Interferon-γ-primed monocytoid cell lines: optimizing their use for in vitro detection of bacterial pyrogens

Abstract

In order to reduce animal testing for quality control of pharmaceutical agents intended for parenteral use, the Limulus amebocyte lysate (LAL) assay is now being accepted in many cases as an alternative to measuring pyrogenic activity of samples in rabbits. However, since the LAL test is specific for cell wall components from Gram-negative bacteria and is sometimes difficult to perform in samples containing large amounts of protein, this alternative still leaves a considerable diagnostic gap. Here, we have optimized a previously established test based on assessing the formation of neopterin or nitrite in interferon-γ-treated human (THP-1) or murine (J774A.1, RAW264.7) monocytoid cell lines, respectively, in response to bacterial pyrogens. Optimal results were obtained either with THP-1 cells in serum-containing media and using a high concentration of interferon-γ (IFN-γ) or with RAW264.7 cells in serum-free media and independent of the IFN-γ dose. Results were significantly correlated with those obtained by another cell-culture-based assay in which formation of tumor necrosis factor-α by THP-1 1G3 cells was assessed. Also in RAW264.7 murine monocytoid cells, formation of nitrite and of tumor necrosis factor-α in response to a variety of samples was correlated. Samples shown to be pyrogenic in rabbits in a previous study were unambiguously detected with the test presented here. As expected, the LAL test was negative with cell-free supernatants from Staphylococcus aureus, but nevertheless a strong activation of IFN-γ-primed monocytoid cells was observed. Gel filtration experiments suggest that this activity is mediated by compounds of various molecular masses (6.5 to >66 kDa). Taken together, these results indicate that the use of monocytoid cell 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 further reduce animal testing for pyrogens.