Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.
Objectives: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor–deficient mice.
Methods: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.
Measurements and Main Results: Estrogen receptor-α knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.
Conclusions: These data suggest that estrogen receptor-α is a critical regulator of airway hyperresponsiveness in mice.
lung function; asthma; hyperreactivity; M2 muscarinic receptor; estrogen receptor
The relative contributions of large and small airways to hyperresponsiveness in asthma have yet to be fully assessed. This study used a mouse model of chronic allergic airways disease to induce inflammation and remodelling and determine whether in vivo hyperresponsiveness to methacholine is consistent with in vitro reactivity of trachea and small airways. Balb/C mice were sensitised (days 0, 14) and challenged (3 times/week, 6 weeks) with ovalbumin. Airway reactivity was compared with saline-challenged controls in vivo assessing whole lung resistance, and in vitro measuring the force of tracheal contraction and the magnitude/rate of small airway narrowing within lung slices. Increased airway inflammation, epithelial remodelling and fibrosis were evident following allergen challenge. In vivo hyperresponsiveness to methacholine was maintained in isolated trachea. In contrast, methacholine induced slower narrowing, with reduced potency in small airways compared to controls. In vitro incubation with IL-1/TNFα did not alter reactivity. The hyporesponsiveness to methacholine in small airways within lung slices following chronic ovalbumin challenge was unexpected, given hyperresponsiveness to the same agonist both in vivo and in vitro in tracheal preparations. This finding may reflect the altered interactions of small airways with surrounding parenchymal tissue after allergen challenge to oppose airway narrowing and closure.
Rationale: Leukotriene B4 (LTB4) is a rapidly synthesized, early leukocyte chemoattractant that signals via its cell surface receptor, leukotriene B4 receptor 1 (BLT1), to attract and activate leukocytes during inflammation. A role for the LTB4–BLT1 pathway in allergen-induced airway hyperresponsiveness and inflammation is not well defined. Objectives: To define the role of the LTB4 receptor (BLT1) in the development of airway inflammation and altered airway function. Methods: BLT1-deficient (BLT1−/−) mice and wild-type mice were sensitized to ovalbumin by intraperitoneal injection and then challenged with ovalbumin via the airways. Airway responsiveness to inhaled methacholine, bronchoalveolar lavage fluid cell composition and cytokine levels, and lung inflammation and goblet cell hyperplasia were assessed. Results: Compared with wild-type mice, BLT1−/− mice developed significantly lower airway responsiveness to inhaled methacholine, lower goblet cell hyperplasia in the airways, and decreased interleukin (IL)-13 production both in vivo, in the bronchoalveolar lavage fluid, and in vitro, after antigen stimulation of lung cells in culture. Intracellular cytokine staining of lung cells revealed that bronchoalveolar lavage IL-13 levels and numbers of IL-13+/CD4+ and IL-13+/CD8+ T cells were also reduced in BLT1−/− mice. Reconstitution of sensitized and challenged BLT1−/− mice with allergen-sensitized BLT1+/+ T cells fully restored the development of airway hyperresponsiveness. In contrast, transfer of naive T cells failed to do so. Conclusion: These data suggest that BLT1 expression on primed T cells is required for the full development of airway hyperresponsiveness, which appears to be associated with IL-13 production in these cells.
airway responsiveness; cytokines; lipid mediators; lung inflammation; T cells
Background: Respiratory syncytial virus (RSV) infection can cause bronchial hyperresponsiveness and asthma exacerbations. In mice it results in airway inflammation and airway hyperresponsiveness. Since viral factors influencing these responses are not well defined, a study was undertaken to investigate the role of secreted G protein of human RSV in determining virulence, inflammatory responses, and changes in lung function.
Methods: BALB/c mice were infected with a spontaneous mutant of RSV deficient in secreted G protein (RSV-ΔsG) or with wild type RSV (RSV-WT). Viral titres, numbers of pulmonary inflammatory cells, and concentrations of interferon (IFN)-γ, interleukin (IL)-4, IL-5 and IL-10 in bronchoalveolar lavage (BAL) fluid were determined. Airway function was assessed at baseline and following methacholine provocation using barometric whole body plethysmography.
