T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression ofcyclin-dependent kinase inhibitors

Objective. Peripheral blood progenitor cells (PBPC) mobilized by granulocyt e colony-stimulating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemotherapy for hematologic malignancies. Surprisingly , no exacerbation of acute graft-vs-host disease has been observed despite a 10-fold higher T-cell content in PBPC compared with bone marrow allograft s. Because G-CSF can suppress T-cell proliferation in response to mitogens and enhance their activation-induced apoptosis, we examined the molecular m echanisms underlying G-CSF-induced immune dysfunction. Materials and Methods. Normal allogeneic lymphocytes were challenged with p hytohemagglutinin in the presence of serum collected after G-CSF administra tion (postG) to healthy PBPC donors, and the expression of key components o f the cell cycle and apoptotic machineries was investigated by flow cytomet ry and Western blotting. Results. Lymphocyte stimulation was associated with collapse of mitochondri al transmembrane potential, hypergeneration of reactive oxygen intermediate s, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arre sted in a G(1)-Like phase of the cell cycle, as measured by G(1)-phase cycl in expression and bromodeoxyuridine (BrdUrd) incorporation. Cell tracking e xperiments confirmed the occurrence of a lower number of population doublin gs in postG compared with preG cultures. Unexpectedly, the phosphorylation state of the protein encoded by the retinoblastoma susceptibility gene (pRB ) was unaltered in postG cultures, and the inhibition of cell cycle progres sion occurred without the recruitment of the cyclin-dependent kinase inhibi tors p15(INK4B), p16(INK4A), and p27(Kip1). We eventually evaluated the abi lity of antioxidant/cytoprotectant agents to prevent the G-CSF-induced mito chondrial dysfunction and inhibition of cell cycle progression. Of interest , both N-acetylcysteine and amifostine reduced apoptotic cell death by 45% on average, inhibited the activation/processing of caspase-3, and increased BrdUrd incorporation in postG cultures. Conclusions. Based on these experimental findings, a model is proposed in w hich T-cell activation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associated with a molecular phenotype mimicking the G(1 )-S transition and consisting of pRB phosphorylation, lack of CDKI recruitm ent, and reduced cyclin-E expression. The putative relationship between lym phocyte mitogenic unresponsiveness and apoptosis induction would occur at t he level of key molecules shared by the cell cycle and apoptotic machinerie s. Whether the G-CSF-mediated modulation of lymphocyte functions in vitro i s beneficial in transplantation medicine remains to be determined. (C) 2001 International Society for Experimental Hematology. Published by Elsevier S cience Inc.