Development Of An Eosinophilic Bronchitis Mouse Model And The Effects Of Eosinophil Activation On Airway Hyperresponsiveness In Eosinophilic Bronchitis Mice

ObjectivePart 1:To develop an animal model of eosinophilic bronchitis in mice.Part 2:To explore the effects of eosinophil activation on airway hyperresponsiveness.MethodsPart 1A total of 80 female specific pathogen free BALB/c female mice were divided into four groups:polymyxin B group,normal saline group,polymyxin B+N-formyl-methionyl-leucyl-phenylalanine(fMLP)group,normal saline+fMLP group.All the groups were given corresponding nasal drops for 21 days.The former two groups were given 12 μ1 polymyxin B(5 mg/ml,dissolved in distilled water)or normal saline once a day by transnasal administration respectively.Besides the above,the latter two groups were given 10 μ1 fMLP(0.05 mg/ml,dissolved in acetic acid)once by transnasal administration 3 hours after polymyxin B or normal saline administration on the 21st day.Ten mice in each group were measured airway responsiveness(inspiratory and expiratory airway resistance and lung compliance)3 hours after the last transnasal administration in the former two groups and 24 hours after the last transnasal administration in the latter two groups.Cells in bronchoalveolar lavage fluid(BALF)of the remaining ten mice were collected and analyzed by absolute different cell counts.HE and Chromotrope 2R staining observed the infiltration of inflammatory cells in lung tissues.Part 2A total of 80 female specific pathogen free BALB/c female mice were divided into four groups,and the methods of group dividing and model making were the same as the first part.Within 24 hours after the last transnasal administration,Serum was collected after heart puncture and centrifugation.BALF was obtained through trachea cannula and then centrifuged to collect supernatant.Lung tissue homogenate was also collected.Eosinophil peroxidase(EPX),eosinophil cationic protein(ECP)and eosinophil chemotactic factor(ECF)levels in serum,BALF and lung tissue homogenate were measured by emzyme linked immunosorbent assay(ELISA).Immunohistochemistry staining was used to detect the expression levels of EG2 in the lung tissues,and the average optical density(OD)value represented the expression.The morphology of low-density eosinophils was observed under electron microscope.ResultsPart 1The total cell counts of BALF in polymyxin B group were significantly higher than the normal saline group[29.50(3.25)×104/ml vs 15.25(2.25)×104/ml,P<0.001],especially eosinophil counts[11.76(6.02)×104/ml vs 0.12(1.08)×104/ml,P<0.001],but no significant difference in macrophage,lymphocyte and neutrophil counts.Similarly,the total cell counts and eosinophil counts of BALF in polymyxin B+fMLP group were significantly higher than the normal saline+fMLP group[27.00(3.50)×104/ml vs 14.00(2.50)×104/ml,10.17(6.15)×104/ml vs 0.17(0.96)×104/ml,respectively,all P<0.001].However,no significant differences oftotal cell counts and differential cell counts existed in polymyxin B and polymyxin B+fMLP group.The comparison of the dose-response curve to intravenous methacholine(Ach)showed that no significant differences of inspiratory and expiratory airway resistance and lung compliance existed in polymyxin B group and normal saline group.Compared to the former two groups,there was no increase in inspiratory resistance and expiratory resistance,and no decrease lung compliance in normal saline+fMLP group.However,with the Ach concentration increasing,inspiratory and expiratory resistance in polymyxin B+fMLP group were significantly higher than the other three groups,the lung compliance was significantly lower than the other three groups(all P<0.001).Lung sections examined by HE and Chromotrope 2R staining showed that the bronchial inflammation of the polymyxin B and polymyxin B+fMLP group were significantly worse than the other two groups,and there were more inflammatory cells,especially eosinophils infiltrated around the airways.However,there was no such inflammatory reaction in normal saline and normal saline+fMLP group.Part 2The EPX,ECP and ECP levels in serum,BALF and lung tissues of the polymyxin B group were similar to those of normal saline group without significant difference.Compared to the former two groups,there were no significant differences of the EPX,ECP and ECP levels in serum,BALF and lung tissues of normal saline+fMLP group.However,the EPX,ECP and ECP levels in serum,BALF and lung tissues of the polymyxin B+fMLP group were all significantly higher than the other three groups(all P<0.001).The immunohistochemical OD staining values of EG2 innormal saline group,polymyxin B group,normal saline+fMLP group,polymyxin B+fMLP group were(0.0618±0.0201),(0.1298±0.0268),(0.0608±0.0211)and(0.1876±0.0329),respectively.The EG2 protein expression of polymyxin B+fMLP group was significantly higher than the other three groups(all P<0.001),and the EG2 protein expression of polymyxin B group was higher than the normal saline and normal saline+fMLP group(all P<0.001).But no significant difference of EG2 protein expression existed in the normal saline and normal saline+fMLP group(P=1.000).Under the electron microscope,there was no obvious eosinophil infiltration in normal saline and normal saline+fMLP group,while there was obvious eosinophil infiltration in polymyxin B and polymyxin B+fMLP group.Most of the eosinophils in the polymyxin B group had intact cell membrane,hemogeneous-density granule,clear center key,and occasionally vacuoles can be seen after degranulation into low-density eosinophils.Meanwhile,more activated eosinophils were observed in polymyxin B+fMLP group,which is characterized by disintegration of cell membrane,dropout and density decrease of center key and cytoplasmic vacuoles after degranulation.ConclusionPart 1:A mouse model of eosinophilic bronchitis can be successfully developed by transnasal administration of polymyxin B.Part 2:The eosinophil activation plays an important role in the occurrence of airway hyperresponsiveness(AHR).