杂色曲霉素暴露对机体免疫调节细胞的影响及其可能机制的研究
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摘要
杂色曲霉素(Sterigmatocystin, ST)是由杂色曲霉、构巢曲霉等产生的一种具有致癌性的代谢产物。杂色曲霉素产生菌及ST在世界各地粮食及饲料中的污染比较普遍,甚至在潮湿居所的地毯中也检出了ST的污染。研究表明,饲喂含有ST的饲料或灌喂ST均可诱发实验动物肺腺癌的发生。ST可以诱发体外培养人胚胃粘膜细胞的恶性转化。除致癌作用外,ST对免疫细胞的增殖、凋亡、细胞因子分泌、抗原递呈等均有一定影响。
免疫系统是机体抵御外界因素侵害的重要屏障,许多致病因素可通过削弱甚至摧毁机体的免疫功能而致病。某些致癌因素也可通过直接和间接作用造成机体免疫细胞改变,特别是免疫监视机能的改变而发挥致癌作用。目前有关ST诱发免疫损伤的机制研究多集中在淋巴细胞细胞的增殖、凋亡、细胞因子分泌等直接的免疫反应方面,对免疫调节细胞的研究甚少。因此,在整体水平和细胞水平上探讨给予ST作用对机体免疫调节功能的影响及其机制对进一步揭示ST的致癌机理和其它生物效应具有一定学术意义。
调节性T淋巴细胞是机体免疫系统的重要组成部分,在防止自身免疫,保持机体的自身耐受性中发挥着决定性作用,但同时也具有减弱机体对肿瘤和感染免疫反应的作用。FoxP3阳性表达是鉴定调节性T淋巴细胞的重要标志。树突状细胞(DCs)是机体免疫系统中一种重要的抗原递呈细胞(APC),可与T、B和NK细胞相互作用,并促进其激活、分化和产生效应;诱导或调节初级或次级免疫反应。浆细胞样树突状细胞(pDCs)是树突状细胞家族中的成员之一,在慢性炎症及自身免疫性疾病的发生发展中发挥着重要的作用。钙调神经磷酸酶(calcineurin, CaN)是Ca2+依赖的信号转导途径的关键蛋白,在体内参与了多种淋巴细胞的激活;皮肤T细胞趋化因子27 (Ccl27)是肿瘤相关T淋巴细胞的重要调节因子,它们的活性变化与多种皮肤疾病的发生发展有关。
迄今为止,文献中尚未见有关ST对调节性T淋巴细胞和浆细胞样树突状细胞,以及对CaN和Ccl27表达影响的报道。为进一步探讨ST对机体免疫调节功能的影响及其可能机制,本研究在前期实验研究的基础上,在整体水平上观察了ST急性作用对BALB/c小鼠外周血、胸腺、脾脏和皮肤中FoxP3+调节性T淋巴细胞和CD123+/BDCA2+浆细胞样树突状细胞的影响,以及小鼠皮肤中CaN和Ccl27表达变化;在细胞水平上研究了ST急性作用对人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响,同时探讨了MAPK信号转导通路在ST影响FoxP3+调节性T淋巴细胞中的作用。
本研究论文共分为三个部分:
1杂色曲霉素对BALB/c小鼠外周血及免疫器官中免疫调节细胞的影响研究
目的:探讨腹腔注射ST对BALB/c小鼠外周血单个核细胞及免疫器官中FoxP3+调节性T淋巴细胞和浆细胞样树突状细胞的影响。
方法:按照3μg/kg、30μg/kg、300μg/kg、3000μg/kg经腹腔注射一次性给予雄性BALB/c小鼠ST处理,眼球放血收集小鼠外周静脉全血,采用聚蔗糖-泛影葡胺密度梯度离心法分离小鼠外周静脉血单个核细胞;处死小鼠,胸腺、脾脏组织完整取材。采用FCM检测小鼠外周血单个核细胞、胸腺和脾脏内CD4+、CD8+和FoxP3+T淋巴细胞百分率;免疫组化检测小鼠胸腺和脾脏内FoxP3+调节性T细胞及CD123+/BDCA2+浆细胞样树突状细胞数量变化情况;蛋白免疫印迹和RT-PCR方法检测FoxP3、CD123和BDCA2在蛋白和mRNA水平的影响。
结果:
1.1 ST对小鼠外周血单个核细胞中CD4+、CD8+和FoxP3+T淋巴细胞的影响
FCM检测结果显示,ST处理24 h,与溶剂对照组相比,不同剂量ST对小鼠外周血单个核细胞中CD4+和CD8+T淋巴细胞百分率均没有明显影响。但随ST浓度的增加,FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(r=0.862, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,与溶剂对照组相比,小鼠外周血单个核细胞FoxP3 mRNA表达明显升高(P<0.05);并且与ST浓度有明显剂量依赖关系(r=0.944, n=5, P<0.01)。
1.2 ST对小鼠胸腺和脾脏内FoxP3+T淋巴细胞的影响
FCM检测结果显示,ST处理24 h,与溶剂对照组相比,不同剂量ST对小鼠胸腺CD4+ T淋巴细胞百分率没有明显影响,但小剂量ST(3μg/kg)处理组小鼠胸腺CD8+ T淋巴细胞百分率可见明显下降(P<0.05)。3和30μg/kg ST处理组脾脏CD4+和CD8+ T淋巴细胞百分率均有明显升高(P<0.05)。而300μg/kg和3000μg/kg ST处理组胸腺和脾脏CD4+和CD8+ T淋巴细胞百分率均无明显变化。但各不同剂量ST处理均可明显提高胸腺和脾脏FoxP3+ T淋巴细胞的百分率,随ST浓度的增加,胸腺和脾脏FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(P<0.05)。
免疫组化结果显示,ST 30μg/kg、300μg/kg和3000μg/kg剂量组胸腺内淋巴细胞FoxP3+T淋巴细胞标记指数明显高于相应溶剂对照组(P<0.05);ST各处理组脾脏内FoxP3+ T淋巴细胞标记指数也均明显高于相应溶剂对照组(P<0.05)。在0～3000μg/kg ST浓度范围内,胸腺和脾脏FoxP3+T淋巴细胞数与ST处理浓度有明显剂量依赖关系(胸腺: r=0.831, n=5, P<0.01;脾: r=0.873, n=5, P<0.01)。
Western印迹检测结果显示,与溶剂对照组相比,在0～3000μg/kg ST浓度范围内,胸腺和脾脏淋巴细胞FoxP3表达量逐渐升高,与ST浓度有明显剂量依赖关系(胸腺: r=0.658, n=5, P<0.01;脾: r=0.621, n=5, P<0.01)。RT-PCR检测结果表明,给予ST作用24 h后,小鼠胸腺和脾脏淋巴细胞FoxP3 mRNA表达明显升高(P<0.05)。
1.3 ST对小鼠胸腺和脾脏内浆细胞样树突状细胞的影响
免疫组织化学染色结果显示,30、300和3000μg/kg ST组胸腺细胞CD123阳性标记指数明显低于溶剂对照组(P<0.05);而在脾脏细胞CD123阳性标记指数明显高于相应溶剂对照组(P<0.05);ST中、高剂量处理组胸腺细胞BDCA2阳性标记指数明显低于相应溶剂对照组(P<0.05);而各ST处理组脾脏细胞BDCA2阳性标记指数均明显高于相应溶剂对照组(P<0.05);小鼠胸腺和脾脏CD123+和BDCA2+细胞数的变化与ST的处理浓度存在明显剂量依赖关系。
Western印迹检测结果显示,与溶剂对照组相比,在0～3000μg/kg ST浓度范围内,胸腺淋巴细胞CD123和BDCA2蛋白的表达量逐渐降低,并与ST浓度呈显著负相关关系(CD123: r=-0.825, n=5, P<0.01; BDCA2:r=-0.831, n=5, P<0.01);而脾脏淋巴细胞CD123和BDCA2蛋白的表达量则逐渐升高(CD123: r=0.819, n=5, P<0.01; BDCA2: r=0.756, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,小鼠胸腺细胞CD123 mRNA表达较溶剂对照组明显降低(P<0.05),并与ST浓度有明显剂量依赖关系(r=-0.944, n=5, P<0.01);而脾脏细胞的CD123 mRNA则均较溶剂对照组明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.906, n=5, P<0.01)。
可见ST对胸腺和脾脏浆细胞样树突状细胞的影响具有器官特异性,不同器官影响不同。
2 ST对小鼠皮肤组织CaN、Ccl27表达以及FoxP3+调节性T淋巴细胞影响的研究
目的:探讨一次性腹腔注射给予ST对小鼠皮肤组织CaN和Ccl27表达,以及FoxP3+调节性T淋巴细胞浸润的影响。
方法:实验动物模型同前一部分,全层皮肤1×1cm2取材。取部分皮肤组织4%多聚甲醛固定4 h后,常规石蜡切片制备,按S-P法进行免疫组织化学染色检测小鼠皮肤组织CaN+、Ccl27+、FoxP3+细胞数量变化;取100 mg皮肤组织加入500μL蛋白裂解液,提取皮肤组织总蛋白,Western blot方法检测CaN、Ccl27、FoxP3蛋白的表达;部分皮肤组织液氮冻存,提取组织RNA,RT-PCR方法检测CaN、Ccl27、FoxP3 mRNA的表达情况。
结果:
2.1皮肤组织病理学变化
病理形态学观察可见各ST处理组动物与溶剂对照组相比,皮肤组织结构均无明显病理性改变。
2.2 ST对小鼠皮肤组织细胞CaN和Ccl27表达的影响
免疫组织化学染色结果显示,30、300和3000μg/kg组CaN阳性标记指数明显高于溶剂对照组(P<0.05),并且与ST的处理浓度存在剂量依赖关系(r=0.752, n=5, P<0.01);而Ccl27阳性标记指数与相应溶剂对照组相比,均没有明显差别。
Western blot检测结果显示,与溶剂对照组相比,随着ST处理浓度的增加,CaN蛋白表达逐渐升高,其中30,300,3000μg/kg ST组蛋白表达升高尤为明显(P<0.05),并与ST的处理浓度存在正相关关系(r=0.931, n=5, P<0.01)。同时,ST 30,300和3000μg/kg处理后相对Ccl27蛋白相对表达量与溶剂对照组相比有升高趋势,但没有统计学意义。
RT-PCR检测结果表明,给予ST作用24h后,小鼠表皮细胞CaN mRNA的表达明显高于溶剂对照组,并且与ST浓度有明显剂量依赖关系(r=0.843, n=5, P<0.01);但Ccl27 mRNA的表达没有明显变化。
2.3 ST对小鼠皮肤内FoxP3+T淋巴细胞的影响
免疫组化染色结果表明,ST 300μg/kg和3000μg/kg处理组FoxP3+T淋巴细胞标记指数分别为3.30±0.675和4.70±0.949,明显高于相应溶剂对照组(1.40±0.966, P<0.05),并且在ST 0～3000μg/kg浓度范围内,随着ST处理浓度的升高,FoxP3+T淋巴细胞标记指数也逐渐增高(r=0.757, n=5, P<0.01)。
Western blot检测结果显示,腹腔给予ST作用24 h后,小鼠皮肤组织FoxP3蛋白的表达量明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.781, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,小鼠皮肤组织FoxP3 mRNA的表达明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.749, n=5, P<0.01)。
3杂色曲霉素对体外培养人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响及其可能的机制研究
