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Background information: During phagocytosis, neutrophils internalize pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40phox, p47phox, p67phox and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide important attachment site for numerous proteins, including several oxidase subunits. Results: We investigated the kinetics of p47phox and Rac2 phagosomal membrane recruitment. Both subunits are known to interact with anionic phospholipids; we therefore addressed the role of PS in this recruitment. Phagosomal accumulation of p47phox and Rac2 tagged with fluorescent proteins was analyzed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localization and to decrease PS accessibility. During phagocytosis of opsonized zymosan, p47phox and constitutively active Rac2G12V briefly translocated to the phagosomal membrane while ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47phox recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first seven minutes following the phagosome closure. Conclusions: These results suggest that p47phox and Rac2 accumulate only transiently at the phagosome at the onset of NADPH activity and detach from the phagosome before the end of ROS production. Furthermore, lactC2, by masking PS, interfered with the phagosomal recruitment of p47phox and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation.

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67phox during phagocytosis by videomicroscopy. p67phox is involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67phox-citrine, expressed in myeloid PLB-985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67phox-citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67phox-citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67phox is stable in this complex. Furthermore, the time of the presence of p67phox-citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67phox-citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67phox for ROS production on the level of individual phagosomes.

Twenty years after the Chernobyl accident, 137-Caesium (137-Cs) is the main residual radionuclide found in contaminated areas. 137-Cs progressively integrated food chains until man, inducing a chronic contamination of large populations through daily ingestion of small quantities of 137-Cs. Some studies indicated an accumulation of 137-Cs in the thyroid, the heart and muscles. It was then suggested that 137-Cs contamination could lead to some pathologies observed within populations living onto contaminated areas. In order to determine the possible influence of Cs137 onto the immune system, we developed a mouse model of chronic contamination through drinking water with a concentration of 20 KBq.l-1 of 137-Cs. This corresponds to a daily ingestion of 6 KBq.day-1.kg of body weight, ten times higher than the highest measurements made after the Chernobyl accident. Parent animals were contaminated before mating and during gestation. After weaning, contamination of offspring was continued through drinking water. Gamma counts indicated that 137-Cs was found in all tissue tested at a concentration between 2 and 20 Bq/g of tissue. However, a progressive accumulation of 137-Cs was observed in muscles and kidney, and to a lesser extend in the heart and in femurs, mainly until 9 weeks of age. The immune system was evaluated by several means, including phenotypic and functional analyses. Phenotypic analysis of blood cells did not showed significant changes in both CD4+ and CD8+ T lymphocytes, in CD4+CD25+ Tregs, or in B220+ B lymphocytes between control and 137-Cs contaminated animals. Intrathymic differentiation was evaluated by phenotypic analysis of thymocyte subpopulation and by T cell excision circle (Trec) detection. However, no significant changes in intra-thymic differentiation were observed whatever the age of animals. Three functional tests were also used to evaluate the immune system, response of splenocytes to a mitogenic stimulation, response of splenocytes in a mixed lymphocyte reaction test, and specific response to a vaccination against tetanus toxoid. However, no significant changes were observed in 137-Cs contaminated animals as compared to control animals, even in 35 weeks-old animals. Overall, these results suggest that 137-Cs may accumulate over time in some organs such as the heart, the kidney, the femurs and muscles. However, our results also demonstrate that 137-Cs ingestion does not induce a significant modification of the immune system. This suggests that the pathologies associated with the immune system observed within populations living onto contaminated territories might not be attributed solely to the 137-Cs internal contamination.