Accumulation of Cytochrome b558 at the Plasma Membrane: Hallmark of Oxidative Stress in Phagocytic Cells.

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.). To study these effects, we used ROS induced by gamma radiation from low (0.2 Gy) to high (25 Gy) dose levels on PLB-985 cells from a myeloid cell line differentiated to neutrophil-like cells that are considered a good alternative to neutrophils. We determined a much longer lifetime of PLB-985 cells than that of neutrophils, which, as expected, decreased by increasing the irradiation dose. In the absence of any secondary stimulus, a very low production of ROS is detected with no significant difference between irradiated and non-irradiated cells. However, in phagocytosing cells, irradiation doses above 2 Gy enhanced oxidative burst in PLB-985 cells. Whatever the irradiation dose, NADPH oxidase devoid of its cytosolic regulatory units is observed at the plasma membrane in irradiated PLB-985 cells. This result is different from that observed for irradiated neutrophils in which irradiation also induced a translocation of regulatory subunits suggesting that the signal transduction mechanism or pathway operate differently in both cells.

Radiation-induced reactive oxygen species partially assemble neutrophil NADPH oxidase.

Neutrophils are key cells from the innate immune system that destroy invading bacteria or viruses, thanks mainly to the non-mitochondrial reactive oxygen species (ROS) generated by the enzyme NADPH oxidase. Our aim was to study the response of neutrophils to situations of oxidative stress with emphasis on the impact on the NADPH oxidase complex. To mimic oxidative stress, we used gamma irradiation that generated ROS (OH•, O2•- and H2O2) in a quantitative controlled manner. We showed that, although irradiation induces shorter half-lives of neutrophil (reduced by at least a factor of 2), it triggers a pre-activation of surviving neutrophils. This is detectable by the production of a small but significant amount of superoxide anions, proportional to the dose (about 3 times that of sham). Investigations at the molecular level showed that this ROS increase was generated by the NADPH oxidase enzyme after neutrophils irradiation. The NADPH oxidase complex undergoes an incomplete assembly which includes p47phox and p67phox but excludes the G-protein Rac. Importantly, this irradiation-induced pre-activation is capable of considerably improving neutrophil reactivity. Indeed, we have observed that this leads to an increase in the production of ROS and the capacity of phagocytosis, leading to the conclusion that radiation induced ROS clearly behave as neutrophil primers.