Antigen Presenting Ability Of Eosinophil Subpopulation In Murine Allergic Asthma Model

Objective:Eosinophils?Eos?are important cells in the process of immune response and anaphylaxis.They are bactericidal and antiparasitic.Once at the site of injury,eosinophils can release the contents of the granules,causing tissue damage,promoting the progression of inflammation,and inducing asthma symptoms such as mucus secretion and airway constriction by various inflammatory mediators.Eosinophils have long been considered as terminal effector cells and play an important role in allergic asthma.Recent studies found that eosinophil also ascts as immunomodulator of allergic pulmonary inflammation.And eosinophils expressed antigen-presenting related molecules,MHC-II,CD40,CD80 and so on,suggesting that eosinophils may play an active role as antigen-presenting cells.Our aim is to investigate the different antigen-presenting capacity of lung eosinophil subpopulation.Murine allergic asthma model was constructed by immunizing with OVA/papain to investigate whether the pulmonary eosinophil subpopulation in mice with allergic asthma possesses the ability to present antigens and induce T cell activation and proliferation.Methods:1.Female C57BL/6 mice were sensitized intranasally with OVA/Papain on days 0 and2,then the mice were immunized again on the 10th day to further strengthen the inflammatory response.Finally,they were challenged intranasally with ovalbumin?OVA?for three days on days 17,18,and 19.Mice were sacrificed on day 21,and phenotypic characteristics of mouse lung eosinophils were analyzed by flow cytometry.2.As described in section 1,mice were immunized with OVA/papain intranasally to induce an allergic asthma model in mice.Mice were sacrificed at different time points?day0,1,3,10,17,21?to demonstratethe the kinetics of eosinophils.The number and percentage of eosinophil subpopulations in lung,bronchoalveolar lavage fluid?BALF?and blood were detedted by flow cytometry.3.As described in section 1,mice immunized with OVA/Papain intranasally were sacrificed at different time points?0,1,3,10,17,21 days?.Flow cytometry was used to analyze the expression of MHC-II,CD80,and CD86 in eosinophil subsets in lungs of mice.4.Fluoresence labeled antigen OVA-FITC was used to administrate naive mice?D0?and allergic asthmatic mice?D21?.Mice were sacrificed 12 hours later,and the ability of eosinophil subsets in antigen phagocytosing in vivo was detected by Flow cytometry.Antigen phagocytic ability of rEos and iEos in lung and bronchoalveolar lavage fluid?BALF?were all analyzed.Lung cells of naive mice?D0?and allergic asthmatic mice?D21?were cultured in 200?l medium per well.And 10?g OVA-FITC were added in each well.After 6 hours the ability of rEos cell subsets and iEos cell subsets in phagocytosing antigen in vitro was examined.5.Sorted rEos,iEos,and DCs from lungs of allergic asthmatic mice were cocultured with e450-labeled na?ve OT-II T cells in vitro.Four days later,flow cytometry was used to detect the activation and proliferation of T cells.Results:1.Only rEos cells could be detected in the lungs of naive C57BL/6 mice that have not been immunized.Its phenotype is SSC^hiSiglec-F^miCD125^miCD10^-CD62L^hi.In the mouse model of allergic asthma induced by OVA/Papain,iEos were also present in addition to the rEos cell subsets.Its phenotype is SSC^hiSiglec-F^hiCD125^hiCD101⁺CD62L^lo.2.In the steady state,there are mainly rEos cells in the lungs of mice,and the iEos cells were rarely detected.However,along with repeated OVA/papian immunizations,the number and percentage of iEos cell subsets in the lungs of mice increased significantly.There were only iEos cell subsets detected in BALF and the number of iEos cells increased rapidly with the development of inflammation.Migrating eosinophils in blood demo-nstrated the rEos-like phenotype,and levels variated with the development of asthma.3.In murine allergic asthma model,both rEos and iEos cells weakly expressed MHC-II,but the expression of MHC-II in iEos is higher than that in rEos cell subsets in the later phase of asthma.The expression of CD80 in iEos cell subsets was significantly higher than that in rEos cells,but the expression of CD86 in both groups was very low.4.REos and iEos showed distinct patterns of antigen phagocytosing.In vivo experiments demonstrated that iEos in BALF and lung tissue could engulf OVA-FITC,but rEos could not acquire OVA-FITC.Both rEos cell subsets and iEos cell subsets could phagocytose antigens in vitro,and there was no significant difference in their ability to engulf antigens.5.After four days of co-culture with rEos and iEos in vitro,T cells were activated and proliferated.And T cells activated by iEos showed higher proliferation level.Conclusion:In murine allergic asthma model induced by OVA/Papain,two eosinophil subpopula-tions were identified and showed different phenotype and migration patterns.iEos(SC^hiSiglec-F^hii CD125^hiCD101⁺CD62L^lo)could inliltrate into BALF,while rEos(SSC^hii Siglec-F^miCD125^miCD101^-CD62L^hi)resided in lung parenchyma.Both rEos and iEos could engulf antigens in vitro,however only iEos acquired antigens in vivo.In vitro coculture experiments suggested that both rEos cell subsets and iEos cell subsets were able to induce activation and proliferation of na?ve T cell,but iEos cell subsets were more competent.