目的:探讨一次性给予ST处理对人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响及其可能的机制
方法:
采用聚蔗糖-泛影葡胺密度梯度离心法分离人外周静脉血单个核细胞(HPBMCs),培养48 h后,用10% 1640培养基调整细胞浓度为(1~2)×108个/L,接种于细胞培养瓶中。更换2%低血清1640培养基,实验组分别给予ST 100μg/L、500μg/L、1000μg/L和2000μg/L处理,溶剂对照组和对照组分别给予DMSO (0.2 mL/L)和生理盐水处理,继续细胞培养24 h。收集细胞,采用FCM方法检测HPBMCs中CD4+、CD8+和FoxP3+T淋巴细胞百分率;采用Western blot方法检测HPBMCs中FoxP3、JNK、ERK、p38蛋白表达情况和JNK、ERK和p38的磷酸化水平。采用RT-PCR方法检测HPBMCs中FoxP3、JNK、ERK和p38 mRNA的表达情况。
取培养48 h后HPBMCs用10% 1640调整细胞浓度为(1~2)×108个/L,接种于接种于培养瓶。实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂预处理+ST处理组。细胞培养24 h后,更换2%低血清1640培养基,阻断剂组分别加入MAPK特异性阻断剂SP600125 (1μM)、PD98059 (50μM)和SB203580 (0.5μM)。30 min后ST处理组和阻断剂预处理+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组给予DMSO (0.1 mL/L)和生理盐水处理。HPBMCs培养24 h后离心收细胞,采用Western blot方法检测细胞内FoxP3蛋白表达和JNK、ERK、p38磷酸化水平。采用RT-PCR方法检测HPBMCs中FoxP3 mRNA的表达情况。
结果:
3.1 ST对体外培养人外周血单个核细胞中CD4+、CD8+和FoxP3+T淋巴细胞影响
FCM检测结果显示,ST处理24 h后,与溶剂对照组相比,不同剂量ST对人外周血单个核细胞中CD4+和CD8+T淋巴细胞百分率均没有明显影响。但ST处理可明显提高FoxP3+T淋巴细胞的百分率,并且在0～2000μg/L浓度范围内,随着ST浓度的增加,FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(r=0.920, n=3, P<0.01)。
3.2体外培养人外周血单个核细胞FoxP3蛋白表达的变化情况
Western检测结果表明,在0～2000μg/L ST浓度范围内,随着ST处理浓度的升高,细胞内FoxP3蛋白表达逐渐升高(r=0.868, n=3, P<0.01)。
3.3体外培养人外周血单个核细胞FoxP3 mRNA的变化情况
RT-PCR检测表明,给予ST作用24 h后,与溶剂对照组相比,体外培养人外周血单个核细胞FoxP3 mRNA表达明显升高(P<0.05);并且与ST浓度有明显剂量依赖关系(r=0.793, n=3, P<0.01)。
3.4 ST对人外周血单个核细胞中MAPK信号传导通路的影响
3.4.1 ST对细胞内JNK、ERK和p38表达的影响
Western检测结果表明,与溶剂对照组相比,不同浓度ST作用于人外周血单个核细胞24 h,对细胞内JNK、ERK和p38蛋白的表达没有明显影响。但在0～2000μg/L ST浓度范围内,随着ST处理浓度的升高,细胞内JNK和ERK的磷酸化水平也逐渐升高(JNK: r=0.831, n=3, P<0.01;ERK: r=0.687, n=3, P<0.01),而p38的磷酸化水平则随着ST处理浓度升高而逐渐降低(r=-0.661, n=3, P<0.01)。
RT-PCR结果显示,与溶剂对照组相比,不同浓度ST作用24 h,对人外周血单个核细胞内JNK、ERK和p38 mRNA的表达也没有明显影响。
3.4.2 MAPK信号传导通路特异性阻断剂对ST诱导的细胞内JNK、ERK和p38表达变化的影响
Western检测结果表明,SP600125和PD98059分别能够明显阻断ST诱导的细胞内JNK和ERK的磷酸化水平升高作用(P<0.05)。1000μg/L ST和SB203580 (0.5μM)单独处理,均可明显抑制细胞内p38的磷酸化水平(P<0.05),0.5μM SB203580预处理可使1000μg/L ST诱导的p38的磷酸化水平降低更显著(P<0.05)。
3.5 MAPK信号传导通路对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
3.5.1 JNK信号传导通路的激活对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明, SP600125预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达则明显高于溶剂对照组(P<0.05),而与1000μg/L ST处理组没有明显差别(P>0.05)。
RT-PCR结果显示,SP600125预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达与1000μg/L ST处理组也没有明显差别(P>0.05),同样明显高于溶剂对照组(P<0.05)。
结果提示,SP600125预处理对ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用没有明显影响。
3.5.2 ERK信号传导通路的激活对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明,PD98059预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达较1000μg/L ST处理组明显降低(P<0.05),而与溶剂对照组没有差别(P>0.05)。
RT-PCR结果显示,PD98059预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达也明显低于1000μg/L ST处理组(P<0.05),而与溶剂对照组相比没有明显差异(P>0.05)。
结果提示,PD98059预处理可以阻断ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用。
3.5.3 p38信号传导通路的受抑制对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明,与溶剂对照组相比,SB203580处理组细胞内FoxP3蛋白的表达量明显升高(P<0.05),SB203580预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达也明显高于溶剂对照组,同时较1000μg/L ST处理组也明显升高(P<0.05)。
RT-PCR结果显示,SB203580预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达明显高于1000μg/L ST处理组和溶剂对照组(P<0.05)。
结果提示,SB203580预处理可以促进ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用。
结论:
1.一次腹腔注射ST处理24 h,可使BALB/c小鼠外周血单个核细胞中FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在mRNA水平的表达。
2.一次腹腔注射ST处理24 h,可使BALB/c小鼠胸腺和脾脏内FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在蛋白和mRNA水平的表达。
3.腹腔注射ST对浆细胞样树突状细胞的影响具有器官特异性。可使BALB/c小鼠胸腺内CD123+/BDCA2+浆细胞样树突状细胞数明显减少,而脾脏内CD123+/BDCA2+浆细胞样树突状细胞数明显增多。同时可以下调BALB/c小鼠胸腺内CD123和BDCA2蛋白及CD123 mRNA的表达;而上调小鼠脾脏内CD123和BDCA2蛋白及CD123 mRNA的表达。
4.一次腹腔注射ST处理24 h,可使BALB/c小鼠皮肤CaN在蛋白和mRNA水平上的表达增高;但对Ccl27的表达没有明显影响。
5.一次腹腔注射ST处理24 h,可使BALB/c小鼠皮肤FoxP3+调节性T淋巴细胞增多,并且上调FoxP3在蛋白和mRNA水平上的表达。
6.一次性给予ST处理24 h,可使体外培养人外周血单个核细胞中FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在蛋白和mRNA水平的表达。
7.一次性给予ST处理24 h,可以激活体外培养人外周血单个核细胞JNK和ERK信号转导通路,同时抑制p38信号转导通路。
8.给予JNK特异性阻断剂SP600125预处理对ST诱导FoxP3的表达升高没有影响;给予ERK特异性阻断剂PD98059预处理可以阻断ST对FoxP3表达升高作用;给予p38特异性阻断剂SB203580预处理对ST诱导的FoxP3表达升高有明显的协同作用。ERK和p38信号转导通路可能参与介导ST诱导体外培养人外周血单个核细胞中FoxP3表达升高的作用。
Sterigmatocystin (ST) is the carcinogenic metabolite producted by Aspergillus versicolor, Aspergillus nidulans etc. Contaminations of ST and it producing fungi are quite commonly seen in grains and animal diets all over the world. ST could be detected even in carpet dust from damp dwellings. Our previous study showed that both foodstuffs containing ST and oral administration of ST could induce adenocarcinoma of lung in NIH mice. ST could induce malignant transformation of human fetal gastric and lung cells in vitro. And studies both in vivo and in vitro showed that apart from its carcinogenic effects, ST could affect the proliferation, apoptosis and secretion of cytokines as well as the antigen presentation mechanisms of human and experimental animal immunocytes.
As we all know that immune system plays a key role in the protection of human body against injuries of different environmental factors. Wide variety of etiological factors, especially biological factors, cause human diseases by weakening or destroying immune fuctions. Some extrinic factors could also affect carcinogenesis by its direct or indirect injury effects on immune mechnisms, especially by their injury effects on immune survillence. The impairment of immune system provides important foundation for the carcinogenesis processes. Currently, studies on the mechanism of ST induced damage to the immune system have centered on the direct immune injuries, such as those on proliferation, apoptosis and cytokine secretion of lymphocytes. Few studies involved in the immune regulatory cells. Therefore, it is of very important significance to explore the effect of ST on the immune regulatory cells so as to have a better understanding of the possible carcinogenic effects and other putative the biological effects of ST on human beings.
Regulatory T cells (Tregs) are important subpopulation of T lymphocyte in immune system. Tregs are thought to be essential for maintaining tolerance to self-antigens recognised by autoreactive T cells that escape deletion in the thymus. An absence of functional Tregs has been associated with several autoimmune diseases. Tregs play important roles suppressing effective immune surveillance of carcinogen-induced tumours in intact animals. Naturally occurring Tregs specifcally express the transcription factor Foxp3 (forkhead box P3), a member of the fork-head/winged-helix family of transcription factors. Foxp3 is a master regulator of Treg development and function. Dendritic cell (DC) is an important antigen presenting cell (APC), which may interact with T, B and NK cells to promote their activation, differentiation and producing effects to induce or regulate primary or secondary immune response. Plasmacytoid dendritic cell (pDCs) is a member of DC family, and is of key importance in the development of chronic inflammation and autoimmune disease.
As a downstream target of the T cell receptor and key protein in the Ca2+-dependent signal transduction pathway,Calcineurin (CaN) participates in the activation process of various lymphocytes. Cutaneous T cell-attracting chemokine 27 (Ccl27) is an important regulatory factor for the tumor-related T lymphocytes. The changes in the activity alteration of CaN and Ccl27 are thought to be closely associated with the development of various kind of skin diseases. Up to now, no studies on the possible effects of ST on Tregs, pDCs and CaN, Ccl27 expression have been seen.
The aim of the present study is to further evaluate the putative effects of ST on the immunoregulary cells and to explore its possible mechanisms. Based on previous studies, we observed the effects of ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells of peripheral blood, thymus, spleen as well as the skin tissue, and the expression of CaN and Ccl27 in the skin was also evaluated in mice.The effects of ST on the FoxP3+ regulatory T cells in human peripheral blood mononuclear cells and on MAPK signal transduction pathway were studied.
The study includes three parts:
1 Effects of sterigmatocystin on the immunoregulary cells in peripheral blood and immune organs of BALB/c mice
Objective: To study the effects of the intraperitoneal injecting ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells in peripheral blood mononuclear cell and immune organs in BALB/c mice.
Methods: Male BALB/c mice were intraperitoneally injected with 3μg/kg, 30μg/kg, 300μg/kg and 3000μg/kg ST for 24 hours and the whole blood in peripheral vein was collected by eyeball enucleation. The murine peripheral blood mononuclear cells (MPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. Thymus and spleen were taken after all the mice were sacrificed. The percentage of CD4+/CD8+ T cells and FoxP3+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The changes of FoxP3+ regulatory T cells and CD123+/BDCA2+ plasmacytoid dendritic cells number in thymus and spleen were studied with immunohistochemical staining. The expressions of FoxP3, CD123 and BDCA2 at protein and mRNA levels were measured by Western blot and RT-PCR.
Results:
1.1 Effect of ST on CD4+, CD8+ and FoxP3+ regulatory T lymphocyte in MPBMCs
The results of FCM showed that no significant difference was found in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups with different dosage of ST in comparison with solvent control group. However, the percentage of FoxP3+ T cells was significant increased as the concentration of ST increases (r=0.862, n=5, P<0.01).
RT-PCR analysis showed that FoxP3 was increased at mRNA level in MPBMCs(P<0.05), and there was a significant dose-effect correlation between ST dosage and the expression of FoxP3 at mRNA level.
1.2 Effect of ST on FoxP3+ regulatory T lymphocyte in thymus and spleen in BALB/c mice
The results of FCM showed that 24 hours after ST treatment, no significant differences were found in in the percentage of CD4+ T cells among control, solvent control and all ST treatment groups. However, in thymus, the percentage of CD8+ T cells was decreased in small dosage ST (3μg/kg) treatment group (P<0.05). While in spleen, the increases in percentage of CD4+ and CD8+ T cells was noted in small dosage ST (3 and 30μg/kg) treatment groups (P<0.05) but not in high dosage ST (300 and 3000μg/kg) treatment groups. Attentively, FCM results showed that the percentages of FoxP3+ T cells in all ST treatment groups were significantly increased both in thymus and spleen. A dose-effect correlation was found between ST dosage and the percentages of FoxP3+ T cells (P<0.05).
In ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups, the immunohistochemical labelling index of FoxP3+ cells in thymus was obviously higher than that in solvent control group (P<0.05). And in spleen, FoxP3+ T cells in all ST treatment group were higher as compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (thymus: r=0.831, P<0.01; spleen: r=0.873, P<0.01).
The results of Western blotting showed that as compared with solvent control, the expressions of FoxP3 at protein level in all ST treatment groups were significantly increased. Within the ST dosage range from 3μg/kg to 3000μg/kg, positive correlation was found between the ST dosage and FoxP3 protein expression (thymus: r=0.658, P<0.01; spleen: r=0.621, P<0.01). The results of RT-PCR showed that FoxP3 was increased at mRNA level in murine thymus and spleen after different dosages of ST exposure for 24 hrs (P<0.05).
1.3 Effect of ST on plasmacytoid dendritic cells in thymus and spleen of BALB/c mice
In comparison with solvent controls, the immunohistochemical labelling index of CD123+ cells in ST 30μg/kg, 300μg/kg, 3000μg/kg treatment groups were obviously lower in thymus and higher in spleen(P<0.05). And the immunohistochemical labelling index of BDCA2+ cells in thymus in 30μg/kg, 300μg/kg, 3000μg/kg ST treatment groups were obviously reduced, but in spleen, that in all ST groups were increased compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of CD123~+ and BDCA2+ cells.
Western blotting results showed that, compared with solvent control group, a significant negative correlation between ST dosage and the expression of CD123 and BDCA2 at protein level in thymus could be found (CD123: r=-0.825, n=5, P<0.01; BDCA2: r=-0.831, n=5, P<0.01) within the dosage range from 3μg/L to 3000μg/kg. But in spleen, a significant positive correlation between ST dosage and the expression of CD123 and BDCA2 at protein level was seen (CD123: r=0.819, n=5, P<0.01; BDCA2: r=0.756, n=5, P<0.01).
The results of RT-PCR suggested that CD123 was decreased at mRNA level in murine thymus and was increased in spleen in all ST treatment groups (P<0.01). Results from dosage-dependent studies indicated that there was a significant dose-effect correlation between ST dosage and the expression of CD123 mRMA both in thymus and spleen (thymus: r=-0.944, n=5, P<0.01; spleen: r=0.906, n=5, P<0.01).
Thus,the results in this part suggestted that the effects of ST on pDCs be organ sprcific. The effects are totally different in central and peripheral lymphpoid oagans.
2 Effects of sterigmatocystin on the expression of CaN and Ccl27 and FoxP3~+ regulatory T lymphocyte in the skin of BALB/c mice
Objective: To evaluate the effects of single intraperitoneal administration of ST on expression of CaN and Ccl27 and the infiltration of FoxP3~+ regulatory T lymphocyte in the skin of BALB/c mice. Methods: The treatment of experimental animals was the same as in the first part. The full-thick skin specimen were obtained from the mice. Representative tissues specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin and sectioned. The changes in the number of CaN+, Ccl27+ and FoxP3~+ cells in skin tissues was studied with immunohistochemical staining method. Fresh skin tissues (100mg) for Western Blot was first homogenized in 500μL lysis buffer and then the total protein was extracted from the skin tissues and stored at -80℃. The expression of CaN, Ccl27 and FoxP3 protein was determined by Western blot. The expression of CaN, Ccl27 and FoxP3 at mRNA level was detected by semi-quantitative RT-PCR.
Results:
2.1 Effects of ST on the histopathological changes in the skin in BALB/c mice
No significant pathologial changes could be found in the skin in all the ST groups as compared with the controls.
2.2 Effects of ST on the expression of CaN and Ccl27 in the skin tissues in BALB/c mice
The immunohistochemical labelling index of CaN+ cells of the skin in ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups were obviously higher than that in solvent control group (P<0.05). And within the dosage range from 0 to 3000μg/kg, the number of CaN+ cells was correlated with ST dosage (r=0.752, n=5, P<0.01). But the immunohistochemical labelling index of Ccl27+ cells in ST treatment groups were not different from that in solvent control group.