Result: Following infection with RSV-ΔsG, viral titres were increased 50-fold compared with RSV-WT. Influx of eosinophils and macrophages to the lung and concentrations of IFN-γ and IL-10 in BAL fluid were also significantly higher following infection with RSV-ΔsG. Airway function, both at baseline and after methacholine provocation, was significantly decreased following infection with RSV-ΔsG compared with RSV-WT.
Conclusion: Secreted G protein is likely to be a regulatory factor in RSV infection limiting infectivity of the virus, inflammatory responses in the lungs, and reduction in lung function.
L-selectin is a cell adhesion molecule, which mediates leukocyte rolling on bronchopulmonary endothelium. Previous studies in a murine model of allergic airways disease have shown that L-selectin plays a role in the regulation of airway hyperresponsiveness in asthma via mechanisms independent of inflammation. Airway remodeling has been shown to modulate airway hyperresponsiveness independently of inflammation.
Our aim was to determine if L-selectin influenced airway hyperresponsiveness via modulation of structural changes as a result of airway remodeling.
A chronic ovalbumin-induced allergic airways disease model was applied to L-selectin-deficient mice and wild-type control mice. The development of airway inflammation was assessed by examining leukocyte influx into bronchoalveolar lavage fluid. Airway remodeling changes were determined via histology and morphometric analysis of lung tissue sections, and the development of airway hyperresponsiveness was assessed by invasive plethysmography.
Total cell counts, but not individual differential cell counts, were reduced in the ovalbumin-treated L-selectin-deficient mice compared to wildtype ovalbumin-treated mice. L-selectin-deficient mice had significantly reduced epithelial thickness and smooth muscle thickness. Airway hyperresponsiveness was abrogated in ovalbumin treated L-selectin-deficient mice compared to wild-type controls.
L-selectin plays an important role in regulating airway remodeling in an animal model of chronic allergic airways disease. Abrogated airway hyperresponsiveness may be related to reduced remodeling changes in L-selectin-deficient mice. L-selectin represents a potential target for novel asthma treatment for airway remodeling and airway hyperresponsiveness.
asthma; L-selectin; airway hyperresponsiveness; airway remodeling
Genome-wide screening and positional cloning have linked neuropeptide S receptor 1 (NPSR1) with asthma and airway hyperresponsiveness. However, the mechanism by which NPSR1 regulates pulmonary responses remains elusive. Because neuropeptide S and its receptor NPSR1 are expressed in brain regions that regulate respiratory rhythm, and Npsr1-deficient mice have impaired stress and anxiety responses, we aimed to investigate whether neuropeptide S and NPSR1 regulate respiratory function through a central-mediated pathway. After neuropeptide S intracerebroventricular administration, respiratory responses of wildtype and Npsr1-deficient mice were monitored by whole-body or invasive plethysmography with or without serial methacholine inhalation. Airway inflammatory and hyperresponsiveness were assessed in allergen-challenged (ovalbumin or Aspergillus fumigatus) Npsr1-deficient mice. Analysis of breathing patterns by whole-body plethysmography revealed that intracerebroventricular neuropeptide S, as compared with the artificial cerebral spinal fluid control, increased respiratory frequency and decreased tidal volume in an NPSR1-dependent manner but did not affect enhanced pause. Following serial methacholine inhalation, intracerebroventricular neuropeptide S increased respiratory frequency in wildtype mice, but not Npsr1-deficient mice, and had no effect on tidal volume. Intracerebroventricular neuropeptide S significantly reduced airway responsiveness to methacholine as measured by whole-body plethysmography. Npsr1 deletion had no impact on airway inflammation or hyperresponsiveness in ovalbumin- or Aspergillus fumigatus-induced experimental asthma. Our results demonstrate that neuropeptide S and NPSR1 regulate respiratory function through a central nervous system-mediated pathway.
Respiration; brain; neuropeptide S; neuropeptide S receptor 1; panting; stress
Rationale: Allergically inflamed mice exhibit airway hyperresponsiveness to inhaled methacholine, which computer simulations of lung impedance suggest is due to enhanced lung derecruitment and which we sought to verify in the present study.
