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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 409-416, (2002)
© 2002 American Thoracic Society

Original Article

Anti–Interleukin-9 Antibody Treatment Inhibits Airway Inflammation and Hyperreactivity in Mouse Asthma Model

Gang Cheng, Masafumi Arima, Kyoko Honda, Hirokuni Hirata, Fukiko Eda, Nozomi Yoshida, Fumiya Fukushima, Yoshiki Ishii and Takeshi Fukuda

Department of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, Tochigi, Japan

Correspondence and requests for reprints should be addressed to Takeshi Fukuda, M.D., Ph.D., Departments of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, Mibu-machi, Shimotsuga-gun, Tochigi, 321–0293, Japan. E-mail: cheng{at}dokkyomed.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous in vitro and in vivo studies in both animals and patients with asthma have shown that interleukin (IL)-9 is an important inflammatory mediator in asthma. To examine the effects of IL-9 antagonism on airway inflammation, ovalbumin-sensitized BALB/c mice were intravenously given anti–IL-9 antibody or an isotype-matched control antibody 30 minutes before challenge with aerosolized ovalbumin. Airway response to methacholine was measured, and samples of bronchoalveolar lavage fluid (BALF) were obtained 24 hours after the last antigen challenge. Lung tissue was harvested and examined histopathologically. After ovalbumin challenge, there were significant increases in airway hyperreactivity, the numbers of inflammatory cells in lung, and IL-4, IL-5, and IL-13 production in BALF. Treatment with anti–IL-9 antibody significantly prevented airway hyperreactivity in response to methacholine inhalation. Blockade of IL-9 reduced the numbers of eosinophils (0.3 ± 0.1 x 105 and 23.6 ± 0.5 x 105/ml, anti–IL-9 antibody/control immunoglobulin G) and lymphocytes (0.2 ± 0.2 x 105 and 0.8 ± 0.1 x 105/ml) in BALF. Anti–IL-9 antibody treatment also reduced the concentrations of IL-4 (from 70.6 ± 4.6 to 30.8 ± 5.2 pg/ml), IL-5 (from 106.4 ± 12 to 54.4 ± 6.6 pg/ml), and IL-13 (from 44.2 ± 7.6 to 30.1 ± 5.5 pg/ml) in BALF. Macrophage-derived cytokine expression in the airways was also decreased by IL-9 blockade. Taken together, our findings emphasize the importance of IL-9 in the pathogenesis of asthma and suggest that blockade of IL-9 may be a new therapeutic strategy for bronchial asthma.

Key Words: interleukin-9 • eosinophils • asthma • airway hyperreactivity • mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atopic asthma is a chronic inflammatory disorder of the airways characterized by reversible obstruction and bronchial hyperresponsivess (1). Asthmatic airway inflammation is associated with mucosal infiltration by T helper (Th) 2-type CD4+ T cells and eosinophils (2, 3). Although the precise mechanism of this response remains unclear, available evidence indicates that complex interactions between these cells lead to the production of cytokines and other inflammatory proteins participating in the pathogenesis of asthma (4, 5). Th2 cells produce interleukin (IL)-4 and IL-5, cytokines implicated in the persistence of inflammation, and the mechanism linking inflammation to altered airway responsiveness (6). Recent studies have suggested that Th2 cell-derived IL-9 is also involved in allergic response (7–9). Mapping studies in mice and humans have provided evidence that IL-9 is a candidate gene for asthma (10, 11).

IL-9 is a T-cell–derived cytokine with pleiotropic effects on various types of cells (12). IL-9 is apparently produced in vitro and in vivo by CD4+ T cells, primarily those of the Th2 subset (13, 14). IL-9 is also expressed by human eosinophils and mast cells (15). IL-9 stimulates the proliferation of activated T cells, enhances the production of immunoglobulin (Ig) E by B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. In addition, IL-9 has been shown to affect IgE-mediated response by increasing expression of the {alpha} chain of high-affinity IgE receptors (16, 17).

Data from animal models of airway inflammation further support an important role of IL-9 in the asthmatic response. Genetic mapping studies in mice demonstrate linkage between the regulation of airway hyperresponsiveness and the IL-9 locus (11). Studies have shown that recombinant IL-9 introduced into the lungs of naive mice results in lung eosinophilia (9). Expression of IL-9 in the lungs of transgenic mice increases the IgE level; causes airway inflammation, mucus hypersecretion, and mast cell hyperplasia; and dramatically increases bronchial hyperresponsiveness (7, 8). Recently, Shimbara and colleagues have reported increased levels of IL-9 and its receptor in allergic asthma (18). Taken together, these findings suggest that IL-9 plays an essential role in allergic diseases.

