Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.
Our understanding of the pathogenesis of atopic dermatitis (AD) and its relationship to asthma remains incomplete. Herein, we describe a murine model of epicutaneous (EC) sensitization to the protein allergen, chicken egg albumin, ovalbumin (OVA), which results in a rise in total and OVA-specific serum IgE and leads to the development of a dermatitis characterized by infiltration of CD3(+) T cells, eosinophils, and neutrophils and by local expression of mRNA for the cytokines IL-4, IL-5, and interferon-gamma. A single exposure of the EC sensitized mice to aerosolized OVA induced eosinophilia in the bronchoalveolar lavage fluid and airway hyperresponsiveness to intravenous methacholine as assessed by measurement of pulmonary dynamic compliance (Cdyn). These results suggest a possible role for EC exposure to antigen in atopic dermatitis and in the development of allergic asthma.
Inhalation of antigen in immunized mice induces an infiltration of eosinophils into the airways and increased bronchial hyperreactivity as are observed in human asthma. We employed a model of late-phase allergic pulmonary inflammation in mice to address the role of leukotrienes (LT) in mediating airway eosinophilia and hyperreactivity to methacholine. Allergen intranasal challenge in OVA-sensitized mice induced LTB4 and LTC4 release into the airspace, widespread mucus occlusion of the airways, leukocytic infiltration of the airway tissue and broncho-alveolar lavage fluid that was predominantly eosinophils, and bronchial hyperreactivity to methacholine. Specific inhibitors of 5- lipoxygenase and 5-lipoxygenase-activating protein (FLAP) blocked airway mucus release and infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation. The role of leukotrienes or eosinophils in mediating airway hyperresponsiveness to aeroallergen could not be established, however, in this murine model.
Asthma is characterized by bronchial inflammation causing increased airway hyperresponsiveness and eosinophilia. The interaction between airway epithelium and inflammatory mediators plays a key role in the asthmatic pathogenesis. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid found in apples and many berries, on inflammation in human airway epithelial BEAS-2B cells. Nontoxic kaempferol at ≤20 μM suppressed the LPS-induced IL-8 production through the TLR4 activation, inhibiting eotaxin-1 induction. The in vivo study explored the demoting effects of kaempferol on asthmatic inflammation in BALB/c mice sensitized with ovalbumin (OVA). Mouse macrophage inflammatory protein-2 production and CXCR2 expression were upregulated in OVA-challenged mice, which was attenuated by oral administration of ≥10 mg/kg kaempferol. Kaempferol allayed the airway tissue levels of eotaxin-1 and eotaxin receptor CCR3 enhanced by OVA challenge. This study further explored the blockade of Tyk-STAT signaling by kaempferol in both LPS-stimulated BEAS-2B cells and OVA-challenged mice. LPS activated Tyk2 responsible for eotaxin-1 induction, while kaempferol dose-dependently inhibited LPS- or IL-8-inflamed Tyk2 activation. Similar inhibition of Tyk2 activation by kaempferol was observed in OVA-induced mice. Additionally, LPS stimulated the activation of STAT1/3 signaling concomitant with downregulated expression of Tyk-inhibiting SOCS3. In contrast, kaempferol encumbered STAT1/3 signaling with restoration of SOCS3 expression. Consistently, oral administration of kaempferol blocked STAT3 transactivation elevated by OVA challenge. These results demonstrate that kaempferol alleviated airway inflammation through modulating Tyk2-STAT1/3 signaling responsive to IL-8 in endotoxin-exposed airway epithelium and in asthmatic mice. Therefore, kaempferol may be a therapeutic agent targeting asthmatic diseases.
We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4+ lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.
Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.
Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.
Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.
Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.
airway hyperresponsiveness; chronic asthma; cytokine; dendritic cells; eosinophil
Background and Objective. The features of asthma are airway inflammation, reversible airflow obstruction, and an increased sensitivity to bronchoconstricting agents, termed airway hyperresponsiveness (AHR), excess production of Th2 cytokines, and eosinophil accumulation in the lungs. To investigate the antiasthmatic potential of hesperidin as well as the underlying mechanism involved, we studied the inhibitory effect and anti-inflammatory effect of hesperidin (HPN) on the production of Th2 cytokines, eotaxin, IL-17, -OVA-specific IgE in vivo asthma model mice.
