Free cholesterol loading of macrophages induces apoptosis involving the Fas pathway

Macrophage death is an important feature of atherosclerosis, but the cellul ar mechanism for this process is largely unknown. There is increasing inter est in cellular free cholesterol (FC) excess as an inducer of lesional macr ophage death because macrophages accumulate large amounts of FC in vitro, a nd 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 a nd DNA fragmentation, indicative of an apoptotic mechanism. Incubation of t he cells with Z-DEVD-fluoromethylketone blocked these events, indicating de pendence upon effector caspases, Macrophages from mice with mutations in ei ther Fas or Fas ligand (FasL) demonstrated substantial resistance to FC-ind uced apoptosis, and FC-induced death in wild-type macrophages was blocked b y an anti-Fast antibody. FC loading had no effect on the expression of cell -surface Fas but caused a small yet reproducible increase in cell-surface F ast. To determine the physiological significance of this finding, unloaded and FC-loaded Fas-deficient macrophages, which can only present Fast, were compared for their ability to induce apoptosis in secondarily added Fas-bea ring macrophages. The FC-loaded macrophages were much more potent inducers of apoptosis than the unloaded macrophages, and this effect was almost comp letely blocked by an inhibitory anti-Fast antibody, In summary, during the early stages of FC loading of macrophages, a fraction of cells exhibited bi ochemical changes that are indicative of apoptosis. An important part of th is event is BC-induced activation of Fast that leads to Fas-mediated apopto sis, In light of recent in vivo findings that show that apoptotic macrophag es in atherosclerotic lesions express both Fas and Fast, we present a cellu lar model of Fas-mediated death in lesional foam cells.