Neutrophils Are Involved In The Recruitment Of Th17Cells In The Airway In Asthmatic Murine

Objective1. To observe chemokines CCL2and CCL20expression changes in miceBronchoalveolar Lavage(BALF) and serum of the normal group and asthma group. Toexplore the possible correlation between the chemokines CCL2,CCL20and thenumber of neutrophils in BALF.2. To observe the proportion of peripheral Th17cells in the normal group and asthmagroup.To observe the expression changes of receptors CCR2and CCR6on thesurface of Th17cells.3. To investigate the roles of chemokines CCL2and CCL20in the recruitment of Th17cells in vitro experiments.Materials and methods20BALB/c mice were randomly divided into2groups,normal control and asthmaticgroup.Mice were sensitized and nebulized in a sealed box with ovalbumin(OVA) liguid asasthmatic models group. The histologic changes in airway remodeling in asthmaticconditions were analazed by using hematoxylin-eosin staining. The cell numbers anddifferential counts in BALF cell pellet were completed. The content of CCL2、CCL20inboth BALF supernatant and serum were determined by enzyme-linked immunosorbentassay(ELISA).The ratio of peripheral Th17cells and their surface CCR2or CCR6receptor expression levels were detected by flow cytometry. The spleen CD4+T cells wereisolated by MACS via a negative selection principle, and then cultured in RPMI1640medium, supplemented with10%low endotoxin fetal bovine serum, treated with IL-6、TGF-1、anti-IFN-、anti-CD3、anti-CD28、IL-23、IL-2、anti-IL-4for Th17cellsdifferentiation, After four days. The chemotactic effect of CCL2、CCL20on Th17cells were observed by Transwell chamber.Results1. On the count of inflammatory cells and differential cells in bronchoalveolar lavagefluid (BALF), the total number of cells and the number of eosinophils in asthmaticmice in BALF were higher than the normal control group, the difference between thetwo groups are statistically significant [（68±18）×10~4/ml vs（33±5）×10~4/ml,(42±14)×10~4/ml vs (5±2)×10~4/ml](n=10, P＜0.01),(n=10, P<0.01). The number oflymphocytes in asthmatic group were also higher than those in normal controlgroup,the difference between the two groups are statistically significant (5±1vs3±1)(n=10,P＜0.05).And neutrophils in asthmatic group were also higher than those innormal control group,the difference are statistically significant.2. Compared to the normal mice,the CCL2and CCL20in the serum of asthmatic micewere increased,And the CCL2in the BALF was increased significantly in asthmaticmurine,but the CCL20in the BALF was decreased significantly in asthmatic murine,3. The content of CCL2in BALF positively correlated to the neutrophil cell number,aswell as the CCL20negatively correlated to it. Both of the differences are statisticsignificant.4. The frequency of Th17cells in the peripheral blood from asthmatic mice modelswere significantly higher than those of normal control groups(4.65±0.85%vs0.89±0.53%）(n=10, P＜0.01）。And compared to normal mice, the expressions ofCCR2and CCR6on the surface of Th17cells were significant increased(1.69±0.68%vs0.56±0.37%）（n=10，P＜0.01),（1.80±1.04%vs0.77±0.51%)(n=10，P＜0.01)，respectively).We may assumed that either CCL2or CCL20had a chemotactic effect onTh17cells cultured in vivo.5. CCL2and CCL20are involved in the recruitment of murine Th17cells that wereinduced and differentiated in vitro culture.Compared to the group that didn’t containchemotactic factors,both of the difference were statistically significant（7.44±1.44%vs 0.33±0.05%）（n=6, P＜0.05）,（5.40±0.64%vs0.33±0.05%）（n=6, P＜0.05）。ConclusionOur results revealed the CCL2and CCL20expressions change associated with moreneutrophils in BALF,positively or negatively.Asthmatic mice had higher proportions ofTh17cells in peripheral blood as well as higher expression of CCR2and CCR6on thesurface of them.Either CCL2or CCL20has a significant in chemotactic effect on Th17cells cultured in vitro.And we speculated that,in asthmaic mice,the neutrophils may beinvolved in the recruitment of Th17cells in the airway through the roles of CCL2.