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

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

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

“…MCP-1 has a crucial role in intestinal chronic inflammation characterized by a macrophage infiltration; its protein expression was augmented in the mucosa of patients with IBD [34]. Contradictory results come from other studies on human monocytic U937 and THP-1 that reported the high capability of 20 mM 7b-OH to induce IL-8 secretion, but not MCP-1 [35,36]. These data agree with our previous results on differentiated CaCo-2 cells that reported a significant up-regulation of both expression and synthesis of IL-8 by 7b-OH; however undifferentiated intestinal cells behave differently.…”

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

“…In U937 and THP-1 cells, stimulation of the secretion of various chemokines involved in the recruitment of immunocompetent cells at the subendothelial level, namely MCP-1, MIP-1ß, TNF-α, IL-1ß and IL-8, was also reported, mainly in the presence of 7ß-hydroxycholesterol and 25-hydroxycholesterol (54). Treatment of aortic rat smooth muscle cells with 25-hydroxycholesterol or 22R-hydroxycholesterol causes the accumulation of group IIA secretory phospholipase A(2) and increases enzyme activity (55).…”

“…Similarly, little information is available on oxysterolactivated metabolic pathways at the translational and posttranslational levels. However, in 7ß-hydroxycholesteroltreated U937 and THP-1 cells, it has been clearly established that IL-8 secretion is associated with activation of the MEK/ERK1/2 signaling pathway (54). Interestingly, in the presence of PD98059, a potent inhibitor of MEK, IL-8 secretion was totally counteracted, whereas no decrease in the IL-8 mRNA level or intracellular IL-8 was observed (54).…”

Inflammation-related gene expression by lipid oxidation-derived products in the progression of atherosclerosis

“…The same study demonstrated that IL-8 secretion was associated with activation of the ERK1/2 signaling pathway [31]. The oxysterols 7-K and 25-OH have also been observed to enhance IL-1 , IL-6, IL-8 and TNF-mRNA and secretion levels, in a dose-dependent manner, although to different extents.…”

“…Further, in human monocytic cells, 7 -OH and 25-OH, but also to a lesser extent 7-K, are potent in vitro inducers of MCP-1, IL-8, IL-1 , tumor necrosis factor-(TNF-), macrophage inflammatory protein-1 (MIP-1 ), as well as of other inflammatory molecules [31]. The same study demonstrated that IL-8 secretion was associated with activation of the ERK1/2 signaling pathway [31].…”