The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Abstract

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular crosstalk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the preincubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.

Induction of apoptosis in circulating angiogenic cells by microparticles

“…Arachidonic acid is a major mediator of the cellular effects of microparticles (32,(37)(38)(39). We previously demonstrated that microparticles shuttle arachidonic acid from leukocytes to fibroblasts (30).…”

“…Fibroblasts do not take up microparticles (23,32). However, when microparticles interact with fibroblasts, they form pseudopodia-like processes; after prolonged contact, they are re-released from the cell membrane of fibroblasts without detectable morphologic changes (Distler JHW, et al: unpublished observations).…”

“…The activity of acid sphingomyelinase was inhibited using phosphatidylinositol 3,5-bisphosphate, a specific inhibitor of acid sphingomyelinase (32,33). CACs were incubated with phosphatidylinositol 3,5-bisphosphate in concentrations of 10 M for 1 hour.…”

“…We previously showed that in contrast to fibroblasts, macrophages take up microparticles (23,29,32). To investigate whether CACs can take up microparticles as well, and whether this is an active process, CACs were incubated with PKH26-labeled microparticles at 37°C or at 4°C for 2 hours.…”

Critical issues in mucosal immunity for HIV-1 vaccine development

“…80 Otherwise, a successful vaccine will need to overcome the loss of CD4 + T cells brought on by cell death and overcome any immune downregulation by cell death products.…”