Methods: BALB/c mice were sensitized and challenged with ovalbumin to induce allergic inflammation; the control mice were sensitized but received no challenge. The mice were then challenged with inhaled methacholine and respiratory system impedance tracked for the following 10 minutes. Respiratory elastance (H) was estimated from each impedance measurement. One group of mice was ventilated with 100% O2 during this procedure and another group was ventilated with air. After the procedure, the mice were killed and ventilated with pure N2, after which the trachea was tied off and the lungs were imaged with micro-computed tomography (micro-CT).
Results: H was significantly higher in allergic mice than in control animals after methacholine challenge. The ratio of H at the end of the measurement period between allergic and nonallergic mice ventilated with O2 was 1.36, indicating substantial derecruitment in the allergic animals. The ratio between lung volumes determined by micro-CT in the control and the allergic mice was also 1.36, indicative of a corresponding volume loss due to absorption atelectasis. Micro-CT images and histograms of Hounsfield units from the lungs also showed increased volume loss in the allergic mice compared with control animals after methacholine challenge.
Conclusions: These results support the conclusion that airway closure is a major component of hyperresponsiveness in allergically inflamed mice.
asthma; micro-computed tomography; input impedance; lung derecruitment; lung volume
The relationship between airway structural changes (remodeling) and airways hyperresponsiveness (AHR) is unclear. Asthma guidelines suggest treating persistent asthma with inhaled corticosteroids and long acting β-agonists (LABA). We examined the link between physiological function and structural changes following treatment fluticasone and salmeterol separately or in combination in a mouse model of allergic asthma.
BALB/c mice were sensitized to intraperitoneal ovalbumin (OVA) followed by six daily inhalation exposures. Treatments included 9 daily nebulized administrations of fluticasone alone (6 mg/ml), salmeterol (3 mg/ml), or the combination fluticasone and salmeterol. Lung impedance was measured following methacholine inhalation challenge. Airway inflammation, epithelial injury, mucus containing cells, and collagen content were assessed 48 hours after OVA challenge. Lungs were imaged using micro-CT.
Results and Discussion
Treatment of allergic airways disease with fluticasone alone or in combination with salmeterol reduced AHR to approximately naüve levels while salmeterol alone increased elastance by 39% compared to control. Fluticasone alone and fluticasone in combination with salmeterol both reduced inflammation to near naive levels. Mucin containing cells were also reduced with fluticasone and fluticasone in combination with salmeterol.
Fluticasone alone and in combination with salmeterol reduces airway inflammation and remodeling, but salmeterol alone worsens AHR: and these functional changes are consistent with the concomitant changes in mucus metaplasia.
Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined.
We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and TH2-driven inflammatory responses.
Wild-type and PDE4B−/− mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and TH2 cytokine production were determined in cultured bronchial lymph node cells.
Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased TH2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, TH2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the TH1 cytokine IFN-γ was not affected in PDE4B−/− mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation.
By relieving a cAMP-negative constraint, PDE4B plays an essential role in TH2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment.
Asthma; PDE4B; TH2 cytokines; airway hyperresponsiveness; airway inflammation; cAMP signaling
Serum-derived hyaluronan (HA)-associated proteins (SHAPs), the heavy chains of inter-α-trypsin inhibitor, covalently bind to HA to form the SHAP-HA complex. The SHAP-HA complex is involved in the pathophysiology of inflammatory diseases, including rheumatoid arthritis. We investigated whether this complex is also involved in airway allergy.
SHAP-HA-deficient (bikunin knockout, KO) mice and wild-type (WT) mice were immunized twice by intraperitoneal injection of ovalbumin (OVA) and exposed to aerosol OVA for 30 min each day for 2 weeks. Twenty-four hours after the final OVA challenge, airway responsiveness to inhaled methacholine (MCh) was measured, and analysis of bronchoalveolar lavage fluid (BALF) and lung histological studies were done.
Compared to WT mice, KO mice showed higher airway hyperresponsiveness to inhaled MCh and higher late-phase responses to OVA whereas the early-phase responses were similar. Cell differentials of BALF showed an increased number of macrophages and neutrophils in KO mice. Furthermore, decreased concentrations of soluble tumor necrosis factor receptor-1 (sTNFR1) were found in BALF from KO mice whereas the levels of Th1 and Th2 cytokines were not different from WT mice. Immunochemical study of the lung tissues revealed stronger staining of sTNFR1 in KO than in WT mice.
Our results suggest that in this murine asthma model, the SHAP-HA complex has an inhibitory role in the development of airway hyperresponsiveness and allergic airway inflammation which may be attributed, at least in part, to negative feedback mechanisms exerted by sTNFR1, the shedding of which from the cell surface might also be promoted by the SHAP-HA complex.
Serum-derived hyaluronan-associated proteins; Hyaluronan; Soluble tumor necrosis factor receptor-1; Interleukin-12p40; Asthma; Airway hyperresponsiveness
Considerable variation exists in the protocols used to induce hyperresponsiveness in murine models of allergic sensitisation. We examined the effect of varying the number of antigen exposures at challenge on the development of methacholine responsiveness in systemically sensitised mice.
BALB/c mice were sensitised with ovalbumin (OVA), challenged with 1, 3 or 6 OVA aerosols. Lung function was measured using low frequency forced oscillations and partitioned into components representing the airways (Raw) and lung parenchyma (tissue damping (G) and tissue elastance (H)). Responsiveness to inhaled methacholine (MCh), inflammatory cell profile and circulating IgE were assessed 24 and 48 hours after challenge. The threshold dose of MCh required to elicit a detectable response (sensitivity) and response to 30 mg.mL-1 (maximal response) were determined for each compartment.
Sensitivity; All three OVA protocols resulted in an increased sensitivity to MCh in Raw but not in G or H. These responses where present at 24 and 48 hrs, except 1 OVA aerosol in which changes had resolved by 48 hrs. Maximal response; 1 OVA aerosol increased maximal responses in Raw, G and H at 24 hrs, which was gone by 48 hrs. Three OVA aerosols increased responses in H at 48 hrs only. Six OVA challenges caused increases in Raw, G and H at both 24 and 48 hrs. Eosinophils increased with increasing antigen challenges. IgE was elevated by OVA sensitisation but not boosted by OVA aerosol challenge.
The pattern of eosinophilia, IgE and MCh responsiveness in mice was determined by antigen dose at challenge. In this study, increased sensitivity to MCh was confined to the airways whereas increases in maximal responses occurred in both the airway and parenchymal compartments. The presence of eosinophilia and IgE did not always coincide with increased responsiveness to inhaled MCh. These findings require further systematic study to determine whether different mechanisms underlie airway and parenchymal hyperresponsiveness post antigen challenge.
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
Overactivation of nuclear factor κB (NF-κB) orchestrates airway eosinophilia, but does not dampen airway hyperresponsiveness in asthma. NF-κB repression by arsenic trioxide (As2O3) contributes to apoptosis of eosinophils (EOS) in airways. Here we provide evidence that As2O3 abrogates allergen (OVA)-induced airway eosinophilia by modulating the expression of IκBα, an NF-κB inhibitory protein, and decreases the airway hyperresponsiveness.
Using a murine model of asthma, the airway hyperresponsiveness was conducted by barometric whole-body plethysmography. Airway eosinophilia, OVA-specific IgE in serum, and chemokine eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid were measured by lung histology, Diff-Quick staining, and ELISA. Chemokine-induced EOS chemotactic activity was evaluated using EOS chemotaxis assay. Electrophoretic mobility shift assay and Western blot analysis were performed to assess pulmonary NF-κB activation and IκBα expression, respectively.
As2O3 attenuated the allergen-induced serum IgE, chemokine expression of eotaxin and RANTES, and the EOS recruitment in bronchoalveolar lavage fluid, which is associated with an increased IκBα expression as well as a decreased NF-κB activation. Also, As2O3 suppressed the chemotaxis of EOS dose-dependently in vitro. Additionally, As2O3 significantly ameliorated the allergen-driven airway hyperresponsiveness, the cardinal feature underlying asthma.