The results of Western blot showed that as compared with solvent control, the expression of CaN at protein level was increased. A significant positive correlation could be found between ST dosage and the expression of CaN at protein level in the skin (r=0.931, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg. But there was no difference of the expression of Ccl27 protein in ST treatment groups as compared with solvent control group.
Similar to the results with immunohistochemical and Western blot, the results of RT-PCR showed that CaN was increased at mRNA level in murine skin after ST treatment for 24 h (P<0.01). And the expression of CaN mRMA in the skin had a significant dose-effect correlation with ST dosage (r=0.843, n=5, P<0.01). But no changes in Ccl27 mRNA expression could be found. 2.3 Effects of ST on FoxP3+ regulatory T lymphocyte in the skin tissues of BALB/c mice
Immunohistochemical staining results showed that the positive labelling index of FoxP3+ cells in skin tissue of 300μg/kg and 3000μg/kg ST treatment groups were 3.30±0.675 and 4.70±0.949 respectively,which was obviously higher than that in solvent control group (1.40±0.966, P<0.05, P<0.05). And within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (r=0.757, n=5, P<0.01).
The results of Western blotting showed that, in comparison with solvent control, a significant dose-effect correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.781, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg.
RT-PCR results showed that FoxP3 was increased at mRNA level in murine skin after exposure to different dosages of ST for 24 h (P<0.05). Results from concentration-dependent studies indicated that there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.749, n=5, P<0.01).
3 Effects of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cell in vitro and the possible mechanisms
Objective: To evaluate the effects of single treatment of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cells in vitro and to explore the potential mechanisms.
Methods:
The human peripheral blood mononuclear cells (HPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. After culture for 48 h, HPBMCs were harvestd, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). The medium of HPBMCs was replaced by new 1640 medium supplemented with 2% FCS 24 h later. Then the cells in ST groups were respectively treated with ST in different concentration of 100μg/L, 500μg/L, 1000μg/L and 2000μg/L, while their counterparts in solvent control and control group were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The percentage of CD4+/CD8+ T cells and FoxP3~+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The protein expression of FoxP3, JNK, ERK, p38 and the phosphorylation of JNK, ERK, p38 in HPBMCs treated with ST were determined by Western blot. The expression of FoxP3, JNK, ERK and p38 mRNA of HPBMCs cells was detected by RT-PCR.
HPBMCs were harvestd 48 h after culture, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). HPBMCs were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of HPBMCs cells was replaced by new 1640 medium supplemented with 2% FCS 24 h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (inhibitor of JNK), 50μM PD98059 (inhibitor of ERK), 0.5μM SB203580 (inhibitor of p38) and 1μM LY-294002 (inhibitor of PI3K) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The phosphorylation of JNK, ERK, p38 and the expression of FoxP3 protein in HPBMCs treated with ST were determined with Western blot. And the expression of FoxP3 mRNA of HPBMCs was detected by RT-PCR.
Results:
3.1 Effect of ST on CD4+, CD8+ and FoxP3~+ regulatory T lymphocyte in HPBMCs in vitro
The results of FCM showed that no differences were observed in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups after exposure to different dosages of ST for 24 h and solvent control group. However, the percentage of FoxP3~+ T cells was significant increased in all ST treated groups and within ST dosage range from 0 to 2000μg/L, the percentage of FoxP3~+ T cells increased as the ST dosage increased (r=0.920, n=3, P<0.01).
3.2 Effect of ST on the expression of FoxP3 protein in HPBMCs in vitro
The results of Western blot showed that as compared with solvent control, FoxP3 protein expressions were in all ST treatment groups. A significant positive correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.868, n=3, P<0.01) within the dosage range from 0 to 2000μg/L.
3.3 Effects of ST on the expression of FoxP3 mRNA in HPBMCs in vitro
The results of RT-PCR showed that the expression of FoxP3 mRNA in HPBMCs in all ST treatmen groups was increased compared with solvent control group (P<0.05), and there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.793, n=3, P<0.01).
3.4 Effects of ST on MAPK signal transduction pathway in HPBMCs
3.4.1 Effects of ST on the expression and phosphorylation of JNK, ERK and p38
The results of Western blot indicated that the expression of JNK, ERK and p38 protein in HPBMCs was not influenced by the ST treatment. No difference in the expression level of JNK, ERK and p38 was found between all the ST treatment groups and solvent control group (DMSO) (P>0.05). The phosphorylated JNK and ERK protein in ST treatment groups was significantly increased as compared with corresponding solvent control groups (p-JNK: r=0.831, n=3, P<0.01; p-ERK: r=0.687, n=3, P<0.01). A significant negative correlation between ST dosages and the phosphorylation of p38 was found (r=-0.661, n=3, P<0.01).
The results of RT-PCR confirmed that no difference in the expression of JNK, ERK and p38 mRNA in HPBMCs was found between all the ST treatment groups and corresponding solvent control group (P>0.05).
3.4.2 Effects of inhibitors on the expression of JNK, ERK and p38 induced by ST
The results of Western blot showed that SP600125 and PD98059 pretreatment could block the increase in the phosphorylation of JNK and ERK induced by ST treatment n HPBMCs in vitro (P<0.05). The phosphorylation of p38 in HPBMCs could be inhibited by either ST 1000μg/L or SB203580 (0.5μM). And SB203580 (0.5μM) pretreatment could further decrease the the phosphorylation decrease of p38 by ST (P<0.05).
3.5 Effects of MAPK signal transduction pathway on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST in vitro
3.5.1 Effects of JNK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
Western blot results showed that though the expression level of FoxP3 protein in SP600125+1000μg/L ST treated cells was significantly higher than that in solvant control group (P<0.05), but it was not different from that in 1000μg/L ST treatment cells (P>0.05).
The findings of RT-PCR confirmed the same result for the expression of FoxP3 mRNA as for the expression of FoxP3 protein.
Thus, the results in this study suggested that SP600125 had no effect on the increase of expression level of FoxP3 at protein and mRNA level induced by ST.
3.5.2 Effects of ERK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
The results of Western blot showed that the expression level of FoxP3 protein in PD98059+1000μg/L ST treatment group was significantly lower than that in 1000μg/L ST group (P<0.05). While there was no difference in the expression level of FoxP3 between PD98059+1000μg/L ST treatment group and solvant control group (P>0.05).
RT-PCR results revealed that the expression level of FoxP3 mRNA in PD98059+ST treatment group also was significantly lower than that in ST group (P<0.05), and was no different from that in solvent control group.
The results in this study indicated that the increase in the expression level of FoxP3 at protein and mRNA level induced by ST could be blocked by PD98059.
3.5.3 Effects of p38 signal transduction pathway inhibition on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
The results of Western blot showed that SB203580 treatment could increase the expression level of FoxP3 protein compared with the solvant control group, and the combination treatment of SB203580 and ST could induce a significant increase of the FoxP3 protein expression as compared with both solvant control group and ST groups (P<0.05).
The results of RT-PCR confirmed that the expression of FoxP3 mRNA in SB203580+ST treatment group was significantly increased as compared with that in solvent control group and ST group (P<0.05).
The results in this study suggested that SB203580 could promote the increase in the expression of FoxP3 at protein and mRNA level induced by ST.
Conclusions:
1. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at mRNA level in peripheral blood mononuclear cells in BALB/c mice.
2. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern both in thymus and spleen of BALB/c mice.
3. The effects of ST on pDCs are organ sprcific. The effect is totally different in central and peripheral lymphpoid oagan in BALB/c mice. Intraperitoneal administration of ST could significantly decrease the number of CD123+/BDCA2+ plasmacytoid dendritic cells in thymus, but increase that in spleen. The expression of CD123 and BDCA2 protein and CD123 mRNA was decreased in thymus while increased in spleen.
4. Intraperitoneal administration of ST could significantly increase the expression of CaN at protein and mRNA level in murine skin, but had no effect on that of Ccl27.
5. Intraperitoneal administration of ST could significantly increase the infiltration of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern in murine skin.
6. ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in HPBMCs.
7. ST could activate JNK and ERK signal trusduction pathway, but inhibit p38 signal trusduction pathway in HPBMCs in vitro.
8. JNK signal transduction pathway inhibitor SP600125 pretreatment had no effect on the increase of FoxP3 expression induced by ST. But ERK signal transduction pathway inhibitor PD98059 pretreatment could block the increase of FoxP3 expression induced by ST. And p38 signal transduction pathway inhibitor SB203580 had synergistic effect with ST on the increase of FoxP3 expression. ERK and p38 signal transduction pathway may be involved in FoxP3+ expression in HPBMCs in vitro.
