Decreased inducibility of TNF expression in lipid-loaded macrophages

BACKGROUND: Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. RESULTS: In macrophages incubated with acetylated low density lipoprotein (ac-LDL) for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1β, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-κB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARγ. In contrast, oxidized LDL stimulated AP-1 and PPARγ but inhibited NF-κB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. CONCLUSIONS: Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages

7β-Hydroxycholesterol induces Ca 2+ oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells

In the present study, we characterize the early cytotoxic effects of 7β-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7β-hydroxycholesterol induced Ca 2+ oscillations with a frequency of ≈0.3–0.4 min −1. A few hours later, thapsigargin-sensitive Ca 2+ pools were depleted, indicating that 7β-hydroxycholesterol perturbs intracellular Ca 2+ homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7β-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7β-hydroxycholesterol and cholesterol-5α,6α-epoxide, as determined by TUNEL staining. Addition of TNFα (10 ng/ml) and IFNγ (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7β-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4–8 h as determined by [ 35S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca 2+ and ERK signals preceded the ultrastructural changes induced by 7β-hydroxycholesterol