These findings demonstrate an essential role of NF-κB in airway eosinophilia, and illustrate a potential dissociation between airway inflammation and hyperresponsiveness. As2O3 likely exerts its broad anti-inflammatory effects by suppression of NF-κB activation through augmentation of IκBα expression in asthma.
The multifunctional surface protein CD38 acts as a receptor with ecto-enzymatic activity, hydrolyzing NAD to generate several products known to exhibit Ca2+-mobilizing properties. Although CD38 is a convenient marker of immune cell development, and an indicator of progression for several diseases, it is not restricted to the immune compartment. To determine the potentially multilayered involvement of CD38 in allergen-induced airway inflammation and hyperreactivity, we dissected the potential role of CD38 as a regulator of immunity, but also pulmonary function. CD38-deficient and wild-type (WT) mice were sensitized and airway challenged with ovalbumin, and subsequently analyzed regarding their level of airway hyperresponsiveness (AHR) in response to methacholine. Parameters of lung inflammation were also analyzed. Similar sets of measurements were obtained from reciprocal bone marrow swapping experiments between CD38−/− and WT mice. Mice lacking CD38 exhibit strongly reduced AHR, which is accompanied by a decrease in typical hallmarks of pulmonary inflammation, including eosinophilia and lymphocytic lung infiltrates, as well as Th2-cytokine levels (IL-4, -5, and -13). Antigen-specific immunoglobulin (Ig)E and IgG1 antibody titers are substantially reduced, consistent with CD38 being crucial for mounting a primary humoral systemic immune response. Reconstitution of lethally irradiated, lung-shielded, CD38-deficient mice with WT bone marrow does not restore WT levels of airway hyperreactivity, nor mucus secretion. The opposite experiment, transferring CD38−/− bone marrow into WT mice, also shows reduced AHR levels. These studies demonstrate that CD38 not only acts as a key modulator of the immune response, but also plays an equally important role as an intrinsic pulmonary component.
airway hyperreactivity; pulmonary inflammation; CD38 knockout mouse; bone marrow chimera
The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17β-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1–dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.
estrogen; sex; airway hyperresponsiveness; EFS; neuronal activation
hyperresponsiveness and airway inflammation are distinctive features of
asthma. Evaluation of nitric oxide (NO) levels in expired air have been
proposed as a reliable method for assessing the airway inflammatory
events in asthmatic subjects. A study was undertaken to evaluate
whether airway hyperresponsiveness is related to levels of exhaled NO.
steroid-naive atopic children with mild intermittent asthma of mean
(SD) age 11.8 (2.3) years and 28 age matched healthy controls were
studied to investigate whether baseline lung function or airway
hyperresponsiveness is related to levels of exhaled NO. Airway
responsiveness was assessed as the dose of methacholine causing a 20%
decrease in forced expiratory volume in one second (FEV1)
from control (PD20 methacholine) and exhaled NO levels were
measured by chemiluminescence analysis of exhaled air.
asthmatic children had significantly higher NO levels than controls
(mean difference 25.87 ppb (95% CI 18.91 to 32.83); p<0.0001) but
there were no significant differences in lung function parameters
(forced vital capacity (FVC), FEV1 (%pred), and forced
expiratory flows at 25-75% of vital capacity (FEF25-75%)). In the asthmatic group exhaled NO levels were not significantly correlated with baseline lung function values or
results suggest that levels of exhaled NO are not accurate predictors
of the degree of airway responsiveness to inhaled methacholine in
children with mild intermittent asthma.
Immunized mice after inhalation of specific antigen have the following characteristic features of human asthma: airway eosinophilia, mucus and Th2 cytokine release, and hyperresponsiveness to methacholine. A model of late-phase allergic pulmonary inflammation in ovalbumin-sensitized mice was used to address the role of the alpha4 integrin (CD49d) in mediating the airway inflammation and hyperresponsiveness. Local, intrapulmonary blockade of CD49d by intranasal administration of CD49d mAb inhibited all signs of lung inflammation, IL-4 and IL-5 release, and hyperresponsiveness to methacholine. In contrast, CD49d blockade on circulating leukocytes by intraperitoneal CD49d mAb treatment only prevented the airway eosinophilia. In this asthma model, a CD49d-positive intrapulmonary leukocyte distinct from the eosinophil is the key effector cell of allergen-induced pulmonary inflammation and hyperresponsiveness.
