Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.

Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8 -/-macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells.

In sepsis, several cell types (e.g., lymphocytes) undergo apoptosis and have the potential to harm the host if not cleared by professional phagocytes. Apoptotic cells display "eat me" signals such as phosphatidylserine that can be readily recognized by phagocytes. For full engulfment of these cells, binding to integrin alpha(v)beta(3), mediated by the bridging protein, milk fat globule epidermal growth factor-factor VIII (MFG-E8), is necessary. We hypothesized that, in sepsis, phagocytosis of apoptotic cells is impaired due to decreased MFG-E8 expression and that adoptive transfer of exosomes containing MFG-E8 is beneficial. Sepsis was induced in rats by cecal ligation and puncture (CLP) and MFG-E8 expression assessed by Western blot 20 h later. Dendritic cells were generated from bone marrow cells, and secreted exosomes were collected and injected into CLP animals. Plasma cytokines (enzyme-linked immunosorbent assay) and thymocyte apoptosis (TC-Ao, annexin V) were assessed. The ability of peritoneal macrophages from septic animals to engulf apoptotic cells was determined in an ex vivo phagocytosis assay. A 10-day survival study was conducted. Cecal ligation and puncture reduced MFG-E8 protein levels in the spleen and liver by 48% and 70%, respectively, and increased TC-Ao by 1.6-fold. Injection of MFG-E8-containing exosomes, however, led to a 33% reduced detection of TC-Ao, without directly inhibiting apoptosis. In fact, peritoneal macrophages from exosome-treated rats displayed a 2.8-fold increased ability to phagocytose apoptotic thymocytes. Inhibition of MFG-E8 before injection of exosomes completely abrogated the enhanced phagocytosis. Treatment with bone marrow dendritic cell-derived exosomes also reduced plasma tumor necrosis factor alpha and interleukin (IL)-6 levels and improved survival from 44% to 81%. We conclude that, by providing the indispensable factor MFG-E8 for complete engulfment of apoptotic cells, these exosomes lead to an attenuation of the systemic inflammatory response and overall beneficial effect in sepsis.