Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants.

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production.

Potentiation of Toll-like receptor-induced cytokine production by (1-->3)-beta-D-glucans: implications for the monocyte activation test.

The monocyte activation test (MAT) has been introduced as an alternative for the detection of pyrogens in pharmaceuticals with the rabbit pyrogen test or the Limulus amebocyte lysate (LAL) test. The basis of the MAT is that pyrogens, via Toll-like receptors (TLRs) expressed on monocytes, stimulate cytokine production. Here, we report that, at concentrations that did not induce whole blood cytokine production when tested separately, (1-->3)-beta-D-glucans powerfully co-stimulated cytokine production (IL-6/IL-8) induced by ligands for TLR1/2, TLR2/6, TLR4, and TLR5.