发热样温度对朗格汉斯细胞表面抗原递呈相关分子及Toll-like受体表达的影响

A study of the effects of molecules associated with antigen presentation and Toll-like receptors on Langerhans cells at a fever-like temperature

目的探讨发热样温度对朗格汉斯细胞(Langerhans cells,LCs)表面抗原递呈相关分子CD40、CD80、CD86、HLA-DR以及Toll-like受体(Toll-like receptor,TLR)-3、TLR-4表达的影响。方法采用Ficoll淋巴细胞分离液分离PBMCs,CD14免疫磁珠分离CD14+细胞。应用含细胞因子(rhGM-CSF 1 000IU/ml,rhIL-4 1 000IU/ml,TGF-β10ng/ml)的RPMI-1640培养基诱导培养CD14+细胞,第3d补加细胞因子,培养7d后的细胞采用流式细胞术检测表面Langerin,鉴定细胞为LCs。将LCs分别置于37℃、39.5℃、41℃、43℃5%CO2细胞培养箱中处理30min,37℃过夜恢复培养后台盼蓝染色法检测LCs的存活率,流式细胞术检测LCs表面抗原递呈相关分子CD40、CD80、CD86、HLADR以及TLR-3和TLR-4的表达情况。结果细胞因子诱导培养7d后细胞出现典型的树枝状突起,表达Langerin,表明LCs诱导成功。发热样温度处理后LCs存活率从37℃时的96.86%分别降至39.5℃时的90.09%、41℃时的86.11%和43℃时的76.78%。检测两份样品的LCs表面分子的变化。结果显示39.5℃处理的LCs表面CD86、HLADR、TLR-4的表达较37℃对照组显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加1.38、1.3和1.78倍;CD40、CD80、TLR-3的表达差异无统计学意义(P>0.05)。41℃处理的LCs表面TLR-3及TLR-4的表达显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加18.96和15.75倍;CD40、CD80、CD86、HLA-DR的表达差异无统计学意义(P>0.05)。43℃处理的LCs表面CD40、CD80、CD86的表达显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加5.32、1.36和1.63倍;HLA-DR、TLR-3、TLR-4表达显著变化(P>0.05)。结论发热样温度处理能增强LCs部分表面抗原递呈相关分子CD40、CD80、CD86、HLA-DR以及TLR-3、TLR-4的表达。其中43℃处理能显著增加CD40的表达,41℃处理能显著增加TLR-3及TLR-4的表达。

Objective To investigate the expression of molecules associated with antigen presentation （CD40, CD80, CD86, and HLA-DR） and Toll-like receptors （receptor and -4） in Langerhans cells （LCs） at a fever-like temperature. Methods PBMCs were separated and purified using Ficoll gradient centrifugation and CD14＋ cells were separated using CD14 MicroBeads. CD14＋ cells were cultured with complete RPMI-1640 medium containing cytokines （rhGM-CSF 1 000 IU/ml, rhIL-4 1 000 IU/ml, and TGF-α 10 ng/ml）, and differentiation into LCs was induced after seven days. Plates containing LCs were placed separately in an incubator at 37℃, 39.5℃, 41℃, and 43＇C for 30 minutes. Cells were then cultured at 37℃ overnight to allow them to recover. The survival rates were examined with Trypan Blue staining and the surface molecules （CD40, CD80, CD86, HLA-DR, TLR-3, and TLR-4） were examined with flow cytometry. Results Cells displayed typical branch-like projections and the expression of langerin was noted, indicating the successful induction of LCs. The survival rate of LCs at a fever-like temperature decreased from 96. 86% at 37℃ to 90. 09% at 39. 5℃, 86.11% at 41℃ , and 76.78% at 43℃. Two samples were examined. The level of expression of CD86, HLA-DR, and TLR-4 in LCs at 39.5℃ increased in comparison to that in the control group （P〈0.05）, and the mean percentage of CD86, HLA-DR, and TLR-4 positive cells increased 1.38, 1.3, and 1.78-fold, respectively, in comparison to the control group. There were no statistical differences in the level of expression of CD40, CD80, and TLR-3. In LCs treated at 41 C , the level of expression of TLR-3 and TLR-4 increased significantly （P -〈0.05）, and the mean percentage of positive cells increased 18.96 and 15.75-fold, respectively, in comparison to the control group. There were no statistical differences in the level of expression of CD40, CD80, CD86, and HLA DR. In LCs treated at 43 ℃ , the level of expression of CD40, CD80, and CD86 increased significantly （P〈0.05）, and the mean percentage of positive cells increased 5. 32, 1.36, and 1.63-fold, respectively, in comparison to the control group. There were no statistically differences in the level of expression of HLA-DR, TLR-3, and TLR-4. Conclusion The survival rate of LCs decreased with an increase in temperature. Some molecules associated with antigen presentation （e. g. CD40, CD80, CD86, and HLA-DR） and Toll like receptors （receptor-3 and 4） on LCs increased at a fever-like temperature. The level of expression of CD40 increased markedly in LCs at 43 ℃ while the level of expression of TLR-3 and TLR 4 increased markedly in LCs at 41℃.