Volume 12 Supplement 5
Downregulation of IL-6 and preserved reactive oxygen species production in human monocytes tolerized by lipopolysaccharide and challenged with Toll-like receptor agonists and whole Gram-negative and Gram-positive bacteria
© Fernandes et al; licensee BioMed Central Ltd. 2008
Published: 18 November 2008
Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge of LPS. This mechanism is believed to be involved in the downregulation of cellular responses observed in patients with severe sepsis and septic shock. The aim of the present investigation was to evaluate the induction of tolerance in monocytes of healthy volunteers, in whole blood, after LPS exposition in vitro, assessed by intracellular cytokine detection and reactive oxygen species (ROS) generation.
Peripheral blood cells were conditioned with small doses of LPS for 18 hours and challenged with different agonists of Toll-like receptors (macrophage-activating lipopeptide-2, flagellin and LPS) and whole Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) killed bacteria. For detection of intracellular IL-6, samples of whole blood were stimulated for 6 hours. Monocytes were identified by forward-scatter and side-scatter parameters and CD14-positive staining. The samples were stained to verify the intracellular production of IL-6 on monocytes by flow cytometry. For induction of ROS, whole blood was stimulated for 30 minutes with LPS, P. aeruginosa and S. aureus. ROS production was measured by flow cytometry, using 2',7'-dichlorfluorescein-diacetate detection.
The conditioning with increasing doses of LPS resulted in lower intracellular detection of IL-6 in monocytes after the challenge with LPS. A similar effect was observed with macrophage-activating lipopeptide-2, P. aeruginosa, and S. aureus, but not with flagellin. LPS conditioning with 15 ng/ml LPS, on the other hand, resulted in preserved or increased production of ROS in monocytes after challenge with LPS, P. aeruginosa and S. aureus.
The phenomenon of tolerance involves a complex regulation, in which the production of proinflammatory cytokines, such as IL-6, is diminished, whereas the production of ROS is preserved or even increased.
This article is published under license to BioMed Central Ltd.