Warbrick E V, Dearman R J, Basketter D A, Gerberick G F, Ryan C A, Kimber I
Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK.
J Appl Toxicol. 1998 May-Jun;18(3):205-13. doi: 10.1002/(sici)1099-1263(199805/06)18:3<205::aid-jat502>3.0.co;2-n.
It has been shown previously that cytokine secretion patterns characteristic of the activation of discrete responses by functional subsets of T cells of type 1 and type 2, respectively, are elicited following topical exposure of BALB/c strain mice to chemical contact and respiratory allergens. In order to investigate if the differences in protein profiles are paralleled by changes in steady-state mRNA levels we have now investigated cultured draining lymph node cell (LNC) cytokine mRNA expression profiles by reverse transcriptase-polymerase chain reaction (RT-PCR) under conditions where divergent cytokine secretion is observed. Mice were exposed topically by repeated application of the respiratory allergen trimellitic anhydride (TMA) or of the contact allergen 2,4-dinitrochlorobenzene (DNCB). An elevation in the expression of mRNA for interleukin 4 (IL-4) and interleukin 10 (IL-10) by LNC from both TMA- and DNCB-treated animals was observed within 6 h of culture, reaching maximal levels after 72 h. Relative mRNA levels for both of these type 2 cytokines were considerably higher in cultured cells derived from TMA-exposed mice, compared with those from DNCB-treated animals. Transient low levels of the type 1 cytokine interferon y (IFN-gamma) were observed in response to treatment with TMA, whereas a substantial upregulation of IFN-gamma gene expression was seen from 24 h onwards in cultured LNC derived from DNCB-exposed mice. Changes in cytokine mRNA in allergen-activated LNC preceded protein production, although the kinetic profiles were similar. These data suggest that the divergent cytokine secretion profiles exhibited by mice treated by repeated topical exposure to contact and respiratory allergens are controlled primarily at the level of transcription. The RT-PCR methodology described herein may be more sensitive for the detection of cytokines expressed in low copy number, such as IL-4, where previously it has been found necessary to stimulate LNC with mitogen to elicit measurable levels of protein secretion. However, this technique was not found to offer major practical advantages when compared with protein detection methods (enzyme-linked immunosorbent assays) for the routine predictive characterization of chemicals as a function of cytokine 'fingerprinting'.
先前的研究表明，将BALB/c品系小鼠局部暴露于化学接触性变应原和呼吸道变应原后，分别可引发1型和2型T细胞功能亚群激活的离散反应所特有的细胞因子分泌模式。为了研究蛋白质谱的差异是否与稳态mRNA水平的变化平行，我们现在通过逆转录聚合酶链反应（RT-PCR）研究了培养的引流淋巴结细胞（LNC）细胞因子mRNA表达谱，实验条件为观察到不同的细胞因子分泌情况。通过反复局部应用呼吸道变应原偏苯三酸酐（TMA）或接触性变应原2,4-二硝基氯苯（DNCB）使小鼠暴露。在培养6小时内，观察到来自TMA和DNCB处理动物的LNC中白细胞介素4（IL-4）和白细胞介素10（IL-10）的mRNA表达升高，72小时后达到最高水平。与来自DNCB处理动物的细胞相比，来自TMA暴露小鼠的培养细胞中这两种2型细胞因子的相对mRNA水平要高得多。在用TMA处理后，观察到1型细胞因子干扰素γ（IFN-γ）出现短暂的低水平，而在来自DNCB暴露小鼠的培养LNC中，从24小时起就观察到IFN-γ基因表达的显著上调。变应原激活的LNC中细胞因子mRNA的变化先于蛋白质产生，尽管动力学曲线相似。这些数据表明，通过反复局部暴露于接触性和呼吸道变应原处理的小鼠所表现出的不同细胞因子分泌谱主要在转录水平受到控制。本文所述的RT-PCR方法对于检测低拷贝数表达的细胞因子（如IL-4）可能更敏感，以前发现有必要用有丝分裂原刺激LNC以引发可测量水平的蛋白质分泌。然而，与蛋白质检测方法（酶联免疫吸附测定）相比，该技术在作为细胞因子“指纹识别”功能的化学物质常规预测表征方面未发现具有主要实际优势。
