Repeated Airway Challenges Research Articles

Maternal allergic asthma during pregnancy alters fetal lung and immune development in sheep: potential mechanisms for programming asthma and allergy.

Experimental maternal allergic asthma in sheep provides an experimental model in which to test impacts on progeny. Fetuses from allergic asthmatic ewes had fewer surfactant-producing cells in lungs. A greater proportion of lymphocytes from thymus were CD44 positive in fetuses from allergic asthmatic ewes than in controls. These changes to fetal development might contribute to poor neonatal lung function and increased risk of allergy seen in offspring of pregnancies complicated by asthma. Asthma is prevalent in pregnancy and increases the risk of disease in offspring, including neonatal respiratory distress and childhood asthma and allergy, but the mechanisms are not understood. We hypothesized that fetal lung structure and immune phenotype in late gestation fetal sheep would be impaired in our sheep model of maternal allergic asthma during pregnancy. Singleton-bearing ewes were either sensitized before pregnancy to house dust mite (HDM, allergic, n=7) or were non-allergic (control, n=5). The ewes were subsequently subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Tissues were collected at 140±1days gestational age (term, ∼147days). The density of type II alveolar epithelial cells (surfactant protein C-immunostained) in the lungs was 30% lower in fetuses from allergic ewes than in controls (P<0.001), but tissue-to-air space ratio and numbers of leucocytes and macrophages were not different between groups. The proportion of CD44+ lymphocytes in the fetal thymus was 3.5-fold higher in fetuses from allergic ewes than in control ewes (P=0.043). Fewer surfactant-producing type II alveolar epithelial cells may contribute to the increased risk of neonatal respiratory distress in infants of asthmatic mothers, suggesting that interventions to promote lung maturation could improve their neonatal outcomes. If the elevated lymphocyte expression of CD44 persists postnatally, this would confer greater susceptibility to allergic diseases in progeny of asthmatic mothers, consistent with observations in humans. Further experiments are needed to evaluate postnatal phenotypes of progeny and investigate potential interventions.

Airway glycomic and allergic inflammatory consequences resulting from keratan sulfate galactose 6-O-sulfotransferase (CHST1) deficiency.

Siglec-F is a pro-apoptotic receptor on mouse eosinophils that recognizes 6'-sulfated sialyl Lewis X and 6'-sulfated sialyl N-acetyl-lactosamine as well as multivalent sialyl N-acetyl-lactosamine structures on glycan arrays. We hypothesized that attenuation of the carbohydrate sulfotransferase 1 (CHST1) gene encoding keratan sulfate galactose 6-O-sulfotransferase, an enzyme likely required for 6'-sulfation of some of these putative Siglec-F glycan ligands, would result in decreased Siglec-F lung ligand levels and enhanced allergic eosinophilic airway inflammation. Tissue analysis detected CHST1 expression predominantly not only in parenchymal cells but not in airway epithelium, the latter being a location where Siglec-F ligands are located. Western blotting of lung extracts with Siglec-F-Fc fusion proteins detected ≈500 kDa and ≈200 kDa candidate Siglec-F ligands that were not appreciably altered in CHST1-/- lungs compared with normal mouse lungs. Characterization of the O-linked glycans of lung tissue and bronchoalveolar lavage fluid detected altered sialylation but minimal change in sulfation. Eosinophilic airway inflammation was induced in wild-type (WT) and CHST1-/- mice via sensitization to ovalbumin (OVA) and repeated airway challenge. After OVA sensitization and challenge, Siglec-F ligands on airway cells, and numbers of eosinophils and neutrophils accumulating in the airways, both increased to a similar degree in WT and CHST1-/- mouse lungs, while macrophages and lymphocytes increased significantly more in CHST1-/- mouse airway compared with normal mouse lungs. Therefore, keratan sulfate galactose 6-O-sulfotransferase does not contribute to the synthesis of glycan ligands for Siglec-F in the airways, although its absence results in exaggerated accumulation of airway macrophages and lymphocytes.

Development of an experimental model of maternal allergic asthma during pregnancy.