Allergic asthma is on the rise in developed countries. A common characteristic of allergens is that they contain intrinsic protease activity, and many have been shown to activate protease-activated receptor (PAR)-2 in vitro. The role for PAR-2 in mediating allergic airway inflammation has not been assessed using a real world allergen.
Mice (wild type or PAR-2-deficient) were sensitized to German cockroach (GC) feces (frass) or protease-depleted GC frass by either mucosal exposure or intraperitoneal injection and measurements of airway inflammation (IL-5, IL-13, IL-17A, and IFNγ levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed.
Following systemic sensitization, GC frass increased airway hyperresponsiveness, Th2 cytokine release, serum IgE levels, cellular infiltration and mucin production in wild type mice. Interestingly, PAR-2-deficient mice had similar responses as wild type mice. Since these data were in direct contrast to our finding that mucosal sensitization with GC frass proteases regulated airway hyperresponsiveness and mucin production in BALB/c mice (Page et. al. 2007 Resp Res 8:91), we backcrossed the PAR-2-deficient mice into the BALB/c strain. Sensitization to GC frass could now occur via the more physiologically relevant method of intratracheal inhalation. PAR-2-deficient mice had significantly reduced airway hyperresponsiveness, Th2 and Th17 cytokine release, serum IgE levels, and cellular infiltration compared to wild type mice when sensitization to GC frass occurred through the mucosa. To confirm the importance of mucosal exposure, mice were systemically sensitized to GC frass or protease-depleted GC frass via intraperitoneal injection. We found that removal of proteases from GC frass had no effect on airway inflammation when administered systemically.
We showed for the first time that allergen-derived proteases in GC frass elicit allergic airway inflammation via PAR-2, but only when allergen was administered through the mucosa. Importantly, our data suggest the importance of resident airway cells in the initiation of allergic airway disease, and could make allergen-derived proteases attractive therapeutic targets.
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.
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.
Objectives: To determine the contribution of TGF-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
lung; mice; hypersensitivity; cytokines
Rationale: Arhgef1 is an intracellular protein, expressed by hematopoietic cells, that regulates signaling by both G protein–coupled receptors and RhoA, and, consequently, is required for appropriate migration and adhesion of diverse leukocyte populations.
Objectives: To evaluate a possible contribution for Arhgef1 in the development of airway inflammation and airway hyperreactivity.
Methods: Arhgef1-deficient (Arhgef1−/−) and wild-type (WT) mice were sensitized and airway challenged, followed by measurement of airway responsiveness to inhaled methacholine. Inflammation was assessed by several parameters that included flow cytometric analysis and histology. Arhgef1-deficient recipients were reconstituted with WT T lymphocytes before sensitization and challenge, and again measured for airway responsiveness and inflammation. Cytokine production in response to specific antigen was measured in cultures of isolated leukocytes from lung and spleen and compared with the levels generated in lung and spleen explant cultures.
Measurements and Main Results: Arhgef1−/− mice display significantly reduced airway hyperreactivity, Th2 cytokine production, and lung inflammation, despite intact systemic immunity. After airway challenge of Arhgef1−/− mice, antigen-specific T cells were present in mutant lungs, but were found to interact with CD11c+ cells at a significantly reduced frequency. Adoptive transfer of WT T cells into Arhgef1−/− mice restored airway hyperreactivity and inflammation.
