| Part 1.Expression and clinical significance of the soluble B7-H3 in plasma of children with bronchial asthmaObjective: To study the expression level of soluble B7-H3(s B7-H3)in the peripheral blood of children with bronchial asthma,and analyze its correlation with the expression of other cytokines.To explore the abnormal expression of s B7-H3 in children with bronchial asthma and its clinical significance.Methods: Twenty-three cases of children with bronchial asthma hospitalized in department of respiration in Children’s hospital of Soochow University from June 2013 to May 2015 were enrolled in this study.Fourteen children hospitalized for surgery selective operation were enrolled in control group.Peripheral blood was collected in children with bronchial asthma within 24 hours after hospitalization and 24 hours before discharged and was divided into acute stage group and convalescence groups.Blood was also collected from children in control group before operation.The level of s B7-H3,interleukin-4,IFN-gamma and interleukin-17 in plasma were detected by Enzyme-linked immunosorbent assay.The correlation between the level of s B7-H3 and other cytokines were analyzed.Results: The level of s B7-H3 and interleukin-4 in plasma was increased in children with asthma of acute episode,which was significantly higher than that in recovery period and in control group(P=0.049,P<0.001).After treatment,most children with asthma had a decreased level of s B7-H3 and interleukin-4(72.9% and 87.0%,respectively),but the overall level was still higher than that in control group(P=0.028).The level of IFN-gamma in plasma was decreased in children with asthma of acute episode,which was significantly lower than that in recovery period and in control group(P=0.018,P=0.001).After treatment,73.9% children with asthma had a decreased level of IFN-gamma,but the overall level was still lower than that in control group(P=0.028).The level of interleukin-17 in plasma had no significantly difference among three groups(P=0.084).There was of good coherence between the level of s B7-H3 and interleukin-4 in plasma in children with asthma after treatment(Kappa=0.596,P=0.002).Meanwhile,s B7-H3 had a poor consistency with IFN-gamma and interleukin-17(Kappa=-0.232 and 0.055,P>0.05).Conclusion: Asthmatic children have an immunity imbalance of Th1/Th2 in vivo.The expression of s B7-H3 was abnormally elevated in children with asthma of acute episode.s B7-H3 may have a positive regulatory role to Th2 cells.Part 2.The effect of recombinant B7-H3 in murine model of asthmaObjective: To study the effect of recombinant B7-H3 in murine model of asthma.To study the immune mechanism of B7-H3 in asthma.To study the status of B7-H3 in the imbalance of Th1/Th2.Methods: Specific-pathogen-free,C57BL/6 mice(6 weeks old)were divided into three groups: control group(C group),asthmatic group(N group)and the recombinant mouse B7-H3 treatment group(NB group).For the N group and NB group,ovalbumin(OVA)was intraperitoneally administered on days 0,7and 14 and they were challenged with aerosolized 1% OVA on days 22,24,26,28.The normal control group mice were administered normal saline(NS)intraperitoneally on the same days and inhaled NS in a similar manner.Additionally,for the NB group,50 ug recombinant mouse B7-H3 was administered intraperitoneally on days 0,3,7,10,14 after the first OVA injection.Twenty-four hours after last challenge all mice were killed after anesthetized and plasma was collected.All mice underwent bronchoalveolar lavage(BAL),and bronchoalveolar lavage fluid(BALF)was collected for cell count and cellular profile count.The level of IFN-gamma,interleukin-4,interleukin-5 and interleukin-17 in BALF was measured by Enzyme-linked immunosorbent assay(ELISA).Mice lung tissue sections were stained with HE,PAS and immunohistochemical(B7-H3),to observe the infiltration of inflammatory cells and eosinophils,mucus secretion and the expression of B7-H3 in lung tissue.Results: The total cell number and the percentage of eosinophils in BALF in N group was significantly higher than that in C group(P=0.004),and the mice in NB group had the highest total cell number and percentage of eosinophils in BALF,which was significantly higher than that in N group and C group(P<0.05).In plasma and BALF of N group,the level of IFN-gamma was lower than that in C group,but the level of interleukin-4,interleukin-5 and interleukin-17 was higher than that in C group(P<0.05).After using recombinant B7-H3,the level of IFN-gamma,interleukin-4 and interleukin-5 in the plasma and BALF in NB group has the varying degree ascension to compare the N group.But there was no significantly difference of interleukin-17 between N group and NB group(P>0.05).HE and PAS stain showed that there was no inflammatory cells infiltration and mucus production in lung in C goup.Large number of inflammatory cells was found around the airway and mucus was overproduced in the airway.Administration of recombinant mouse B7-H3 significantly accelerated eosinophil infiltration and mucus overproduction in lung tissues.Immunohistochemistry showed B7-H3 did not expressed in lung of C group,but was highly expressed in the lung of N group.B7-H3 was expressed highest in the lung of NB group,which was significantly higher than that in C group and N group(P<0.05).Conclusion: Administration of recombinant B7-H3 could augment both Th1 and Th2 characterized cytokines and aggravate pathological changes of lung tissue in asthma.Part 3.The role of Toll-like receptor 2 for the effect of B7-H3 in murine model of asthmaObjective: To reveal the role of B7-H3 and Toll-like receptor 2(TLR2)in murine model of asthma and their relationship.To further clarify the immune mechanism of B7-H3 in asthma.Methods: Specific-pathogen-free,C57BL/6 mice(6 weeks old)were divided into five groups: the normal control group(C group),the wild-type group with asthma(N group),the recombinant mouse B7-H3 treatment group(NB group),the TLR2-deficient mice group with asthma(T group),and the TLR2-deficient mice with recombinant mouse B7-H3 treatment group(TB group).For the N group,NB group,T group and TB group,ovalbumin(OVA)was intraperitoneally administered on days 0,7and 14 and they were challenged with aerosolized 1% OVA on days 22,24,26,28.The normal control group mice were administered normal saline(NS)intraperitoneally on the same days and inhaled NS in a similar manner.Additionally,for the NB group and TB group,50 ug recombinant mouse B7-H3 was administered intraperitoneally on days 0,3,7,10,14 after the first OVA injection.Twenty-four hours after last challenge all mice were killed after anesthetized and plasma was collected.All mice underwent bronchoalveolar lavage(BAL),and bronchoalveolar lavage fluid(BALF)was collected for cell count and cellular profile count.The level of IFN-gamma,interleukin-4,interleukin-5 and interleukin-17 in BALF was measured by Enzyme-linked immunosorbent assay(ELISA).Mice lung tissue sections were stained with HE,PAS and immunohistochemical(B7-H3 and NF-kappa B),to observe the infiltration of inflammatory cells and eosinophils,mucus secretion and the expression of B7-H3 and NF-kappa B in lung tissue.Result: The total cell number and the percentage of eosinophils in BALF in T group was significantly higher than that in C group(P=0.025 and 0.006,respectively),but was significantly lower than that in N group(P=0.004).And the mice in TB group had a higher total cell number and percentage of eosinophils in BALF compared with T group(P=0.006 and 0.004,respectively).Compared with N group,the level of interleukin-4 and interleukin-5 in plasma and BALF in T group was significantly lower,but the level of IFN-gamma in plasma and BALF in T group was significantly higher(P<0.05).T group had the same level of cytokines in plasma and BALF compared with C group(P>0.05),except that it had a lower level of IFN-gamma in BALF(P=0.006).After using recombinant B7-H3,the level of interleukin-4,interleukin-5 and IFN-gamma in the plasma in TB group has the varying degree ascension to compare the T group.Lung tissue pathology staining showed that inflammatory cells were found around the airway of mice in T group,but was slighter than that in N group.Mucus overproduction was found in lung tissues of T group,which was similar to N group.Administration of recombinant mouse B7-H3 significantly accelerated mucus overproduction in lung tissues of TB group.Immunohistochemistry showed B7-H3 was expressed highest in the lung of NB group and TB group,which was significantly higher than that in C group,N group and T group(P<0.05),followed by N group and T group.B7-H3 did not expressed in lung of C group.The expression of NF-kappa B in lung tissue of C group,T group and TB group was similar,which was significantly lower than that in N group and NB group(P=0.004).Conclusion: TLR2 receptor is closely related to the occurrence and development of asthma.TLR2 deletion blocks the development of asthma.The B7-H3–mediated effects in asthmatic model are independent of TLR2 signaling,at least,not only depends on the TLR2 signaling. |
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