A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens.

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. Copyright 2010 Elsevier B.V. All rights reserved.