Role of Abl in airway hyperresponsiveness and airway remodeling

Background

Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.

Methods

To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.

Results

The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.

Conclusions

These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.     

Thioredoxin suppresses airway hyperresponsiveness and airway inflammation in asthma

Thioredoxin (TRX) is a 12-kDa redox (reduction/oxidation)-active protein that has a highly conserved site (-Cys-Gly-Pro-Cys-) and scavenges reactive oxygen species. Here we examined whether exogenously administered TRX modulated airway hyperresponsiveness (AHR) and airway inflammation in a mouse asthma model. Increased AHR to inhaled acetylcholine and airway inflammation accompanied by eosinophilia were observed in OVA-sensitized mice. Administration of wild-type but not 32S/35S mutant TRX strongly suppressed AHR and airway inflammation, and upregulated expression of mRNA of several cytokines (e.g., IL-1alpha, IL-1beta, IL-1 receptor antagonist, and IL-18) in the lungs of OVA-sensitized mice. In contrast, TRX treatment at the time of OVA sensitization did not improve AHR or airway inflammation in OVA-sensitized mice. Thus, TRX inhibited the asthmatic response after sensitization, but did not prevent sensitization itself. TRX and redox-active protein may have clinical benefits in patients with asthma.     

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

Background

Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease.

Methods

We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy.

Results

The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks.

Conclusion

These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR.

     

Inhibition of LPS-induced airway hyperresponsiveness and airway inflammation by LPS antagonists

To determine whether the inflammatory effects of inhaled endotoxin could be prevented, we pretreated mice with synthetic competitive antagonists (975, 1044, and 1287) for lipopolysaccharide (LPS) before a LPS inhalation challenge. In preliminary studies, we found that these LPS antagonists did not act as agonists in vitro (THP-1 cells) or in vivo (after intratracheal instillation of 10 microg) and that these compounds (at least 1 microg/ml) effectively antagonized the release of tumor necrosis factor-alpha by LPS-stimulated THP-1 cells. Pretreatment of mice with 10 microg of either 1044 or 1287 resulted in a decrease in the LPS-induced airway hyperreactivity. Moreover, pretreatment of mice with 10 microg of 975, 1044, or 1287 resulted in significant reductions in LPS-induced lung lavage fluid concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline. Using residual oil fly ash to induce airway inflammation, we found that the action of the LPS antagonists was specific to LPS-induced airway disease. These results suggest that LPS antagonists may be an effective and potentially safe treatment for endotoxin-induced airway disease.     

Upper airway and respiratory muscle responses to continuous negative airway pressure

To determine upper airway and respiratory muscle responses to nasal continuous negative airway pressure (CNAP), we quantitated the changes in diaphragmatic and genioglossal electromyographic activity, inspiratory duration, tidal volume, minute ventilation, and end-expiratory lung volume (EEL) during CNAP in six normal subjects during wakefulness and five during sleep. During wakefulness, CNAP resulted in immediate increases in electromyographic diaphragmatic and genioglossal muscle activity, and inspiratory duration, preserved or increased tidal volume and minute ventilation, and decreased EEL. During non-rapid-eye-movement and rapid-eye-movement sleep, CNAP was associated with no immediate muscle or timing responses, incomplete or complete upper airway occlusion, and decreased EEL. Progressive diaphragmatic and genioglossal responses were observed during non-rapid-eye-movement sleep in association with arterial O2 desaturation, but airway patency was not reestablished until further increases occurred with arousal. These results indicate that normal subjects, while awake, can fully compensate for CNAP by increasing respiratory and upper airway muscle activities but are unable to do so during sleep in the absence of arousal. This sleep-induced failure of load compensation predisposes the airways to collapse under conditions which threaten airway patency during sleep. The abrupt electromyogram responses seen during wakefulness and arousal are indicative of the importance of state effects, whereas the gradual increases seen during sleep probably reflect responses to changing blood gas composition.     

Ebselen suppresses late airway responses and airway inflammation in guinea pigs

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.     

IL-10 reduces grain dust-induced airway inflammation and airway hyperreactivity.

To determine whether interleukin-10 (IL-10) could alter the development of grain dust-induced airway disease, we pretreated mice with either saline or IL-10 intravenously, exposed the mice to an inhalation challenge with corn dust extract (CDE), and measured inflammation and the development of airway hyperreactivity. Pretreatment with IL-10, in comparison to saline, reduced the concentration and percentage of polymorphonuclear cells in the lavage fluid 30 min after the inhalation challenge with CDE (P < 0. 05). In comparison to saline-treated mice, IL-10 did not significantly alter the degree of airway hyperreactivity 30 min after the exposure to CDE. IL-10-treated mice lavaged 18 h after challenge with CDE also exhibited a lower percentage of polymorphonuclear cells in the lavage fluid (P < 0.05) and had significantly less airway hyperreactivity than did mice pretreated with the saline placebo (P < 0.05). These findings indicate that exogenous IL-10 is effective in reducing airway inflammation and airway hyperreactivity due to the inhalation of CDE.     

TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 cells represent a distinct population of CD4(+) Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of T(H)17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4(+) T cells from DO11.10 OVA-specific TCR-transgenic mice to a T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma.     

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.     