免疫系统是机体抵御外界因素侵害的重要屏障,许多致病因素可通过削弱甚至摧毁机体的免疫功能而致病。某些致癌因素也可通过直接和间接作用造成机体免疫细胞改变,特别是免疫监视机能的改变而发挥致癌作用。目前有关ST诱发免疫损伤的机制研究多集中在淋巴细胞细胞的增殖、凋亡、细胞因子分泌等直接的免疫反应方面,对免疫调节细胞的研究甚少。因此,在整体水平和细胞水平上探讨给予ST作用对机体免疫调节功能的影响及其机制对进一步揭示ST的致癌机理和其它生物效应具有一定学术意义。
调节性T淋巴细胞是机体免疫系统的重要组成部分,在防止自身免疫,保持机体的自身耐受性中发挥着决定性作用,但同时也具有减弱机体对肿瘤和感染免疫反应的作用。FoxP3阳性表达是鉴定调节性T淋巴细胞的重要标志。树突状细胞(DCs)是机体免疫系统中一种重要的抗原递呈细胞(APC),可与T、B和NK细胞相互作用,并促进其激活、分化和产生效应;诱导或调节初级或次级免疫反应。浆细胞样树突状细胞(pDCs)是树突状细胞家族中的成员之一,在慢性炎症及自身免疫性疾病的发生发展中发挥着重要的作用。钙调神经磷酸酶(calcineurin, CaN)是Ca2+依赖的信号转导途径的关键蛋白,在体内参与了多种淋巴细胞的激活;皮肤T细胞趋化因子27 (Ccl27)是肿瘤相关T淋巴细胞的重要调节因子,它们的活性变化与多种皮肤疾病的发生发展有关。
迄今为止,文献中尚未见有关ST对调节性T淋巴细胞和浆细胞样树突状细胞,以及对CaN和Ccl27表达影响的报道。为进一步探讨ST对机体免疫调节功能的影响及其可能机制,本研究在前期实验研究的基础上,在整体水平上观察了ST急性作用对BALB/c小鼠外周血、胸腺、脾脏和皮肤中FoxP3+调节性T淋巴细胞和CD123+/BDCA2+浆细胞样树突状细胞的影响,以及小鼠皮肤中CaN和Ccl27表达变化;在细胞水平上研究了ST急性作用对人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响,同时探讨了MAPK信号转导通路在ST影响FoxP3+调节性T淋巴细胞中的作用。
本研究论文共分为三个部分:
1杂色曲霉素对BALB/c小鼠外周血及免疫器官中免疫调节细胞的影响研究
目的:探讨腹腔注射ST对BALB/c小鼠外周血单个核细胞及免疫器官中FoxP3+调节性T淋巴细胞和浆细胞样树突状细胞的影响。
方法:按照3μg/kg、30μg/kg、300μg/kg、3000μg/kg经腹腔注射一次性给予雄性BALB/c小鼠ST处理,眼球放血收集小鼠外周静脉全血,采用聚蔗糖-泛影葡胺密度梯度离心法分离小鼠外周静脉血单个核细胞;处死小鼠,胸腺、脾脏组织完整取材。采用FCM检测小鼠外周血单个核细胞、胸腺和脾脏内CD4+、CD8+和FoxP3+T淋巴细胞百分率;免疫组化检测小鼠胸腺和脾脏内FoxP3+调节性T细胞及CD123+/BDCA2+浆细胞样树突状细胞数量变化情况;蛋白免疫印迹和RT-PCR方法检测FoxP3、CD123和BDCA2在蛋白和mRNA水平的影响。
结果:
1.1 ST对小鼠外周血单个核细胞中CD4+、CD8+和FoxP3+T淋巴细胞的影响
FCM检测结果显示,ST处理24 h,与溶剂对照组相比,不同剂量ST对小鼠外周血单个核细胞中CD4+和CD8+T淋巴细胞百分率均没有明显影响。但随ST浓度的增加,FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(r=0.862, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,与溶剂对照组相比,小鼠外周血单个核细胞FoxP3 mRNA表达明显升高(P<0.05);并且与ST浓度有明显剂量依赖关系(r=0.944, n=5, P<0.01)。
1.2 ST对小鼠胸腺和脾脏内FoxP3+T淋巴细胞的影响
FCM检测结果显示,ST处理24 h,与溶剂对照组相比,不同剂量ST对小鼠胸腺CD4+ T淋巴细胞百分率没有明显影响,但小剂量ST(3μg/kg)处理组小鼠胸腺CD8+ T淋巴细胞百分率可见明显下降(P<0.05)。3和30μg/kg ST处理组脾脏CD4+和CD8+ T淋巴细胞百分率均有明显升高(P<0.05)。而300μg/kg和3000μg/kg ST处理组胸腺和脾脏CD4+和CD8+ T淋巴细胞百分率均无明显变化。但各不同剂量ST处理均可明显提高胸腺和脾脏FoxP3+ T淋巴细胞的百分率,随ST浓度的增加,胸腺和脾脏FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(P<0.05)。
免疫组化结果显示,ST 30μg/kg、300μg/kg和3000μg/kg剂量组胸腺内淋巴细胞FoxP3+T淋巴细胞标记指数明显高于相应溶剂对照组(P<0.05);ST各处理组脾脏内FoxP3+ T淋巴细胞标记指数也均明显高于相应溶剂对照组(P<0.05)。在0～3000μg/kg ST浓度范围内,胸腺和脾脏FoxP3+T淋巴细胞数与ST处理浓度有明显剂量依赖关系(胸腺: r=0.831, n=5, P<0.01;脾: r=0.873, n=5, P<0.01)。
Western印迹检测结果显示,与溶剂对照组相比,在0～3000μg/kg ST浓度范围内,胸腺和脾脏淋巴细胞FoxP3表达量逐渐升高,与ST浓度有明显剂量依赖关系(胸腺: r=0.658, n=5, P<0.01;脾: r=0.621, n=5, P<0.01)。RT-PCR检测结果表明,给予ST作用24 h后,小鼠胸腺和脾脏淋巴细胞FoxP3 mRNA表达明显升高(P<0.05)。
1.3 ST对小鼠胸腺和脾脏内浆细胞样树突状细胞的影响
免疫组织化学染色结果显示,30、300和3000μg/kg ST组胸腺细胞CD123阳性标记指数明显低于溶剂对照组(P<0.05);而在脾脏细胞CD123阳性标记指数明显高于相应溶剂对照组(P<0.05);ST中、高剂量处理组胸腺细胞BDCA2阳性标记指数明显低于相应溶剂对照组(P<0.05);而各ST处理组脾脏细胞BDCA2阳性标记指数均明显高于相应溶剂对照组(P<0.05);小鼠胸腺和脾脏CD123+和BDCA2+细胞数的变化与ST的处理浓度存在明显剂量依赖关系。
Western印迹检测结果显示,与溶剂对照组相比,在0～3000μg/kg ST浓度范围内,胸腺淋巴细胞CD123和BDCA2蛋白的表达量逐渐降低,并与ST浓度呈显著负相关关系(CD123: r=-0.825, n=5, P<0.01; BDCA2:r=-0.831, n=5, P<0.01);而脾脏淋巴细胞CD123和BDCA2蛋白的表达量则逐渐升高(CD123: r=0.819, n=5, P<0.01; BDCA2: r=0.756, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,小鼠胸腺细胞CD123 mRNA表达较溶剂对照组明显降低(P<0.05),并与ST浓度有明显剂量依赖关系(r=-0.944, n=5, P<0.01);而脾脏细胞的CD123 mRNA则均较溶剂对照组明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.906, n=5, P<0.01)。
可见ST对胸腺和脾脏浆细胞样树突状细胞的影响具有器官特异性,不同器官影响不同。
2 ST对小鼠皮肤组织CaN、Ccl27表达以及FoxP3+调节性T淋巴细胞影响的研究
目的:探讨一次性腹腔注射给予ST对小鼠皮肤组织CaN和Ccl27表达,以及FoxP3+调节性T淋巴细胞浸润的影响。
方法:实验动物模型同前一部分,全层皮肤1×1cm2取材。取部分皮肤组织4%多聚甲醛固定4 h后,常规石蜡切片制备,按S-P法进行免疫组织化学染色检测小鼠皮肤组织CaN+、Ccl27+、FoxP3+细胞数量变化;取100 mg皮肤组织加入500μL蛋白裂解液,提取皮肤组织总蛋白,Western blot方法检测CaN、Ccl27、FoxP3蛋白的表达;部分皮肤组织液氮冻存,提取组织RNA,RT-PCR方法检测CaN、Ccl27、FoxP3 mRNA的表达情况。
结果:
2.1皮肤组织病理学变化
病理形态学观察可见各ST处理组动物与溶剂对照组相比,皮肤组织结构均无明显病理性改变。
2.2 ST对小鼠皮肤组织细胞CaN和Ccl27表达的影响
免疫组织化学染色结果显示,30、300和3000μg/kg组CaN阳性标记指数明显高于溶剂对照组(P<0.05),并且与ST的处理浓度存在剂量依赖关系(r=0.752, n=5, P<0.01);而Ccl27阳性标记指数与相应溶剂对照组相比,均没有明显差别。
Western blot检测结果显示,与溶剂对照组相比,随着ST处理浓度的增加,CaN蛋白表达逐渐升高,其中30,300,3000μg/kg ST组蛋白表达升高尤为明显(P<0.05),并与ST的处理浓度存在正相关关系(r=0.931, n=5, P<0.01)。同时,ST 30,300和3000μg/kg处理后相对Ccl27蛋白相对表达量与溶剂对照组相比有升高趋势,但没有统计学意义。
RT-PCR检测结果表明,给予ST作用24h后,小鼠表皮细胞CaN mRNA的表达明显高于溶剂对照组,并且与ST浓度有明显剂量依赖关系(r=0.843, n=5, P<0.01);但Ccl27 mRNA的表达没有明显变化。
2.3 ST对小鼠皮肤内FoxP3+T淋巴细胞的影响
免疫组化染色结果表明,ST 300μg/kg和3000μg/kg处理组FoxP3+T淋巴细胞标记指数分别为3.30±0.675和4.70±0.949,明显高于相应溶剂对照组(1.40±0.966, P<0.05),并且在ST 0～3000μg/kg浓度范围内,随着ST处理浓度的升高,FoxP3+T淋巴细胞标记指数也逐渐增高(r=0.757, n=5, P<0.01)。
Western blot检测结果显示,腹腔给予ST作用24 h后,小鼠皮肤组织FoxP3蛋白的表达量明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.781, n=5, P<0.01)。
RT-PCR检测结果表明,给予ST作用24 h后,小鼠皮肤组织FoxP3 mRNA的表达明显升高(P<0.05),并与ST浓度有明显剂量依赖关系(r=0.749, n=5, P<0.01)。
3杂色曲霉素对体外培养人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响及其可能的机制研究
目的:探讨一次性给予ST处理对人外周血单个核细胞中FoxP3+调节性T淋巴细胞的影响及其可能的机制
方法:
