Abstract
Macrophage death is an important feature of atherosclerosis, but the cellular mechanism for this process is largely unknown. There is increasing interest in cellular free cholesterol (FC) excess as an inducer of lesional macrophage death because macrophages accumulate large amounts of FC in vivo, and FC loading of macrophages in culture causes cell death. In this study, a cell culture model was used to explore the cellular mechanisms involved in the initial stages of FC-induced macrophage death. After 9 h of FC loading, some of the macrophages exhibited externalization of phosphatidylserine and DNA fragmentation, indicative of an apoptotic mechanism. Incubation of the cells with Z-DEVD-fluoromethylketone blocked these events, indicating dependence upon effector caspases. Macrophages from mice with mutations in either Fas or Fas ligand (FasL) demonstrated substantial resistance to FC-induced apoptosis, and FC-induced death in wild-type macrophages was blocked by an anti-FasL antibody. FC loading had no effect on the expression of cell-surface Fas but caused a small yet reproducible increase in cell-surface FasL. To determine the physiological significance of this finding, unloaded and FC-loaded Fas-deficient macrophages, which can only present FasL, were compared for their ability to induce apoptosis in secondarily added Fas-bearing macrophages. The FC-loaded macrophages were much more potent inducers of apoptosis than the unloaded macrophages, and this effect was almost completely blocked by an inhibitory anti-FasL antibody. In summary, during the early stages of FC loading of macrophages, a fraction of cells exhibited biochemical changes that are indicative of apoptosis. An important part of this event is FC-induced activation of FasL that leads to Fas-mediated apoptosis. In light of recent in vivo findings that show that apoptotic macrophages in atherosclerotic lesions express both Fas and FasL, we present a cellular model of Fas-mediated death in lesional foam cells.
Highlights
Possible causes of macrophage death in atherosclerotic lesions include cholesterol-induced cytotoxicity [12, 13], growth factor deprivation [14], and exposure to oxidized lipoproteins and other arterial wall factors such as inflammatory cytokines and nitric oxide [7, 15]
In light of recent in vivo findings that show that apoptotic macrophages in atherosclerotic lesions express both Fas and Fas ligand (FasL), we present a cellular model of Fasmediated death in lesional foam cells
This laboratory and others have been interested in death caused by excess cellular free cholesterol (FC),1 because macrophages in advanced lesions have been shown to accumulate large amounts of FC (16 –19), and FC loading of cultured macrophages is a potent inducer of cell death [12, 13]
Summary
Materials—Falcon tissue culture plastic ware used in these studies was purchased from Fisher Scientific. On the day of the experiment, the cells were washed three times with warm phosphate-buffered saline and incubated for the indicated times in 0.2 ml of 1% FBS in Dulbecco’s modified Eagle’s medium (w/v) alone or with 100 g of acetyl-LDL/ml and 10 g of compound 58035/ml, either in combination or separately as described previously [30]. To determine cell-surface Fas expression, cells were incubated for 30 min at 4 °C with a hamster anti-Fas monoclonal antibody or with non-immune hamster IgG at a concentration of 0.06 g/106 cells. To determine FasL expression, cells were incubated with 10 g/ml rabbit anti-FasL antibody or non-immune rabbit IgG followed by the addition of fluorescein isothiocyanate-conjugated antirabbit IgG in 2% FBS in RPMI (1:50) for an additional 30 min at 4 °C. Statistics—Results are presented as mean Ϯ S.E. (n ϭ 5), and the absence of error bars in the figures signifies S.E. values that are smaller than the graphic symbols
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