Active sensitization of guinea-pig airways in vivo enhances in vivo and in vitro responsiveness

A plethysmographic method was employed to assess the airway resistance of conscious, free-breathing guinea-pigs. Using this method animals sensitized by inhalation of ovalbumin and appropriate controls were assessed for their responsiveness to histamine and methacholine in vivo. The cough frequency on exposure to citric acid mist in the two groups was also assessed. Tracheal spirals from these animals were subsequently tested for their responsiveness to histamine, methacholine and prostaglandin D2 in vitro. Sensitization increased responsiveness to histamine, methacholine and citric acid in vivo but only histamine responses were affected in vitro. These changes were accompanied by a significant eosinophilia in the airways as assessed by bronchoalveolar lavage. We conclude that sensitization of the airways to ovalbumin results in responsiveness changes in bronchial smooth muscle accompanied by signs of airway inflammation.     

Association of airway pentosidine levels with bronchodilator response mediated by salbutamol administration in asthmatic patients

BackgroundRecently, increased levels of pentosidine, an intermolecular cross-linking type of advanced glycation end products, are observed in the airways of asthmatic patients. This study was designed to determine whether differences in bronchodilator response among individuals with asthma are attributable to pentosidine levels in their airways.MethodsFifty-six asthmatic patients (21 with airway obstruction, 35 without airway obstruction) and 10 normal controls were included in this study. For asthmatic patients, we evaluated the spontaneous reversibility of airway obstruction or the reversibility that can be obtained after methacholine provocation. And we also measured pentosidine levels and percentage of sputum eosinophils in induced sputum, and exhaled nitric oxide (NO) levels.ResultsThe pentosidine levels did not significantly differ between the two asthmatic subgroups with and without airway obstruction. In asthmatic patients without airway obstruction, airway hyperresponsiveness to methacholine (PC20 methacholine) was significantly correlated with sputum eosinophils and exhaled NO levels. In contrast, PC20 methacholine was not significantly correlated with pentosidine levels. In asthmatic patients with or without airway obstruction, bronchodilator response was not significantly correlated with sputum eosinophils and exhaled NO levels. However, bronchodilator response was closely correlated with pentosidine levels (asthmatics without airway obstruction: r = ?0.54, p = 0.002; asthmatics with airway obstruction: r = ?0.48, p = 0.03).ConclusionsOur results showed that pentosidine might be a potential biomarker reflecting the reduced bronchodilator response in asthma. This study will provide new insights into the mechanisms underlying persistent airway obstruction.     

烟草雾吸入导致慢阻肺机制的实验研究——大鼠Clara细胞结构及其分泌蛋白的变化

为探讨烟草雾吸入诱导的慢性阻塞性肺疾病 (COPD)大鼠气道Clara细胞及其分泌蛋白 (又称Clara细胞 10kDa蛋白 ,CC10 )表达的变化 ,采用自制的染毒箱 ,使大鼠吸入烟草雾制作COPD模型 ,对Clara细胞进行电镜及免疫组化观察 ,并测定大鼠气道阻力及呼吸系统总顺应性。COPD大鼠终末及呼吸性细支气管Clara细胞数量减少 ,胞浆内滑面内质网扩张 ;终末及呼吸性细支气管上皮细胞CC10表达减少 ;呼吸功能检查显示气道阻力增加、呼吸系统总顺应性下降 ;气道上皮细胞CC10阳性细胞百分率与气道阻力呈负相关。COPD大鼠终末及呼吸性细支气管上皮细胞Clara细胞数量减少 ,气道上皮细胞CC10表达减少 ,CC10阳性细胞百分率与气道阻力呈负相关、与动态顺应性呈正相关。这可能与大鼠气道阻力增加及呼吸系统总顺应性下降有关     

烟草雾吸入导致慢阻肺机制的实验研究：大鼠Clara细胞结构 …

为探讨烟草雾吸入诱导的慢性阻塞性肺疾病（ＣＯＰＤ）大鼠气道Ｃｌａｒａ细胞及其分泌蛋白（又称Ｃｌａｒａ细胞１０ｋＤａ蛋白，ＣＣ１０）表达的变化，采用自制的染毒箱，使大鼠吸入烟草雾制作ＣＯＰＤ模型，对Ｃｌａｒａ细胞进行电镜及免疫组化观察，并测定大鼠气道阻力及呼吸系统总顺应性。ＣＯＰＤ大鼠终末及呼吸性细支气管Ｃｌａｒａ细胞数量减少，胞浆内滑面内质网扩张；终末及呼吸性细支气管上皮细胞ＣＣ１０表达减少；呼吸     

Bronchodilating effects of the anesthetic ketamine in an in vitro guinea pig preparation

Ketamine, a dissociative anesthetic, is capable of reducing airway resistance and has proved useful in anesthetizing surgical patients with acute or chronic bronchospasm. To determine if ketamine alters smooth muscle tone, the relative responses of large and small airways of the same animal were studied by comparing the pharmacologic reactivity of tracheal smooth muscle strips with that of a specially prepared perfused bronchial tree. Trachealis and bronchial smooth muscle were found to react to methacholine and histamine in a concentration-dependent manner and have similar sensitivities. Ketamine, by itself, in the concentration range 10⁻⁸–10⁻³ M did not alter resting tone as compared to epinephrine, which reduced baseline tone. In tissues precontracted with histamine or methacholine at ED₅₀ doses, ketamine inhibited smooth muscle contraction. In a second series of experiments, the dose-dependent contraction of smooth muscle to histamine and methacholine was reevaluated in the presence of ketamine. Both tissue sensitivity and maximum contractile response to these agonists were reduced by ketamine at 10⁻⁴ M. These data indicate that ketamine alters the in vitro response of guinea pig airways to agonists associated with the asthmatic state. Although ketamine does not reduce airway tone in nonstimulated tissues, its effects on agonist-induced contraction of airway tissues in vitro are consistent with clinical observations that ketamine relieves bronchospasm.     

Serial distribution of bronchoconstriction in normal subjects. Methacholine versus histamine

We compared the relative sites of airway responses to methacholine and histamine aerosols in normal subjects by simultaneous assessment of respiratory resistance (Rrs) and anatomic dead space (VD). Methacholine (12.5 mg/ml) or histamine (12.5 mg/ml) was continuously inhaled during tidal breathing until a nearly twofold increase in Rrs was observed. Large airway response was determined by VD, and overall airway response was determined by Rrs. Small airways response was inferred from Rrs when the change in VD was slight. Inhalation of methacholine and histamine increased Rrs with decreased VD, but the decrease in VD was significantly greater with methacholine than with histamine for an equivalent change in Rrs (p less than 0.01). Inhalation of atropine (5 mg/ml) decreased Rrs with a simultaneous increase in VD. After inhalation of atropine, time required for a twofold increase in Rrs by histamine inhalation was prolonged, and histamine did little decrease VD. These results suggest that the predominant site of cholinergically mediated constriction is the large airways, and that histamine constricts the large airways in part via a cholinergic reflex mechanism and constricts the small airways via its direct action in normal subjects.     

Cartilaginous airway wall dimensions and airway resistance in cystic fibrosis lungs.

It is not clear how airway pathology relates to the severity of airflow obstruction and increased bronchial responsiveness in cystic fibrosis (CF) patients. The aim of this study was to measure the airway dimensions of CF patients and to estimate the importance of these dimensions to airway resistance using a computational model. Airway dimensions were measured in lungs obtained from CF patients who had undergone lung transplantation (n=12), lobectomy (n=1), or autopsy (n=4). These dimensions were compared to those of airways from lobectomy specimens from 72 patients with various degrees of chronic obstructive pulmonary disease (COPD). The airway dimensions of the CF and COPD patients were introduced into a computational model to study their effect on airway resistance. The inner wall and smooth muscle areas of peripheral CF airways were increased 3.3- and 4.3-fold respectively compared to those of COPD airways. The epithelium was 53% greater in height in peripheral CF airways. The sensitivity and maximal plateau resistance of the computed dose/response curves were substantially increased in the CF patients compared to COPD patients. The changes in airway dimensions of cystic fibrosis patients probably contribute to the severe airflow obstruction, and to increased bronchial responsiveness, in these patients.     

