Interferon-gamma-primed monocytoid cell lines: optimizing their use for invitro detection of bacterial pyrogens

In order to reduce animal testing for quality control of pharmaceutical age nts intended for parenteral use, the Limulus amebocyte lysate (LAL) assay i s now being accepted in many cases as an alternative to measuring pyrogenic activity of samples in rabbits. However, since the LAL test is specific fo r cell wall components from Gram-negative bacteria and is sometimes difficu lt to perform in samples containing large amounts of protein, this alternat ive still leaves a considerable diagnostic gap. Here, we have optimized a p reviously established test based on assessing the formation of neopterin or nitrite in interferon-gamma-treated human (THP-1) or murine (J774A.1, RAW2 64.7) monocytoid cell lines, respectively, in response to bacterial pyrogen s. Optimal results were obtained either with THP-1 cells in serum-containin g media and using a high concentration of interferon-gamma (IFN-gamma) or w ith RAW264.7 cells in serum-free media and independent of the IFN-gamma dos e. Results were significantly correlated with those obtained by another cel l-culture-based assay in which formation of tumor necrosis factor-alpha by THP-1 1G3 cells was assessed. Also in RAW264.7 murine monocytoid cells, for mation of nitrite and of tumor necrosis factor-alpha in response to a varie ty of samples was correlated. Samples shown to be pyrogenic in rabbits in a previous study were unambiguously detected with the test presented here. A s expected, the LAL test was negative with cell-free supernatants from Stap hylococcus aureus, but nevertheless a strong activation of IFN-gamma-primed monocytoid cells was observed. Gel filtration experiments suggest that thi s activity is mediated by compounds of various molecular masses (6.5 to >66 kDa). Taken together, these results indicate that the use of monocytoid ce ll lines and the detection of metabolites which are triggered in the course of immunostimulation could fill the gap left by the LAL test and help to f urther reduce animal testing for pyrogens. (C) 2000 Elsevier Science B.V. A ll rights reserved.