Comparison In Characteristics Of Airway Inflammation Between Nonasthmatic Eosinophilic Bronchitis And Asthma

Background and ObjectiveEosinophilic bronchitis (EB) is an important etiological factor inducing chronic cough. The analysis of induced sputum and bronchial mucosal biopsies from EB patients showed that the airway inflammation was similar to that seen in asthma. However, it remains open to question why no functional abnormalities associated with asthma was observed in EB patients and what its underlying mechanisms are contributing to the pathophysiology of asthma. In the present study, we investigate the airway pathological characteristics of EB and use the comparative proteomics technique to explore at the molecular level differences between eosinophilic bronchitis and bronchial asthma for identifying idiotype proteins associated with the airway hyperresponsiveness and the reversible obstruction. Furthermore, we try to establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness based on our clinical work.Results and conclusion1. The pathological features of the airway inflammation in EB and asthma are similar. It is an airway inflammatory disorder characterized by infiltration of various inflammation cells (eosinophils, T lymphocytes and mast cells). However, the intensity of airway inflammation in EB is milder, and the extent is more limited than that in asthma.2. By comparative proteomics technique, we have succeeded in the identification of two proteins differentially expressed in the EB, asthma and normal group, and we found that GSTT1 might be closely related to the development of airway responsiveness.3. We established a preliminary EB animal model successfully. The intensity of airway inflammation in this EB model was milder than that in the asthma model. Our model established a foundation for the further research.PartⅠComparison in Immunopathology between Eosinophilic Bronchitis and AsthmaObjectiveTo investigate the airway pathological characteristics of eosinophilic bronchitis (EB) in comparison with asthmaMethodsInduced sputum analysis was performed in 20 cases with EB, 24 cases with bronchial asthma and 10 cases of healthy subjects. Differential cell counts in induced sputum, fiber optic bronchoscopy, and bronchoalveolar lavage (BAL) were performed, followed by cell counting, and classification. The biopsy specimens of bronchial mucosa were taken from the carinae, the gap between the left lingual lobe and the anterior segment (B8) of the right and lower lung. The biopsy specimens of the control group were just taken from the gap of lingual lobe. Pathological features of the biopsy specimens were observed by Light microscopy, and the thickness of the basement membrane of the epithelial mucosa was measured. Using immunohistochemical analysis, the distribution of inflammatory cells (Eosinophils, Mast cell, T lymphocyte) in epithelial and subepithelial layer was observed and the distribution density of the cells was calculated.Results1. The percentage of eosinophils in induced sputum of EB group (10.13%(20.19)) was lower than that of asthma group ((26.00%(32.97)) (P<0.05). The percentages of eosinophils in induced sputum of EB and asthma group were significantly higher than that of control group( (0.25%(0.50) )(P<0.001).2. The percentage of eosinophils in BALF of EB group (1.63% (3.81)) was significantly lower than that of asthma group (11.75%(15.70)) (P<0.001), and the percentages of eosinophils in BALF of EB group and asthma group were significantly higher than that of control group (0.38%(1.00)) (P<0.05).3. The thickness of the basement membrane of bronchial mucosa at the gap of the lingual lobe in the asthma group (4.73μm(2.61)) was significantly greater than that in the control group (3.23μm(1.35)) (P<0.001) and the EB group {3.41μm (1.89)} (P<0.01). However, there was no difference between the EB group and asthma group (P<0.05). The thickness of the basement membrane of bronchial mucosa at the anterior section of the right and lower lobe in the asthma group (5.36μm(3.65)) was significantly greater than that in the EB group (3.53μm (3.18)) (P<0.05).4. The distribution densities of Eos in the subepithelial layer of bronchial mucosa of the lingual lobe in the asthma group (8.90(52.88) piece/mm2) were significantly higher than that in EB group (0.00(10.40) piece/mm2) and the control group (0.00(0.00) piece/mm2) (P<0.05).5.The distribution density of T lymphocytes cells in the epithelial layer of bronchial mucosa of the lingual lobe in the EB group (475.76(244.75)piece/mm2) and the asthma group (583.29(507.69) pieces/mm2) was significantly higher than that in the control group (260.71(430.31)piece/mm2) (P<0.05). There were no significant differences between the EB group and the asthma group in any site (P>0.05).6.The distribution density of mast cells in the epithelial layer of bronchial mucosa of the lingual lobe in in the EB group (168.80(314.28)piece/mm2) and the asthma group (252.78(545.68) pieces/mm2) was significantly higher than that in the control group (0.00(13.61)piece/mm2) (P<0.05). In the subepithelial layer of bronchial mucosa of the lingual lobe in in the EB group (218.44(383.65)piece/mm2) and the asthma group (197.22(208.54) pieces/mm2) was significantly higher than that in the control group (66.07(45.92)piece/mm2) (P<0.05). There were no significant differences between the EB group and the asthma group in any site (P>0.05).Conclusions1. The pathological features of the airway inflammation in EB and asthma are similar. In EB patients, it is an airway inflammatory disorder characterized by infiltration of various inflammation cells (eosinophils, T lymphocytes and mast cells).2. The intensity of airway inflammation in EB is milder, and the extent was more limited, than that in asthma.PartⅡThe Initial Proteomics Analysis Between