Influence in vitro of IL-3/Epo fusion proteins compared with the combination of IL-3 plus Epo in enhancing the proliferation of single isolated erythroid and multipotential progenitor cells from human umbilical cord blood and adult bone marrow.
Human interleukin-3/erythropoietin (IL-3/Epo) fusion protein have been constructed, expressed, and tested for biological activity. These fusion proteins were previously shown to be active on erythroid progenitors (BFU-E) from unseparated human bone marrow. We evaluated if these fusion proteins could stimulate erythroid and multipotential progenitor cells directly at the single-cell level. Two IL-3/Epo fusion proteins containing short (SL-3E, two amino acids) and long (LL-3E, 23 amino acids) linker sequences as well as a short linker Epo/IL-3 sequence (SL-E3, three amino acids) were tested. Highly enriched CD34 or BFU-E enriched CD34 CD33- cells from human umbilical cord blood or CD34 HLA-DR+CD33- cells from normal adult bone marrow were sorted as single cells into single wells. The combination of Epo plus IL-3 synergized to enhance the proliferation of BFU-E and multipotential progenitors (CFU-GEMM) in comparison to the individual effects of these cytokines. The three fusion proteins also enhanced proliferation of BFU-E and CFU-GEMM at the single-cell level and were at least as active as the combination of Epo and IL-3, demonstrating that IL-3/Epo fusion proteins directly stimulate proliferation of BFU-E and CFU-GEMM and that biological activity of IL-3 and Epo in vitro can be maintained when these proteins are fused. The activity of the combination of Epo and IL-3 or the fusion proteins was partially neutralized by preincubation with monoclonal antibodies to either Epo or IL-3 and was neutralized by greater than 90% by the combination of both antibodies, suggesting that the Epo and IL-3 components of the fusion proteins were both involved in the enhancing activity of these proteins. Additionally, use of monoclonal antibody to the human Epo receptor completely blocked the stimulating/enhancing activity of Epo alone, Epo plus IL-3, or the fusion proteins for stimulation of colony formation by BFU-E and CFU-GEMM but not for granulocyte-macrophage progenitors (CFU-GM), suggesting that the enhancing effects of the fusion proteins are most likely mediated, at least in part, by the Epo receptor.
Lu
L
L
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Xiao
M
M
Li
Z H
ZH
Jolliffe
L K
LK
Jones
S
S
Broxmeyer
H E
HE
Weich
N
N
D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D015672:Erythroid Precursor Cells / Q000166:cytology*; D004920:Erythropoiesis / Q000187:drug effects*; D004921:Erythropoietin / Q000008:administration & dosage*; D005312:Fetal Blood / Q000166:cytology*; D006410:Hematopoiesis / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin