Comparative profiling of selected pattern recognition receptor (PRR) pathways reveals functional heterogeneity of monocytic models in the monocyte activation test

The monocyte activation test (MAT) detects pyrogenic contamination from the cytokine response of human monocytes and is used as an alternative to the rabbit pyrogen test. PBMC are the reference cell source for the MAT; however, donor variability and limited supply have led to frequent substitutions with the cell lines THP-1, HL-60, and U937. Consequently, whether these lines reproduce PBMC behavior beyond that of endotoxins across receptor pathways remains unknown. In this study, we compared the IL-6 responses of PBMCs and cell lines to 10 pyrogenic stimuli acting through Toll-like receptors (TLRs) and NOD2. All four systems were assayed in parallel, and each stimulus was tested over a full dose range. Basal transcript levels of the corresponding receptors were measured using RT-qPCR in the same cells and compared with publicly available expression data. Detection limits, precision estimates, and endotoxin equivalent potencies were reported for each stimulus. PBMCs responded to every stimulus in the panel and produced the highest maximal IL-6 for most pathways; the cell lines responded only at higher ligand concentrations and over the compressed dose-response ranges. These results indicate that the relationship between primary and immortalized monocytic systems depends on the receptor engaged, rather than a fixed reduction in sensitivity. Performance against endotoxins does not predict performance against non-endotoxin pyrogens, which are the contaminants that MAT is used to detect.