Decreased inducibility of TNF expression in lipid-loaded macrophages

Abstract

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-1beta, 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-kappaB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARgamma. In contrast, oxidized LDL stimulated AP-1 and PPARgamma but inhibited NF-kappaB, 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.

Figure 1

Decreased inducibility of TNF mRNA in macrophages incubated with ac-LDL. Macrophages were cultured for 48 h in the presence of 5% LPDS ± 50 μg/mL ac-LDL. Half of the dishes were then stimulated with TNF (10 ng/mL) for 2 h. TNF mRNA was analysed by Northern blotting, using β-actin as loading control. Lanes: 1, Control; 2, TNF; 3, ac-LDL; 4, ac-LDL + TNF. Data are representative of three separate experiments.

Figure 2

Lipid loading does not decrease inducibility of NF-κB but inhibits AP-1. Macrophages were cultured for 48 h in the presence of 5% LPDS ± 50 μg/mL ac-LDL. The cells were then stimulated with TNF (10 ng/mL) for 2 h. Nuclear extracts were prepared and transcription factor activities (lanes 1–5, NF-κB; lanes 6–10, AP-1) were determined in the extracts. The arrowheads indicate the positions of specific complexes. Lanes: 1 and 6, probe only; 2 and 7, control; 3 and 8, TNF; 4 and 9, ac-LDL; 5 and 10, ac-LDL + TNF. Similar results were obtained in five independent experiments.

Figure 3

Comparison of the effects of different lipoproteins on AP-1. Macrophages were cultured for 48 h in serum-free medium (SF, lanes 2–3) or in the presence of 5% LPDS ± 50 μg/mL LDL or 50 μg/mL ac-LDL. Half of the dishes were then stimulated with TNF (10 ng/mL) for 2 h. Some dishes were treated for 2 h with ox-LDL (100 μg/mL) before addition of TNF. EMSA was performed as described in methods. The arrowhead indicates the position of AP-1 specific complexes. Lanes: 1, probe only; 2, SF; 3, SF + TNF; 4, Control (5% LPDS); 5, TNF; 6, LDL; 7, LDL + TNF; 8, ac-LDL; 9, ac-LDL + TNF; 10, ox-LDL; 11, ox-LDL + TNF. Data are representative of three independent experiments.

Figure 4

Effects of ox-LDL on NF-κB and AP-1 binding activity. Macrophages were incubated for 48 h in serum-free medium without GM-CSF and subsequently treated for 2 h with copper-oxidized LDL (ox-LDL, 100 μg/mL) or LDL stored for one month at 4°C without EDTA (old LDL, 100 μg/mL). Half of the cells were simultaneously stimulated with TNF (10 ng/mL). The same nuclear extracts were analysed for NF-κB (grey bars) and AP-1 (black bars) binding activity by EMSA followed by densitometry. Data are representative of two independent experiments.

Figure 5

Specificity tests for NF-κB binding. EMSA was performed using extracts from macrophages stimulated with TNF for 2 h. Unlabeled probe in excess (as indicated below) was used as specific competitor. The mutant competitor (see Methods), had one point mutation in the NF-κB binding site. The antibodies (2 μg/sample) were added after the radioactive probe. After addition of antibodies, incubation was continued for 1 h on ice. Lanes: 1, probe only; 2, control (TNF, 10 ng/mL, 2 h); 3, specific competitor (50X excess); 4, specific competitor (100X excess); 5, mutant competitor (50X excess); 6, mutant competitor (100X excess); 7, anti-p65 (RelA); 8, anti-p50 (NFKB1). The arrowheads indicate the positions of specific complexes. The bars mark supershifted bands. Data are representative of four separate experiments.

Figure 6

Specificity tests for AP-1 binding. EMSA was performed using extracts from macrophages stimulated with TNF for 2 h. Unlabeled probe in excess (as indicated below) was used as specific competitor. The mutant competitor (see Methods), had 2 point mutations in the AP-1 binding site, respectively. The antibodies (2 μg/sample) were added after the radioactive probe. After addition of antibodies, incubation was continued for 1 h on ice. Lanes: 1, probe only; 2, control (TNF, 10 ng/mL, 2 h); 3, specific competitor (20X excess); 4, specific competitor (40X excess); 5, mutant competitor (20X excess); 6, mutant competitor (40X excess); 7, anti-c-Fos; 8, anti-c-Jun (rabbit); 9, anti-c-Jun (goat); 10, anti-JunB; 11, anti-JunD. The arrowheads indicate the positions of specific complexes. The bars mark supershifted bands. Data are representative of four separate experiments.

Figure 7

Lipid loading does not induce activation of PPAR-γ. Macrophages were cultured for 48 h in the presence of 5% LPDS ± 50 μg/mL LDL or ac-LDL. Half of the cells were then stimulated with TNF (10 ng/mL) for 2 h. Nuclear extracts were prepared and PPAR-γ binding activities were determined in the extracts. The arrowhead indicates the position of specific complexes. Lanes: 1, control; 2, TNF; 3, LDL; 4, LDL + TNF; 5, ac-LDL; 6, ac-LDL + TNF; 7, ox-LDL; 8, ox-LDL + TNF. Similar data were obtained in three separate experiments.