脂多糖活化的单核细胞对人Th17细胞分化的影响

Effect of monocytes activated with lipopolysaccharide on human Th17 cell differentiation

目的:拟建立体外人外周血CD4+T细胞与CD14+单核细胞共培养体系,观察脂多糖(LPS)活化的单核细胞,在联合或不联合抗CD3 mAb、不同浓度和时间条件下对诱导Th17细胞分化的影响。方法:采用免疫磁珠法分离纯化健康志愿者外周血CD4+T细胞与CD14+单核细胞,在两者共培养体系中分别单加LPS或抗CD3 mAb、抗CD3 mAb刺激下添加不同浓度LPS刺激培养3 d或同时加入LPS和抗CD3 mAb刺激培养3 10 d。以流式细胞术检测细胞表面分子CD4,胞内细胞因子IL-17及IFN-γ,确定各种刺激培养条件下,诱导生成的Th17及Th1等细胞亚群占CD4+T细胞的百分比。结果:单独采用LPS或抗CD3 mAb刺激,分别仅能诱导(1.30 0.19)%、(1.10 0.21)%CD4+T细胞分泌表达IL-17,而采用LPS联合抗CD3 mAb刺激显著增加Th17频率,可达(2.01 0.46)%(P<0.05)。在抗CD3 mAb刺激条件下,LPS浓度分别为0.1μg/mL,1μg/mL,10μg/mL时,诱导生成Th17细胞频率依次为(1.92 0.21)%、(1.300.37)%、(1.01 0.25)%,随LPS浓度增加,Th17细胞生成显著减少(P<0.05)。LPS体外刺激3 d可诱导(2.130.32)%Th17细胞生成,随时间推移,Th17细胞减少(P<0.05),Th1细胞在6 d达最高频率(17.45 3.04)%,10 d时下降到(11.34 1.29)%。结论:低剂量LPS联合抗CD3mAb活化的单核细胞可在短时间内诱导CD4+T细胞向Th17细胞分化,所建立的共培养体系更加贴近类风湿关节炎等炎症性疾病体内异常免疫应答的微环境。

AIM： To investigate whether monocytes activated with lipopolysaccharide （LPS） have an effect on Thl？ cell differentiation in humans, CD4^＋ T cell and CD14^＋ monocytes activated with LPS were treated in the absence or presence of anti-CD3 mAb with various concentrations at different time points. METHODS： Purification of CD4^＋ T cell and CD14^＋ monocytes were performed by magnetic cell sorting and cultured together. Cultures were stimulated with LPS alone or anti-CD3 mAb alone or LPS plus anti-CD3 mAb for 3 days. In the anti-CD3 mAb stimulation cells were added different concentrations of LPS. Cells were activated under LPS/anti-CD3 costimulation for 3, 6, or 10 days. The percentage of IL-17^＋ T cells and INF-γ^＋ T was determined by flow cytometry. RESULTS： LPS or anti-CD3 mAb alone induced only very low levels of IL-17^＋ T cells, （1.30±0.19）%, （1.10±0.21 ） %, respectively. The percentage was substantially higher in the LPS and anti-CD3 mAb costimulationa as much as（2.01±0.46）%. In the presence of 0.1 μg/mL, 1 μg/mL, 10 μg/mL LPS, the proportion of Th17 reached to （1.92±0.21）%, （1.30±0.37）%, （1.01±0.25）%. Lowconcentration LPS （0. 1 μg/mL） stimulation favored Th17 differentiation. The highest proportion of IL-17^＋ T cells was found at day 3（2. 13±0.32）%, with levels declining at day 6 and day 10, while, Thl at day 6（17.45±3.04）%, declining at day 10. CONCLUSION： Low-concentration LPS stimulation plus anti-CD3 mAb in short term support optimal Th17 generation. Nevertheless, this model closely mimics the environment of rheumatoid arthritis in vivo and proposes an effective model for the generation of human Th17 cells.