T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors.
Peripheral blood progenitor cells (PBPC) mobilized by granulocyte 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 allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens and enhance their activation-induced apoptosis, we examined the molecular mechanisms underlying G-CSF-induced immune dysfunction. Normal allogeneic lymphocytes were challenged with phytohemagglutinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPC donors, and the expression of key components of the cell cycle and apoptotic machineries was investigated by flow cytometry and Western blotting. Lymphocyte stimulation was associated with collapse of mitochondrial transmembrane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arrested in a G(1)-like phase of the cell cycle, as measured by G(1)-phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation. Cell tracking experiments confirmed the occurrence of a lower number of population doublings 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 progression occurred without the recruitment of the cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p27(Kip1). We eventually evaluated the ability of antioxidant/cytoprotectant agents to prevent the G-CSF-induced mitochondrial 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. Based on these experimental findings, a model is proposed in which 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 recruitment, and reduced cyclin-E expression. The putative relationship between lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at the level of key molecules shared by the cell cycle and apoptotic machineries. Whether the G-CSF-mediated modulation of lymphocyte functions in vitro is beneficial in transplantation medicine remains to be determined.
Rutella
S
S
Department of Hematology, Catholic University Medical School, Largo A. Gemelli 8-00168 Rome, Italy. sergiorutella@tin.it
Pierelli
L
L
Rumi
C
C
Bonanno
G
G
Marone
M
M
Sica
S
S
Capoluongo
E
E
Ameglio
F
F
Scambia
G
G
Leone
G
G
D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D004999:Amifostine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors; D005260:Female; D016193:G1 Phase; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D018655:Lymphocyte Count; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology* / Q000648:ultrastructure; D025521:Tumor Suppressor Proteins
D000975:Antioxidants; C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D017382:Reactive Oxygen Species; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D016179:Granulocyte Colony-Stimulating Factor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D004999:Amifostine; D000111:Acetylcysteine