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.
To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor–deficient mice.
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.
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
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
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
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
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
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.
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.
Vitamin D deficiency is associated with disease severity in asthma. We tested whether there is a causal association between vitamin D deficiency, airway smooth muscle (ASM) mass, and the development of airway hyperresponsiveness (AHR). A physiologically relevant mouse model of vitamin D deficiency was developed by raising BALB/c mice on vitamin D‐deficient or ‐replete diets. AHR was assessed by measuring lung function responses to increasing doses of inhaled methacholine. Five‐micron sections from formalin‐fixed lungs were used for ASM measurement and assessment of lung structure using stereological methods. Transforming growth factor (TGF)‐β levels were measured in bronchoalveolar lavage fluid (BALF). Lungs were dissected from embryonic day (E) 17.5 vitamin D‐deficient and ‐replete fetal mice for quantification of ASM density and relative gene expression of TGF‐β signaling pathway molecules. Eight‐week‐old adult vitamin D‐deficient female mice had significantly increased airway resistance and ASM in the large airways compared with controls. Vitamin D‐deficient female mice had a smaller lung volume, volume of parenchyma, and alveolar septa. Both vitamin D‐deficient male and female mice had reduced TGF‐β levels in BALF. Vitamin D deficiency did not have an effect on ASM density in E17.5 mice, however, expression of TGF‐β1 and TGF‐β receptor I was downregulated in vitamin D‐deficient female fetal mice. Decreased expression of TGF‐β1 and TGF‐β receptor I during early lung development in vitamin D‐deficient mice may contribute to airway remodeling and AHR in vitamin D‐deficient adult female mice. This study provides a link between vitamin D deficiency and respiratory symptoms in chronic lung disease.
Vitamin D deficiency caused airway hyperresponsiveness and increased airway smooth muscle mass in the airways of adult female mice. Vitamin D deficiency also reduced transforming growth factor (TGF)‐β1 protein levels in both male and female mice, as well as reduced gene expression of TGF‐β1 and TGF‐β receptor I in female E17.5 fetal pups. These observations may provide a link between vitamin D deficiency and respiratory symptoms in chronic lung disease.
Airway hyperresponsiveness; airway smooth muscle; lung structure; mouse model; vitamin D
Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2–8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M3-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations.
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
Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1–challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein–coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain–dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein–coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.
asthma; airway hyperresponsiveness; inflammation; house dust mite; Der p 1
Rationale: The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)–associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.
Objectives: This study tested whether D6 regulates pulmonary chemokine levels, inflammation, or airway responsiveness during allergen-induced airway disease.
Methods: D6-deficient and genetically matched C57BL/6 mice were sensitized and challenged with ovalbumin. ELISA and flow cytometry were used to measure levels of cytokines and leukocytes, respectively. Mechanical ventilation was used to measure airway reactivity.
Results: The ability of D6 to diminish chemokine levels in the lung was chemokine concentration dependent. CCL17 and CCL22 were abundant in the airway, and their levels were attenuated by D6 when they were within a defined concentration range. By contrast, airway concentrations of CCL3, CCL5, and CCL11 were low and unaffected by D6. Allergen-challenged D6-deficient mice had more dendritic cells, T cells, and eosinophils in the lung parenchyma and more eosinophils in the airway than similarly challenged C57BL/6 mice. By contrast, D6-deficient mice had reduced airway responses to methacholine compared with C57BL/6 mice. Thus, D6 has opposing effects on inflammation and airway reactivity.
Conclusions: The ability of D6 to scavenge chemokines in the lung is dependent on chemokine concentration. The absence of D6 increases inflammation, but reduces airway reactivity. These findings suggest that inhibiting D6 function might be a novel means to attenuate airway responses in individuals with allergic asthma.
chemokines; lung; D6; allergic; transforming growth factor–; β
Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1), a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC) to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL) whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.
Wild-type (WT) BALB/c and eosinophil deficient (PHIL) mice were sensitized to house dust mite (HDM) using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD) and airway methacholine responsiveness (AHR).
Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.
This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.
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
In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.
BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.
AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4+ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.
Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.
