Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression

Objective. Sera from healthy subjects receiving recombinant human granulocy te colony-stimulating factor (rHuG-CSE) to mobilize CD34(+) peripheral bloo d progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigatio n, the effects of rHuG-CSF on the early stages-of lymphocyte activation-ind uced apoptosis and on lymphocyte cell cycle entry were evaluated. Materials and Methods. Sera were obtained from HLA-identical donors receivi ng rHuG-CSF to mobilize CD34(+) PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagg lutinin (PHA) in the presence of serum collected before (preG) or after rHu G-CSF administration (postG), Mitochondrial function, that is, incorporatio n of 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] and generation of reac tive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family m embers (Bcl-2, Bcl-X-L, Bax) were evaluated by multiparameter flow cytometr y. The activation-induced fragmentation of genomic DNA was detected by high ly sensitive LM-PCR assay. Results. CD4(+)DiOC(6)(3)(low) and CD8(+)DiOC(6)(3)(low) T lymphocytes incr eased and reached 32% (range 27%38%) and 20% (range 15%-23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneratio n of ROS could be demonstrated in 65% (range 58%-82%) of CD4(+) T lymphocyt es and in 0.4% (range 0.2%-0.8%) of circulating CD8(+) T cells. rHuG-CSF de termined no alteration of mitochondrial function if added to allogeneic PBM C in vitro, thus suggesting indirect effects mediated by soluble factors; o n the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Delta p si(m)) and hypergeneration of ROS were induced, and lymphocytes were predom inantly arrested in a G(o)-like phase of the cell cycle and displayed genom ic DNA fragmentation. Interestingly, the preincubation of PBMC with a block ing antibody directed against CD95 abrogated the perturbation of lymphocyte Delta psi(m), suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of pos tG serum. Moreover, Bar protein was overexpressed in postG (median fluoresc ence intensity = 180, range 168-186) compared with preG cultures (median fl uorescence intensity = 75, range 68-80; p < 0.01), while no differences in Bcl-2, Bcl-X-L, and c-Myc staining intensity were observed. Conclusions. Our findings demonstrate a humoral-mediated rHuG-CSF-induced d issipation of lymphocyte mitochondrial Delta psi(m); these effects might he mediated by Pax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent inductio n 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. (C) 2000 in ternational Society for Experimental Hematology. Published by Elsevier Scie nce Inc.