EB and asthmaObjectiveWe found that there were a great deal of similarities in pathology and immunpathology between EB and asthma in a previous study, but there still existed many differences. Bronchoaveolar lavage fluid (BALF), which can reflect the changes of airway inflammation directly and objectively, was recognized to be an ideal sample of proteomics in the investigation of airway inflammation diseases. In this study, we used the comparative proteomics technique to explore at the molecular level differences between eosinophilic bronchitis and bronchial asthma for identifying idiotype proteins associated with the airway hyperresponsiveness and reversible obstruction.MethodsThere were five cases of EB, six cases of asthma and six cases of normal subjects respectively. Fiber bronchoscopy, bronchoalveola lavage (BAL), and the BALF were performed in the right and middle lobe. Total protein extracted from the cells in BALF was separated using two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG). After silver nitrate staining, the gel image analysis was carried out using Image Master 2D Elite 5.0 analysis software to identify the proteins differentially expressed in EB, asthma and control group. The differential expression proteins were identified by peptide mass fingerprint (PMF) using matrix-assisted laser desorption /ionization time of flight mass spectrometry (MALDI-TOF-MS), followed by MALDI-TOF/TOF-MS for a secondary PMF, and 4 protein spots were identified.ResultsAs shown in the two-dimensional electrophoresis, there were significant differences in the protein expression in BALF cells from the EB, asthma and normal control group. After comparing the peptide mass fingerprints of various protein spots with standard molecular weight and isoelectric point, two proteins, ferritin (light polypeptide) and Human Glutathione -S -Tranferase T1 (GSTT1), were identified. Ferritin was up-regulated in asthma group compared with the EB and control group. 83.3% in asthma patients and 33.3% in controls had no expression of GSTT1 , while the expression of GSTT1 was relatively higher .Conclusions1. The comparative analysis of two-dimensional electropherogram displayed that the protein expressions in the BALF cells among these three groups were distinct. Identification of the different proteins provided an important method and clue for discovering the protein markers of airway hyperresponsiveness and elucidating the pathogenesis of airway responsiveness.2. By comparative proteomics technique, we have succeeded in the identification of two differentially expressed proteins in the EB, asthma and normal group, and we found that GSTT1 might be closely related to the development of airway responsiveness. PartⅢEstablishment of Eosinophilic Bronchitis Mouse Model and Comparison in Airway Inflammation between Eosinophilic Bronchitis Model and Asthma ModelSegment1 Proper Method for Measuring Airway Responsiveness in Allergic MiceObjectiveTo investigate the applicability of invasive measurement and non-invasive measurement systems in analyzing hypersensitivity of the lower airway in mice.MethodsFemale Balb/c mice 6 weeks of age were obtained and divided randomly into two main groups: airway allergic group and normal control group. Each group was divided again into 2 sub-groups (4 groups in all), n=8, according to the different measurements: non-invasive airway allergic group, non-invasive normal control group, invasive airway allergic group and invasive normal control group. Mice were immunized by an intraperitoneal injection of 10μg OVA (Sigma, USA)1.3mg of aluminum hydroxide gel on days 0 , 7 and 14, followed by daily intranasal challenges on days 28 with 0.2% OVA, which was diluted in sterile normal saline (50 ul OVA solution, 2_ per mouse). The control mice received saline injection and intranasal challenge instead of the OVA solution. The mice were subjected to the following evaluations on days 29 (24 hours after intranasal challenge).The measurement of airway responsiveness was carried out by using invasive measurement system ("RC"system, Buxco,USA) and non-invasive measurement system ("penh"system, Buxco,USA). Measurements were made at concentrations of 0(saline), 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25and 50 mg/ml MCh. In the invasive measurement, AR was assessed as an increase in RL after challenge with aerosolized MCh in anesthetized, tracheotomized and ventilated mice. To measure transpulmonary pressure (Ptp), a water-filled tube was inserted into the esophagus to the level of the midthorax and coupled to a pressure transducer. Changes of lung resistance (RL) and lung dynamic compliance (Cdyn) were measured. In the non-invasive measurement, mice were placed into the chamber and allowed to move freely. Saline and MCh solutions of increasing concentrations were aerosolized and pumped into the chamber. Penh was measured after each aerosolization. After the measurement of airway responsiveness, bronchial alveolus lavage fluid (BALF) and histopathological evaluation of the nose and trachea was performed by a pathologist blinded to treatment of the groups. Epithelial erosions and inflammation were scored using a subjective semiquantitative scale of 0-5 as Underwood described before.Results1. At Mch concentrations of 6.25, 12.5 and 25mg/ml, Penh levels were significantly higher in the non-invasive airway allergic group than that in the non-invasive normal group. Penh200 and Penh 300 in the non-invasive airway allergic group were significantly lower than that in the non-invasive normal group (P<0.001). However, in the invasive measurements, significant bronchoconstriction was not observed in either the invasive airway allergic group or the invasive normal control group.2. In the non-invasive airway allergic group and invasive airway allergic group, no significant