Relationship between airway microvascular leakage, edema, and baseline airway functions

Matheson, Melissa, Ann-Christine Rynell, Melissa McClean,and Norbert Berend. Relationship between airway microvascular leakage, edema, and baseline airway functions. J. Appl. Physiol. 84(1): 77-81, 1998.This study wasdesigned to examine the relationship among microvascular leakage,edema, and baseline airway function. Microvascular leakage was inducedin the airways of anesthetized, tracheostomized New Zealand Whiterabbits (n = 22) by using nebulized N-formyl-methionyl-leucyl-phenylalanine(10 mg) and was measured in the trachea by using the Evans blue dyetechnique. Airway wall thickness was assessed morphometrically in theright main bronchus after Formalin fixation at a pressure of 25 cmH₂O. Areas calculated includedthe mucosal wall area, the adventitial wall area, the total wall area,and the percentage of total wall area consisting of blood vessels. Aneutrophil count was also performed by analyzing numbers of cells inboth the mucosal wall area and the adventitial wall area. Airwayfunction was assessed before and 30 min after challenge withN-formyl-methionyl-leucyl-phenylalanineby determining airway resistance, functional residual capacity,specific airway resistance, and flow-volume and pressure-volume curves(after paralysis of the animals with suxamethonium). The concentration of Evans blue dye in tracheal tissue ranged from 31.3 to 131.2 µg.There was a significant correlation between this concentration and boththe adventitial wall area (P < 0.01)and mucosal neutrophil numbers (P < 0.005). There was no correlation between Evans blue concentration andeither blood vessel area or changes in respiratory physiologyparameters before and after challenge. There was no significantdifference between any respiratory physiology measurements before andafter challenge. We conclude that an increase in microvascular leakagecorrelates with airway edema in the adventitia; however, these airwaychanges have no significant effect on airway elastic or resistiveproperties.

     

Changes in upper airway resistance with lung inflation and positive airway pressure

The influence of pulmonary inflation and positive airway pressure on nasal and pharyngeal resistance were studied in 10 normal subjects lying in an iron lung. Upper airway pressures were measured with two low-bias flow catheters while the subjects breathed by the nose through a Fleish no. 3 pneumotachograph into a spirometer. Resistances were calculated at isoflow rates in four different conditions: exclusive pulmonary inflation, achieved by applying a negative extra-thoracic pressure (NEP); expiratory positive airway pressure (EPAP), which was created by immersion of the expiratory line; continuous positive airway pressure (CPAP), realized by loading the bell of the spirometer; and CPAP without pulmonary inflation by simultaneously applying the same positive extrathoracic pressure (CPAP + PEP). Resistance measurements were obtained at 5- and 10-cmH2O pressure levels. Pharyngeal resistance (Rph) significantly decreased during each measurement; the decreases in nasal resistance were only significant with CPAP and CPAP + PEP; the deepest fall in Rph occurred with CPAP. It reached 70.8 +/- 5.5 and 54.8 +/- 6.5% (SE) of base-line values at 5 and 10 cmH2O, respectively. The changes in lung volume recorded with CPAP + PEP ranged from -180 to 120 ml at 5 cmH2O and from -240 to 120 ml at 10 cmH2O. Resistances tended to increase with CPAP + PEP compared with CPAP values, but these changes were not significant (Rph = 75.9 +/- 6.1 and 59.9 +/- 6.6% at 5 and 10 cmH2O of CPAP + PEP). We conclude that 1) the upper airway patency increases during pulmonary inflation, 2) the main effect of CPAP is related to pneumatic splinting, and 3) pulmonary inflation contributes little to the decrease in upper airways resistance observed with CPAP.     

Cardiotrophin-1 alters airway smooth muscle structure and mechanical properties in airway explants

Induction of hypertrophy and inhibition of apoptosis may be important mechanisms contributing to increased airway smooth muscle (ASM) mass in asthma. Data from our laboratory indicate that cardiotrophin-1 (CT-1) induces hypertrophy and inhibits apoptosis in isolated human ASM cells. To determine whether these novel effects of CT-1 also occur in the airway tissue milieu and to determine whether structural changes are accompanied by functional changes, matched pairs of guinea pig airway explants were treated with or without CT-1 for 7 days, and structural features as well as isometric and isotonic contractile and relaxant mechanical properties were measured. CT-1 (0.2-5 ng/ml) increased both myocyte mass and extracellular matrix in a concentration-dependent fashion. CT-1 (10 ng/ml)-treated tissues exhibited a significant increase in passive tension at all lengths on day 7; at optimal length, passive tension generated by CT-1-treated tissues was 1.72 +/- 0.12 vs. 1.0 +/- 0.1 g for control. Maximal isometric stress was decreased in the CT-1-treated group on day 7 (0.39 +/- 0.10 kg/cm(2)) vs. control (0.77 +/- 0.15 kg/cm(2), P < 0.05). Isoproterenol-induced relaxant potency was reduced in CT-1-treated tissues, log EC(50) being -7.28 +/- 0.34 vs. -8.12 +/- 0.25 M in control, P < 0.05. These data indicate that CT-1 alters ASM structural and mechanical properties in the tissue environment and suggest that structural changes found in the airway wall in asthma are not necessarily associated with increased responsiveness.