Russ Ellis

Allergen-induced murine upper airway inflammation: local and systemic changes in murine experimental allergic rhinitis

The role of inflammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clarified. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper‐ and lower‐airway physiological responsiveness and inflammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Significant nasal symptoms and hyper‐responsiveness appeared after intranasal OVA challenge (P < 0·0001 and P < 0·01, respectively), accompanied with significant nasal mucosal changes in CD4+ cells (P < 0·001), interleukin (IL)‐4+ cells (P < 0·01), IL‐5+ cells (P < 0·01), basophilic cells (P < 0·02) and eosinophils (P < 0·001), in the complete absence of hyper‐responsiveness or inflammatory changes in the lower airway. In the bone marrow, there were significant increases in CD34+ cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL‐5, IL‐3 or granulocyte–macrophage colony‐stimulating factor (GM‐CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony‐forming cells increased significantly in the OVA‐sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal inflammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.

Pathogenesis of Murine Experimental Allergic Rhinitis: A Study of Local and Systemic Consequences of IL-5 Deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5−/−) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5+/+ and IL-5−/− BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5−/− OVA-sensitized mice had significantly higher nasal mucosal CD4+ cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Rα on bone marrow CD34+CD45+ progenitor cells in IL-5−/− mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.

Granulocyte-macrophage colony-stimulating factor, eosinophils and eosinophil cationic protein in subjects with and without mild, stable, atopic asthma

Increasing evidence implicates the eosinophil as an important effector cell in asthma, but little is known regarding its regulation in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to regulate eosinophil function in vitro. We investigated the in vivo role of eosinophils and GM-CSF in mild asthma. We compared the number and function of eosinophils and the presence of GM-CSF in blood, bronchoalveolar lavage (BAL) and biopsy tissue obtained from eight mild, stable, atopic asthmatics and 10 nonasthmatics, five of whom were atopic and five nonatopic. Eosinophils were significantly increased in the blood, BAL and biopsy tissue from asthmatics. Activated eosinophils, assessed by immunostaining for the secreted form of eosinophil cationic protein (EG2), were also increased in asthmatic BAL cells and biopsy tissue. Significant increases in GM-CSF in BAL cells and biopsy tissue from asthmatics were also evident. Significant positive correlations existed between GM-CSF in BAL and EG2, and GM-CSF in biopsy tissue and BAL and biopsy eosinophils. Airway responsiveness was also significantly positively correlated with eosinophil number and activation, and with GM-CSF. These results demonstrate that there are increased numbers of activated eosinophils and GM-CSF is increased in patients with mild asthma. Furthermore, GM-CSF is correlated with eosinophil number and function in vivo and these indices are significantly correlated with airway function. These findings emphasize the importance of eosinophils, potentially regulated in vivo by GM-CSF, in contributing to the disordered airway function evident even in mild asthma.

Influence of airway wall stiffness and parenchymal tethering on the dynamics of bronchoconstriction

Understanding how tissue remodeling affects airway responsiveness is of key importance, but experimental data bearing on this issue remain scant. We used lung explants to investigate the effects of enzymatic digestion on the rate and magnitude of airway narrowing induced by acetylcholine. To link the observed changes in narrowing dynamics to the degree of alteration in tissue mechanics, we compared our experimental results with predictions made by a computational model of a dynamically contracting elastic airway embedded in elastic parenchyma. We found that treatment of explanted airways with two different proteases (elastase and collagenase) resulted in differential effects on the dynamics of airway narrowing following application of ACh. Histological corroboration of these different effects is manifest in different patterns of elimination of collagen and elastin from within the airway wall and the surrounding parenchyma. Simulations with a computational model of a dynamically contracting airway embedded in elastic parenchyma suggest that elastase exerts its functional effects predominately through a reduction in parenchymal tethering, while the effects of collagenase are more related to a reduction in airway wall stiffness. We conclude that airway and parenchymal remodeling as a result of protease activity can have varied effects on the loads opposing ASM shortening, with corresponding consequences for airway responsiveness.

Morphometric Analysis of Mouse Airways After Chronic Allergen Challenge

Understanding the mechanisms of airway remodeling in chronic allergic conditions such as asthma is increasingly dependent on the use of animal models. Techniques for quantifying structural changes are required that are reproducible and responsive and that can be applied to different staining techniques in both human and animal airway tissues. Here, we characterize a morphometric technique to quantify changes in extracellular matrix and contractile tissue as two indices of airway remodeling in mice. Specific aims were to establish the optimum projection beneath the epithelium to assess remodeling changes and to determine whether such changes are reproducible within different areas of the lung. Finally, based on the variance within measurements, we calculated sample size requirements for research applications of this technique. BALB/c mice were sensitized to ovalbumin and studied after chronic allergen challenge. Lungs were formalin fixed and sectioned were then assayed for extracellular matrix or contractile tissue using morphometric/colorimetric techniques. In this model, the optimum projected distance to measure changes in extracellular matrix or contractile tissue was 20 μm beneath the epithelium; projecting beyond this depth resulted in decreased ability to detect allergen-induced changes (signal) because of increased irrelevant staining of surrounding parenchymal tissue (noise). The technique was responsive, because an allergen-induced signal was detected in all airway sections and all lung regions studied (p < 0.05). The power of this analysis was such that allergen-induced changes can be reliably (>80% power) detected using 8 to 10 mice. This morphometric technique provides a valid and objective method to assess structural changes in the airways of mice after chronic allergen exposure.

Comparison of Aerosol and Intranasal Challenge in a Mouse Model of Allergic Airway Inflammation and Hyperresponsiveness

Background: The aim was to optimize antigen challenge for induction of airway hyperresponsiveness (AHR) and inflammation in BALB/c mice sensitized to ovalbumin (OVA). Comparisons were made between mice challenged with OVA either as an aerosol or intranasally. The protocol that induced maximal AHR in BALB/c mice was thereafter tested in C57BL/6 mice. Method: Methacholine responsiveness was measured using the flexiVent® system to assess AHR. Inflammatory responses were investigated by histology and cell counts in bronchoalveolar lavage (BAL) fluid. Results: 48 h after challenge with 1 or 6% OVA aerosols, there were similar increments in AHR and BAL cells, predominantly eosinophils. When comparing the effect of 1% OVA aerosol on AHR and cell infiltration at 24 and 48 h after challenge, the responses were similar. At 24 h, intranasal OVA administration (20–200 µg) caused a dose-dependent increase in AHR. BAL cells were increased by all intranasal OVA doses and to a greater extent than after 1% OVA aerosol challenge but without any dose dependency. Histological examination confirmed that there was an increase of eosinophils in lung tissue following either challenge. In C57BL/6 mice, baseline tissue elastance was the only functional outcome that was increased after intranasal OVA challenge. Even though the AHR response was negligible in C57BL/6 mice, a similar infiltration of BAL cells was observed in both strains. Conclusion: Intranasal challenge was more effective than aerosol challenge at inducing both AHR and airway inflammation in BALB/c mice. Although intranasal challenge caused airway inflammation in C57BL/6 mice, this strain is not optimal for studying AHR.

Regional differences in the pattern of airway remodeling following chronic allergen exposure in mice

BackgroundAirway remodeling present in the large airways in asthma or asthma models has been associated with airway dysfunction in humans and mice. It is not clear if airways distal to the large conducting airways have similar degrees of airway remodeling following chronic allergen exposure in mice. Our objective was to test the hypothesis that airway remodeling is heterogeneous by optimizing a morphometric technique for distal airways and applying this to mice following chronic exposure to allergen or saline.MethodsIn this study, BALB/c mice were chronically exposed to intranasal allergen or saline. Lung sections were stained for smooth muscle, collagen, and fibronectin content. Airway morphometric analysis of small (0–50000 μm2), medium (50000 μm2–175000 μm2) and large (>175000 μm2) airways was based on quantifying the area of positive stain in several defined sub-epithelial regions of interest. Optimization of this technique was based on calculating sample sizes required to detect differences between allergen and saline exposed animals.ResultsFollowing chronic allergen exposure BALB/c mice demonstrate sustained airway hyperresponsiveness. BALB/c mice demonstrate an allergen-induced increase in smooth muscle content throughout all generations of airways, whereas changes in subepithelial collagen and fibronectin content are absent from distal airways.ConclusionWe demonstrate for the first time, a systematic objective analysis of allergen induced airway remodeling throughout the tracheobronchial tree in mice. Following chronic allergen exposure, at the time of sustained airway dysfunction, BALB/c mice demonstrate regional differences in the pattern of remodeling. Therefore results obtained from limited regions of lung should not be considered representative of the entire airway tree.

In Vivo Role of Platelet-Derived Growth Factor–BB in Airway Smooth Muscle Proliferation in Mouse Lung

Airway smooth muscle (ASM) hyperplasia in asthma likely contributes considerably to functional changes. Investigating the mechanisms behind proliferation of these cells may lead to therapeutic benefit. Platelet-derived growth factor (PDGF)-BB is a well known ASM mitogen in vitro but has yet to be directly explored using in vivo mouse models in the context of ASM proliferation and airway responsiveness. To determine the in vivo influence of PDGF-BB on gene transcripts encoding contractile proteins, ASM proliferation, and airway physiology, we used an adenovirus overexpression system and a model of chronic allergen exposure. We used adenovirus technology to selectively overexpress PDGF-BB in the airway epithelium of mice. Outcome measurements, including airway physiology, real time RT-PCR measurements, proliferating cell nuclear antigen staining, and airway smooth muscle quantification, were performed 7 days after exposure. The same outcome measurements were performed 24 hours and 4 weeks after a chronic allergen exposure model. PDGF-BB overexpression resulted in airway hyperresponsiveness, decreased lung compliance, increased airway smooth muscle cell numbers, positive proliferating cell nuclear antigen-stained airway smooth muscle cells, and a reduction in genes encoding contractile proteins. Chronic allergen exposure resulted in elevations in lung lavage PDGF-BB, which were observed in conjunction with changes in gene transcript expression encoding contractile proteins and ASM proliferation. We demonstrate for the first time in vivo that PDGF-BB induces ASM hyperplasia and changes in lung mechanics in mice and that, during periods of allergen exposure changes in lung, PDGF-BB are associated with changes in airway structure and function.

Concurrent dual allergen exposure and its effects on airway hyperresponsiveness, inflammation and remodeling in mice.

SUMMARY Experimental mouse models of asthma have broadened our understanding of the mechanisms behind allergen-induced asthma. Typically, mouse models of allergic asthma explore responses to a single allergen; however, patients with asthma are frequently exposed to, and tend to be allergic to, more than one allergen. The aim of the current study was to develop a new and more relevant mouse model of asthma by measuring the functional, inflammatory and structural consequences of chronic exposure to a combination of two different allergens, ovalbumin (OVA) and house dust mite (HDM), in comparison with either allergen alone. BALB/c mice were sensitized and exposed to OVA, HDM or the combination of HDM and OVA for a period of 10 weeks. Following allergen exposure, airway responsiveness was measured using the flexiVent small animal ventilator, and mice were assessed for indices of airway inflammation and remodeling at both 24 hours and 4 weeks after the final allergen exposure. Mice exposed to the HDM-OVA combination exhibited increased numbers of inflammatory cells in the bronchoalveolar lavage (BAL) when compared with mice exposed to a single allergen. Mice exposed to HDM-OVA also exhibited an elevated level of lung tissue mast cells compared with mice exposed to a single allergen. Following the resolution of inflammatory events, mice exposed to the allergen combination displayed an elevation in the maximal degree of total respiratory resistance (Max RRS) compared with mice exposed to a single allergen. Furthermore, trends for increases in indices of airway remodeling were observed in mice exposed to the allergen combination compared with a single allergen. Although concurrent exposure to HDM and OVA resulted in increased aspects of airway hyperresponsiveness, airway inflammation and airway remodeling when compared with exposure to each allergen alone, concurrent exposure did not result in a substantially more robust mouse model of allergic asthma than exposure to either allergen alone.

Bone marrow progenitors in allergic airways diseases: studies in canine and human models.

In a canine model of Ascaris suum-inducible bronchial hyperresponsiveness, we previously demonstrated that bone marrow-derived myeloid progenitors rise within 24 h of allergen inhalation; this effect is abolished by pretreatment with inhaled budesonide. We now report that this allergen-induced bone marrow response is observable in human asthmatics, and involves increases in both neutrophil-macrophage and eosinophil-basophil progenitors, within 6 h of allergen inhalation, as measured either by hematopoietic colony assays or by flow cytometric analyses of CD34+, IL-3R alpha+, and/or IL-5-responsive cell populations. In dogs, but not in humans, a transferrable serum hematopoietic activity accounts for the marrow response to inhaled allergen. These findings suggest that allergen-induced increases in bone marrow progenitor formation depend either on a serum hematopoietic factor(s) released after allergen challenge, or upon constitutive marrow upregulation of specific progenitors in allergic airway disease. Further studies to characterize the serum hematopoietic factor(s) and to determine the nature of any atopy-related progenitor profile are in progress.