采用聚蔗糖-泛影葡胺密度梯度离心法分离人外周静脉血单个核细胞(HPBMCs),培养48 h后,用10% 1640培养基调整细胞浓度为(1~2)×108个/L,接种于细胞培养瓶中。更换2%低血清1640培养基,实验组分别给予ST 100μg/L、500μg/L、1000μg/L和2000μg/L处理,溶剂对照组和对照组分别给予DMSO (0.2 mL/L)和生理盐水处理,继续细胞培养24 h。收集细胞,采用FCM方法检测HPBMCs中CD4+、CD8+和FoxP3+T淋巴细胞百分率;采用Western blot方法检测HPBMCs中FoxP3、JNK、ERK、p38蛋白表达情况和JNK、ERK和p38的磷酸化水平。采用RT-PCR方法检测HPBMCs中FoxP3、JNK、ERK和p38 mRNA的表达情况。
取培养48 h后HPBMCs用10% 1640调整细胞浓度为(1~2)×108个/L,接种于接种于培养瓶。实验分为5组,即对照组、溶剂对照组、ST处理组、阻断剂组和阻断剂预处理+ST处理组。细胞培养24 h后,更换2%低血清1640培养基,阻断剂组分别加入MAPK特异性阻断剂SP600125 (1μM)、PD98059 (50μM)和SB203580 (0.5μM)。30 min后ST处理组和阻断剂预处理+ST处理组分别给予1000μg/L ST,溶剂对照组和对照组给予DMSO (0.1 mL/L)和生理盐水处理。HPBMCs培养24 h后离心收细胞,采用Western blot方法检测细胞内FoxP3蛋白表达和JNK、ERK、p38磷酸化水平。采用RT-PCR方法检测HPBMCs中FoxP3 mRNA的表达情况。
结果:
3.1 ST对体外培养人外周血单个核细胞中CD4+、CD8+和FoxP3+T淋巴细胞影响
FCM检测结果显示,ST处理24 h后,与溶剂对照组相比,不同剂量ST对人外周血单个核细胞中CD4+和CD8+T淋巴细胞百分率均没有明显影响。但ST处理可明显提高FoxP3+T淋巴细胞的百分率,并且在0～2000μg/L浓度范围内,随着ST浓度的增加,FoxP3+T淋巴细胞百分率呈逐渐增高的趋势(r=0.920, n=3, P<0.01)。
3.2体外培养人外周血单个核细胞FoxP3蛋白表达的变化情况
Western检测结果表明,在0～2000μg/L ST浓度范围内,随着ST处理浓度的升高,细胞内FoxP3蛋白表达逐渐升高(r=0.868, n=3, P<0.01)。
3.3体外培养人外周血单个核细胞FoxP3 mRNA的变化情况
RT-PCR检测表明,给予ST作用24 h后,与溶剂对照组相比,体外培养人外周血单个核细胞FoxP3 mRNA表达明显升高(P<0.05);并且与ST浓度有明显剂量依赖关系(r=0.793, n=3, P<0.01)。
3.4 ST对人外周血单个核细胞中MAPK信号传导通路的影响
3.4.1 ST对细胞内JNK、ERK和p38表达的影响
Western检测结果表明,与溶剂对照组相比,不同浓度ST作用于人外周血单个核细胞24 h,对细胞内JNK、ERK和p38蛋白的表达没有明显影响。但在0～2000μg/L ST浓度范围内,随着ST处理浓度的升高,细胞内JNK和ERK的磷酸化水平也逐渐升高(JNK: r=0.831, n=3, P<0.01;ERK: r=0.687, n=3, P<0.01),而p38的磷酸化水平则随着ST处理浓度升高而逐渐降低(r=-0.661, n=3, P<0.01)。
RT-PCR结果显示,与溶剂对照组相比,不同浓度ST作用24 h,对人外周血单个核细胞内JNK、ERK和p38 mRNA的表达也没有明显影响。
3.4.2 MAPK信号传导通路特异性阻断剂对ST诱导的细胞内JNK、ERK和p38表达变化的影响
Western检测结果表明,SP600125和PD98059分别能够明显阻断ST诱导的细胞内JNK和ERK的磷酸化水平升高作用(P<0.05)。1000μg/L ST和SB203580 (0.5μM)单独处理,均可明显抑制细胞内p38的磷酸化水平(P<0.05),0.5μM SB203580预处理可使1000μg/L ST诱导的p38的磷酸化水平降低更显著(P<0.05)。
3.5 MAPK信号传导通路对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
3.5.1 JNK信号传导通路的激活对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明, SP600125预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达则明显高于溶剂对照组(P<0.05),而与1000μg/L ST处理组没有明显差别(P>0.05)。
RT-PCR结果显示,SP600125预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达与1000μg/L ST处理组也没有明显差别(P>0.05),同样明显高于溶剂对照组(P<0.05)。
结果提示,SP600125预处理对ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用没有明显影响。
3.5.2 ERK信号传导通路的激活对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明,PD98059预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达较1000μg/L ST处理组明显降低(P<0.05),而与溶剂对照组没有差别(P>0.05)。
RT-PCR结果显示,PD98059预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达也明显低于1000μg/L ST处理组(P<0.05),而与溶剂对照组相比没有明显差异(P>0.05)。
结果提示,PD98059预处理可以阻断ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用。
3.5.3 p38信号传导通路的受抑制对ST诱导人外周血单个核细胞中FoxP3+调节性T细胞增多的影响
Western检测结果表明,与溶剂对照组相比,SB203580处理组细胞内FoxP3蛋白的表达量明显升高(P<0.05),SB203580预处理+1000μg/L ST处理组细胞内FoxP3蛋白的表达也明显高于溶剂对照组,同时较1000μg/L ST处理组也明显升高(P<0.05)。
RT-PCR结果显示,SB203580预处理+1000μg/L ST处理组细胞内FoxP3 mRNA的表达明显高于1000μg/L ST处理组和溶剂对照组(P<0.05)。
结果提示,SB203580预处理可以促进ST诱导的FoxP3在蛋白和mRNA水平表达升高的作用。
结论:
1.一次腹腔注射ST处理24 h,可使BALB/c小鼠外周血单个核细胞中FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在mRNA水平的表达。
2.一次腹腔注射ST处理24 h,可使BALB/c小鼠胸腺和脾脏内FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在蛋白和mRNA水平的表达。
3.腹腔注射ST对浆细胞样树突状细胞的影响具有器官特异性。可使BALB/c小鼠胸腺内CD123+/BDCA2+浆细胞样树突状细胞数明显减少,而脾脏内CD123+/BDCA2+浆细胞样树突状细胞数明显增多。同时可以下调BALB/c小鼠胸腺内CD123和BDCA2蛋白及CD123 mRNA的表达;而上调小鼠脾脏内CD123和BDCA2蛋白及CD123 mRNA的表达。
4.一次腹腔注射ST处理24 h,可使BALB/c小鼠皮肤CaN在蛋白和mRNA水平上的表达增高;但对Ccl27的表达没有明显影响。
5.一次腹腔注射ST处理24 h,可使BALB/c小鼠皮肤FoxP3+调节性T淋巴细胞增多,并且上调FoxP3在蛋白和mRNA水平上的表达。
6.一次性给予ST处理24 h,可使体外培养人外周血单个核细胞中FoxP3+调节性T淋巴细胞数明显增多,并且上调FoxP3在蛋白和mRNA水平的表达。
7.一次性给予ST处理24 h,可以激活体外培养人外周血单个核细胞JNK和ERK信号转导通路,同时抑制p38信号转导通路。
8.给予JNK特异性阻断剂SP600125预处理对ST诱导FoxP3的表达升高没有影响;给予ERK特异性阻断剂PD98059预处理可以阻断ST对FoxP3表达升高作用;给予p38特异性阻断剂SB203580预处理对ST诱导的FoxP3表达升高有明显的协同作用。ERK和p38信号转导通路可能参与介导ST诱导体外培养人外周血单个核细胞中FoxP3表达升高的作用。
Sterigmatocystin (ST) is the carcinogenic metabolite producted by Aspergillus versicolor, Aspergillus nidulans etc. Contaminations of ST and it producing fungi are quite commonly seen in grains and animal diets all over the world. ST could be detected even in carpet dust from damp dwellings. Our previous study showed that both foodstuffs containing ST and oral administration of ST could induce adenocarcinoma of lung in NIH mice. ST could induce malignant transformation of human fetal gastric and lung cells in vitro. And studies both in vivo and in vitro showed that apart from its carcinogenic effects, ST could affect the proliferation, apoptosis and secretion of cytokines as well as the antigen presentation mechanisms of human and experimental animal immunocytes.
