Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3h. Total RNA was isolated from lung tissues at 6 and 24h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5-fold at 6h post-ozone exposure (t-test, p < .05). At 24h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < .05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24h post-ozone exposure (q < .05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function.

Ozone is a common urban air pollutant. Children are particularly vulnerable to developing adverse respiratory health effects from ozone exposure. Ozone can cause tissue damage and may play a role in sensitization to allergens. We examined the effect of postnatal ozone exposure on the development of house dust mite (HDM) induced allergic airway disease in newborn mice. Newborn BALB/c mice (2-4 days old) were exposed to ozone (1000 ppb) or filtered air (FA) for 3 hrs followed 6 hrs later by intranasal challenge with HDM; the latter was repeated 3 times a week for 3 consecutive weeks. Lung dendritic cells (DC) were isolated 24 hrs after the first HDM challenge to examine their phenotype and accessory function. Airway responses were assessed 24hrs after the last HDM exposure. Airway inflammation was examined by measuring cytokine levels and by enumerating inflammatory cell types recovered in the BAL fluid. Airway function was assessed by invasive method, measuring changes in lung resistance in response to increasing doses of inhaled methacholine. Postnatal ozone exposure did not alter lung DC phenotypes or expression of DC accessory molecules (MHC-II, CD80, CD86, and OX40L). Airway inflammation indicated by levels of IL-4, IL-5 and IL-13 in the BAL, HDM specific serum antibody levels and production of cytokines by restimulated lymphocytes was unaffected by ozone exposure. However, compared to FA, postnatal ozone significantly enhanced HDM induced airway sensitivity to methacholine. Postnatal ozone increases airway sensitivity to methacholine induced by HDM allergen challenge without increasing allergic sensitization.

RATIONALE: We previously reported endogenous estrogen negatively regulates airway hyperresponsiveness (AHR) induced by repeated allergen inhalation without adjuvant, unmasking a gender difference in the development of AHR (Swasey CH et al., Proc Am Thorac Soc 2006;3:A822). The objective is to define the regulatory role of estrogen on development of AHR. METHODS: Female and male BALB/c mice were exposed to 1% ovalbumin (OVA) aerosol via the airways on 10 consecutive days. Twenty-four hours after the last OVA exposure, airway responsiveness to inhaled methacholine (MCh) was determined. An estrogen antagonist, ICI182,780 (10 mg/kg), and 17β-estradiol (E2, 100 μg/kg) were intraperitoneally administered 1 hour before the determination of AHR. To investigate the effects of estrogen on airway contractility, tracheas were isolated, mounted in Harvard chambers, and pre-incubated with E2 (1-100 nM). The responses to carbachol (CCh) and electrical field stimulation (EFS) were monitored. RESULTS: OVA-exposed male mice showed significant (p<0.01) increases in airway responsiveness to inhaled MCh, but AHR could not be detected in female mice. Administration of the antagonist ICI182,780 to female mice resulted in a significant (p<0.01) increase in response to MCh, but only after repeated OVA inhalation. Administration of E2 to male mice significantly (p<0.01) suppressed AHR to MCh. In in vitro experiments, E2 dose-dependently inhibited airway responses detected by EFS, but not CCh. CONCLUSION: These results demonstrate that estrogen is a potent inhibitor of the development of AHR at the prejunctional level, but does not affect cholinergic smooth muscle contractility directly in response to inhaled MCh after 10-day allergen exposure without adjuvant.

RATIONALE Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. OBJECTIVE To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. METHODS AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. RESULTS The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. CONCLUSION This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.

The mechanisms by which respiratory syncytial virus (RSV) infection causes airway hyperresponsiveness (AHR) are not fully established. We hypothesized that RSV infection may alter the expression of airway sensory neuropeptides, thereby contributing to the development of altered airway function. BALB/c mice were infected with RSV followed by assessment of airway function, inflammation, and sensory neuropeptide expression. After RSV infection, mice developed significant airway inflammation associated with increased airway resistance to inhaled methacholine and increased tracheal smooth muscle responsiveness to electrical field stimulation. In these animals, substance P expression was markedly increased, whereas calcitonin gene-related peptide (CGRP) expression was decreased in airway tissue. Prophylactic treatment with Sendide, a highly selective antagonist of the neurokinin-1 receptor, or CGRP, but not the CGRP antagonist CGRP(8-37), inhibited the development of airway inflammation and AHR in RSV-infected animals. Therapeutic treatment with CGRP, but not CGRP(8-37) or Sendide, abolished AHR in RSV-infected animals despite increased substance P levels and previously established airway inflammation. These data suggest that RSV-induced airway dysfunction is, at least in part, due to an imbalance in sensory neuropeptide expression in the airways. Restoration of this balance may be beneficial for the treatment of RSV-mediated airway dysfunction.

Sensory neuropeptides are localized to airway nerves and endocrine cells in both human and animal species and may participate in the development of airway inflammation and hyperresponsiveness (AHR). We used a mouse model to identify the changes that occur in calcitonin gene-related peptide (CGRP) expression in the airways during development of allergic inflammation and to investigate the potential role of this neuropeptide in modulating AHR. In sensitized mice, allergen challenge induced eosinophilic airway inflammation and AHR and resulted in considerable depletion of CGRP in neuroepithelial bodies and submucosal nerve plexuses without altering the overall density of airway nerve fibers. This depletion was subsequent to the development of airway inflammation and was prevented by anti-very late antigen-4 and anti-interleukin-5 treatments, which blocked airway eosinophilia and abolished AHR. Administration of CGRP to sensitized and challenged mice resulted in the normalization of airway responsiveness to inhaled methacholine, an effect that was neutralized by the receptor antagonist CGRP(8-37). These data demonstrate that replacement of CGRP following its depletion in allergic mice can reverse the changes in airway responsiveness and suggest that CGRP may have potential for the treatment of allergic AHR.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M-2 muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

A dysfunction of pathways that normally cause contraction or relaxation of airways has been proposed to explain heightened levels of responsiveness produced by various insults to the airway. For example, we previously reported (4) that infection of cotton rats with the human respiratory syncytial virus (HRSV) leads to a significant decrease in an airway's nonadrenergic noncholinergic inhibitory (NANCi) response shortly after the infection. In the present study we addressed the more chronic effects of HRSV infection on airway function in young ferrets during a period of rapid somatic growth. Animals 1 wk old received HRSV or uninfected cell culture medium intranasally. In vitro studies of airway function were performed on tracheal smooth muscle (TSM) segments at 4, 8, and 24 wk of age. To evaluate neurally mediated contractile responses, frequency-response curves to electrical field stimulation (EFS) were performed with results expressed in terms of the frequency causing 50% of the maximal contractile response (ES50). In addition, contractile responses of TSM to methacholine (MCh) were also assessed with results expressed as the concentration needed to produce 50% of the maximal contractile response (EC50). To gauge NANCi responses, TSM was contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. Relaxant responses to EFS were assessed at frequencies from 5 to 30 Hz, with results expressed as mean percent relaxation. We found increased contractile responses to EFS in infected animals compared with that in the control group in both 4- and 8-wk old animals (p = 0.001 and p = 0.008, respectively). This difference had resolved by 24 wk of age. There was no difference in TSM responses to MCh between the groups at any age. Although there were no NANCi responses in 4-wk-old ferrets from either group, NANCi responses were significantly decreased in 8-wk-old ferrets previously infected with HRSV in the first week of life (p = 0.0001). A significant difference persisted (p = 0.008), albeit to a lesser degree, at 24 wk of age. These findings demonstrate that HRSV produces prolonged alterations of TSM function in ferret airways in vitro.

We studied the effects of recurrent aspiration of milk on neural control of airways in young developing rabbits. Beginning at 1 week of age, rabbits received 0.5 ml/kg of whole milk or sterile physiologic saline intranasally while under light methoxyflourane anesthesia 5 days a week for a period of 3 weeks. At 4 and 8 weeks of age, in vitro studies of contractile and relaxant responses of tracheal smooth muscle (TSM) segments were evaluated. To assess the neurally mediated contractile responses, frequency response curves to electrical field stimulation (EFS) were performed with results expressed in terms of frequency of EFS causing 50% of the maximal contractile response (ES(50)) values. In addition, the contractile responsiveness of TSM to methacholine (MCh) as reflected by the concentration causing 50% of the maximal contractile response (EC(50)) values was also determined to evaluate the underlying cholinergic reactivity of this segment of airway. To assess nonadrenergic noncholinergic inhibitory (NANCi) responses, experiments were performed on TSM contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. EFS was delivered to the contracted tissue at stimulation frequencies ranging from 5 to 30 Hz with results expressed as mean percent relaxation, Recurrent aspiration of milk but not saline increased EFS-induced contractile responses, as shown by significantly lower ES(50) values compared with the control group: P=0.02 and P=0.001 at 4 and 8 weeks of age, respectively. TSM responsiveness to MCh was no different between the two groups, suggesting that alterations in prejunctional mechanisms of neural control were most likely responsible for the increased contractile response to EFS. The NANCi responses were significantly decreased by milk aspiration at both 4 and 8 weeks of age, with the abnormalities less pronounced at the later time point. These findings demonstrate that repeated aspiration of milk leads to abnormal mechanisms of neural control within airways of developing rabbits. While aspiration of milk altered both contractile and relaxant responses to EFS, the former abnormalities became more pronounced with time while the latter appeared to be resolving. These observations suggest that injury to an airway early in development does not necessarily resolve with time but may persist, with functional abnormalities becoming more pronounced even after the airway insult has ceased. (C) 1997 Wiley-Liss, Inc.

The effects of local versus systemic treatment with soluble IL-4 receptors (sIL-4R) were tested in a model of allergen-induced immediate hypersensitivity responses in BALB/c mice. Mice sensitized through the airways to ovalbumin (OVA) by ultrasonic nebulization once a week for 4 weeks developed increased serum anti-OVA IgE and IgG1 antibody titers and these were accompanied by immediate-type skin test responses to the allergen. These responses were also associated with the development of increased airway responsiveness (AR) as monitored by electrical field stimulation of tracheal smooth muscle preparations in vitro. Sensitized mice, treated by intraperitoneal injections of sIL-4R (150 micrograms/injection) administered in parallel to the sensitization protocol, developed significant suppression of anti-OVA IgE, anti-OVA IgG1 antibody production and of immediate cutaneous hypersensitivity responses. Airway responsiveness was normalized to some extent. Total IgE production was only slightly reduced. These effects were comparable to the findings following intraperitoneal injection of monoclonal anti-IL-4 antibody. Administration of sIL-4R via the airways was also effective in inhibiting the development of immediate hypersensitivity responses, including IgE production, and was more potent in normalizing airway responsiveness. These effects were achieved at lower concentrations than needed for systemic treatment. These data suggest that delivery of sIL-4R via the airways can effectively modulate the development of immediate hypersensitivity and airway hyperresponsiveness in response to aerosolized allergen.

The ability of subcutaneous pretreatment with an immunogenic peptide derived from Fel d I, the major cat protein, to suppress the development of allergic responses was examined in a mouse model of antigen-induced sensitization. BALB/c mice exposed to aerosolized Fel d I chain 1 peptide developed antigen-specific IgE responses, immediate cutaneous reactivity to the peptide, and increased airway responsiveness (AR). Both subcutaneous and intraperitoneal administration of the peptide prior to sensitization caused a 50% reduction in cutaneous reactivity which was associated with a decrease in serum anti-Fel d I chain 1 IgE and IgG1 antibody responses and an increase in specific IgG. Pretreatment with the peptide also suppressed spleen and lymph node proliferative responses to the peptide. However, only subcutaneous peptide injections could prevent the development of increased AR. Transfer of spleen cells from subcutaneously peptide-treated mice to sensitized recipients reduced serum antigen-specific IgE and IgG1 antibody responses and skin test reactivity, and prevented alterations in AR. These data suggest that IgE (and IgG1) responses and airway hyperresponsiveness induced by allergen sensitization via the airways can be modulated by subcutaneous administration of peptide. Further, the results define a model for investigating the modulatory effects of subcutaneous administration of immunogenic peptides or protein on an ongoing allergic response.

In a proportion of atopic asthmatics, exposure to a relevant antigen is followed by chronic inflammation in the airways leading to altered airway responsiveness (AR). However, the mechanisms underlying the development of airway hyperresponsiveness still remain unclear. To elucidate the relationship between IgE-mediated reactions and airway hyperresponsiveness, a murine model of passive sensitization and airway challenge with ovalbumin (OVA) was developed using anti-OVA IgE and IgG antibodies from murine B cell hybridomas. Passive sensitization by intravenous injection of anti-OVA IgE resulted in immediate cutaneous hypersensitivity and, after airway challenge with OVA on two consecutive days, increased AR in BALB/c and SJL mice. Increased numbers of eosinophils were observed in bronchoalveolar lavage fluid, in cells extracted from the lungs, and in the peribronchial areas of BALB/c mice passively sensitized with IgE and challenged through the airways compared with nonsensitized mice. Eosinophil peroxidase activity was also elevated in lung tissue from these mice. Passive sensitization with anti-OVA IgG1 but not IgG2a or IgG3 was similarly associated with development of skin test reactivity and increased AR after airway challenge, accompanied by an increase in eosinophils in bronchoalveolar lavage fluid. These data suggest that IgE/IgG1-mediated reactions together with local challenge with antigen can result in allergic inflammation resulting in altered airway function.

A dysfunction of the nonadrenergic noncholinergic inhibitory (NANCi) system has been invoked as a possible mechanism underlying or contributing to altered airway function. In the present study we assessed whether human respiratory syncytial virus (HRSV) infection affects the airways' neurally mediated contractile and relaxant (NANCi) responses in vitro. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from young adult cotton rats, a well-established model for HRSV infection. To assess NANCi responses, TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit, atropine (1 x 10(-6) M) and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation (EFS) was applied at stimulation frequencies ranging from 5 to 30 Hz. The NANCi responses were measured and expressed as the mean (+/- SE) percent relaxation. To evaluate neurally mediated contractile responses, full frequency response curves (0.5-30 Hz) to EFS were also performed. We found significantly decreased NANCi responses in TSM segments obtained from infected cotton rats (n = 12) compared with control animals (n = 9) (P < 0.002). Furthermore, the contractile responses to EFS were increased in infected animals compared with the control group (P = 0.0001). These findings demonstrate that HRSV infection leads to an enhanced contractile response to EFS and a significant decrease in NANCi response in cotton rat airways in vitro. This disruption of the neural control of airways may lead to the development of altered airway function.