changes in the cell number or cell differentials in BALF were observed. Furthermore, the score of the epithelial erosions and inflammation was not different between these two groups.3. The nasal mucosa showed a mild infiltration of inflammatory cells in airway allergic groups and the mean number of nasal itching in airway allergic groups was significantly greater than that in normal control groups at 30mM-histamine concentration. ConclusionsOur study showed that invasive and non-invasive measurement might lead to the different results for airway responsiveness in allergic mice. We think one reason was that the airway inflammation of the allergic mouse was comparatively mild, and this lead to no apparent bronchoconstriction in lower airway. Another possibility was that in the non-invasive measurements, inhalation exposure includes nasal and gastro-intestinal uptake, and it might not necessarily represent a change in the lower respiratory tract. The increased airway responsiveness in the non-invasive approach is related with obstruction of upper airway. We suggest it is necessary to use the invasive measurement first to measure the lower airway hyperresponsiveness of the allergic mice.Segment 2 Establishment of Eosinophilic Bronchitis Mouse Model without HyperresponsivenessObjectiveTo establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness.MethodsFemale Balb/c mice were obtained and randomly divided into 4 groups: airway allergic aerosolized group (aerosolized group), airway allergic intranasal challenge group 1(intranasal challenge group 1), airway allergic intranasal challenge group 2 (intranasal challenge group 2) and normal control group (control group). Mice were immunized as before, then the aerosolized group was aerosolized on day 28 with 0.2% OVA. The intranasal challenge group 1 was followed by intranasal challenge on day 28 with 0.2% OVA while the intranasal challenge group 2 was followed by intranasal challenge on days 28, 29 with 0.2% OVA. The Control group was sensitized and challenged with diluents. Twenty four hours after the latest challenge, the measurement of airway responsiveness was carried out using the invasive measurement system ("RC"system, Buxco, USA). After the measurement, a pathologist blinded to the treatment of the groups performed bronchial alveolus lavage fluid (BALF).Results1. At Mch concentrations of 25 mg/ml, RL% in the intranasal challenge group 2 was higher than that in the control group(P<0.05). No difference in airway reactivity between the aerosolized group and the control group and the same was found between the intranasal challenge group 1 and the control group.2. The percentage of BAL macrophages decreased significantly in all OVA immunized mice when compared with the control group and the percentage of BAL lymphocytes, neutrophils and eosinophils increased significantly in all OVA immunized mice when compared with the control group.3. The percentage of macrophages in BAL in the aerosolized group was higher than that in the intranasal challenge group 1 and group 2 while the percentage of eosinophils in BAL ((7.40±3.41)%) was lower than that in the intranasal challenge group 1 ((25.33±10.79)%)and group 2 ((39.73±6.02)%).ConclusionsBy changing the antigen immunizing and challenge methods, it is possible to establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness. We achieved the goal successfully. Segment 3: The Airway Inflammation Characteristics of Eosinophilic bronchitis Mouse Model and the Comparison between Eosinophilic Bronchitis Model and Asthma ModelObjectiveTo observe and compare the airway inflammation characteristics of the eosinophilic bronchitis mouse model and the mice model of asthma.MethodsFemale Balb/c mice were obtained and divided randomly into 3 groups: eosinophilic bronchitis group (EB group), asthma group and normal control group (control group). Mice were immunized as before. EB group was followed by intranasal challenges on day 28 with 0.2% OVA and asthma group was followed by intranasal challenges on days 28, 29, 30 with 0.2% OVA. The control mice received saline injection and intranasal challenge instead of the OVA solution. Twenty four hours after the latest intranasal challenge,the measurement of airway responsiveness in every group was carried out using the invasive measurement system ("RC"system, Buxco, USA) and a pathologist blinded to the treatment of the groups performed bronchial alveolus lavage fluid (BALF) and histopathological evaluation of the trachea and lung. Twelve and 48 hours after the latest intranasal challenge,the measurement of airway responsiveness was carried out using the invasive measurement system ("RC"system, Buxco, USA) and bronchial alveolus lavage fluid (BALF) performed in EB group.Results1. Twelve, 24 and 48 hours after intranasal challenge, no difference of airway reactivity were found between the EB group and the control group. In contrast, at Mch concentrations of 12.5, 25, 50mg/ml, RL% in the asthma group was higher than that in the control group(P<0.05). 2. Twenty four hours after intranasal challenge, the percentage of BAL eosinophils and the total cell number in BALF of the asthma group ((42.15±13.35)%, (7.59±1.33)×105/ml) were higher than that in EB group ((25.33±10.79)%, (2.86±0.55)×105/ml)). Forty eight hours after intranasal challenge, the percentage of BAL eosinophils increased significantly when compared with that after 12 and 24 hours in EB group. Furthermore, the score of the epithelial erosions and inflammation in asthma group was signicantly higher than that in EB group(P<0.05)。Conclusions1. Airway inflammation in the EB model became more severe as time went by, while significant bronchoconstriction was not observed during our observation period.2. The intensity of airway inflammation in the EB model was milder than that in asthma model.