The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 M C6-ceramide induced significant H 2 O 2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H 2 O 2 production was observed in C6-ceramide-treated U937 °c ells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-B and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H 2 O 2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H 2 O 2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramideinduced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.

Two types of opsonic phagocytosis have been defined depending on the receptor engaged: FcγRs mediate type I phagocytosis of IgG-coated particles; complement receptor 3 (CR3) mediates type II phagocytosis of complement-coated particles. In addition to opsonic phagocytosis, CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites. Using Chinese hamster ovary cells stably expressing human CR3, we studied CR3-mediated ingestion of nonopsonized particles, Z or M. kansasii, compared with opsonized zymosan (OZ). We show that 1) while OZ sinks into cells, Z is engulfed by pseudopodia as visualized by electron microscopy; 2) in contrast to OZ, nonopsonic phagocytosis of Z and M. kansasii depends on Rac and Cdc42 but not on Rho activity; and 3) CR3-mediated phagocytosis of Z depends on the kinase activity of the Src family tyrosine kinase Hck, while OZ internalization does not. Therefore, CR3 mediates type I phagocytosis under nonopsonic conditions and type II under opsonic conditions. This is the first evidence that a single receptor can mediate both types of phagocytosis depending on the ligand used.

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or β-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N-acetyl-d-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.

The subjective experience of color by synesthetes when viewing achromatic letters and numbers supposedly relates to real color experience, as exemplified by the recruitment of the V4 color center observed in some brain imaging studies. Phenomenological reports and psychophysics tests indicate, however, that both experiences are different. Using functional magnetic resonance imaging, we tried to precise the degree of coactivation by real and synesthetic colors, by evaluating each color center individually, and applying adaptation protocols across real and synesthetic colors. We also looked for structural differences between synesthetes and nonsynesthetes. In 10 synesthetes, we found that color areas and retinotopic areas were not activated by synesthetic colors, whatever the strength of synesthetic associations measured objectively for each subject. Voxel-based morphometry revealed no white matter (WM) or gray matter difference in those regions when compared with 25 control subjects. But synesthetes had more WM in the retrosplenial cortex bilaterally. The joint coding of real and synesthetic colors, if it exists, must therefore be distributed rather than localized in the visual cortex. Alternatively, the key to synesthetic color experience might not lie in the color system.