Phospho- AKT Is Not Sufficient To Delay Neutrophil Apoptosis.

Neutrophils play an important role in the innate immune response and usually are the first cells to defend the organism against infections. The lifetime of neutrophils is tightly regulated and once the mature cells left the bone marrow most of them undergo apoptosis within 48 hours. The rate of neutrophil apoptosis can be modulated by a variety of cytokines, including granulocyte colony-stimulating factor (G-CSF). To investigate the involvement of the G-CSF receptor in the neutrophil susceptibility to apoptosis we utilized mice bearing truncation mutations on the G-CSF receptor (G-CSFR) and also the G:EpoR mutation, where the entire cytoplasmic (signaling) domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR). We examined the role of AKT-mTOR pathway in neutrophil apoptosis. Bone marrow neutrophils from wild type (C57BL/6) and mutant mice where cultured in presence or absence of G-CSF and/or Rapamycin (a specific mTor inhibitor); the apoptosis and the level of phosphorylation of AKT and ERK were evaluated. Neutrophils extracted from G:EpoR mice do not respond to G-CSF treatment and undergo apoptosis within 24 hours, although WT neutrophils can be partially protected against apoptosis when treated with G-CSF. Interestingly, G-CSF treatment leads to activation of Akt and Erk kinases, even in G:EpoR neutrophils. Furthermore, the addition of Rapamycin had no effect in the apoptosis of WT neutrophils in presence or absence of G-CSF. To further elucidate the role of the AKT-mTOR pathway, Sca+Lin- WT bone marrow cells were utilized in an in vitro G-CSF dependent granulocytic differentiation assay. The experiments showed that the mTor pathway is important for the early stages of myeloid differentiation but has little influence in the late stages, and also in the life span of neutrophils. Interestingly, over expression of a constitutively active form of AKT (myr-AKT) in Sca+Lin- cells impaired proliferation even though the differentiation was not affected. To overcome this, a tetracycline response element (TRE) conditional expression system was utilized. These experiments showed that myr-AKT expression does not rescue neutrophils from apoptosis. In conclusion, our results indicate that after treatment with G-CSF; AKT and ERK are phosphorylated in WT and G:EpoR neutrophils, despite this, G-CSF does not suppress G:EpoR neutrophil apoptosis while WT neutrophils can have their apoptosis partially delayed. We also found that mTor is involved in the early stages of G-CSF dependent myeloid differentiation and its inhibition can partially arrest differentiation. Conversely, rapamycin does not affect neutrophil apoptosis, in presence or not of G-CSF. In concordance with these results overexpression of a constitutive active form of AKT, myr-AKT does not rescue neutrophils from apoptosis and it is also detrimental to early stages of granulocytic proliferation. These data strongly suggest that the AKT-mTor pathway while important in early myeloid differentiation and proliferation does not play a role in cytokine suppression of neutrophil apoptosis.