Despite substantial evidence linking IL-9 to the etiology of asthma, to our knowledge, few studies have assessed the feasibility of using IL-9 antagonist to treat asthma. To test the therapeutic potential of IL-9 blockage in asthma, we examined the effect of exogenously administered anti–IL-9 antibody in a murine model of allergen-induced asthma. Airway inflammation was induced in ovalbumin-sensitized mice by aerosolized ovalbumin–antigen challenge. Twenty-four hours after the last challenge, airway inflammation, cytokine levels in bronchoalveolar lavage fluid (BALF), and hyperreactivity to methacholine were evaluated. We found that anti–IL-9 treatment inhibited eosinophil and lymphocyte infiltration; IL-4, IL-5, and IL-13 production in BALF; macrophage-derived cytokine (MDC) production in bronchial epithelium; and airway hyperreactivity to methacholine after allergen challenge.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Specific pathogen-free male BALB/c mice (6 to 8 weeks old; SLS, Shizuoka, Japan) were used in all experiments. The study protocol was reviewed and approved by the Dokkyo University School of Medicine Committee on Animal Care and complies with National Institutes of Health Guidelines for Animal Care.

Sensitization and Antigen Challenge of Mice
BALB/C mice were sensitized and challenged, as described previously (19). Briefly, mice were sensitized by intraperitoneal injections of ovalbumin (Sigma, St. Louis, MO) (8 µg per mouse) adsorbed with aluminum hydroxide (Wako Pure Chemical Industries, Osaka, Japan) on Days 0 and 5. Twelve days after the last injection, the mice were challenged with aerosolized ovalbumin or with saline, used as control. For blocking experiments, mice also received 20 µg per mouse of neutralizing polyclonal antibody against murine IL-9 (anti–IL-9 antibody). This antibody was produced in goats immunized with purified, insect cell-line Sf21-derived, recombinant mouse IL-9. IL-9–specific IgG was purified by affinity chromatography of mouse IL-9 (42409; Genzyme Techne, Cambridge, MA). Anti–IL-9 antibody was administered intravenously 30 minutes before ovalbumin provocation. As control, ovalbumin-challenged mice were given the same amount of goat immunoglobulin control IgG (Dako, Japan) by intravenous injection.

Determination of Airway Response
Airway responsiveness to inhaled methacholine in conscious, spontaneously breathing mice was measured by barometric whole-body plethysmography (Buxco, Troy, NY) as described previously (20–22). Mice were placed in the main chamber, and baseline readings were obtained and averaged for 3 minutes. Aerosolized saline or methacholine in increasing concentrations (3 to 25 mg/ml) was nebulized through an inlet of the main chamber for 3 minutes, and readings were taken and averaged for 3 minutes after each nebulization. Airway reactivity was expressed as enhanced pause values for each concentration of methacholine.

Bronchoalveolar Lavage
After determination of enhanced pause, BALF samples were obtained. Animals were anesthetized with pentobarbital, and the lungs were lavaged three times with 1.5 ml saline. Total cells and cell differentials were counted using a hemocytometer and Diff-Quik stain (International Reagent Corp., Osaka, Japan), respectively. The supernatant was stored at -70°C.

Histologic Examination of Lung and Immunocytochemistry
Lungs were fixed in formalin and embedded in paraffin. Three-µm thick sections were stained with hematoxylin and eosin.

Paraffin sections were dewaxed. Endogenous peroxidase activity was quenched with hydrogen peroxide, and sections were incubated with thymus- and activation-regulated chemokine (TARC) (1:500) or MDC (1:500) antibody or isotype-matched control (1:1,000) antibody overnight at 4°C. Antibodies were detected with biotinalated rabbit anti-goat immunoglobulin and then with streptavidin peroxidase complex prepared according to the manufacturer's instructions (both from the Dako kit). The sections were flooded with peroxidase substrate solution before counterstaining with hematoxylin.

