title: Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression.
abstract: Sera from healthy subjects receiving recombinant human granulocyte colony-stimulating factor (rHuG-CSF) to mobilize CD34(+) peripheral blood progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigation, the effects of rHuG-CSF on the early stages of lymphocyte activation-induced apoptosis and on lymphocyte cell cycle entry were evaluated. Sera were obtained from HLA-identical donors receiving rHuG-CSF to mobilize CD34(+) PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagglutinin (PHA) in the presence of serum collected before (preG) or after rHuG-CSF administration (postG). Mitochondrial function, that is, incorporation of 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] and generation of reactive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family members (Bcl-2, Bcl-X(L), Bax) were evaluated by multiparameter flow cytometry. The activation-induced fragmentation of genomic DNA was detected by highly sensitive LM-PCR assay.CD4(+)DiOC(6)(3)(low) and CD8(+)DiOC(6)(3)(low) T lymphocytes increased and reached 32% (range 27%-38%) and 20% (range 15%-23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneration of ROS could be demonstrated in 65% (range 58%-82%) of CD4(+) T lymphocytes and in 0.4% (range 0.2%-0. 8%) of circulating CD8(+) T cells. rHuG-CSF determined no alteration of mitochondrial function if added to allogeneic PBMC in vitro, thus suggesting indirect effects mediated by soluble factors; on the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Deltapsi(m)) and hypergeneration of ROS were induced, and lymphocytes were predominantly arrested in a G(0) -like phase of the cell cycle and displayed genomic DNA fragmentation. Interestingly, the preincubation of PBMC with a blocking antibody directed against CD95 abrogated the perturbation of lymphocyte Deltapsi(m), suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of postG serum. Moreover, Bax protein was overexpressed in postG (median fluorescence intensity = 180, range 168-186) compared with preG cultures (median fluorescence intensity = 75, range 68-80; p < 0.01), while no differences in Bcl-2, Bcl-X(L), and c-Myc staining intensity were observed. Our findings demonstrate a humoral-mediated rHuG-CSF-induced dissipation of lymphocyte mitochondrial Deltapsi(m); these effects might be mediated by Bax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent induction of mitochondrial permeability transition. Whether immune dysfunction will favorably impact on incidence and severity of acute graft vs host disease after allogeneic PBPC transplantation remains to be determined.
authors.affiliation: Department of Hematology, Catholic University, Rome, Italy. sergiorutella@tin.it
authors.forename: S
authors.initials: S
authors.lastname: Rutella
authors.forename: C
authors.initials: C
authors.lastname: Rumi
authors.forename: L
authors.initials: L
authors.lastname: Pierelli
authors.forename: R
authors.initials: R
authors.lastname: Morosetti
authors.forename: S
authors.initials: S
authors.lastname: Sica
authors.forename: G
authors.initials: G
authors.lastname: Bonanno
authors.forename: G
authors.initials: G
authors.lastname: Scambia
authors.forename: G
authors.initials: G
authors.lastname: Leone
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D001790:Blood Physiological Phenomena; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D053938:DNA Fragmentation; D003864:Depression, Chemical; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006086:Graft vs Host Disease / Q000209:etiology; D016179:Granulocyte Colony-Stimulating Factor / Q000009:adverse effects / Q000494:pharmacology*; D019650:Hematopoietic Stem Cell Mobilization / Q000009:adverse effects; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects; D006801:Humans; D019394:Ki-67 Antigen / Q000032:analysis; D008213:Lymphocyte Activation / Q000187:drug effects; D016131:Lymphocyte Subsets / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000187:drug effects*; D018384:Oxidative Stress; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000502:physiology
chemical_list: C494826:BAX protein, human; D019394:Ki-67 Antigen; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D016179:Granulocyte Colony-Stimulating Factor