Jennifer Wattie

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.

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.

Effects of allergen on airway narrowing dynamics as assessed by lung-slice technique

Asthma is characterised by an excessive airway narrowing in response to a variety of stimuli, called airway hyperresponsiveness (AHR). Previous comparisons between mouse strains have shown that increased velocity of airway narrowing correlates with baseline airway responsiveness. These data prompted the investigation into models of induced AHR to see whether airway narrowing dynamics correlated with in vivo responsiveness. In an attempt to reproduce some of the features of asthma, BALB/c mice were sensitised and subjected to either brief or chronic periods of allergen exposure. Brief exposure involved two challenges with intranasal chicken egg ovalbumin (OVAin). Chronic exposure involved six 2-day periods of OVAin challenges, each separated by 12 days. Control mice received intranasal saline challenges. Outcomes included videomicrometry of lung slices (magnitude and velocity of airway narrowing), in vivo respiratory physiology measurements and histological staining with morphometric analysis. Neither brief nor chronic allergen exposure resulted in greater airway narrowing and increased velocity compared with saline controls. Structural changes in the airway, such as goblet cell hyperplasia, subepithelial fibrosis and increased contractile tissue, were detected in mice chronically challenged with allergen. In conclusion, increased responsiveness to methacholine following allergen challenge may not be due to an intrinsic change to the smooth muscle per se, but rather to other changes in the lung, which ultimately manifest as an increase in respiratory resistance.

Goblet Cell Rebound and Airway Dysfunction with Corticosteroid Withdrawal in a Mouse Model of Asthma

RATIONALE Although corticosteroids are highly effective at preventing allergen-induced increases in goblet cell numbers, we observed in unpublished experiments a rebound increase in goblet cell numbers in mice after the simultaneous withdrawal of corticosteroid and cessation of exposure to allergen that reached levels greater than those observed in mice exposed to allergen alone, without corticosteroid treatment. OBJECTIVES To formally explore the goblet cell hyperplasia rebound observed after corticosteroid withdrawal in allergen-exposed mice to determine the mechanism responsible for this previously undescribed pathology. METHODS Mice airways were assessed for mucin-containing goblet cells after exposure to varying durations of allergen and corticosteroid. MEASUREMENTS AND MAIN RESULTS We confirmed that the simultaneous withdrawal of corticosteroid and cessation of exposure to allergen resulted in a goblet cell hyperplasia rebound that reached levels greater than those observed in allergen-exposed corticosteroid naive mice. Importantly, the goblet cell rebound was associated with a significant airway dysfunction greater than that observed in allergen-exposed corticosteroid naive mice. The goblet cell hyperplasia rebound is independent of the type of corticosteroid or allergen and was associated with an increased level of bronchoalveolar lavage IL-13. Inhibition of IL-13, but not CD4+ T cells, completely inhibited the goblet cell hyperplasia rebound and, critically, the associated airway dysfunction. CONCLUSIONS These findings suggest that certain corticosteroid treatment regimes may actually potentiate airway remodeling and dysfunction in patients with asthma and lead to increased exacerbations and worsening of asthma symptoms.

Budesonide prevents but does not reverse sustained airway hyperresponsiveness in mice

Despite the effectiveness of corticosteroids at resolving airway inflammation, they are only moderately effective at attenuating airway hyperresponsiveness (AHR). The extent to which corticosteroids are able to reverse or inhibit the development of sustained AHR is not known. The present study aimed to determine whether budesonide can resolve and or prevent the development of sustained AHR in mice. Mice were chronically exposed to allergen and treated with budesonide either: 1) briefly during the final weeks of exposure to allergen; 2) prolonged concurrently throughout exposure to allergen; or 3) delayed following final exposure to allergen. AHR was assessed 24 h (brief treatment) or 4 weeks (prolonged concurrent and delayed treatments) following final exposure to allergen. Brief budesonide intervention significantly attenuated the inflammation-associated AHR assessed immediately following final exposure to allergen. Similarly, prolonged concurrent budesonide treatment prevented the development of sustained AHR. Delayed budesonide intervention, however, did not resolve sustained AHR. In conclusion, the early introduction and, importantly, the persistence of corticosteroid treatment prevented the development of sustained airway hyperresponsiveness; however, the inability of corticosteroids to reverse established airway dysfunction indicates a limitation in their use for the complete, long-term management of airway hyperresponsiveness.

The effects of an inhaled corticosteroid on oxygen radical production by bronchoalveolar cells after allergen or ozone in dogs.

Both ozone and allergen inhalation increase the capacity to produce oxygen radicals by bronchoalveolar lavage cells in dogs. The purpose of these studies was to determine whether inhaled corticosteroids inhibits these increases in oxygen radical production from bronchoalveolar lavage cells. Six random source dogs were studied after dry air or ozone inhalation (3 ppm, 30 min). Seven random source dogs were studied after diluent or allergen inhalation. The dogs inhaled budesonide (2.74 mg/day) or lactose powder, twice daily for 7 days before ozone and allergen. 90 min after ozone or dry air, and 24 h after Ascaris suum or diluent a bronchoalveolar lavage was carried out. Spontaneous luminol-enhanced chemiluminescence was measured from bronchoalveolar lavage cells (4 x 10(6) cells) for 10 min, followed by a measurement of phorbol myristate acetate (PMA 2.4 micromol/l) stimulated chemiluminescence for 10 min. Both ozone and allergen inhalation caused an increase in PMA stimulated chemiluminescence (P<0.05). Budesonide pretreatment inhibited ozone-induced (P<0.008), but not allergen-induced PMA stimulated chemiluminescence (P>0.90). Both ozone and allergen inhalation caused an increase in the bronchoalveolar lavage neutrophils. Budesonide pretreatment significantly inhibited the ozone-induced (P=0.007), but not the ascaris-induced neutrophil influx (P=0.93). These results demonstrate that ozone, but not allergen, stimulated oxygen radical release and neutrophil influx are attenuated by inhaled corticosteroids. This suggests that luminol-enhanced chemiluminescence from bronchoalveolar lavage cells measures oxygen radicals derived from neutrophils, and that ozone-and allergen-induced bronchoalveolar lavage neutrophilia are caused by different mechanisms.

Lung-homing of endothelial progenitor cells and airway vascularization is only partially dependant on eosinophils in a house dust mite-exposed mouse model of allergic asthma.

Background 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. Methods 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). Results 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. Conclusions 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.

Electrophysiological effects of erythromycin, but lack of mechanical effects, in airway smooth muscle

The antibiotic erythromycin has been shown to modulate a variety of electrophysiological and mechanical responses in many cell types. We investigated whether it did so in airway smooth muscle using standard patch clamp, fura-2 fluorimetric and organ bath techniques. Erythromycin (10(-4) M) evoked a small transient inward current with reversal potential and time-course similar to that of the Ca2+-dependent Cl- currents seen in these cells. Unlike its effects in other cell types, however, it did not alter basal [Ca2+]i, voltage-dependent Ca2+ currents, nor mechanical tone at rest, nor the corresponding responses to cholinergic stimulation (membrane currents; release of internally sequestered Ca2+, nor contractions evoked by neural stimulation or exogenously added cholinergic agonist). In conclusion, erythromycin does exert interesting electrophysiological actions in airway smooth muscle, but does not alter mechanical activity as it has been shown to do elsewhere.

In vitro airway responsiveness of Flinders sensitive and resistant line rats.

Recently, we reported that freely moving Flinders sensitive line rats (FSL, selectively bred for their cholinergic hyperresponsiveness) are more susceptible to allergen-induced airway hyperresponsiveness than their control counterparts-Flinders resistant line (FRL) rats. In this study the two Flinders lines were compared for responsiveness of excised tracheal and primary bronchial smooth muscle in vitro. FSL tissues were slightly but significantly more sensitive to cholinergic stimulation than FRL tissues (slightly lower EC(50) value for carbachol) but the FRL tissues were more responsive, exhibiting larger amplitude of response. Surprisingly, previous exposure to allergen challenge was accompanied by reduced in vitro responses to spasmogens in both rat lines. We conclude that FSL and FRL airways do not differ greatly with respect to sensitivity to cholinergic stimulation in vitro and that inflamed airways show reduced in vitro responses to spasmogens. The discrepancy between the in vivo and in vitro findings suggests that responsiveness of airway smooth muscle involves regulation from both proximal and distal sites.

Induction of bystander tolerance and immune deviation after Fel d 1 peptide immunotherapy

Background Treatment of patients with cat allergy with peptides derived from Fel d 1 (the major cat allergen) ameliorated symptoms of cat allergy in phase 2 clinical trials. Objective We sought to demonstrate that the tolerance induced by Fel d 1 peptide immunotherapy can be exploited to reduce allergic responses to a second allergen, ovalbumin (OVA), in mice sensitized dually to OVA and Fel d 1. Methods Induction of tolerance to OVA was achieved through simultaneous exposure to both allergens after peptide treatment. Functional tolerance to each allergen was assessed in a model of allergic airways disease in which treated mice were protected from eosinophilia, goblet cell hyperplasia, and TH2 cell infiltration. Results Suppression of allergic responses to cat allergen challenge was associated with significant increases in numbers of CD4+CD25+Foxp3+ T cells, IL‐10+ cells, and CD19+IL‐10+ B cells, whereas the response to OVA was associated with a marked reduction in numbers of TH2 cytokine–secreting T cells and less prominent changes in outcomes associated with immune regulation. Conclusions These observations suggest that immune tolerance induced by peptide immunotherapy can be used experimentally to treat an allergic response to another allergen and that the molecular mechanisms underlying induction of tolerance to a treatment‐specific allergen and a bystander allergen might be different. Graphical abstract Figure. No Caption available.