Ovalbumin-Specific T-cell Response In Vitro
Mice spleen cells were isolated and cultured as described previously (23) with anti–IL-9 antibody or control IgG during stimulation with ovalbumin (10 µg/ml) for 24 hours. IL-4, IL-5, and IL-13 concentrations in cell culture supernatants were determined by enzyme-linked immunosorbent assay.

Bone Marrow Mast Cell Response
Mice bone marrow mast cells were isolated and cultured as described previously (24). Bone marrow mast cells were then cultured with 0.25 µM ionomycin during stimulation with anti–IL-9 antibody or control IgG for 24 hours.

Measurement of BALF Cytokines
Cytokine concentrations in the BALF supernatants were measured by enzyme-linked immunosorbent assay kits (R&D, Minneapolis, MN) according to the manufacturer's instructions.

Cell Culture of Human Bronchial Epithelial Cells
A BEAS-2B cell line was derived from human bronchial epithelium and transformed by an adenovirus, 12-SV40 hybrid virus, which was a generous gift from Dr. Curtis Harris (National Institutes of Health, Bethesda, MD). Cells were cultured in F12/Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) in 25 cm2 tissue culture flasks at 37°C in 5% CO2. Only cells that reached 80–90% confluence were used for experiments.

Stimulation of BEAS-2B Cells with IL-9 and Tumor Necrosis Factor-{alpha}
To assess the effect of certain stimulants on MDC gene expression, we treated BEAS-2B cell monolayers with control medium, various concentrations of IL-9 (1–30 ng/ml), tumor necrosis factor-{alpha} (TNF-{alpha}; 30 ng/ml); or combinations of these stimuli. Cultures were harvested after stimulation for 48 hours and underwent reverse transcription-polymerase chain reaction analysis. MDC was measured in the supernatants by using a solid-phase sandwich enzyme-linked immunosorbent assay (R&D).

Total RNA was extracted from the BEAS-2B cells using Trisolv, according to the manufacturer's instructions (Biotec Laboratories, Houston, TX). Semiquantitative reverse transcription-polymerase chain reaction was performed to determine the relative quantities of MDC mRNA, using a modified method as described elsewhere (25). The sequences of primers were from the coding regions of human genes as follows: MDC, 5'-TACAGACTGCACTCCTGGTTGTCC-3' and 5'-TTCTGGCGGG GAGCAGCTATAATG-3'. The level of MDC expression was quantified by calculating the ratio of the densitometric reading of the band for MDC to that for ß-actin from the same cDNA.

Statistical Analysis
Data are expressed as means ± SEM. The statistical significance of differences between groups was assessed by analysis of variance; p values of less than 0.05 were considered to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cellular Changes in BALFs after Ovalbumin Challenge
Total cell numbers in BALFs were significantly increased 24 hours after ovalbumin inhalation compared with saline inhalation. The increase of total cell numbers was associated with eosinophils, lymphocytes, and neutrophils (Figure 1) .



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  		Figure 1. Effects of anti–IL-9 on eosinophil and lymphocyte accumulation in BALF. The number of inflammatory cells in BALF was determined 24 hours after the last challenge, as described in METHODS. Data represent means ± SEM (n = 6). *Significant differences (p < 0.05). **Significant differences (p < 0.01) between sensitized and challenged control groups and sensitized and challenged anti–IL-9 treatment groups.

 
Effect of Anti–IL-9 Treatment on Lung Inflammation
As compared with control IgG, treatment with anti–IL-9 antibody significantly inhibited the increase in total cell numbers in BALF (Figure 1). Eosinophils, lymphocytes, and neutrophils decreased after anti–IL-9 antibody treatment (p < 0.01).

Histological Examination of Lung
Evidence of inflammatory cell infiltration and the effects of anti–IL-9 treatment were further investigated by histologically examining lung sections stained with hematoxylin and eosin. Intraperitoneal sensitization and aerosol challenge with ovalbumin increased the numbers of eosinophils and lymphocytes in the lung tissue (Figure 2) . Anti–IL-9 treatment abolished eosinophil and lymphocyte infiltration in the lung, whereas control IgG treatment did not.