As we all know that immune system plays a key role in the protection of human body against injuries of different environmental factors. Wide variety of etiological factors, especially biological factors, cause human diseases by weakening or destroying immune fuctions. Some extrinic factors could also affect carcinogenesis by its direct or indirect injury effects on immune mechnisms, especially by their injury effects on immune survillence. The impairment of immune system provides important foundation for the carcinogenesis processes. Currently, studies on the mechanism of ST induced damage to the immune system have centered on the direct immune injuries, such as those on proliferation, apoptosis and cytokine secretion of lymphocytes. Few studies involved in the immune regulatory cells. Therefore, it is of very important significance to explore the effect of ST on the immune regulatory cells so as to have a better understanding of the possible carcinogenic effects and other putative the biological effects of ST on human beings.
Regulatory T cells (Tregs) are important subpopulation of T lymphocyte in immune system. Tregs are thought to be essential for maintaining tolerance to self-antigens recognised by autoreactive T cells that escape deletion in the thymus. An absence of functional Tregs has been associated with several autoimmune diseases. Tregs play important roles suppressing effective immune surveillance of carcinogen-induced tumours in intact animals. Naturally occurring Tregs specifcally express the transcription factor Foxp3 (forkhead box P3), a member of the fork-head/winged-helix family of transcription factors. Foxp3 is a master regulator of Treg development and function. Dendritic cell (DC) is an important antigen presenting cell (APC), which may interact with T, B and NK cells to promote their activation, differentiation and producing effects to induce or regulate primary or secondary immune response. Plasmacytoid dendritic cell (pDCs) is a member of DC family, and is of key importance in the development of chronic inflammation and autoimmune disease.
As a downstream target of the T cell receptor and key protein in the Ca2+-dependent signal transduction pathway,Calcineurin (CaN) participates in the activation process of various lymphocytes. Cutaneous T cell-attracting chemokine 27 (Ccl27) is an important regulatory factor for the tumor-related T lymphocytes. The changes in the activity alteration of CaN and Ccl27 are thought to be closely associated with the development of various kind of skin diseases. Up to now, no studies on the possible effects of ST on Tregs, pDCs and CaN, Ccl27 expression have been seen.
The aim of the present study is to further evaluate the putative effects of ST on the immunoregulary cells and to explore its possible mechanisms. Based on previous studies, we observed the effects of ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells of peripheral blood, thymus, spleen as well as the skin tissue, and the expression of CaN and Ccl27 in the skin was also evaluated in mice.The effects of ST on the FoxP3+ regulatory T cells in human peripheral blood mononuclear cells and on MAPK signal transduction pathway were studied.
The study includes three parts:
1 Effects of sterigmatocystin on the immunoregulary cells in peripheral blood and immune organs of BALB/c mice
Objective: To study the effects of the intraperitoneal injecting ST on the FoxP3+ regulatory T cells and plasmacytoid dendritic cells in peripheral blood mononuclear cell and immune organs in BALB/c mice.
Methods: Male BALB/c mice were intraperitoneally injected with 3μg/kg, 30μg/kg, 300μg/kg and 3000μg/kg ST for 24 hours and the whole blood in peripheral vein was collected by eyeball enucleation. The murine peripheral blood mononuclear cells (MPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. Thymus and spleen were taken after all the mice were sacrificed. The percentage of CD4+/CD8+ T cells and FoxP3+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The changes of FoxP3+ regulatory T cells and CD123+/BDCA2+ plasmacytoid dendritic cells number in thymus and spleen were studied with immunohistochemical staining. The expressions of FoxP3, CD123 and BDCA2 at protein and mRNA levels were measured by Western blot and RT-PCR.
Results:
1.1 Effect of ST on CD4+, CD8+ and FoxP3+ regulatory T lymphocyte in MPBMCs
The results of FCM showed that no significant difference was found in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups with different dosage of ST in comparison with solvent control group. However, the percentage of FoxP3+ T cells was significant increased as the concentration of ST increases (r=0.862, n=5, P<0.01).
RT-PCR analysis showed that FoxP3 was increased at mRNA level in MPBMCs(P<0.05), and there was a significant dose-effect correlation between ST dosage and the expression of FoxP3 at mRNA level.
1.2 Effect of ST on FoxP3+ regulatory T lymphocyte in thymus and spleen in BALB/c mice
The results of FCM showed that 24 hours after ST treatment, no significant differences were found in in the percentage of CD4+ T cells among control, solvent control and all ST treatment groups. However, in thymus, the percentage of CD8+ T cells was decreased in small dosage ST (3μg/kg) treatment group (P<0.05). While in spleen, the increases in percentage of CD4+ and CD8+ T cells was noted in small dosage ST (3 and 30μg/kg) treatment groups (P<0.05) but not in high dosage ST (300 and 3000μg/kg) treatment groups. Attentively, FCM results showed that the percentages of FoxP3+ T cells in all ST treatment groups were significantly increased both in thymus and spleen. A dose-effect correlation was found between ST dosage and the percentages of FoxP3+ T cells (P<0.05).
In ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups, the immunohistochemical labelling index of FoxP3+ cells in thymus was obviously higher than that in solvent control group (P<0.05). And in spleen, FoxP3+ T cells in all ST treatment group were higher as compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (thymus: r=0.831, P<0.01; spleen: r=0.873, P<0.01).
The results of Western blotting showed that as compared with solvent control, the expressions of FoxP3 at protein level in all ST treatment groups were significantly increased. Within the ST dosage range from 3μg/kg to 3000μg/kg, positive correlation was found between the ST dosage and FoxP3 protein expression (thymus: r=0.658, P<0.01; spleen: r=0.621, P<0.01). The results of RT-PCR showed that FoxP3 was increased at mRNA level in murine thymus and spleen after different dosages of ST exposure for 24 hrs (P<0.05).
1.3 Effect of ST on plasmacytoid dendritic cells in thymus and spleen of BALB/c mice
In comparison with solvent controls, the immunohistochemical labelling index of CD123+ cells in ST 30μg/kg, 300μg/kg, 3000μg/kg treatment groups were obviously lower in thymus and higher in spleen(P<0.05). And the immunohistochemical labelling index of BDCA2+ cells in thymus in 30μg/kg, 300μg/kg, 3000μg/kg ST treatment groups were obviously reduced, but in spleen, that in all ST groups were increased compared with solvent controls (P<0.05). Moreover, within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of CD123~+ and BDCA2+ cells.
Western blotting results showed that, compared with solvent control group, a significant negative correlation between ST dosage and the expression of CD123 and BDCA2 at protein level in thymus could be found (CD123: r=-0.825, n=5, P<0.01; BDCA2: r=-0.831, n=5, P<0.01) within the dosage range from 3μg/L to 3000μg/kg. But in spleen, a significant positive correlation between ST dosage and the expression of CD123 and BDCA2 at protein level was seen (CD123: r=0.819, n=5, P<0.01; BDCA2: r=0.756, n=5, P<0.01).
The results of RT-PCR suggested that CD123 was decreased at mRNA level in murine thymus and was increased in spleen in all ST treatment groups (P<0.01). Results from dosage-dependent studies indicated that there was a significant dose-effect correlation between ST dosage and the expression of CD123 mRMA both in thymus and spleen (thymus: r=-0.944, n=5, P<0.01; spleen: r=0.906, n=5, P<0.01).
Thus,the results in this part suggestted that the effects of ST on pDCs be organ sprcific. The effects are totally different in central and peripheral lymphpoid oagans.
2 Effects of sterigmatocystin on the expression of CaN and Ccl27 and FoxP3~+ regulatory T lymphocyte in the skin of BALB/c mice
Objective: To evaluate the effects of single intraperitoneal administration of ST on expression of CaN and Ccl27 and the infiltration of FoxP3~+ regulatory T lymphocyte in the skin of BALB/c mice. Methods: The treatment of experimental animals was the same as in the first part. The full-thick skin specimen were obtained from the mice. Representative tissues specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin and sectioned. The changes in the number of CaN+, Ccl27+ and FoxP3~+ cells in skin tissues was studied with immunohistochemical staining method. Fresh skin tissues (100mg) for Western Blot was first homogenized in 500μL lysis buffer and then the total protein was extracted from the skin tissues and stored at -80℃. The expression of CaN, Ccl27 and FoxP3 protein was determined by Western blot. The expression of CaN, Ccl27 and FoxP3 at mRNA level was detected by semi-quantitative RT-PCR.
Results:
2.1 Effects of ST on the histopathological changes in the skin in BALB/c mice
No significant pathologial changes could be found in the skin in all the ST groups as compared with the controls.
2.2 Effects of ST on the expression of CaN and Ccl27 in the skin tissues in BALB/c mice
The immunohistochemical labelling index of CaN+ cells of the skin in ST 30μg/kg, 300μg/kg and 3000μg/kg treatment groups were obviously higher than that in solvent control group (P<0.05). And within the dosage range from 0 to 3000μg/kg, the number of CaN+ cells was correlated with ST dosage (r=0.752, n=5, P<0.01). But the immunohistochemical labelling index of Ccl27+ cells in ST treatment groups were not different from that in solvent control group.
The results of Western blot showed that as compared with solvent control, the expression of CaN at protein level was increased. A significant positive correlation could be found between ST dosage and the expression of CaN at protein level in the skin (r=0.931, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg. But there was no difference of the expression of Ccl27 protein in ST treatment groups as compared with solvent control group.