Maternal asthma during pregnancy adversely affects pregnancy outcomes but identification of the cause/s, and the ability to evaluate interventions, is limited by the lack of an appropriate animal model. We therefore aimed to characterise maternal lung and cardiovascular responses and fetal-placental growth and lung surfactant levels in a sheep model of allergic asthma. Immune and airway functions were studied in singleton-bearing ewes, either sensitised before pregnancy to house dust mite (HDM, allergic, n = 7) or non-allergic (control, n = 5), and subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Maternal lung, fetal and placental phenotypes were characterised at 140 ± 1 days gestational age (term, ∼147 days). The eosinophil influx into lungs was greater after HDM challenge in allergic ewes than after saline challenge in control ewes before mating and in late gestation. Airway resistance increased throughout pregnancy in allergic but not control ewes, consistent with increased airway smooth muscle in allergic ewes. Maternal allergic asthma decreased relative fetal weight (-12%) and altered placental phenotype to a more mature form. Expression of surfactant protein B mRNA was 48% lower in fetuses from allergic ewes than controls, with a similar trend for surfactant protein D. Thus, allergic asthma in pregnant sheep modifies placental phenotype, and inhibits fetal growth and lung development consistent with observations from human pregnancies. Preconceptional allergen sensitisation and repeated airway challenges in pregnant sheep therefore provides an animal model to identify mechanisms of altered fetal development and adverse pregnancy outcomes caused by maternal asthma in pregnancy.

Histamine H4 receptor antagonism diminishes existing airway inflammation and dysfunction via modulation of Th2 cytokines

BackgroundAirway remodeling and dysfunction are characteristic features of asthma thought to be caused by aberrant production of Th2 cytokines. Histamine H4 receptor (H4R) perturbation has previously been shown to modify acute inflammation and Th2 cytokine production in a murine model of asthma. We examined the ability of H4R antagonists to therapeutically modify the effects of Th2 cytokine production such as goblet cell hyperplasia (GCH), and collagen deposition in a sub-chronic model of asthma. In addition, effects on Th2 mediated lung dysfunction were also determined.MethodsMice were sensitized to ovalbumin (OVA) followed by repeated airway challenge with OVA. After inflammation was established mice were dosed with the H4R antagonist, JNJ 7777120, or anti-IL-13 antibody for comparison. Airway hyperreactivity (AHR) was measured, lungs lavaged and tissues collected for analysis.ResultsTherapeutic H4R antagonism inhibited T cell infiltration in to the lung and decreased Th2 cytokines IL-13 and IL-5. IL-13 dependent remodeling parameters such as GCH and lung collagen were reduced. Intervention with H4R antagonist also improved measures of central and peripheral airway dysfunction.ConclusionsThese data demonstrate that therapeutic H4R antagonism can significantly ameliorate allergen induced, Th2 cytokine driven pathologies such as lung remodeling and airway dysfunction. The ability of H4R antagonists to affect these key manifestations of asthma suggests their potential as novel human therapeutics.

Airway Management for Infants with Severe Micrognathia Having Mandibular Distraction Osteogenesis

We retrospectively reviewed the charts of seven neonates and infants with severe micrognathia and upper airway obstruction who underwent mandibular distraction osteogenesis as an alternative to long-term tracheostomy at the Children's Hospital at Westmead, Sydney, from 2004 to 2007. All patients required a variety of airway interventions at an early age and had poor feeding or failure to thrive. These children had other anomalies and required many investigations and procedures and presented repeated airway challenges for the anaesthetist. The mean number of anaesthetics per child in this series was 6.7 (range 4 to 13). Under anaesthesia, four patients had grade 4 laryngoscopy, one was grade 3 but two were only grade 2. Patients with a preoperative grade 4 laryngoscopy were very likely to need fibreoptic endotracheal intubation. Duration of intubation after surgery for mandibular distraction was a mean of 8.17 days (range 1 to 19). Three were extubated in the operating theatre and three in intensive care. Five patients had an improved laryngoscopy grade after completion of mandibular distraction and one remained grade four The remaining patient had a tracheostomy from birth.

IL-10- and IL-12-Independent Down-Regulation of Allergic Sensitization by Stimulation of CD40 Signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.

Common vaccine antigens inhibit allergen‐induced sensitization and airway hyperresponsiveness in a murine model

Co-vaccination with cellular pertussis vaccine down-regulates allergic sensitization to diphtheria and tetanus antigens. Using a murine model, we investigated whether vaccination with diphtheria/tetanus toxoids, administered separately or simultaneously with the whole cell vaccine of Bordetella pertussis, inhibits subsequent allergen-induced immune and inflammatory responses. BALB/c-mice were vaccinated intracutaneously with a combination of diphtheria and tetanus toxoids or a combination of diphtheria and tetanus toxoids with a whole cell vaccine of B. pertussis (three times, days -21 to -7) prior to systemic sensitization (days 1-14) and repeated airway challenges (days 28-30) with ovalbumin. Compared with negative controls, systemic sensitization and airway allergen challenges induced high serum levels of allergen-specific IgE, predominant Th2-type cytokine production, airway inflammation and development of in vivo airway hyperreactivity. Vaccination with diphtheria and tetanus toxoids prior to sensitization suppressed IgE formation and development of eosinophilic airway inflammation. Co-vaccination with a whole cell pertussis vaccine inhibited allergen sensitization, airway inflammation and development of in vivo airway hyperreactivity. Prevention was due to an allergen-specific and general shift from a predominant Th2 towards a predominant Th1 immune response. Vaccination with diphtheria and tetanus toxoids alone or in combination with whole cell pertussis vaccine prior to allergen sensitization prevented allergen-induced Th2 immune responses. Vaccine antigens may down-regulate allergic responses to a range of common allergens.

Dynamics of dendritic cell migration and the subsequent induction of protective immunity in the lung after repeated airway challenges by nontypeable Haemophilus influenzae outer membrane protein

To determine the dynamics of dendritic cell (DCs) migration and their role in recurrent infections by nontypeable Haemophilus influenzae (NTHi), the migration of mature DC into pulmonary lymph nodes (LN) and the development of a P6-specific immune response and bacterial clearance in the lung were examined after repeated airway challenges with outer membrane protein (OMP) at 1-week intervals in mice. Although the migration of mature DC into the pulmonary LN is attenuated after repeated airway challenge with OMP, the in vitro P6-specific T cell proliferation in the cultured pulmonary LN cells was enhanced and was subsequently linked to the development of P6-specific IgA production and the development of protective immunity in the airway of mice.

Induction of allergy and autoreactivity after immunization with a foreign allergen and the respective autoantigen

Rationale Allergy and autoimmunity are traditionally considered as two entities related to Th2- or Th1-dominated immune responses. This study was aimed to assess whether allergy and autoimmunity may coexist following immunization with a foreign allergen, the human NAC (hu-NAC), respectively the self-antigen, the murine NAC (mu-NAC). Methods BALB/c-mice were intraperitoneally immunized with the mu-NAC, respectively hu-NAC, a component of the nascent-associated polypeptide complex (NAC), identified as an IgE autoantigen in patients with atopic dermatitis. Immunization was followed by repeated airway challenge and the phenotype of these mice was analyzed. Results Immunization to hu-NAC induced an anti-NAC IgE, IgG1 and IgG2a antibody response. Antibody binding to mu-NAC in lungs and skin tissue was detected by nitrocellulose-blotted mouse skin and lung extracts probed with sera of hu-NAC immunized mice. Antigen challenge of sensitized mice resulted in airway inflammation with eosinophils and neutrophils accompanied by airway hyperresponsiveness to metacholin. In parallel, perivasculitis of lung veins was detected. Hu-NAC immunized mice developed immediate-type hypersensitivity responses to both human and self NAC. But also sensitization to the murine NAC induced autoreactive T-cells, detected by proliferation, airway hyperresponsiveness to metacholin, a similar airway inflammation as well as an positive skin test to murine NAC. Conclusions These data demonstrate that sensitization to an foreign allergen mimicking a self-antigen induced a mixed pattern of TH1 and TH2 responses, confirmed by comparable results with the self-antigen. Furthermore allergy and autoreactivity coexist in both models and this may represent a major pathomechanism involved in the maintenance of chronic forms of allergy.

Exposure to endotoxin and allergen in early life and its effect on allergen sensitization in mice

Background: Exposure to endotoxins, allergens, or both in early life might regulate the development of tolerance to allergens later in life. Objective: We investigated whether continuous exposure of infant mice to aerosolized endotoxin, allergen, or both inhibits subsequent allergen-induced immune and inflammatory responses. Methods: Infant BALB/c mice were pre-exposed to aerosolized endotoxin, ovalbumin (OVA), or both (3 times a week for the first 4 weeks of life) before systemic sensitization (days 1-14) and repeated airway challenge (days 28-30) with OVA. Results: Compared with that seen in negative control animals, systemic sensitization and airway allergen challenges induced high serum levels of allergen-specific IgE (0.7 ± 0.09 vs 0.02 ± 0.01 OD units), predominant T H2-type cytokine production (IL-5 by splenic mononuclear cells in vitro, 1.2 ± 0.2 vs 0.04 ± 0.06 ng/mL), airway inflammation (bronchoalveolar lavage fluid leukocytes, 125 ± 15 vs 64 ± 7/μL; eosinophils, 28 ± 5 vs 1 ± 0/μL) and development of in vivo airway hyperreactivity (maximal enhanced pause, 11 ± 1.9 vs 4 ± 0.2). Pre-exposure with LPS before sensitization increased production of specific IgG2a (67 ± 10 vs 32 ± 5 U/mL) but failed to prevent T H2-mediated immune responses. Pre-exposure with OVA or with OVA plus LPS completely suppressed allergen sensitization, airway inflammation, and development of in vivo airway hyperreactivity; values were similar to those of negative control animals. Inhibition was due to allergen-specific T-cell anergy indicated by omitted allergen-specific T H2 and T H1 immune responses. In addition, combined exposure to endotoxin and allergen induced a general shift toward an unspecific T H1 immune response. Conclusion: Exposure with endotoxins before allergen sensitization is not able to induce unresponsiveness but might decrease the susceptibility for sensitization to a variety of common allergens. (J Allergy Clin Immunol 2003;112:389-96.)

Development of eosinophilic airway inflammation and airway hyperresponsiveness requires interleukin-5 but not immunoglobulin E or B lymphocytes.

We previously defined a role for B cells and allergen-specific immunoglobulins in the development of allergic sensitization, airway inflammation, and airway hyperresponsiveness (AHR), using a 10-d protocol in which allergen exposure occurred exclusively via the airways, without adjuvant. In the present protocol, normal and B-cell-deficient (microMt(-/-)) mice were sensitized intraperitoneally to ovalbumin (OVA) and challenged with OVA via the airways in order to examine the requirements for AHR with this protocol. T-cell activation (antigen-specific proliferative responses and Th2-type cytokine production) and eosinophil infiltration in the peribronchial regions of the airways, with signs of eosinophil activation and degranulation, occurred in both experimental groups. In contrast to the 10-d protocol, increased in vivo airway responsiveness to methacholine and in vitro tracheal smooth-muscle responses to electrical field stimulation were observed in both normal and B-cell-deficient mice, and these responses were inhibited by anti-interleukin (IL)-5 administration before airway challenge. These data show that IL-5, but not B cells or allergen-specific IgE, are required for eosinophil airway infiltration and the development of AHR following allergen/alum sensitization and repeated airway challenge with allergen. These results emphasize that the use of different sensitization and challenge protocols can influence the requirements for development of AHR.

Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice.

Allergic sensitization in asthma develops as a consequence of complex interactions between T cells and antigen-presenting cells. We have developed several in vivo models to study allergen-specific T cell and B cell function and their relevance to allergic airway hyperresponsiveness (AHR), focusing on the role of the costimulatory molecules CD80 and CD86. Treatment of mice with anti-CD86, but not anti-CD80, significantly inhibited increased serum levels of ovalbumin (OA)-specific IgE and IgG1, airway eosinophilia, and AHR both after 10 d of OA aerosol exposure (in the absence of adjuvant) and after intraperitoneal sensitization followed by repeated airway challenges. Inhibition of AHR was associated with decreased IL-4 and IL-5 levels in the BAL fluid of sensitized mice, suggesting impaired Th2 function in anti-CD86-treated animals. This effect was not seen when mice received treatment only before allergen challenge, indicating that anti-CD86 acts through inhibition of allergic sensitization and not simply by inhibiting the influx of inflammatory cells. These data suggest that the CD86 costimulatory ligand plays a major role in the development of allergic inflammation and AHR in allergen-challenged mice. Further, this study demonstrates that T-B cell interactions during allergic sensitization are amenable to therapeutic manipulation and that selective blockade of accessory signals can be an effective means for modulating distinct T cell functions.

Role of IgE in the development of allergic airway inflammation and airway hyperresponsiveness--a murine model.

The importance of IgE in airway inflammation and development of AHR in allergen-sensitized mice has been compared and contrasted in different models of sensitization and challenge. Using different modes of sensitization in normal and genetically manipulated mice after anti-IgE treatment, we have been able to distinguish the role of IgE under these different conditions. Striking differences in the three sensitization protocols were delineated in terms of the role of allergen-specific IgE, extent of eosinophilic airway inflammation, and development of AHR (Table 1). The highest levels of IgE and eosinophil infiltration (approximately 20-fold increases) were achieved after systemic sensitization with allergen (plus adjuvant) followed by repeated airway challenge. Passive sensitization with allergen-specific IgE followed by limited airway challenge induced a modest eosinophilic inflammatory response in the airways despite high levels of serum IgE. Exposure to allergen exclusively via the airways also resulted in a modest serum IgE response and a limited eosinophilic inflammatory response (approximately fourfold increases). Under all of these conditions, inhibition of IL-5-mediated eosinophilic airway inflammation was associated with attenuation of AHR. In contrast, the differences in the responses to the different modes of allergen exposure were associated with differences in the requirements for IgE in the development of AHR (Table 1). In the two models associated with mild eosinophil infiltration (passive sensitization and exclusive airway exposure), IgE was required for the development of AHR but did not substantially enhance airway inflammation on its own. However, IgE-allergen interaction was able to enhance T-cell function in vitro and induce T-cell expansion in vivo. In mice systemically sensitized and challenged via the airways, IgE (or IgE-mediated mast-cell activation) was not required for T-cell activation, eosinophilic inflammation and activation in the airways, or development of AHR. This was most clearly seen in B-cell-deficient and mast-cell-deficient, low-IgE-responder mouse strains (B6, B10) and in anti-IgE-treated high-IgEresponder mice (BALB/c). At the same time, we confirmed the importance of IgE in the induction of immediate-type hypersensitivity (mast-cell activation, immediate cutaneous hypersensitivity, passive cutaneous and systemic anaphylaxis). These differences were also highlighted by the means used to detect altered airway function. Passive sensitization and limited airway challenge or exclusive airway exposure to allergen over 10 days elicited changes in airway function that could be detected only in tracheal smooth-muscle preparations exposed to EFS. In contrast, systemic sensitization followed by repeated airway challenge resulted not only in changes in the contractile response to EFS but also in increased responsiveness to inhaled MCh. Thus, these results distinguish not only the differential involvement of IgE and eosinophil numbers but also their contribution to the readouts used to monitor airway function. Based on these studies, we conclude that IgE plays an important role in the development of airway inflammation and AHR under conditions in which limited IL-5-mediated eosinophilic airway infiltration is induced. In conditions where a robust eosinophilic inflammation of the airways is elicited, IgE (and IgE-mediated mast-cell activation) does not appear to be essential for airway inflammation and the development of AHR, detected as increased responsiveness to inhaled MCh. These findings reveal the potential importance of differential targeting in the treatment of allergic diseases with a predominance of IgE-mediated symptoms, e.g., allergic rhinitis and conjunctivitis, where anti-IgE may be an effective therapy, compared to those diseases with a predominant inflammatory component, e.g., AHR in atopic bronchial asthma, where anti-inflammatory or anti-IL-5 therapy may be more beneficial.

Airway refractoriness to adenosine 5′-monophosphate after repeated inhalation

Adenosine-5′-monophosphate (AMP) is a bronchoconstrictor agonist in subjects with atopic and nonatopic asthma. In this study we have investigated the effect of repeated airway challenge with AMP in 10 atopic, subjects without asthma. At the first visit, subjects had repeated inhalation challenges with increasing doubling concentrations of AMP, and the provocation concentration required to reduce FEV 1 by 20% of baseline was determined (PC 20 AMP). When the FEV 1 had returned to within 5% of the baseline, the AMP challenge test was repeated on a further one or two occasions. When the maximum concentration of AMP (400 mg/ml) failed to produce a PC 20, a bronchial provocation test with histamine was performed and the PC 20 was determined. On the second visit, the effect of three consecutive inhalation challenge tests with histamine on the airway response to this agonist was determined. On the third visit, the duration of induced refractoriness to AMP was assessed. With the first AMP challenge, all subjects demonstrated bronchoconstriction with a geometric mean (GM) PC 20 of 332 mg/ml. With a second AMP challenge, five of the 10 subjects became refractory and failed to achieve a PC 20. In the remaining five subjects, the GM PC 20 increased from 110 to 583 mg/ml. With a third AMP challenge, these subjects also failed to achieve a PC 20. The initial GM PC 20 histamine for the group was 22.9 mg/ml, which did not change significantly with two further histamine challenges or when the airways were refractory to AMP, suggesting that loss of AMP effect was not related to loss of contractility of airway smooth muscle or down regulation of H 1-histamine receptors. Once it was induced, refractoriness to AMP lasted approximately 4 hours. We conclude that with repeated challenge the airways became refractory to AMP compatible with depletion of mast cell mediators or tachyphylaxis at the level of cell-surface adenosine receptors.