Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.