Cultivation and biological characterization of splenic CD11clow CD45RBhing dendritic cells derived from endotoxin tolerance mice / 中华传染病杂志

ABSTRACT

Objective To isolate and culture splenic CD11clow CD45RBhigh dendritic cells (DC) derived from endotoxin tolerance (ET) mice and investigate its biological characterization.Methods Mice weighed 20 to 25 gram were completely randomized into two groups including ET group and control group with 6 each.ET mice were modeled by intraperitoneal injection of low-dose lipopolysaccharide (LPS) for several days (pretreated with LPS 0.1 μg/mouse for 5 d).Mice in control group were given the same volume of normal saline (NS).CD11clowCD45RBhighDC were isolated from spleen by magnetic activated cell sorting (MACS).The immunological phenotypes were detected by flow cytometry.The suppressive capacity of CD11clow CD45RBhigh DC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in allogenic mixed T cells reaction.The expressions of interleukin (IL)-10 and IL-12 produced by CD11clow CD45RBhigh DC were measured by enzyme-linked immunosorbent assay (ELISA).Statistical significance was analyzed through one-way analysis of variance (ANOVA).The homogeneity of variances was detected by Levene test.If variances were homogeneous,the least significant difference (LSD) test was used.If not,Dunnett T3 test was applied.Results The consistence of CD1 1 clow CD45RBhigh DC in control group was 30 ％,reaching the amount of (5.30±0.12) × 105/mouse ;In ET group,the percentage of CD11clow CD45RBhighDC achieved 80 ％ and the production was (1.20 ± 0.13) × 106/mouse the difference was statistically significant (t=3.23,P＜0.01).The cellar morphology in two groups showed no obvious difference.Compared to expression levels of all cell phenotypes (histocompatibility complex-Ⅱ,CD40 and CD80) in normal mice,the cell surface expression levels of CD11clowCD45RBhigh DC in ET mice were much lower.The difference in two groups was statistically significant.Splenic CD11clowCD45RBhighDC derived from ET mice with cell concentration of 1∶ 10,1∶50and 1∶100 had more obvious prohibitory effects on allogenic T cells (t1∶0 =1.36,P1∶10 ＜0.01,t1∶50 =2.49,P1∶50 ＜0.01,1∶100 =1.88,Pm00 ＜0.01).Secretion of IL-10 produced by CD11clowCD45RBhighDC of ET mice was significantly increased (t1∶0=13.63,P1∶10 ＜0.01,t1∶50 =13.45,P1∶50 ＜0.01,t1∶00 =9.31,P1∶00 ＜0.01),but the expression of IL-12 was lower (t1∶0 =2.62,P1∶0 =0.02,1∶∶50 =2.74,P1∶0=0.02,t1∶100 =2.99,P1∶100 =0.01).Conclusion Splenic CD11clow CD45RBhigh DC from ET mice have weaker ability of antigen presenting and allogeneic lymphocytes proliferation stimulating than those from normal mice.