the bronchoalveolar lumen at 3 h after provocation with OVA (74.3 ± 23.6 x 10³/ml of BAL fluid, P < 0.001; Figure 2A) was remarkable. In contrast to the significant neutrophilic influx, the numbers of eosinophils and lymphocytes were not altered by allergen provocation as early as 3 h after provocation (Figure 1A). It was only at 12 and 24 h after provocation with OVA that a significant increase in bronchial eosinophil and lymphocyte counts was observed (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 2. Bronchial responses in mice after acute allergen provocation. Mice were first sensitized (Days 1–13) and chronically exposed to nebulized OVA (Days 33–40). On Day 50, an acute provocation was performed by inhalation of nebulized OVA. (A) Cell counts on MGG-stained cytospin preparations of BAL fluid of mice killed before (open bars) and at 3 h after provocation (filled bars). Neutrophils (Neutro), eosinophils (Eosino), lymphocytes (Lympho), and monocytes (Mono) were differentiated. (B) mRNA levels for IL-17, IL-4, IL-5, IL-10, IL-13, IL-12 p40, IFN-, and IL-1ß were measured in lung tissue using real time quantitative RT-PCR before (open bars) and at 3 h after provocation (filled bars). Data were expressed as means ± SEM for each group (n = 12). *P < 0.005 versus mice killed before provocation.

The OVA-mediated nature of the observed bronchial neutrophilic influx and IL-17 mRNA induction after provocation with OVA of mice with experimental asthma was illustrated by extensive pilot studies. Negligible bronchial neutrophilic influx and minute amounts of IL-17 mRNA were induced by OVA inhalation (200 mg/ml for 10 min) in nonsensitized mice, as well as after inhalation of nebulized BSA (200 mg/ml for 10 min) by mice with OVA-induced eosinophilic airway inflammation (data not shown).

To exclude a major contribution of endotoxins contaminating the OVA solution to the bronchial neutrophilic influx and IL-17 mRNA induction, experiments were performed in which sensitized and OVA-challenged mice were exposed on Day 50 to nebulized LPS for 10 min at 5 µg/ml, i.e., the concentration of endotoxins present in an OVA solution of 200 mg/ml (measured with the Limulus Amebocyte Lysate test from BioWhittaker, Walkersville, MD). Exposure to this concentration of nebulized endotoxin did not induce neutrophilic influx nor IL-17 mRNA in the airways (data not shown).

Effect of Anti–IL-17 mAb Administration before Acute Provocation on Bronchial Responses after Provocation
When anti–IL-17 mAb were injected intraperitoneally 30 min before OVA provocation on Day 50, this treatment markedly altered the bronchial influx of granulocytes following allergen provocation. Neutrophilic influx into the bronchial lumen was significantly reduced by anti–IL-17 mAb compared with the control (P < 0.05; Figure 3A) . Interestingly, anti–IL-17 mAb inhibited the bronchial neutrophilic influx in a manner equally as potent as did dexamethasone therapy (46.3 ± 12.9 and 40.1 ± 10.1 x 10⁴/ml of BAL fluid, respectively; Figure 3A). In line with the observation of reduction of bronchial neutrophilic influx by anti–IL-17 mAb, we found less MPO activity in BAL fluid (55.8 ± 5.5 versus 97.8 ± 21.4 OD; Figure 3B) and smaller MPO-positive infiltrates in bronchial tissue (25.3 ± 5.2 x 10^-2 versus 75.3 ± 7.9 x 10^-2 mm, P < 0.05; Figures 4A and 4B) . Also in the circulation, percentages of neutrophils in the white cell population were reduced after administration of anti–IL-17 mAb (44.4 ± 1.2% versus 50.2 ± 1.3%, P < 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 3. Effects of anti–IL-17 therapy on bronchial inflammation after OVA provocation. Mice that had been sensitized (Days 1–13) and challenged with OVA (Days 33–40) were injected with control Ig (open bars), anti–IL-17 mAb (gray bars), or dexamethasone (black bars) 30 min before inhalation of nebulized OVA on Day 50 and killed at 3 h after provocation. (A) Neutrophils (Neutro), eosinophils (Eosino), lymphocytes (Lympho), and monocytes (Mono) were counted on MGG-stained cytospin preparations of BAL fluid. (B) The activity of MPO and EPO were measured in BAL fluid of both groups of mice. (C) Levels of IL-5 and IFN- were measured in BAL fluid and serum using sandwich ELISA in both groups. *P < 0.05 compared with control mice (n = 10–12 mice/group); one representative experiment out of two is shown.