Methods. In this paper, BALB/c mice were systemically sensitized to ovalbumin (OVA) followed intratracheally, intraperitoneally, and by aerosol allergen challenges. We investigated the effect of HPN on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production and OVA-specific IgE production in a mouse model of asthma. Results. In BALB/c mice, we found that HPN-treated groups had suppressed eosinophil infiltration, allergic airway inflammation, and AHR, and these occurred by suppressing the production of IL-5, IL-17, and OVA-specific IgE. Conclusions. Our data suggest that the therapeutic mechanism by which HPN effectively treats asthma is based on reductions of Th2 cytokines (IL-5), eotaxin, OVA-specific IgE production, and eosinophil infiltration via inhibition of GATA-3 transcription factor.
This study was conducted to determine if oral administration of the novel herbal medicine, MA, and its Lactobacillus acidophilus fermented product, MA128, have therapeutic properties for the treatment of asthma. Asthma was induced in BALB/c mice by systemic sensitization to ovalbumin (OVA) followed by intratracheal, intraperitoneal, and aerosol allergen challenges. MA and MA128 were orally administered 6 times a week for 4 weeks. At 1 day after the last ovalbumin exposure, airway hyperresponsiveness was assessed and samples of bronchoalveolar lavage fluid, lung cells, and serum were collected for further analysis. We investigated the effect of MA and MA128 on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, OVA-specific IgE production, and Th1/Th2 cytokine production in this mouse model of asthma. In BALB/c mice, we found that MA and MA128 treatment suppressed eosinophil infiltration into airways and blood, allergic airway inflammation and AHR by suppressing the production of IL-5, IL-13, IL-17, Eotaxin, and OVA-specific IgE, by upregulating the production of OVA-specific Th1 cytokine (IFN-γ), and by downregulating OVA-specific Th2 cytokine (IL-4) in the culture supernatant of spleen cells. The effectiveness of MA was increased by fermentation with Lactobacillus acidophilus.
Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.
Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN blocks the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. We have used a mouse model for asthma to determine the effect of PI3K inhibitors and PTEN on allergen-induced bronchial inflammation and airway hyperresponsiveness. PI3K activity increased significantly after allergen challenge. PTEN protein expression and PTEN activity were decreased in OVA-induced asthma. Immunoreactive PTEN localized in epithelial layers around the bronchioles in control mice. However, this immunoreactive PTEN dramatically disappeared in allergen-induced asthmatic lungs. The increased IL-4, IL-5, and eosinophil cationic protein levels in bronchoalveolar lavage fluids after OVA inhalation were significantly reduced by the intratracheal administration of PI3K inhibitors or adenoviruses carrying PTEN cDNA (AdPTEN). Intratracheal administration of PI3K inhibitors or AdPTEN remarkably reduced bronchial inflammation and airway hyperresponsiveness. These findings indicate that PTEN may play a pivotal role in the pathogenesis of the asthma phenotype.
Persistent activation of nuclear factor κB (NF-κB) has been associated with the development of asthma. Galangin, the active pharmacological ingredient from Alpinia galanga, is reported to have a variety of anti-inflammatory properties in vitro via negative regulation of NF-κB. This study aimed to investigate whether galangin can abrogate ovalbumin- (OVA-) induced airway inflammation by negative regulation of NF-κB. BALB/c mice sensitized and challenged with OVA developed airway hyperresponsiveness (AHR) and inflammation. Galangin dose dependently inhibited OVA-induced increases in total cell counts, eosinophil counts, and interleukin-(IL-) 4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. Galangin also attenuated AHR, reduced eosinophil infiltration and goblet cell hyperplasia, and reduced expression of inducible nitric oxide synthase and vascular cell adhesion protein-1 (VCAM-1) levels in lung tissue. Additionally, galangin blocked inhibitor of κB degradation, phosphorylation of the p65 subunit of NF-κB, and p65 nuclear translocation from lung tissues of OVA-sensitized mice. Similarly, in normal human airway smooth muscle cells, galangin blocked tumor necrosis factor-α induced p65 nuclear translocation and expression of monocyte chemoattractant protein-1, eotaxin, CXCL10, and VCAM-1. These results suggest that galangin can attenuate ovalbumin-induced airway inflammation by inhibiting the NF-κB pathway.