Conclusions: These data demonstrate that T cells depend on Arhgef1 to promote lung inflammation. Moreover, a deficiency in Arhgef1 results in reduced T cell–CD11c+ antigen-presenting cell interaction, and likely underscores the inability of Arhgef1−/− mice to mount an adaptive immune response to airway challenge.
airway hyperreactivity; cytokines; lung inflammation; T cells
Chronic airway inflammation is a hallmark of asthma, an immune-based disease with great societal impact. Honokiol (HNK), a phenolic neurotransmitter receptor (GABAA) agonist purified from magnolia, has anti-inflammatory properties, including stabilization of inflammation in experimentally-induced arthritis. The present study tested the prediction that HNK could inhibit the chronic inflammatory component of allergic asthma. C57Bl/6 mice sensitized to and challenged with ovalbumin (OVA) had increased airway hyperresponsiveness to methacholine challenge and eosinophilia compared to naïve controls. HNK-treated mice showed a reduction in airway hyperresponsiveness as well as a significant decrease in lung eosinophilia. Histopathology studies revealed a marked drop in lung inflammation, goblet cell hyperplasia, and collagen deposition with HNK treatment. Antigen recall responses from HNK-treated mice showed decreased pro-inflammatory cytokines in response to OVA, including TNF-α, IL-6, Th1, and Th17-type cytokines, despite an increase in Th2-type cytokines. Regulatory cytokines IL-10 and TGF-β were also increased. Assessment of lung homogenates revealed a similar pattern of cytokines, with a noted increase in the number of FoxP3+ cells in the lung. HNK was able to alter B- and T-lymphocyte cytokine secretion in a GABAA dependent manner. These results indicate that symptoms and pathology of asthma can be alleviated even in the presence of increased Th2 cytokines, and that neurotransmitter agonists such as HNK have promise as a novel class of antiinflammatory agents in the treatment of chronic asthma.
Inflammation; mouse; cytokines; asthma
Rationale: Severe respiratory syncytial virus (RSV) bronchiolitis has been associated with deficient IFN-γ production in humans, but the role of this cytokine in determining the outcome of reinfection is unknown.
Objectives: To define the role of IFN-γ in the development of RSV-mediated airway hyperresponsiveness (AHR) and lung histopathology in mice.
Methods: Wild-type (WT) and IFN-γ knockout mice were infected with RSV in the newborn or weaning stages and reinfected 5 weeks later. Airway responses were assessed on Day 6 after the primary or secondary infection.
Measurements and Main Results: Both WT and IFN-γ knockout mice developed similar levels of AHR and airway inflammation after primary infection. After reinfection, IFN-γ knockout mice, but not WT mice, developed AHR, airway eosinophilia, and mucus hyperproduction. Intranasal administration of IFN-γ during primary infection but not during reinfection prevented the development of these altered airway responses on reinfection in IFN-γ knockout mice. Adoptive transfer of WT T cells into IFN-γ knockout mice before primary infection restored IFN-γ production in the lungs and prevented the development of altered airway responses on reinfection. Treatment of mice with IFN-γ during primary neonatal infection prevented the enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction on reinfection.
Conclusions: IFN-γ production during primary RSV infection is critical to the development of protection against AHR and lung histopathology on reinfection. Provision of IFN-γ during primary infection in infancy may be a potential therapeutic approach to alter the course of RSV-mediated long-term sequelae.
respiratory syncytial virus; interferon-γ; asthma; airway hyperresponsiveness; mice
Background: Smooth muscle contraction is one of the hallmarks of asthma. A recently developed pyridine derivative, Y-27632, a selective Rho kinase inhibitor, has been reported to inhibit the smooth muscle contraction of human and animal trachea in ex vivo systems but its effect in animal models of airway hyperresponsiveness (AHR) has not been examined. The purpose of this study was to evaluate the effect of Y-27632 in a murine model of allergic and virally induced AHR.
Methods: Baseline lung resistance and methacholine induced AHR were measured in mice sensitised to ovalbumin (OVA) and also in mice infected with respiratory syncytial virus (RSV) following ovalbumin sensitisation (OVA/RSV).
Results: Time course and dose ranging experiments indicated that 30 mg/kg Y-27632 given by gavage 2 hours before methacholine challenge significantly reduced baseline lung resistance and prevented AHR in OVA sensitised mice. Y-27632 also suppressed AHR induced by the bronchospastic agent serotonin in OVA sensitised mice and prevented methacholine induced AHR in OVA/RSV mice.
Conclusions: These results suggest that the signalling pathway mediated through Rho kinase may have an important role in bronchial smooth muscle tone in allergen induced and virus induced AHR and should be considered as a novel target for asthma treatment.
In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs.