Very low density lipoprotein potentiates tumor necrosis factor-α expression in macrophages
Introduction
Even without prior oxidative modification, chylomicron remnants, VLDL and IDL can be taken up in large amounts by macrophages. Lipid accumulation in macrophages leads to formation of foam cells, a distinct cell type found in fatty streaks and more advanced atherosclerotic lesions. Apart from effects on the endothelial expression of cell adhesion molecules [1], the effects of intracellular lipid accumulation on inflammatory gene expression have not been extensively studied.
VLDL (Sf 20 to 60) and IDL (Sf 12 to 20) have been shown to be independently associated with the presence, severity, and progression of atherosclerosis [2], [3]. VLDL and VLDL remnants have been found in atherosclerotic plaques [4].
Increased levels of TNF have been detected in young survivors of myocardial infarction [5]. TNF expression in humans is also elevated in atheroma [6] and in macrophages from atherosclerotic lesions [7]. Arbustini et al. [8] observed that TNF expression was highest in lipid-rich regions of atherosclerotic lesions.
It was recently reported that, compared to normolipidemic controls, patients with familial hypercholesterolemia exhibit significantly lower TNF production in blood stimulated ex vivo by LPS [9]. In contrast, the same investigators observed that LPS treatment of blood from hypertriglyceridemic patients led to production of more TNF than did the same treatment of blood from control subjects, and they also found that lowering of serum triacylglycerol decreased the TNF levels [9]. These findings suggest that VLDL and other triacylglycerol-rich lipoproteins (but not LDL) augment production of TNF, and this conclusion is supported by results obtained in hypercholesterolemic rabbits [10]. We have previously shown that treatment of LDL has a suppressive effect on TNF expression in macrophages, likely mediated by downregulation of transcription factor AP-1 [11]. In the present study, we examined how VLDL influences TNF expression. Mitogen-activated protein kinase (MAPK) pathways are known to regulate TNF expression in macrophages [12]. Using U0126, a specific inhibitor of MEK1/2 activity, we studied MAPK involvement in TNF expression induced by LPS and VLDL.
Section snippets
Materials
[γ-32P]ATP (185 TBq/mmol/L) was purchased from Amersham Biosciences (Buckinghamshire, UK). The oligonucleotides for mobility shift assays of AP-1 and NF-κB were obtained from Promega (Madison, WI) and Santa Cruz Biotechnology (Santa Cruz, CA). TNF was from R&D Systems (Minneapolis, MN). Tris–buffered saline (pH 7.2) was from Pierce (Rockford, IL) and Ficoll-Paque was purchased from Amersham Biosciences and was used according to the instructions of the manufacturer. Macrophage-SFM culture medium
Dose response study
Cells were treated with 0, 5, 10, 25, 50, 75 and 100 μg/mL VLDL for 8 h, and thereafter, medium was replaced with VLDL-free medium, and the cells were further incubated for 12 h in the absence or presence of 1 μg/mL LPS. The culture medium was collected, and TNF secretion was analyzed by ELISA. The VLDL concentration of 75 μg/mL gave the strongest, statistically significant potentiation of TNF secretion (Fig. 1A). This concentration was therefore used in subsequent experiments.
Effect of VLDL on LPS-induced TNF synthesis
In our preliminary
Discussion
Our results show that VLDL potentiates LPS-induced TNF expression in macrophages, as determined by Northern blotting and analysis of TNF in culture medium by ELISA. Activation of MEK1/2 and AP-1 was observed in cells treated with VLDL alone, but was much stronger in cells treated with LPS and VLDL. TNF expression was strongly inhibited by U0126, a specific inhibitor of MEK1/2 activity. MEK1/2 has been shown to be involved in AP-1 activation [20]. Accordingly, U0126 inhibited the VLDL-induced
Acknowledgements
This study was supported by grants from the Swedish Research Council (no. 8311), the Swedish Heart and Lung Foundation, the Wallenberg Foundation, the Swedish Medical Society, the Crafoord Foundation, Ernhold Lundström Foundation, Malmö University Hospital, The Royal Physiographic Society, and Lars Hierta Foundation. We thank Anna Larsson and Linda Andersson for expert technical assistance.
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