Mast cells are the main effector cells of immediate hypersensitivity and anaphylaxis. Their role in the development of allergen-induced airway hyperresponsiveness (AHR) is controversial and based on indirect evidence. To address these issues, mast cell–deficient mice (W/W v) and their congenic littermates were sensitized to ovalbumin (OVA) by intraperitoneal injection and subsequently challenged with OVA via the airways. Comparison of OVA-specific immunoglobulin E (IgE) levels in the serum and numbers of eosinophils in bronchoalveolar lavage fluid or lung digests showed no differences between the two groups of mice. Further, measurements of airway resistance and dynamic compliance at baseline and after inhalation of methacholine were similar. These data indicate that mast cells or IgE–mast cell activation is not required for the development of eosinophilic inflammation and AHR in mice sensitized to allergen via the intraperitoneal route and challenged via the airways.
It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue.
To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation.
Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period.
Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment.
Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.
Signal transducers and activators of transcription 6 (STAT6) is essential for interleukin 4–mediated responses, including class switching to IgE and induction of type 2 T helper cells. To investigate the role of STAT6 in allergic asthma in vivo, we developed a murine model of allergen-induced airway inflammation. Repeated exposure of actively immunized C57BL/6 mice to ovalbumin (OVA) aerosol increased the level of serum IgE, the number of eosinophils in bronchoalveolar lavage (BAL) fluid, and airway reactivity. Histological analysis revealed peribronchial inflammation with pulmonary eosinophilia in OVA-treated mice. In STAT6-deficient (STAT6−/−) C57BL/6 mice treated in the same fashion, there were no eosinophilia in BAL and significantly less peribronchial inflammation than in wild-type mice. Moreover STAT6−/− mice had much less airway reactivity than wild-type mice. These findings suggest that STAT6 plays a crucial role in the pathogenesis of allergen-induced airway inflammation.
Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated ovalbumin-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared to sham-infected, ovalbumin-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13 and IFN-γ. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemistry showed eotaxin-1 in the lung macrophages of virus-infected, ovalbumin-treated mice, and confocal fluorescence microscopy revealed co-localization of rhinovirus, eotaxin-1 and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from ovalbumin-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from ovalbumin-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2 and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from ovalbumin-sensitized and -challenged mice reduced eosinophilic inflammation and airway hyperreactivity following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
rodent; T cells; cytokines; chemokines; lung
The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness
(BHR) that characterize asthma is achieved by the regulated accumulation and activation of
different leukocyte subsets in the lung. The development and maintenance of these processes
correlate with the coordinated production of chemokines. Here, we have assessed the role that
different chemokines play in lung allergic inflammation and BHR by blocking their activities
in vivo. Our results show that blockage of each one of these chemokines reduces both lung
leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the
eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES
(regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor
antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR.
Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates
with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators.
These results suggest that different chemokines activate different cellular and molecular pathways
that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their
individual blockage results in intervention at different levels of these processes.
chemokines; allergic inflammation; bronchial hyperresponsiveness; eosinophilia; leukocytes
Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10+CD4+CD25+ and Foxp3+CD4+CD25+) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10+/+, but not IL-10-/- Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.
Phosphoinositide 3-kinase (PI3K)/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, and has been shown to inhibit PI3K/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K/Akt signaling pathway.
Female BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Artesunate dose-dependently inhibited OVA-induced increases in total and eosinophil counts, IL-4, IL-5, IL-13 and eotaxin levels in bronchoalveolar lavage fluid. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, IL-17, IL-33 and Muc5ac in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, artesunate blocked epidermal growth factor-induced phosphorylation of Akt and its downstream substrates tuberin, p70S6 kinase and 4E-binding protein 1, and transactivation of NF-κB. Similarly, artesunate blocked the phosphorylation of Akt and its downstream substrates in lung tissues from OVA-challenged mice. Anti-inflammatory effect of artesunate was further confirmed in a house dust mite mouse asthma model.