View larger version (197K): [in this window] [in a new window]   Figure 4. Histologic sections of inflamed bronchi. Mice that had been sensitized (Days 1–13) and challenged with OVA (Days 33–40) were acutely exposed to nebulized OVA on Day 50. Lungs were removed for histologic examination of bronchi at 3 h after provocation. Bronchial sections of control Ig (A and C) and anti–IL-17–treated mice (B and D) were stained with the classic MPO stain (A and B) and H&E stain (C and D), for visualization of bronchial subepithelial neutrophils (A and B) and eosinophils (C and D) respectively, as indicated with black arrows.

Effect of Anti–IL-17 mAb on IL-5 Production In Vivo
To explain the remarkable effect of neutralizing IL-17 on bronchial eosinophilia after OVA provocation on Day 50, IL-5 levels were measured. Levels of circulating IL-5 were elevated at 3 h after OVA provocation in anti–IL-17–treated mice compared with controls (59.7 ± 5.8 versus 25.0 ± 4.1 pg/ml, respectively, P < 0.05; Figure 3C). Also in BAL fluid, higher levels of IL-5 were found in mice injected with anti–IL-17 mAb before OVA provocation, without reaching levels of significance (P > 0.05; Figure 3C). As far as IL-4 production is concerned, anti–IL-17 mAb enhanced IL-4 levels in serum (19.8 ± 4.1 versus 12.3 ± 2.1 pg/ml) and BAL fluid (42.2 ± 5.0 versus 32.4 ± 2.4 pg/ml) in a nonsignificant way (P > 0.05). In contrast to Th2 cytokines, IFN- levels in BAL fluid were reduced by anti–IL-17 mAb (2.9 ± 1.1 versus 7.0 ± 1.3 pg/ml, P < 0.05) and systemic levels of IFN- were under the detection limit (1.2 pg/ml) in both groups of mice. In OVA-sensitized and OVA-challenged mice, exposure to nebulized saline on Day 50 did not induce detectable production of IL-4, IL-5, and IFN- in BAL fluid, nor in serum of either anti–IL-17 mAb or control mice (data not shown).

Effects of Prolonged Neutralization of IL-17 during Repeated Allergen Inhalation on the Manifestation of Allergic Airway Inflammation
In addition to the effects of acute neutralization of IL-17 before allergen provocation, we studied the effects of prolonged neutralization of IL-17 on bronchial inflammation and BM biology. To this purpose, anti–IL-17 mAb or control Ig were administered to sensitized mice on alternate days from Days 32–40, i.e., during the inhalatory challenge phase with OVA or saline (Days 33–40, Figure 1). On Day 41, less neutrophils were found in BAL fluid of OVA-challenged mice treated with anti–IL-17 mAb (Figure 5A) , whereas the number of eosinophils was equally high in both groups (Figure 5A). Neutralization of IL-17 led to higher production of IL-5 (83.1 ± 12.3 versus 25.0 ± 3.2 pg/ml, P < 0.05; Figure 5B) and to a lesser extent of IL-4 in inflamed bronchi (34.1 ± 9.0 versus 15.0 ± 5.3 pg/ml, P < 0.05; Figure 5B). IFN- levels in BAL fluid were low and not affected by anti–IL-17 mAb (Figure 5B). Serum levels of IL-5, IL-4, and IFN- were below detection limit at the time of analysis (data not shown) and bronchial responsiveness to inhaled Mch was not affected by chronic administration of anti–IL-17 mAb during the inhalatory challenge phase (Figure 5C). In bronchi of saline-challenged mice treated with anti–IL-17 mAb or control Ig, no inflammation was present and no cytokine level was above the detection limit (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 5. Effects of repeated anti–IL-17 mAb administration during the challenge phase on the development of allergic airway inflammation. Mice that had been sensitized to OVA (Days 1–13) were treated with anti–IL-17 mAb (filled bars) or control IgG (open bars) before and during the phase of repeated inhalations of nebulized OVA (Days 33–40). Mice were killed on Day 41 and differential cell counts performed on MGG-stained cytospin preparations of BAL fluid (A). Protein levels of IL-5, IL-4, and IFN- were measured in BAL fluid with sandwich ELISA (B). Using whole body plethysmography, airway responsiveness to incremental doses of MCh was evaluated by measuring Penh values (C) in sensitized mice that had inhaled nebulized OVA or saline (Sal). *P < 0.05 versus control; #P < 0.05 versus Sal; n = 6–12 mice/group; one representative experiment out of two is shown.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effects of chronic anti–IL-17 therapy on granulocyte biology in different compartments. Mice that had been sensitized to OVA (Days 1–13) were treated with anti–IL-17 mAb (filled bars) or control IgG (open bars) before and during the phase of repeated inhalations of nebulized OVA (Days 33–40). Mice were killed on Day 41 and percentages of neutrophils (A) and eosinophils (B) were counted in BM, peripheral blood, and BAL fluid. *P < 0.05 and **P < 0.005 versus control (n = 6 per group); one representative experiment out of two is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We here report for the first time upregulation of IL-17 mRNA in inflamed airways after allergen inhalation. The allergen-specific nature of this observation has been investigated in pilot studies. To exclude a major contribution of endotoxins to the observed IL-17 mRNA induction and bronchial neutrophilic influx after allergen provocation, OVA-sensitized and -challenged mice were exposed on Day 50 to nebulized endotoxins, at a concentration present in the OVA solution used for provocation (see RESULTS). As this exposure did not induce any IL-17 mRNA production or bronchial neutrophilia, the observed responses following the OVA provocation are likely allergen-mediated.