Similar to the results with immunohistochemical and Western blot, the results of RT-PCR showed that CaN was increased at mRNA level in murine skin after ST treatment for 24 h (P<0.01). And the expression of CaN mRMA in the skin had a significant dose-effect correlation with ST dosage (r=0.843, n=5, P<0.01). But no changes in Ccl27 mRNA expression could be found. 2.3 Effects of ST on FoxP3+ regulatory T lymphocyte in the skin tissues of BALB/c mice
Immunohistochemical staining results showed that the positive labelling index of FoxP3+ cells in skin tissue of 300μg/kg and 3000μg/kg ST treatment groups were 3.30±0.675 and 4.70±0.949 respectively,which was obviously higher than that in solvent control group (1.40±0.966, P<0.05, P<0.05). And within the dosage range from 0 to 3000μg/kg, there was a significant dose-effect correlation between ST dosage and the percentage of FoxP3+ T cells (r=0.757, n=5, P<0.01).
The results of Western blotting showed that, in comparison with solvent control, a significant dose-effect correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.781, n=5, P<0.01) within the dosage range from 3μg/kg to 3000μg/kg.
RT-PCR results showed that FoxP3 was increased at mRNA level in murine skin after exposure to different dosages of ST for 24 h (P<0.05). Results from concentration-dependent studies indicated that there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.749, n=5, P<0.01).
3 Effects of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cell in vitro and the possible mechanisms
Objective: To evaluate the effects of single treatment of ST on FoxP3+ regulatory T lymphocyte in human peripheral blood mononuclear cells in vitro and to explore the potential mechanisms.
Methods:
The human peripheral blood mononuclear cells (HPBMCs) were isolated with Ficoll-Meglucamine Diatrizoate density gradient centrifugation. After culture for 48 h, HPBMCs were harvestd, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). The medium of HPBMCs was replaced by new 1640 medium supplemented with 2% FCS 24 h later. Then the cells in ST groups were respectively treated with ST in different concentration of 100μg/L, 500μg/L, 1000μg/L and 2000μg/L, while their counterparts in solvent control and control group were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The percentage of CD4+/CD8+ T cells and FoxP3~+ T cells was determined using Epics-XLⅡflow cytometry with immunofluorescence labeling. The protein expression of FoxP3, JNK, ERK, p38 and the phosphorylation of JNK, ERK, p38 in HPBMCs treated with ST were determined by Western blot. The expression of FoxP3, JNK, ERK and p38 mRNA of HPBMCs cells was detected by RT-PCR.
HPBMCs were harvestd 48 h after culture, centrifuged and resuspended in 1640 medium supplemented with 10% FCS at the concentration of (1~2)×108 cells/L in culture flasks (8 ml). HPBMCs were randomly divided into 5 groups: control, solvent control, ST 1000μg/L, blocking agent and blocking agent +ST 1000μg/L. The medium of HPBMCs cells was replaced by new 1640 medium supplemented with 2% FCS 24 h later. The cells of blocking agent groups were pretreated for 30 min with 1μM SP600125 (inhibitor of JNK), 50μM PD98059 (inhibitor of ERK), 0.5μM SB203580 (inhibitor of p38) and 1μM LY-294002 (inhibitor of PI3K) respectively. Then the cells in blocking agent +ST 1000μg/L group were treated with ST 1000μg/L, while these in solvent control and control groups were incubated with DMSO and saline respectively. Cells were harvested 24 h after ST treatment. The phosphorylation of JNK, ERK, p38 and the expression of FoxP3 protein in HPBMCs treated with ST were determined with Western blot. And the expression of FoxP3 mRNA of HPBMCs was detected by RT-PCR.
Results:
3.1 Effect of ST on CD4+, CD8+ and FoxP3~+ regulatory T lymphocyte in HPBMCs in vitro
The results of FCM showed that no differences were observed in the percentage of CD4+ and CD8+ T cells in MPBMCs between all the ST groups after exposure to different dosages of ST for 24 h and solvent control group. However, the percentage of FoxP3~+ T cells was significant increased in all ST treated groups and within ST dosage range from 0 to 2000μg/L, the percentage of FoxP3~+ T cells increased as the ST dosage increased (r=0.920, n=3, P<0.01).
3.2 Effect of ST on the expression of FoxP3 protein in HPBMCs in vitro
The results of Western blot showed that as compared with solvent control, FoxP3 protein expressions were in all ST treatment groups. A significant positive correlation could be found between ST dosage and the expression of FoxP3 at protein level in the skin (r=0.868, n=3, P<0.01) within the dosage range from 0 to 2000μg/L.
3.3 Effects of ST on the expression of FoxP3 mRNA in HPBMCs in vitro
The results of RT-PCR showed that the expression of FoxP3 mRNA in HPBMCs in all ST treatmen groups was increased compared with solvent control group (P<0.05), and there was a significant positive correlation between ST dosage and the expression of FoxP3 mRNA (r=0.793, n=3, P<0.01).
3.4 Effects of ST on MAPK signal transduction pathway in HPBMCs
3.4.1 Effects of ST on the expression and phosphorylation of JNK, ERK and p38
The results of Western blot indicated that the expression of JNK, ERK and p38 protein in HPBMCs was not influenced by the ST treatment. No difference in the expression level of JNK, ERK and p38 was found between all the ST treatment groups and solvent control group (DMSO) (P>0.05). The phosphorylated JNK and ERK protein in ST treatment groups was significantly increased as compared with corresponding solvent control groups (p-JNK: r=0.831, n=3, P<0.01; p-ERK: r=0.687, n=3, P<0.01). A significant negative correlation between ST dosages and the phosphorylation of p38 was found (r=-0.661, n=3, P<0.01).
The results of RT-PCR confirmed that no difference in the expression of JNK, ERK and p38 mRNA in HPBMCs was found between all the ST treatment groups and corresponding solvent control group (P>0.05).
3.4.2 Effects of inhibitors on the expression of JNK, ERK and p38 induced by ST
The results of Western blot showed that SP600125 and PD98059 pretreatment could block the increase in the phosphorylation of JNK and ERK induced by ST treatment n HPBMCs in vitro (P<0.05). The phosphorylation of p38 in HPBMCs could be inhibited by either ST 1000μg/L or SB203580 (0.5μM). And SB203580 (0.5μM) pretreatment could further decrease the the phosphorylation decrease of p38 by ST (P<0.05).
3.5 Effects of MAPK signal transduction pathway on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST in vitro
3.5.1 Effects of JNK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
Western blot results showed that though the expression level of FoxP3 protein in SP600125+1000μg/L ST treated cells was significantly higher than that in solvant control group (P<0.05), but it was not different from that in 1000μg/L ST treatment cells (P>0.05).
The findings of RT-PCR confirmed the same result for the expression of FoxP3 mRNA as for the expression of FoxP3 protein.
Thus, the results in this study suggested that SP600125 had no effect on the increase of expression level of FoxP3 at protein and mRNA level induced by ST.
3.5.2 Effects of ERK signal transduction pathway activation on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
The results of Western blot showed that the expression level of FoxP3 protein in PD98059+1000μg/L ST treatment group was significantly lower than that in 1000μg/L ST group (P<0.05). While there was no difference in the expression level of FoxP3 between PD98059+1000μg/L ST treatment group and solvant control group (P>0.05).
RT-PCR results revealed that the expression level of FoxP3 mRNA in PD98059+ST treatment group also was significantly lower than that in ST group (P<0.05), and was no different from that in solvent control group.
The results in this study indicated that the increase in the expression level of FoxP3 at protein and mRNA level induced by ST could be blocked by PD98059.
3.5.3 Effects of p38 signal transduction pathway inhibition on the increase of FoxP3+ regulatory T lymphocyte in HPBMCs induced by ST
The results of Western blot showed that SB203580 treatment could increase the expression level of FoxP3 protein compared with the solvant control group, and the combination treatment of SB203580 and ST could induce a significant increase of the FoxP3 protein expression as compared with both solvant control group and ST groups (P<0.05).
The results of RT-PCR confirmed that the expression of FoxP3 mRNA in SB203580+ST treatment group was significantly increased as compared with that in solvent control group and ST group (P<0.05).
The results in this study suggested that SB203580 could promote the increase in the expression of FoxP3 at protein and mRNA level induced by ST.
Conclusions:
1. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at mRNA level in peripheral blood mononuclear cells in BALB/c mice.
2. Intraperitoneal administration of ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern both in thymus and spleen of BALB/c mice.
3. The effects of ST on pDCs are organ sprcific. The effect is totally different in central and peripheral lymphpoid oagan in BALB/c mice. Intraperitoneal administration of ST could significantly decrease the number of CD123+/BDCA2+ plasmacytoid dendritic cells in thymus, but increase that in spleen. The expression of CD123 and BDCA2 protein and CD123 mRNA was decreased in thymus while increased in spleen.
4. Intraperitoneal administration of ST could significantly increase the expression of CaN at protein and mRNA level in murine skin, but had no effect on that of Ccl27.
5. Intraperitoneal administration of ST could significantly increase the infiltration of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in a dose-dependent pattern in murine skin.
6. ST could significantly increase the percentage of FoxP3+ regulatory T cells and the expression of FoxP3+ at protein and mRNA level in HPBMCs.
7. ST could activate JNK and ERK signal trusduction pathway, but inhibit p38 signal trusduction pathway in HPBMCs in vitro.
8. JNK signal transduction pathway inhibitor SP600125 pretreatment had no effect on the increase of FoxP3 expression induced by ST. But ERK signal transduction pathway inhibitor PD98059 pretreatment could block the increase of FoxP3 expression induced by ST. And p38 signal transduction pathway inhibitor SB203580 had synergistic effect with ST on the increase of FoxP3 expression. ERK and p38 signal transduction pathway may be involved in FoxP3+ expression in HPBMCs in vitro.
引文
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