In the present study, we have shown that uptake of ac-LDL by macrophages leads to reduced inducibility of TNF expression. Suppression of cytokine expression was associated with reduced basal and stimulated DNA binding activity of AP-1, a transcription factor known to regulate TNF expression. Even though NF-κB has been shown to regulate the expression of TNF, there are a number of reports [4
] suggesting that AP-1 sites are more important for the activation of this gene. AP-1 has been implicated in the transcriptional regulation of several other cytokines as well, including IL-1β [13
], IL-2 [14
], IL-3 [15
], IL-6 [16
], colony-stimulating factor-1 [17
], and transforming growth factor β1 [18
]. Our results suggest that AP-1 is important for the expression of TNF, even in the presence of high levels of active NF-κB.
LDL oxidized in the presence of Cu2+
has been shown to inhibit LPS or maleylated BSA-induced expression of TNF and IL-1α mRNA in mouse peritoneal macrophages [19
]. In the same study, LDL and ac-LDL suppressed LPS-induced expression of inflammatory cytokines, but the authors thought that these effects could have been due to oxidation of lipoproteins. In the present study, we have shown that ox-LDL and ac-LDL have different effects on the transcription factors NF-κB, AP-1 and PPARγ, indicating that the suppression of cytokine expression by ac-LDL was not due to oxidation of lipoproteins.
Inhibition of NF-κB by oxidized lipoproteins has been demonstrated in several studies [6
]. Other groups [23
] have reported stimulatory effects. In another study [27
], ox-LDL was shown to inhibit NF-κB after long (>20 h) incubations, even though a stimulatory effect was reported for short treatments (4 h). Our data from experiments with macrophages and smooth muscle cells suggests that oxidized LDL inhibits NF-κB during short treatments as well, but we can not completely exclude the possibility that some preparations of LDL or ox-LDL might be stimulatory. Inhibition of NF-κB by ox-LDL could attenuate the inflammatory response to oxidized lipids, but it could also make cells more susceptible to apoptosis [28
], which in vascular context has been shown to induce inflammation [29
]. In any case, the consistent activation of AP-1 by ox-LDL in macrophages and in smooth muscle cells [6
] indicates that AP-1 may in some cases be more important for the direct inflammatory effects of ox-LDL than NF-κB.
In a recent study on THP-1 macrophages, treatment with ox-LDL for 3 days decreased mRNA expression of NF-κB but increased that of c-jun
]. Ox-LDL potentiated LPS-induced TNF expression despite perturbing NF-κB activation [30
], adding further support to the notion that major changes in AP-1 levels are more predictive of changes in TNF expression than minor effects on NF-κB.
Significant amounts of LDL can be taken up by macrophages without prior oxidation. In addition to uptake of native LDL, aggregated LDL and complexes of LDL with proteoglycans, matrix proteins, mast cell granules or antibodies are efficiently taken up by macrophages [31
]. Furthermore, human monocyte-derived macrophages take up massive amounts of VLDL, which has been reported to enter the vascular wall [32
]. Another potential source of non-oxidized lipid is the phagocytosis of cellular remains and extracellular lipid deposits. It can thus be argued that substantial amounts of non-oxidized lipoproteins are taken up by macrophages in the vascular wall. The relative contribution of such lipid uptake to foam cell formation in vivo
is difficult to estimate. We chose ac-LDL instead of VLDL as the means to load macrophages with non-oxidized lipid, because these cells take up VLDL so extensively that long treatments would probably kill the cells as a result of lipid overload.
Recently, patients with familial hypercholesterolemia were reported to have significantly lower TNF production in blood stimulated ex vivo
by LPS, compared to normolipidemic controls [34
]. The study by Thai et al.
] reported suppressive effects of ac-LDL on the mRNA expression of TNF, IL-1α, and MCP-1 in mouse peritoneal macrophages, as well as on macrophage-mediated tumor cytolysis [19
]. In other studies, ac-LDL and LDL have been shown to inhibit natural killer (NK) cell function [35
], and both ox-LDL and LDL decreased TNF mRNA expression in NK cells [36
], providing indirect support for the concept that LDL uptake results in downregulation of the inflammatory functions of monocytes/macrophages. It is important to note that even though the immediate effect of LDL uptake seems anti-inflammatory, accumulation of lipoproteins and foam cells in the vascular wall indirectly results in tissue damage and inflammation that can weaken the atherosclerotic plaque.