| Objective To investigate the role of Gr-1~+CD11b+ myeloid-derived cells in the allergic inflammation related to OVA-induced asthma in mice.Methods The Fraction 2 (Fr2) cells were isolated using Percoll density gradient centrifugation from the bone marrow or the lungs of mice, Gr-1~+CD11b+ myeloid-derived cells were acquired by adhered Fr2 cells and collected the nonadherent cells. The SPF level of female BALB/c mice were randomly divided into three groups: control group, asthma group, asthma / treated group. (1) The asthma group and the asthma/treated group were immunized intraperitoneal injection with OVA-aluminum hydroxide adjuvant on day 0, day 7 and day 14. The immunized mice were exposed to aerosol OVA on day 17, continuing for 7 days. The control group were immunized and challenged with PBS instead of OVA. (2) On day 16 and day 19, Gr-1~+CD11b+ myeloid-derived cells were adoptively transferred into the asthma / treated group mice by the tail vain. (3) The Fr2 cells were isolated using Percoll density gradient centrifugation from the lungs of asthmatic mice, cultured 4 hours in 6-well round-bottom plates at 37℃in 5% CO2. Nonadherent cells were collected, then analyzed the percentage of Gr-1~+CD11b+ cell by flow cytometric analysis. And the cells were stained with Wright-Giemsa in order to characterize the morphology of the Gr-1~+CD11b+ myeloid-derived cells. (4) Gr-1~+ CD11b+ myeloid-derived cells were labeled with CFSE, then adoptively transferred into asthmatic mice or control mice by the tail vain ,the lungs were surgically excised from mice 12 hours after the injection, frozen sections were prepared and analyzed by fluorescence microscopy. The single-cell suspension was prepared, used for flow cytometric analysis of the percentage of CFSE-labeled cells. (5) The percentage of Gr-1~+CD11b+ cells in the lungs of asthmatic mice was dynamic detected by flow cytometry on day 3, day 5 and day 7 during OVA challenged. (6) The Gr-1~+CD11b+ myeloid-derived cells ability of inhibition on anti-CD3/ anti-CD28 induced primary T cell proliferation were detected by MTT colorimetric assay and flow cytometry . (7) Compared the total number of BALF cells and differential cell counting to evaluate the effects of airway inflammation after Gr-1~+CD11b+ myeloid-derived suppressor cells treated. (8) Lung sections were stained with Hematoxylin and eosin to evaluate the histopathological change after Gr-1~+CD11b+ myeloid-derived cells treated. (9) The concentrations of OVA-specific IgE in sera and IL-4 and IFN-γin the supernatant of BALF were assayed by enzyme-linked immunosorbent assay (ELISA). (10) The relative quantity of IL-4 mRNA and IFN-γmRNA in lungs were determined by reverse transcription-polymerase chain reaction (RT-PCR).Results (1)There was a certain amount of Gr-1~+CD11b+ myeloid-derived cells in the lungs of asthmatic mice: the number of fraction 2 cells and the percentage of Gr-1~+ CD11b+ cells in fraction 2 cells in the asthmatic mice were more than control mice.(2) Gr-1~+CD11b+ myeloid-derived cells had the ability of recruitment and accumulation to the lungs of asthmatic mice, and the potential became increased with the development of inflammatory: mice with inflamed lungs had a higher number of CFSE-labeled Gr-1~+CD11b+ myeloid-derived cells compared with control mice; The percentage of Gr-1~+CD11b+ cells in the lungs of asthmatic mice elevated with the times of OVA challenged.(3) Gr-1~+CD11b+ myeloid-derived cells derived from the lungs of asthmatic mice significantly inhibited the anti-CD3/ anti-CD28-induced primary T cell proliferative response, and dose-dependently suppressed the proliferation.(4) Gr-1~+ CD11b+ myeloid -derived cells apparently depressed the airway inflammation: the total number of BALF cells and eosinophilic cells and macrophages in asthma/treated group were significantly lower than the asthma group.(5)Histological analysis indicated the attenuated infiltrations of inflammatory cells in the lungs and bronchial perivascular region and the extent of goblet cell hyperplasia in asthma/treated group compared with asthma group.(6) The concentrations of OVA-specific IgE in serum and IL-4 in the supernatant of BALF were markedly decreased, but the concentration of IFN-γin the supernatant of BALF was increased in asthma/treated group compared with asthma group. In the mRNA level, IFN-γmRNA increased, IL-4 mRNA decreased in asthma/treated group compared with asthma group.Conclusions There is a certain amount of Gr-1~+CD11b+ myeloid-derived cells in the lungs of asthmatic mice, and has some immunosuppressive function; Adoptively transfer -red Gr-1~+CD11b+ myeloid-derived suppressor cells derived from the bone marrow of tumor-bearing mice into the asthmatic mice have a significant effects. Therefore, to explore appropriate strategies to induce the number of Gr-1~+CD11b+ myeloid-derived cells increased and immunosuppressive function enhanced is expected to become a new strategy on prevention and treatment of clinical allergic asthma.
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