Structural abnormalities and inflammation in COPD: a focus on small airways

Chronic obstructive pulmonary disease (COPD) is characterized by poorly reversible airflow limitation associated with airway remodelling and inflammation of both large and small airways. The site of airflow obstruction in COPD is located in the small airways, justifying a focus on this compartment. The structural abnormalities that are found in bronchioles with a diameter less than 2mm are characterized by increased airway wall thickness with peribronchial fibrosis, and by luminal obstruction by mucous exudates. Destruction of alveolar walls, the hallmark of emphysema, may be related to protease-antiprotease imbalance, and to mechanisms involving apoptosis, senescence, and autoimmunity. Cigarette smoke inhalation triggers the recruitment of innate immune cells (neutrophils and macrophages) and putatively adaptive immunity mediated via T and B lymphocytes and lymphoid follicles in the small airways. These data suggest a potential role for therapies that can target remodelling and inflammation in the small airways of patients with COPD.     

The changes in airways structure associated with reduced forced expiratory volume in one second

We compared the structure of the membranous and respiratory bronchioles of resected lungs from 111 patients with a normal predicted forced expiratory volume in one second (FEV1) to the structure of these airways from 45 patients with an FEV1 reduced below the 95% confidence limits for height and age. Membranous and respiratory bronchioles of less than 2 mm in internal diameter were counted and their diameter and wall thickness were measured. The data show that there were more membranous bronchioles of internal diameter less than 0.4 mm in patients with reduced FEV1. The wall thickness of respiratory bronchioles was increased in the obstructed group and there was also an increase in the ratio of wall thickness to lumen diameter in these airways. The walls of membranous bronchioles were not increased in thickness but there was an increase in the ratio of wall thickness to lumen diameter. Although the data is consistent with the hypothesis that airways obstruction in patients with chronic obstructive pulmonary disease is due to thickening of the airway wall and narrowing of the airway lumen, we cannot rule out distortion of the membranous bronchioles by loss of elastic recoil.     

Effect of indomethacin on leukotriene4-induced histamine hyperresponsiveness in asthmatic subjects.

The effect of indomethacin on the capacity of LTE4 to enhance airway histamine responsiveness was evaluated in eight mild asthmatic subjects. Subjects attended the laboratory on three separate pairs of study days when inhalation challenges with methacholine or LTE4 were performed and the airway responses to histamine were measured 4 and 7 h later. An open pair of study days was followed by a pair of study days during ingestion of either placebo or indomethacin capsules. The dose of agonist that produced a 35% fall in specific airways conductance (PD35 SGaw) was obtained by linear interpolation from the logarithmic dose-response curve. Indomethacin treatment did not affect baseline SGaw or methacholine airway responsiveness. However, indomethacin significantly inhibited LTE4-induced histamine hyperresponsiveness. Maximum enhancement of histamine responsiveness by LTE4 on the open and placebo study days was 4.1 +/- 0.9- (mean +/- SEM) and 5.7 +/- 1.2-fold, respectively (p = 0.36). Maximal enhancement on the indomethacin day was 1.68 +/- 0.46, and this was significantly decreased compared with that on the placebo day (p = 0.02). This suggests that LTE4-induced enhanced responsiveness to histamine is mediated in part by cyclooxygenase pathway-derived products.     

Airway hyperreactivity in patients undergoing lung and heart/lung transplantation

To assess airway reactivity in lung transplant patients, three heart/lung, three double lung, and eight single lung transplant patients underwent airway challenge tests. All patients were assessed by methacholine aerosol challenge and thirteen were also assessed by histamine aerosol challenge at least three months after transplant surgery. The airways of patients with bilaterally denervated lungs (heart/lung and double lung transplants) were significantly more reactive to both methacholine and histamine (p less than 0.01) than were the airways of patients with unilaterally denervated (single lung transplants) lungs. Inflammatory changes in the airway mucosal biopsies were minimal in three patients and absent in all others. These studies raise questions about the role of central innervation in the maintenance of normal airway function as well as the mechanisms of action of both methacholine and histamine in causing bronchoconstriction.     

The nature, severity and distribution of obstructive airway lesions in COPD studied by morphometry and computer-assisted 3-D reconstruction

The severity of obstructive bronchial lesions and their distribution in the airway tree were studied in autopsy lungs from ten patients with various types of chronic obstructive pulmonary disease (COPD), where the changes of bronchial dimension were studied by morphometry and the 3-D distribution of obstructive lesions by computer-assisted 3-D reconstruction. A case of paraquat intoxication was added in order to study the airway changes in lungs with early fibrotic changes. In each case, morphometry was performed on microscopic lung sections where the initial diameter of bronchi was estimated from that of the accompanying pulmonary arteries. The varyingly constricted airways and arteries were standardized into a circular state by measuring the perimeter length L of the epithelial basement membrane (BM) and the internal elastic membrane (IEM) with a digital image analyzer; D(br), the anatomical diameter of an airway, and D(pa), that of an artery, were calculated at this state of completely stretched BM or IEM. Rs, the ratio of luminal stenosis by thickened epithelia, was also determined from the area of epithelium simultaneously measured. Three-D reconstruction of airway was performed in cases typically representing different types of obstruction; from microscopic, sometimes macroscopic serial sections, where the 2-D images were inputted into a computer which integrated in its display a 3-D picture. It was shown that in lungs with chronic bronchitis, the bronchial dimension did not significantly differ from that of ordinary lungs. Overt shrinkage of bronchial dimension was demonstrated in chronic obstructive bronchiolitis; in both diffuse panbronchiolitis (DPB) and broncho-bronchiolitis obliterans (BBO), narrowing of the peripheral airways combined with ectasis of the proximal bronchi proved to be a common feature. However, reconstruction disclosed an essential difference between these in the distribution of occlusive lesions, which mainly involved the respiratory bronchioles in DPB, while in BBO, the site of obstruction was from the terminal to slightly upper bronchioles. Also in pulmonary emphysema, the pattern of bronchial dimension was a narrowing in the periphery, both in the centrilobular and panlobular types. Especially worth of attention was that the study disclosed the presence of a type of COPD hitherto poorly defined. In a patient who had a 25-year history of COPD and died of respiratory failure, the lungs, only mildly emphysematous, were shown to have uniformly narrowed bronchioles; also mucus hypersecretion and elevated Rs appeared to have contributed to the obstruction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Large and small airway responses to bronchoconstrictors in the guinea pig and ferret

In the isolated, perfused lung lobe of the ferret we evaluated the bronchoconstrictor response of its airways to methacholine and histamine, pharmacologic agents associated with the asthmatic state. The bronchus of excised lobes was cannulated and needle scarifications were made on the pleural surface to allow perfusate to exit. Lung airways were perfused at constant flow with equilibrated 95% O2/5% CO2, warmed Krebs-Ringers solution. Perfusion pressure was measured as a gauge of airway resistance. A concentration-dependent smooth muscle contraction of the ferret lung lobes was observed to methacholine and histamine. The ED50's of methacholine and histamine were 6.41 x 10(-6) M +/- 1.38 x 10(-6) (SEM) and 6.41 x 10(-6) M +/- 1.38 x 10(-6) (SEM) and 2.39 x 10(-6) M +/- 0.53 x 10(-6) (SEM), respectively. The maximum level of bronchoconstriction developed in the ferret (2.42 mmHg/ml/min +/- 0.28 SEM (resistance units] in response to methacholine, was six times greater than that found for histamine (0.42 mmHg/ml/min +/- 0.05 SEM). Responses to both agonists were less pronounced in the ferret lung preparation than those in a similar lung preparation of guinea pig. Compliance changes in both animals were also evaluated. The ferret did not demonstrate a compliance change in response to histamine as was seen for methacholine, suggesting that resistance changes precede compliance changes, or that the ferret airways are particularly resistant to histamine. Despite a lesser contractile response, the ferret has the advantage of a relatively large lung and long trachea that allow study in several preparations obtained from a single animal. It should prove a useful animal model for study of pulmonary pharmacology.     

Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro

Erythromycin and other antibiotics have been used empirically in the treatment of patients with chronic obstructive pulmonary disease (COPD). We studied whether this empirical role of antibiotics might not be related to a possible direct effect on respiratory glycoconjugate (RGC) secretion. The effect of erythromycin on RGC secretion and hypersecretion was studied in an in vitro preparation of human airways that were secreting [3H]glucosamine respiratory glycoconjugate (RGC), and on a human endometrial adenocarcinoma cell line secreting a glycoconjugate (tumor glycoconjugate = TGC) chemically similar to the RGC secreted by the airways. Erythromycin at 10(-5) M reduced RGC secretion by 35 +/- 4% (n = 9, p less than 0.001) in both human airways and the adenocarcinoma cells, and was increasingly active in the pharmacologic range of 10(-7) to 10(-4) M. The inhibitory effect of erythromycin was maximal within 16 h and was still evident 34 h after incubation. Erythromycin was noted to reduce both spontaneous (baseline) and stimulated RGC secretion (by histamine and methacholine) from airways in culture. The blocking effect appeared to be more selective for histamine than methacholine. These effects were not associated with any toxicity to the tissues and were not associated with the inhibition of protein synthesis. Dexamethasone also inhibited RGC release in both assay systems and exhibited dose-related effects in the physiologic ranges (10(-9) to 10(-5) M). When administered together, erythromycin and dexamethasone had an additive inhibitory effect on RGC secretion (68.0 +/- 3.0%, n = 7, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mannitol and AMP do not induce bronchoconstriction in eosinophilic bronchitis: Further evidence for dissociation between airway inflammation and bronchial hyperresponsiveness

Background and objective: Eosinophilic bronchitis (EB) shares many pathological features with asthma. However, patients with EB do not develop the characteristic physiological abnormalities of asthma: variable airflow obstruction and bronchial hyperresponsiveness (BHR) to a direct bronchial challenge with methacholine. Indirect bronchial challenges with AMP and mannitol are dependent on the presence of airway inflammation, and positive in 10% of asthmatic subjects who have a negative response to methacholine. We have therefore investigated whether subjects with EB are responsive to indirect airway challenge with AMP and mannitol. Methods: Subjects with asthma, EB and healthy controls attended on up to four occasions. After screening, subjects performed bronchial provocation tests to methacholine and then either AMP or mannitol. Each challenge was followed immediately by sputum induction for the measurement of airway inflammation and mast cell‐derived histamine. Results: No subjects with EB responded to either AMP (n = 5) or mannitol (n = 7) while 4/8 and 7/10 subjects with asthma responded to the respective challenges (P = 0.057 for AMP, P = 0.004 for mannitol). There was no difference in induced sputum concentrations of histamine or eosinophil cell counts following methacholine challenge compared with AMP or mannitol. Conclusions: The airways of patients with EB are not responsive to either direct or indirect bronchial challenge. This supports the view that it is the presence of functionally abnormal airway smooth muscle that is the key determinant of BHR in asthma, and that while this may be aggravated by the presence of mucosal airway inflammation, it is not caused by it.     

Interaction between parenchyma and airways in chronic obstructive pulmonary disease and in asthma

The extent of air-space destruction caused by emphysema is very variable in severe chronic obstructive pulmonary disease (COPD), constituting one of the most obvious differences between COPD and asthma. Differences in the static deflation pressure-volume curve between COPD and asthma can easily be shown, but it has been surprisingly difficult to find distinctive mechanical features of impaired airway function caused by air-space destruction. This may be because in mild airway obstruction related to smoking--particularly in younger subjects--emphysema may be absent, and the predominant site of airway narrowing in the smallest bronchi and respiratory bronchioles may be the same as that found in asthma in remission. In more severe obstruction caused by COPD there is almost always very severe intrinsic disease of the airways and this may so dominate the functional abnormality that it is difficult to detect any additional change because of airspace destruction. Overall, few studies have set out to detect specific effects of parenchymal destruction on airway function.     

Is there loss of protective muscarinic receptor mechanism in asthma?

