Multiplexed flow cytometric analyses of pro‐ and anti‐inflammatory cytokines in the culture media of oxysterol‐treated human monocytic cells and in the sera of atherosclerotic patients

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Abstract
Background: Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C‐reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7β‐hydroxycholesterol, 7‐ketocholesterol, and 25‐hydroxycholesterol), and various cytokines. Methods: Different flow cytometric bead‐based assays were used to quantify some cytokines (IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, IL‐12, IL‐13, IL‐17, G‐CSF, GM‐CSF, IFN‐γ, MCP‐1, MIP‐1β, or TNF‐α) in the culture media of oxysterol‐treated U937 and THP‐1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL‐8 mRNA levels, intracellular IL‐8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal‐regulated kinase1/2 (MEK/ERK1/2) signaling pathway. Results: All oxysterols investigated are potent in vitro inducers of MCP‐1, MIP‐1β, TNF‐α, and/or IL‐8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL‐8, IL‐1β, IL‐6, IL‐10, TNF‐α, IL‐12, and MCP‐1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF‐α, and IL‐10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered. Conclusions: Flow cytometric bead‐based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol‐treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages. © 2006 International Society for Analytical Cytology

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