Activated T lymphocytes have been regarded to be the principal source of IL-17 (10, 36). Our finding of rapid induction of mRNA for IL-17 after allergen provocation suggests the presence of an IL-17–producing memory T cell population in inflamed airways. Flow cytometric analysis of CD4⁺ lymphocytes obtained from peribronchial lymph nodes of mice with experimental allergic asthma for intracellular IL-17 showed that these CD4⁺ cells represent a significant source of IL-17 (unpublished observation). From existing literature, IL-17 production does not segregate into a distinct subset of CD4⁺ cells, as IL-17–producing T cell clones belong to the Th1 phenotype in rheumatoid arthritis (37), and both Th1 and Th2 phenotypes after priming in the presence of Borrelia burgdorferi (38) and in allergic contact dermatitis (39). Here, the simultaneous induction of mRNA for IL-17 and Th2, and not Th1 cytokines, suggests that activated Th2 lymphocytes are involved in IL-17 secretion. In addition to T cells, eosinophils in bronchial mucosa of patients with asthma have recently been reported to express IL-17 (2), suggestive of a multicellular origin of IL-17 in allergic airway inflammation.

After demonstration of enhanced expression of IL-17 by OVA inhalation, we studied the functional relevance of IL-17 in experimental allergic asthma in vivo. We first demonstrated that neutralization of IL-17 before acute allergen provocation severely impairs the neutrophilic influx into inflamed airways following allergen inhalation. Therefore, these data illustrate that IL-17 secretion is primarily involved in the allergen-mediated bronchial neutrophilic influx. Previously, Laan and coworkers (26) reported that IL-17 is capable of inducing neutrophil recruiment toward the airways. The study by Ye and colleagues (19) and our study are the first to show that endogenous IL-17 is critical for lung polymorphonuclear cell recruitment in response to a gram-negative pathogen and an allergen, respectively. Taken together, IL-17 plays a pivotal role in the neutrophil migration toward inflamed airways.

Several mechanisms of action may underlie our observation of inhibition of neutrophilic recruitment by anti–IL-17 mAb. First of all, we demonstrated that absence of functional IL-17 resulted in lower neutrophil counts in the BM and less circulating neutrophils, suggestive of reduced allergen-mediated stimulation of neutropoiesis. This sequence of events may result from less IL-17–mediated production of G-CSF and stem cell factor in anti–IL-17 mAb-treated mice (11, 25, 40) and highlights the key role of IL-17 in regulating stress-induced neutropoiesis. As BM responses in mice can occur as early as 2 h after mucosal allergen encounter (41), one could assume that modulation of BM biology by anti–IL-17 mAb may have an impact on the early bronchial cellular infiltrate after allergen provocation. In addition, anti–IL-17 mAb may have impaired the migration of neutrophils toward bronchi due to lower production of chemokines like KC (42), macrophage inflammatory protein-2 (19, 26), and/or granulocyte chemotactic protein-2 (GCP-2), a C-X-C chemokine that is the most potent murine neutrophil-chemoattractive protein (43) and is nearly as effective as human IL-8 in attracting neutrophils (44). We could not, however, detect any change in bronchial production of granulocyte chemotactic protein-2 on protein level after anti–IL-17 therapy (unpublished observation). Alternatively, decreased production of other factors like proinflammatory cytokines IL-1ß and TNF- (28), or impaired upregulation of ICAM-1 (23, 24, 27), may explain the impaired bronchial influx of neutrophils in anti–IL-17–treated mice as well.