We investigated the hypothesis that prior airway muscarinic receptor stimulation (with aerosolized methacholine) would modify the bronchoconstrictor response to histamine, which is, in part, vagally mediated. On four different experiment days, the following combinations of methacholine and histamine inhalation challenges were performed in 15 subjects (nine normal and six asthmatic) in a random fashion: methacholine-histamine, histamine-methacholine; methacholine-methacholine and histamine-histamine. Cumulative provocative dose of each agonist which caused a 50 percent decrease in SGaw was estimated (PD50). The second challenge was performed approximately 1 hour after the first challenge, when SGaw had returned to baseline. In normal subjects, prior muscarinic stimulation with methacholine suppressed the subsequent bronchoconstrictor response to histamine (mean +/- SE PD50 histamine increased from 13.7 +/- 3.1 to 28.4 +/- 7.2 breath units), without modifying the bronchoconstrictor response to methacholine. In asthmatic subjects, prior methacholine exposure failed to modify the bronchoconstrictor responses to histamine and methacholine. In contrast, prior challenge with histamine did not modify the subsequent bronchoconstrictor responses to histamine and methacholine in both normal and asthmatic subjects. Pretreatment with ipratropium bromide attenuated the histamine-induced bronchoconstriction, suggesting that airway effects of histamine, in part, are vagally mediated. These data suggest that prior muscarinic stimulation has a protective effect on histamine-induced bronchoconstriction in normal subjects and the absence of this inhibitory effect in asthmatic patients may represent loss of a protective muscarinic receptor mechanism.     

Large and small airway responses to bronchoconstrictors in the guinea pig and ferret

In the isolated, perfused lung lobe of the ferret we evaluated the bronchoconstrictor response of its airways to methacholine and histamine, pharmacologic agents associated with the asthmatic state. The bronchus of excised lobes was cannulated and needle scarifications were made on the pleural surface to allow perfusate to exit. Lung airways were perfused at constant flow with equilibrated 95% O₂/5% CO₂, warmed Krebs-Ringers solution. Perfusion pressure was measured as a gauge of airway resistance. A concentration-dependent smooth muscle contraction of the ferret lung lobes was observed to methacholine and histamine. The ED₅₀’s of methacholine and histamine were 6.41 × 10^?6 M ± 1.38 × 10^?6 (SEM) and 2.39 × 10^?6 M ± 0.53 × 10^?6 (SEM), respectively. The maximum level of bronchoconstriction developed in the ferret (2.42 mmHg/ml/min ± 0.28 SEM (resistance units)) in response to methacholine, was six times greater than that found for histamine (0.42 mmHg/ml/min ± 0.05 SEM). Responses to both agonists were less pronounced in the ferret lung preparation than those in a similar lung preparation of guinea pig [17]. Compliance changes in both animals were also evaluated. The ferret did not demonstrate a compliance change in response to histamine as was seen for methacholine, suggesting that resistance changes precede compliance changes, or that the ferret airways are particularly resistant to histamine. Despite a lesser contractile response, the ferret has the advantage of a relatively large lung and long trachea that allow study in several preparations obtained from a single animal. It should prove a useful animal model for study of pulmonary pharmacology.     

Nasal hyperresponsiveness to histamine induced by repetitive exposure to cedar pollen in guinea-pigs.

Nasal hyperresponsiveness is one of the characteristic features of the pathogenesis of allergic rhinitis. This study examined whether repetitive inhalation of antigen (Japanese cedar pollen) led to the development of nasal hyperresponsiveness to histamine in sensitized conscious guinea-pigs. Guinea-pigs were repeatedly challenged by pollen inhalation once every week following sensitization by means of intranasal application of pollen extract plus aluminium hydroxide. The upper airways obstruction (increase in specific airway resistance (sRaw)) in response to intranasally instilled histamine was measured as an index of nasal (hyper)responsiveness. The hyperresponsiveness to histamine gradually developed with repeated pollen inhalation challenge, and the airway response at the 20th and 24th challenges was three to four orders of magnitude higher than that in nonsensitized animals. Similar degrees of hyperresponsiveness were observed at 10 h and 2 days after a pollen inhalation challenge, but the hyperresponsiveness had almost disappeared by day 7. The increased responsiveness was suppressed by pretreatment with mepyramine but not with atropine. The maximum sRaw, which was observed 10 min after histamine instillation, was largely blocked by naphazoline. Hyperresponsiveness was hardly observed on methacholine instillation. The present allergic rhinitis model, showing marked nasal hyperresponsiveness to histamine after repeated intranasal allergen challenge in guinea pigs, should be useful for investigating the pathogenesis of allergic rhinitis.