| Part 1.Expression and clinical significance of the soluble B7-H1 in BALF of children with neutrophilc asthmaObjective: To study the expression level of soluble B7-H1(sB7-H1)in the BALF of children with neutrophilc asthma,and analyze its correlation with the expression of Th1、Th2、Th17 type cytokines.Methods:26 cases of children with asthma were collected in department of respiration in Children’s hospital of Soochow University from October 2015 to November 2016,divided into neutrophilc asthma group(group A)and non-neutrophilc asthma group(group B)according to the neutrophil counts of BALF.15 cases of children hospitalized for bronchial foreign body were enrolled in control group(group C).The level of sB7-H1,interleukin-4,interleukin-6,IFN-gamma,interleukin-17 and GM-CSF(granulocytemacrophage colony stimulating factor)in BALF were detected by Enzyme-linked immunosorbent assay.The correlation between the level of s B7-H1 and other cytokines were analyzed.Results: The percentage of lymphocytes and neutrophils in BALF in neutrophilc asthma group were higher than that in non-neutrophilc asthma group(P<0.05),but the percentage of eosinophils in neutrophilc asthma group were lower than that in non-neutrophilc asthma group(P<0.05).The level of sB7-H1,interleukin-6,interleukin-17 and GM-CSF in neutrophilc asthma group was significantly higher than that in non-neutrophilc asthma group and in control group(P<0.05).In non-neutrophilc asthma group,the level of interleukin-4 was higher than that in neutrophilc asthma group and in control group(P=0.014,P<0.001),while the level of IFN-gamma was lower than that in neutrophilc asthma group and in control group(P<0.05).The level of s B7-H1 and the level of GM-CSF,interleukin-6,IFN-gamma,interleukin-17 in BALF has positive correlation of neutrophilc asthma group(r=0.879、P<0.001,r=0.718、P=0.006,r=0.791、P=0.001,r=0.726、P=0.005),and has no correlation with interleukin-4(r=-0.349、P=0.242).The level of interleukin-17 and the level of interleukin-6,GM-CSF in BALF has positive correlation of neutrophilc asthma group(r=0.890、P<0.001,r=0.817、P=0.001),and has no correlation with interleukin-4(r=-0.029、P=0.924).Conclusion: There was a partial difference in the mechanism of neutrophilc asthma and eosinophil-predominant non-neutrophilc asthma development.The development of neutrophilc asthma may be associated with Th17/IL-17 immune imbalance with elevated levels of IL-6、IL-17 and GM-CSF.Eosinophil-predominant non-neutrophilc asthma was characterized by Th1/Th2 immunity imbalance.The level of s B7-H1 was correlated with Th17 cytokines GM-CSF,IL-6 and IL-17 in neutrophilc asthma.sB7-H1 induces IL-6 and GM-CSF secretion through Th17 cytokine IL-17.Part 2.The effect of B7-H1 antibody in murine model of neutrophilc asthmaObjective: To study the effect of B7-H1 antibody in murine model of neutrophilc asthma.To study the immune mechanism of B7-H1 antibody in neutrophilc asthma,and especially the relationship with Th1,Th2 and Th17 cells.Methods:Specific-pathogen-free,female BALB/c mice(6-8 wk old)were divided into four groups,neutrophilc asthma group(group A),PBS(phosphate buffer saline)control group(group B),Ig G group(group C)and the B7-H1 antibody group(group D).Mice in group A,group C and group D were sensitized to ovalbumin(OVA)with lipopolysaccharides(LPS)by intraperitoneally injection on days 0,7,14,then they were exposed to OVA aerosol challenges for 30 minutes on consecutive 8 days on day 22.Mice in group C and group D were given intraperitoneally injection of Ig G and B7-H1 antibody on days 0,3,7,10,14.The control group mice(group B)were administrated PBS intraperitoneally on the same days and inhaled PBS in a similar manner.After 24 hours of last challenge all mice were anesthetized to death to collect plasma.All mice underwent bronchoalveolar lavage(BAL),and bronchoalveolar lavage fluid(BALF)was collected for cell count and cellular profile count.The expression of B7-H1 in lung tissue was measured by flow cytometry.The level of interleukin-4,interleukin-6,interleukin-17,IFN-gamma,TNF-alpha and GM-CSF(granulocyte-macrophage colony stimulating factor)in BALF was measured by Enzyme-linked immunosorbent assay(ELISA).Mice lung tissue sections were stained with HE,PAS and immunohistochemical(B7-H1),to observe the neutrophiles and other inflammatory cells.Results: The total cell number and the percentage of neutrophils in BALF in A group and C group was significantly higher than that in B group(P<0.05).After using B7-H1 antibody,the total cell number and the percentage of neutrophils in BALF in D group was lower than that in A group and C group,but higher than that in B group.The positive rate and fluorescence intensity of B7-H1 in lung tissue in A group and C group were higher than that in B group and D group(P<0.05).In plasma and BALF of A group and C group,the level of interleukin-17,interleukin-4,TNF-alpha and G-CSF were significantly higher than those in B group(P<0.05),but the level of IFN-gamma was higher than that in C group(P<0.05).The level of interleukin-4 in BALF in A group was significantly higher than that in B group(P<0.05).After using B7-H1 antibody,the level of interleukin-17, interleukin-4,TNF-alpha and G-CSF in plasma in D group was lower than that in A group and C group(P<0.05),but the level of these cytokines were higher than B group(P<0.05).the level of interleukin-17 and interleukin-6 in BALF in D group was lower than that in A group(P<0.05)and was no significantly difference in B group(P>0.05),but the level of interleukin-4 and TNF-alpha was higher than that in B group(P<0.05)and was no significantly difference between A group and C group(P>0.05).the level of G-CSF in BALF in D group was higher than B group,but lower than that in A group.Lung tissue pathological stained showed that there was no significant infiltration of inflammatory cells and mucus secretion in lung in B group.Large number of inflammatory cells was found around the airway and mucus was overproduced in the airway in A group and C group.Administration of mouse B7-H1 antibody reducted inflammatory cells and mucus secretion in lung tissues.Immunohistochemistry showed highest expression of B7-H1 in A group and C group,and B7-H1 partly expression in the lung tissue of D group,but almost did not expressed in B group.Conclusion:The murine neutrophilc asthmatic model suggested that the neutrophils aggregation and the development neutrophilc asthma were associated with Th1/Th2/Th17 immunity imbalance.The mouse B7-H1 antibody could reduced airway inflammation,mucus secretion,neutrophils infiltration and B7-H1 expressed in lung tissue.The mouse B7-H1 antibody could also reduced the level of interleukin-17,interleukin-4,tumor necrosis factor-α,G-CSF and interleukin-4 of Th2 and Th17,but had no effect on IFN-gamma of Th1.These results suggest that the development of neutrophilc asthmatic can be partially blocked by using B7-H1 antibody to down-regulating Th2 and Th17 immune responses.The B7-H1 has a positive stimulating effect on Th2 and Th17,which has a positive effect on the development and progression of asthma. |
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