FTY720; Fingolimod; Gilenya; Dermatophagoides pteronyssinus; Apoptosis; Dendritic cells; CD4+ T cells; Asthma; S1P; AAL-R; AAL-S; Sphingosine
Antigen challenge of sensitized guinea pigs decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, potentiating vagally induced bronchoconstriction. Loss of M2 receptor function is associated with the accumulation of eosinophils around airway nerves. To determine whether recruitment of eosinophils via expression of VLA-4 and L-selectin is critical for loss of M2 receptor function, guinea pigs were pretreated with monoclonal antibodies to VLA-4 (HP1/2) or L-selectin (LAM1-116). Guinea pigs were sensitized and challenged with ovalbumin, and M2 receptor function was tested. In controls, blockade of neuronal M2 muscarinic receptors by gallamine potentiated vagally induced bronchoconstriction, while in challenged animals this effect was markedly reduced, confirming M2 receptor dysfunction. Pretreatment with HP1/2, but not with LAM1-116, protected M2 receptor function in the antigen-challenged animals. HP1/2 also inhibited the development of hyperresponsiveness, and selectively inhibited accumulation of eosinophils in the lungs as measured by lavage and histology. Thus, inhibition of eosinophil influx into the lungs protects the function of M2 muscarinic receptors, and in so doing, prevents hyperresponsiveness in antigen-challenged guinea pigs.
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.
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.
Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4–producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate–labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine.
GATA-3; antisense DNA; asthma; T cells; Th2 cytokines
A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.
Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.
Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.
Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.
Pim kinases are a family of serine/threonine kinases whose activity can be induced by cytokines involved in allergy and asthma. These kinases play a role in cell survival and proliferation, but have not been examined, to the best of our knowledge, in the development of allergic disease. This study sought to determine the role of Pim1 kinase in the development of allergic airway responses. Mice were sensitized and challenged with antigen (primary challenge), or were sensitized, challenged, and rechallenged with allergen in a secondary model. To assess the role of Pim1 kinase, a small molecule inhibitor was administered orally after sensitization and during the challenge phase. Airway responsiveness to inhaled methacholine, airway and lung inflammation, cell composition, and cytokine concentrations were assessed. Lung Pim1 kinase concentrations were increased after ovalbumin sensitization and challenge. In the primary allergen challenge model, treatment with the Pim1 kinase inhibitor after sensitization and during airway challenges prevented the development of airway hyperresponsiveness, eosinophilic airway inflammation, and goblet cell metaplasia, and increased Th2 cytokine concentrations in bronchoalveolar fluid in a dose-dependent manner. These effects were also demonstrated after a secondary allergen challenge, where lung allergic disease was established before treatment. After treatment with the inhibitor, a significant reduction was evident in the number of CD4+ and CD8+ T cells and concentrations of cytokines in the airways. The inhibition of Pim1 kinase was effective in preventing the development of airway hyperresponsiveness, airway inflammation, and cytokine production in allergen-sensitized and allergen-challenged mice. These data identify the important role of Pim1 kinase in the full development of allergen-induced airway responses.
airway hyperresponsiveness; inflammation; Pim1 kinase; T cells
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
Airflow in the lungs of patients with allergic asthma is impaired by excessive mucus production and airway smooth muscle contractions. Elevated levels of the cytokines IL-4 and IL-13 are associated with this pathology. In vitro studies have suggested that IL-4 receptor alpha (IL-4Rα) signalling on smooth muscle cells is critical for airway inflammation and airway hyperresponsiveness.
In order to define the contribution of IL-4 and IL-13 to the onset of asthmatic pathology the role of their key receptor IL-4Rα in smooth muscle cells was examined in vivo.
By using transgenic SMC-MHCcreIL-4Rα−/lox mice deficient for IL-4Rα in smooth muscle cells, in vivo effects of impaired IL-4Rα signalling in smooth muscle cells on the outcome of asthmatic disease were investigated for the first time. Allergic asthma was introduced in mice by repeated sensitisation with ovalbumin/aluminium hydroxide on days 0, 7 and 14 followed by intranasal allergen challenge on days 21–23. Mice were investigated for the presence of airway hyperresponsiveness, airway inflammation, allergen specific antibody production, Th2 type cytokine responses and lung pathology.
Airway hyperresponsiveness, airway inflammation, mucus production, Th2 cytokine production and specific antibody responses were unaffected in SMC-MHCcreIL-4Rα−/lox mice when compared to control animals.
The impairment of IL-4Rα on smooth muscle cells had no effect on major aetiological markers of allergic asthma. These findings suggest that IL-4Rα responsiveness in airway smooth muscle cells during the early phase of allergic asthma is not, as suggested, necessary for the outcome of the disease.
Therapies targeting the IL-4Rα might have no direct effect on smooth muscle cells in an allergic asthma response.
Smooth muscle cell; Allergy; Asthma; Cytokine Receptors; IL-4; IL-13; gene-deficient mice
In this investigation we have used a mouse model containing certain phenotypic characteristics consistent with asthma and IL-4- and CD40-deficient mice to establish the role of this cytokine and allergen-specific immunoglobulins in the initiation of airways hyperreactivity and morphological changes to the airways in responses to aeroallergen challenge. Sensitization and aerosol challenge of mice with ovalbumin resulted in a severe airways inflammatory response which directly correlated with the induction of extensive airways damage and airways hyperreactivity to beta-methacholine. Inflammatory infiltrates were primarily characterized by the presence of CD4+ T cells and eosinophils. In IL-4-deficient mice, the recruitment of airways eosinophils was impaired, but not abolished in response to aeroallergen. Moreover, the characteristic airways damage and hyperreactivity normally resulting from allergen inhalation were not attenuated. Induction of these structural and functional changes to the airways occurred in the absence of ovalbumin-specific IgE and IgG1, but IgG2a and IgG3 were detected in the sera of IL-4-deficient mice. CD4+ T cells isolated from both wild-type and IL-4-deficient mice given ovalbumin produced significant levels of IL-5 after in vitro stimulation. Treatment of IL-4-deficient mice with anti-IL-5 mAb before aeroallergen challenge abolished blood and airways eosinophilia, lung damage, and airways hyperreactivity. These results indicate that IL-4 is not essential for the development of IL-5-producing CD4+ T cells or for the induction of eosinophilic inflammation and airways damage and hyperreactivity. In response to sensitization and aerosol challenge, CD40-deficient mice did not produce ovalbumin-specific IgE, IgG isotypes, or IgA, and airways inflammation and hyperreactivity were not attenuated. Our results suggest that allergic airways disease can occur via pathways which operate independently of IL-4 and allergen-specific immunoglobulins. Activation of these pathways is intimately associated with IL-5 and eosinophilic inflammation. Such pathways may play a substantive role in the etiology of asthma.
Allergic sensitization to aeroallergens develops in response to mucosal exposure to these allergens. Allergic sensitization may lead to the development of asthma, which is characterized by chronic airway inflammation. The objective of this study is to describe in detail a model of mucosal exposure to cockroach allergens in the absence of an exogenous adjuvant.
Cockroach extract (CE) was administered to mice intranasally (i.n.) daily for 5 days, and 5 days later mice were challenged with CE for 4 consecutive days. A second group received CE i.n. for 3 weeks. Airway hyperresponsiveness (AHR) was assessed 24 h after the last allergen exposure. Allergic airway inflammation was assessed by BAL and lung histology 48 h after the last allergen exposure. Antigen-specific antibodies were assessed in serum. Lungs were excised from mice from measurement of cytokines and chemokines in whole lung lysate.
Mucosal exposure of Balb/c mice to cockroach extract induced airway eosinophilic inflammation, AHR and cockroach-specific IgG1; however, AHR to methacholine was absent in the long term group. Lung histology showed patchy, multicentric damage with inflammatory infiltrates at the airways in both groups. Lungs from mice from the short term group showed increased IL-4, CCL11, CXCL1 and CCL2 protein levels. IL4 and CXCL1 were also increased in the BAL of cockroach-sensitized mice in the short-term protocol.
Mucosal exposure to cockroach extract in the absence of adjuvant induces allergic airway sensitization characterized by AHR, the presence of Th2 cytokines in the lung and eosinophils in the airways.