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  		Figure 2. Effect of anti–IL-9 treatment on airway inflammation in ovalbumin-treated mice. Lung tissues of ovalbumin-sensitized/challenged mice treated with control IgG (A) or anti–IL-9 antibody (B) were obtained, stained with hematoxylin and eosin, and examined by light microscopy. Leukocyte accumulation in the lung after IL-9 blockade during lung allergic inflammation. (A) Representative sections of lungs isolated from mice treated with control antibody show widespread infiltration of inflammatory cells into the peribronchial region. (B) In the anti–IL-9-treated groups, only small numbers inflammatory cells are observed (original magnification: x200).

 
Anti–IL-9 Antibody Treatment Decreased IL-4, IL-5, and IL-13 Concentrations in BALF
IL-2, IL-12, interferon-{gamma}, IL-4, IL-5, and IL-13 concentrations in BALF were measured by enzyme-linked immunosorbent assay 24 hours after the last challenge (Figure 3) . The production of Th1 cytokine IL-2, IL-12, and interferon-{gamma} in BALF was unchanged by anti–IL-9 treatment (Figures 3A–3C). After sensitization and challenge, the increases in IL-4, IL-5, and IL-13 production in BALF supernatant were significantly suppressed by anti–IL-9 treatment (p < 0.05) (Figures 3D–3F). The control IgG had no apparent effect.



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  		Figure 3. Effect of anti–IL-9 treatment on Th1 and Th2 cytokine production in BALF. IL-5 (A), IL-4 (B), IL-13 (C) and IL-2 (D), IL-12 (E), interferon-{gamma} (F) cytokine concentrations in BALF were assessed 24 hours after the last challenge, as described in METHODS. Marked increases in IL-5 and IL-4 production in BALF after ovalbumin sensitization and challenge were significantly suppressed by treatment with anti–IL-9 as compared with the control IgG-treated groups. Injection of anti–IL-9 antibody also decreased IL-13 production slightly but not significantly as compared with the level in control-treated mice. IL-2, IL-12, and interferon-{gamma} production in BALF was unchanged by anti–IL-9. *Significant differences (p < 0.05) between sensitized and challenged control groups and sensitized and challenged anti–IL-9 treatment groups.

 
Effect of Anti–IL-9 Antibody Treatment on Chemokine Concentrations in BALF
Eotaxin, regulated upon activation, normal T cell expressed and secreted, KC (chemokine related to the human interleukin-8), monocyte chemotactic protein-1, and macrophage inflammatory protein-1{alpha} concentrations were also analyzed by enzyme-linked immunosorbent assay 24 hours after the last challenge (Figure 4) . The levels of none of these chemokines in BALF were affected by anti–IL-9 treatment as compared with the concentrations after treatment with control IgG.



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  		Figure 4. Effect of anti–IL-9 treatment on chemokine production in BALF. Eotaxin (A), RANTES (B), KC (C), monocyte chemotactic protein-1 (MCP-1) (D), and macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) (E) cytokine concentrations in BALF were assessed 24 hours after the last challenge. There were no significant differences for any chemokine. *Significant differences (p < 0.05) between sensitized and challenged control groups and sensitized and challenged anti–IL-9 treatment groups.

 
Detection of MDC and TARC Protein in the Airways by Immunohistochemistry
To confirm the production of MDC and TARC protein in the lung, immunohistochemical staining was performed. TARC and MDC were identified in airway sections of ovalbumin-sensitized and -challenged mice. In these mice, TARC and MDC were localized predominantly in the epithelium (data not shown). In the anti–IL-9 treatment group, MDC expression was significantly lower than that after control IgG treatment (Figure 5) ; TARC expression was unaffected by anti–IL-9 treatment (data not shown).



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  		Figure 5. MDC protein expression in the lung of ovalbumin-treated mice. Lung sections obtained from control IgG (A) and anti–IL-9 antibody (B) treatment groups were stained with polyclonal antibodies against mouse MDC. Positive staining was detected with an avidin-biotin peroxidase staining system that results in a brown reaction product. Sections were counterstained with hematoxylin (blue) for contrast. Protein expression was strongly detected in the airway epithelium of ovalbumin-treated mice (A). In contrast, anti-IL-9 antibody treatment decreased MDC protein expression in the in the airway epithelium (B). Representative results of six different experiments are shown (original magnification: x200).

 
Effect of Anti–IL-9 Treatment on Ovalbumin-Specific T-cell Response In Vitro
To determine whether anti–IL-9 antibody treatment influences systemic immune responses, we analyzed the ovalbumin-specific T-cell response in vitro (Figure 6) . Anti–IL-9 treatment did not affect ovalbumin-induced Th2 cytokine production by mice spleen T cells.



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  		Figure 6. Effect of anti–IL-9 treatment on cytokine release from mice spleen T cells in vitro. Spleen T cells from sensitized and challenged mice were cultured for 24 hours with anti–IL-9 antibody (black bars) or control Ig-G (gray bars) during ovalbumin stimulation. IL-9 antibody had no effect on release of IL-5, IL-4, or IL-13.

 
Effect of Anti–IL-9 Treatment on Mast Cell Response In Vitro
We analyzed the bone marrow mast cell response in vitro (Figure 7) . Anti–IL-9 treatment did not affect ionomycin-induced Th2 cytokine production by mice mast cells.



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  		Figure 7. Effect of anti–IL-9 treatment on cytokine release from bone marrow mast cells in vitro. Mast cells were cultured for 24 hours with ionomycin (0.25 µm) during stimulation with IL-9 antibody (black bars) or control IgG (gray bars). Anti–IL-9 antibody had no effect on release of IL-5, IL-4, or IL-13.

 
Inhibition of Allergen-induced Hyperresponsiveness to Methacholine by Administration of Anti–IL-9 Antibody
Airway reactivity to methacholine was evaluated 24 hours after the last challenge. In the ovalbumin-challenged group, airway hyperreactivity occurred after challenge with methacholine and was associated with a significantly greater increase in enhanced pause than that in the saline group (p < 0.01; Figure 8) . Anti–IL-9 treatment significantly blocked the increased airway reactivity to methacholine. In contrast, control IgG treatment did not alter the methacholine-induced airway hyperresponsiveness.



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  		Figure 8. Inhibition of ovalbumin-induced airway hyperrespnsiveness (AHR) after IL-9 blockade. Mice were first exposed to nebulized saline, followed by increasing doses (3 to 25) mg/ml) of nebulized methacholine for 3 minutes each. Breathing indices were read for 3 minutes after each nebulization, and enhanced pause values were determined. AHR was significantly inhibited by anti–IL-9 treatment (closed triangles) as compared with control IgG treatment (open circles). Saline challenge (closed squares) and ovalbumin challenge (closed circles) values are also presented. *Significant differences (p < 0.05) between sensitized and challenged control groups and sensitized and challenged anti–IL-9 treatment groups.

 
Effects of IL-9 and TNF-{alpha} on Production of MDC in BEAS-2B Cells
BEAS-2B cells incubated with IL-9 (1, 10, 30 ng/ml) or TNF-{alpha} (30 ng/ml) alone for 48 hours showed little or no expression of MDC mRNA (Figure 9A) . MDC was also not detected in the supernatants derived from the same culture conditions (Figure 9B). MDC mRNA was expressed after stimulation with both IL-9 and TNF-{alpha}, and such expression was significantly dependent on the concentration of IL-9. The production of MDC was not detected after stimulation with TNF-{alpha} (30 ng/ml) alone but was detected after stimulation with IL-9 and TNF-{alpha}; there was a trend toward higher MDC production with higher concentrations of IL-9 (Figure 9B).



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  		Figure 9. Effects of IL-9 and TNF-{alpha} on induction of MDC in BEAS-2B cells. (A) Top: Densitometric analysis of reverse transcription-polymerase chain reaction products of each mRNA. Bottom: Representative electrophoresis showing the expression of MDC mRNA. (B) MDC production. ND = detectable. Data are expressed as means ± SEM (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The study was designed to determine whether anti–IL-9 antibody reduces airway inflammation and hyperreactivity in a murine model of allergen-induced asthma. We showed that the blockade of IL-9 by anti–IL-9 antibody given intravenously before allergen challenge abrogated IL-4 and IL-5 secretion, airway hyperreactivity, and eosinophil and lymphocyte accumulation in the lung. Th2-specific chemokine MDC expression was also inhibited by anti–IL-9 treatment.

Eosinophil infiltration has been shown to play an important role in the pathogenesis of asthma. In our study, pulmonary eosinophilia was decreased by anti–IL-9 antibody treatment. This result is supported by the findings of several studies. IL-9 inhibits apoptosis of peripheral human eosinophils in vitro (26), and instillation of IL-9 into the lung significantly increases the eosinophil count in BALF from B6 animals (9). IL-5 and its receptor are thought to play a critical role in the recruitment of eosinophils to the lung in asthma. IL-9 upregulates IL-5R{alpha} chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line (27). IL-5R{alpha} expression in acute myelogenous leukemia (KG-1) cells, which are capable of spontaneously differentiating into granulocytes and eosinophils, is also induced by IL-9 (27). These findings indicate that the inhibition of eosinophil recruitment to the lung associated with anti–IL-9 antibody treatment most likely directly reflects neutralization of IL-9–induced promotion of eosinophil activity. However, the critical role of IL-9 in eosinophil infiltration had remained unclear until now.

Lung eosinophilia results from recruitment of eosinophils from blood. Such recruitment is mediated by the local expression of chemokines that signal eosinophils to migrate from the circulation into the airway tissue. Accumulating evidence suggests that chemokines, especially the C-C subfamily, are involved in both migration and activation of eosinophils during asthmatic response. Chemokines implicated in asthma include eotaxin, RANTES, macrophage inflammatory protein-1{alpha}, monocyte chemotactic protein-1, and monocyte chemotactic protein-4 (28–32). We have confirmed production of these chemokines in BALF, although chemokine levels were unchanged by anti–IL-9 antibody treatment. These data suggest that blockade of IL-9 did not affect C-C chemokine production and indicate that the inhibition of lung eosinophilia is mediated by other cytokines.

We also showed that anti–IL-9 antibody treatment prevented the increase in T lymphocytes in BALF. Recently, several studies have focused on the association between activated CD4+ T lymphocytes, in particular the Th2 subset, and the pathogenesis of asthma (33–36). CD4+ Th2 cells are present and activated in the bronchial wall and lavage fluid of patients with atopic asthma (37, 38). Recruitment of Th2 cells by local chemokines is thought to play a critical role in allergic asthma. Identification of CCR4 as a chemokine receptor selectively expressed in Th2 cells strongly suggests that TARC and MDC, the ligand for CCR4, are key chemokines in the migration of Th2 cells to sites of inflammation associated with allergic disorder (39–41). TARC and MDC blockade in a murine asthma model attenuates airway hyperreactivity and lung inflammation, whereas neutralization of TARC leads to inhibition of hepatic Th2-dominant response (42–44). In our study, both TARC and MDC reactivity were immunohistochemically detected in bronchial epithelium after allergen challenge. However, only MDC expression was decreased by anti–IL-9 treatment. We also found that IL-9 can stimulate MDC expression in bronchial epithelial cells in vitro. MDC in the culture supernatants of bronchial epithelial cells contributed partially to chemotactic activity for TH2 cells not for TH1 cells, suggesting that MDC produced by bronchial epithelial cells may contribute to airway local recruitment of TH2 cytokine-producing T cells. Perhaps inhibition of MDC decreased BALF lymphocytes, possibly Th2 cells, which produce Th2 cytokines such as IL-4, IL-5, and IL-13. These findings indicate that IL-9 may act by initiating MDC production by bronchial epithelial cells.

Neutrophils may also represent an important target for IL-9 because anti–IL-9 antibody also decreased neutrophil infiltration in BALF. It has been reported that human neutrophils from donors with asthma can express IL-9 receptor. In peripheral blood neutrophils, IL-9 induces IL-8 production in a concentration-dependent manner (45). IL-8 has been shown to have chemotactic activity for neutrophils involved in allergic disease. However, IL-8 levels were unchanged in our study. Further studies designed to clarify the effects of IL-9 on neutrophil functions such as signal transduction and cell proliferation are needed.

Mast cells play a central role in immediate allergic reactions. Cross-linking of allergen-specific IgE bound to high-affinity receptors for IgE on mast cells leads to cell activation and release of potent inflammatory mediators (46). In vitro studies have shown that recombinant IL-9 supports the growth of bone marrow–derived mast cell lines and increases survival and proliferation of primary mast cell cultures (47). Moreover, recombinant IL-9 in the presence of kit ligand prolongs the long-term survival of murine bone marrow-derived mast cells (48). We found that mast cell numbers in lung tissue after ovalbumin challenge were unaffected by anti–IL-9 treatment (data not shown). Therefore, the effects of IL-9 on mast cell functions remain to be determined.

IL-4, IL-5, and IL-13 produced by Th2 cells have been postulated to have central roles in the initiation and maintenance of allergic inflammation (49). The development of airway eosinophilia and allergen-induced hyperresponsiveness (AHR) was also associated with increased levels of IL-4, IL-5, and IL-13 in BALF, consistent with development of a Th2-mediated allergic response. Treatment with anti–IL-9 antibody significantly inhibited these increases in IL-4, IL-5, and IL-13 levels in BALF. Recent studies demonstrate that IL-9 can rescue Th2-type T-cell clones from apoptosis induced by deprivation of IL-2 and that the expression of IL-9R{alpha} is necessary for the antiapoptotic signals mediated by IL-9 (50). In vivo studies have shown that selective overexpression of IL-9 in the lung of transgenic mice results in T-cell infiltration (7). Whether the inhibitory effect of anti–IL-9 involves commitment, expansion, recruitment, or activation of lymphocytes remains to be determined. In this study, we found that anti–IL-9 stimulation did not affect Th2 cytokine production by spleen T cells in vitro. We also found that anti–IL-9 stimulation had no effect on Th2 cytokine production by mast cells. These findings suggest that the effect of anti–IL-9 treatment on Th2 cytokine production may involve inhibitory activity not directly mediated by T cells or mast cells, although the number of T cells infiltrating into the lung was suppressed. The inhibition of Th2 cytokine levels may be associated with decreased T cells in the lung. We also showed that MDC, a specific chemoattractant for Th2 cell production, was decreased by IL-9 blockage. We speculate that anti–IL-9 treatment did not influence systemic immune response but acted locally within the airways and adjacent parenchymal tissue to inhibit Th2 cytokine production.

Neutralization of murine IL-9 also inhibited the development of AHR in this study. Recent genetic studies of murine models have identified the IL-9 gene as a candidate gene related to asthma and have shown an association between lung IL-9 expression and bronchial hyperresponsiveness. Genetic studies in different inbred strains of mice have linked bronchial hyperresponsiveness to mouse chromosome 13, which shares homology with human chromosomal region 5q31–q33 and on which the murine homologue of the IL-9 gene is located (11). These studies have shown that reduced expression of IL-9 is associated with lowered airway responsiveness, whereas overexpression of IL-9 results in markedly increased airway responsiveness (10, 11). However, the precise mechanism by which IL-9 alters bronchial responsiveness remains unknown. Several studies have demonstrated a correlation between the degree of inflammation, in particular eosinophilia, and AHR in asthmatic patients. Indeed, anti–IL-9 inhibited both AHR and the associated airway eosinophilia in this study. Our findings thus support an important role of IL-9 in the development of airway hyperreactivity.

During preparation of this article, an important article related to our study appeared. McMillan and colleagues reported that IL-9 deletion had no effect on allergen-induced pulmonary inflammation and airway hyperreactivity (51). It is unknown why the results that were observed in this report and for the IL-9 transgenic mice are not consistent with those observed with IL-9–deficient mice. The same studies about IL-4 were also reported (52, 53), using neutralizing antibody to IL-4, antigen-induced airway eosinophilia and AHR. In contrast, mice deficient in IL-4, antigen-induced eosinophilic inflammation, airway hyperreactivity was not affected. However, we must keep in mind that the findings obtained from their knockout mice cannot always be applied to the actual pathophysiology of the disease because they are mature animals that have lacked the targeted gene since birth. In our studies, we directly assessed the importance of IL-9 in a murine model of asthma using anti–IL-9 antibody and found that anti–IL-9 antibody treatment can abolish the development of airway eosinopilia and AHR. Therefore, although IL-9 may not be critical for development of airway eosinopilia and AHR, IL-9 may play an important role in the subsequent response to allergen challenge in asthma or reinforcement for allergic inflammation.

In conclusion, we have demonstrated that anti–IL-9 antibody given intravenously can significantly attenuate pulmonary eosinophilic inflammation, Th2 cytokine production, Th2 cell-specific chemokine (MDC) expression in the airways, and bronchial hyperresponsiveness. These findings suggest that IL-9 plays a key role in this murine model of allergic asthma. Our results will hopefully improve our understanding of the pathogenesis of asthma and lead to novel therapeutic approaches to the management of this disease.


   Acknowledgments
 
The authors thank Mrs. Kazumi Okazaki for her excellent technical assistance.

Received in original form May 17, 2001; accepted in final form April 17, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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