Fraunhofer-Institut fuer Grenzflaechen- und Bioverfahrenstechnik, Stuttgart, Germany.
J Immunol Methods. 2010 Jun 30;358(1-2):93-103. doi: 10.1016/j.jim.2010.03.020. Epub 2010 Apr 10.
The pattern recognition receptors (PRRs) of the innate immune system are the first defence line of the immune system. Toll-like receptors (TLRs) are the most well known and the best examined of the PR receptors. In the last years TLRs had been studied in different ways resulting in a lot of new insights in the function and signalling pathways of these receptors. However, it was not possible to investigate individual combinations of the TLRs and their specific ligands, because of the complex network in immune signalling resulting in interference with each other. This work shows a new cell-based assay, established for the analysis of single PRRs or heterodimers. For this purpose NIH3T3 (mouse fibroblasts) were stably transfected with the NF-kappaB-inducible reporter gene secreted alkaline phosphatase (SEAP) together with the corresponding combinations of human TLRs and their co-receptors (e.g. TLR1/2, TLR2/6 and TLR4/CD14). The specificity of the respective cell lines was shown by induction with variations of specific and unspecific ligands (immune-stimulating components of microorganisms or synthetic ligands). Analysis via the NF-kappaB-dependent reporter gene SEAP allows a direct way to detect the human TLR-activity. Our results showed that this assay is highly sensitive and specific for the respective ligands. For the synthetic ligands Pam(2)CysSK(4) the assay demonstrates a detection limit of 1 pg/ml for TLR2/6. In summary, this test system allows the investigation of individual human PRR-receptors in a highly specific way, without interference with other immune components opening new avenues for novel insights in the innate immune system and its applications.
模式识别受体 (PRRs) 是先天免疫系统的第一道防线。 Toll 样受体 (TLRs) 是 PRR 中最著名和研究最充分的受体。在过去的几年中，TLRs 已经通过不同的方式进行了研究，从而对这些受体的功能和信号通路有了很多新的认识。然而，由于免疫信号中的复杂网络相互干扰，不可能研究 TLR 及其特定配体的单个组合。这项工作展示了一种新的基于细胞的测定方法，用于分析单个 PRRs 或异二聚体。为此，NIH3T3（小鼠成纤维细胞）与相应的人 TLR 及其共受体（例如 TLR1/2、TLR2/6 和 TLR4/CD14）一起稳定转染 NF-κB 诱导的报告基因分泌型碱性磷酸酶 (SEAP)。通过用特定和非特异性配体（微生物的免疫刺激成分或合成配体）的变化诱导来显示各自细胞系的特异性。通过 NF-κB 依赖性报告基因 SEAP 的分析，可以直接检测人 TLR 活性。我们的结果表明，该测定法对各自的配体高度敏感和特异。对于合成配体 Pam(2)CysSK(4)，该测定法对 TLR2/6 的检测限为 1 pg/ml。总之，该测试系统允许以高度特异性研究单个人类 PRR 受体，而不会与其他免疫成分相互干扰，为先天免疫系统及其应用的新见解开辟了新途径。