Artesunate ameliorates experimental allergic airway inflammation probably via negative regulation of PI3K/Akt pathway and the downstream NF-κB activity. These findings provide a novel therapeutic value for artesunate in the treatment of allergic asthma.
Macrophage migration inhibitory factor (MIF) has recently been forwarded as a critical regulator of inflammatory conditions, and it has been hypothesized that MIF may have a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). Hence, we examined effects of MIF immunoneutralization on the development of allergen-induced eosinophilic inflammation as well as on lipopolysaccharide (LPS)-induced neutrophilic inflammation in lungs of mice. Anti-MIF serum validated with respect to MIF neutralizing capacity or normal rabbit serum (NRS) was administered i.p. repeatedly during allergen aerosol exposure of ovalbumin (OVA)-immunized mice in an established model of allergic asthma, or once before instillation of a minimal dose of LPS into the airways of mice, a tentative model of COPD. Anti-MIF treatment did not affect the induced lung tissue eosinophilia or the cellular composition of bronchoalveolar lavage fluid (BALF) in the asthma model. Likewise, anti-MIF treatment did not affect the LPS-induced neutrophilia in lung tissue, BALF, or blood, nor did it reduce BALF levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha). The present data suggest that MIF is not critically important for allergen-induced eosinophilic, and LPS-induced neutrophilic responses in lungs of mice. These findings do not support a role of MIF inhibition in the treatment of inflammatory respiratory diseases.
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
AHR; dendritic cells; eosinophils; mice; Syk
Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by polymerase chain reaction (PCR), but not by serology and culture, suggesting a low-level Mp involved in chronic asthma. However, the role of such a low-level Mp infection in regulation of allergic inflammation remains unknown.
To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms (i.e., prostaglandin E2 [PGE2] pathway) since PGE2 inhalation before allergen challenge suppressed eosinophil infiltration in human airways.
BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low-dose or high-dose Mp were used to assess IL-4 expression, BAL eosinophil, eotaxin-2 and PGE2 levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine if Mp-induced PGE2 or exogenous PGE2 down-regulates IL-4/IL-13-induced eotaxin-2.
Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE2 levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE2 induction. Exogenous PGE2 inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner.
This study highlights a novel concept on how differing bacterial loads in the lung modify the established allergic airway inflammation, and thus interact with an allergen to further induce Th2 responses. That is: Unlike high-level Mp, low-level Mp fails to effectively induce PGE2 to down-regulate allergic responses (e.g., eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
asthma; Mycoplasma pneumoniae; eotaxin-2; PGE2; alveolar macrophages
The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1f/fTekCre+ (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-β1, and eotaxin levels, as well as decreased expression of TGF-β1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1f/fTekCre− wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-β1.
Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.
BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.
Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.
These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. TNF-α blocking strategies are now being tried in asthma patients. This study investigated whether TNF-α blocking therapy inhibits airway inflammation and airway hyperresponsiveness (AHR) in a mouse model of asthma. We also evaluated the effect of TNF-α blocking therapy on cytokine production and adhesion molecule expression.
Materials and Methods
Ovalbumin (OVA) sensitized BALB/c female mice were exposed to intranasal OVA administration on days 31, 33, 35, and 37. Mice were treated intraperitoneally with soluble TNF-α receptor (sTNFR) during the OVA challenge.
There were statistically significant decreases in the numbers of total cell and eosinophil in bronchoalveolar lavage fluid (BALF) in the sTNFR treated group compared with the OVA group. However, sTNFR-treatment did not significantly decrease AHR. Anti-inflammatory effect of sTNFR was accompanied with reduction of T helper 2 cytokine levels including interleukin (IL)-4, IL-5 and IL-13 in BALF and vascular cell adhesion molecule 1 expression in lung tissue.
These results suggest that sTNFR treatment can suppress the airway inflammation via regulation of Th2 cytokine production and adhesion molecule expression in bronchial asthma.
Asthma; soluble TNF-α receptor; airway inflammation