Unexpectedly, we observed that anti–IL-17 mAb significantly aggravated bronchial eosiniophilic inflammation after allergen provocation. We attribute this phenomenon to enhanced production of IL-5 in serum as well as in bronchi of anti–IL-17 mAb-treated mice. Indeed, circulating IL-5 is one of the major stimuli for eosinopoiesis (41) and migration of eosinophils toward airways (45). In addition to more IL-5 production, absence of IL-17 may interfere with the production of other mediators involved in the chemotaxis of eosinophils toward airways, like RANTES (24).

In view of the upregulation of IL-5 and to a lesser extent of IL-4 by neutralization of IL-17, we tend to speculate that IL-17 is an immunomodulatory cytokine involved in dampening allergic airway inflammation through inhibition of Th2 cytokine secretion. However, we were unable to demonstrate any direct effect of recombinant mouse IL-17 on IL-5 production by lymphocytes in in vitro studies (unpublished observation). Therefore, we think that the mechanism underlying enhanced IL-5 production by administration of anti–IL-17 mAb most likely represents an indirect mechanism.

Interestingly, the reduction of bronchial neutrophilia in anti–IL-17 mAb-treated mice was not associated with beneficial effects on airway hyperresponsiveness. This observation is consistent with data obtained in rat models of allergic asthma (8) and acute respiratory distress syndrome (46), where a reduction of bronchial neutrophilia did not alter bronchial function. We should, however, mention that reduction of neutrophilic inflammation by anti–IL-17 mAb was not absolute, allowing no firm conclusion on the role of neutrophils in the development of BHR. Another reason for the lack of effect of anti–IL-17 mAb on BHR may be that the bronchial eosinophilia, which is still considered to play a role in the manifestation of BHR (1), was equally high in both groups of mice studied.

Whether targeting IL-17 may have therapeutic potential in human airway disease in future remains speculative. It is, however, interesting to mention that blocking anti–IL-17 mAb decreased the bronchial influx of neutrophils in a manner equally as potent as did dexamethasone, a potent anti-inflammatory agent. In view of the nonspecific mode of action of dexamethasone, targeting IL-17 may represent a new, more specific, therapeutic approach for airway diseases with major neutrophilic inflammation like acute severe asthma attacks and chronic obstructive pulmonary diseases (47). However, studies are warranted to dissect the role of neutrophils in allergic airway disease in vivo. In vitro studies show that neutrophilic proteases are important mucin secretagogues (48), mediate activation of epithelial cells, heighten vascular permeability (49), and even activate eosinophils (50). Bronchial neutrophils may also be involved in airway remodeling in asthma, as they represent a potentially important source of transforming growth factor-ß (51). However, it remains to be determined to what extent acute and/or prolonged reduction of bronchial neutrophilia may lead to decreased mucus secretion, bronchial remodeling, and/or symptom relief. Therefore, better insight into the pathophysiologic role of bronchial neutrophils is required before one can delineate the potentially beneficial effect of anti–IL-17 mAb in human airway disease.

In conclusion, we here show for the first time that IL-17 is produced in experimental allergic asthma following allergen inhalation, and that IL-17 orchestrates T cell–dependent granulocyte influx into inflamed airways. Our data reveal that IL-17 is critical for the bronchial recruitment of neutrophils, and may hence contribute to the chronic inflammatory changes associated with allergic asthma.

	Acknowledgments

 
The authors thank E. Dewil, H. Devijvere, and the staff of the animal housing department for taking care of the mice. This work was supported by Grants G.0227.01 and 7.0029.00 (Levenslijn) of the FWO (Fund for Scientific Research) Vlaanderen. P.H. is the recipient of a research fellowship of the FWO Vlaanderen.

Received in original form February 4, 2002

Received in final form July 25, 2002

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