title: Expression of Fas/CD95 and Bcl-2 by primitive hematopoietic progenitors freshly isolated from human fetal liver.
abstract: The cell-surface expression and the functional status of the CD95/Fas antigen on primitive hematopoietic progenitors (PHPs) freshly isolated from human fetal liver (FL) were studied. PHPs were phenotypically defined as CD34++ CD38 -/+ cells. The most immature subfractions of PHPs, CD34++CD38- and CD34+2CD38+ FL cells, expressed CD95, whereas the more mature CD34++CD38++ and CD34+CD38++2 FL cells displayed low CD95 expression. Combinations of cytokines, such as kit ligand (KL) + interleukin-3 or KL + granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated the expression of CD95 on PHPs upon in vitro culture. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) further increased the CD95 expression induced by KL+GM-CSF. The hematopoietic potential of sorted CD34++lineage (lin)- CD95+ versus CD34++ lin-CD95-FL cells was compared by colony-forming unit-culture (CFU-C) assays performed in serum-deprived medium. Lin+ cells were composed of erythrocytes, monocytes, T cells, B cells, and natural killer cells. Our results indicated that both CD95- and CD95+ subsets contained pluripotent progenitors, generating myeloid and erythroid progenitors. The functional status of CD95 and the effects of TNF-alpha and IFN-gamma, cytokines known to induce CD95-mediated apoptosis, were analyzed by incubation of PHPs in the presence of anti-CD95 monoclonal antibodies (MoAbs). The effect of anti-CD95 MoAbs was measured by viable cell counting, flow cytometry, and CFU-C assays. A decrease of CFU-C numbers was observed in the presence of anti-CD95 MoAbs and TNF-alpha and/or IFN-gamma. However, whereas growth factor deprivation induced apoptosis of PHPs, cross-linking of CD95 did not lead to apoptosis of PHPs measured by flow cytometry and viable cell counting. The correlation of increased intracytoplasmic levels of bcl-2 with high levels of cell-surface CD34 and the presence of CD95 on fresh FL cells suggests that bcl-2 may be involved in protecting against CD95-mediated apoptosis of FL PHPs.
authors.affiliation: Ingenex, Inc., Menlo Park, CA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bárcena
authors.forename: S W
authors.initials: SW
authors.lastname: Park
authors.forename: B
authors.initials: B
authors.lastname: Banapour
authors.forename: M O
authors.initials: MO
authors.lastname: Muench
authors.forename: E
authors.initials: E
authors.lastname: Mechetner
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D018952:Antigens, CD34 / Q000032:analysis; D002469:Cell Separation; D015672:Erythroid Precursor Cells / Q000166:cytology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D008099:Liver / Q000196:embryology*; D008214:Lymphocytes / Q000166:cytology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D019089:Stem Cell Factor / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D007377:Interleukin-3; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019089:Stem Cell Factor; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor