title: The effect of recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor on Fc gamma receptor expression and antibody-dependent cellular cytotoxicity of hematopoietic progenitor cells during in vitro myeloid maturation.
abstract: Enriched progenitor cell fractions from human bone marrow were induced to undergo myeloid maturation in culture using recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony stimulating factor (rhGM-CSF). A negative selection method using the murine monoclonal antibodies (MABs) PM81 (anti-CD15), AML-2-23 (anti-CD14), PC251 (anti-CD33), OKT11 (anti-CD2), and SCCL-1 (anti-CD71) and immunomagnetic beads coated with sheep anti-mouse IgG (Dynal A.S., Oslo, Norway) was used to remove the more mature cellular components of mononuclear cells from normal donor bone marrow samples. The resulting fraction of cells contained 35 to 40% CD34-positive cells, and less than 1% of cells expressed the receptors for the constant portion of immunoglobulin G Fc gamma RI or Fc gamma RII. A small population (3-5%) expressed Fc gamma RIII on day 0, and these cells were found by two-color flow cytometry to be primarily natural killer (NK) cells. The level of Fc gamma R expression was determined every 2 to 3 days on aliquots of the differentiating cells. Thirteen percent of the cultured bone marrow cells expressed Fc gamma RII after 48 hours in liquid culture with rhIL-3 and rhGM-CSF. The percent of cells expressing Fc gamma RII increased to a peak of 78% of the gated population on day 10. The mean fluorescence intensity (MFI) remained low for the first 8 to 10 days of culture, but at that time the MFI more than doubled. Fc gamma RI and Fc gamma RIII expression remained low throughout the culture period. The ability of the differentiating cells to perform antibody-dependent cellular cytotoxicity (ADCC) was determined at a single-cell level in a modified plaque assay using monolayers of ox erythrocyte (oxE) target cells. The purified progenitor cells, when placed in oxE monolayers sensitized with polyclonal rabbit anti-oxE antibody (AB), showed no plaque formation over control oxE layers. No increase in ability to generate cytolytic plaques in antibody-sensitized oxE layers was seen compared with unsensitized oxE layers until after 10 days of incubation in liquid culture. At that time, the percent of cells forming plaques in the AB-sensitized oxE layers was 34.4 +/- 10.7% (average +/- standard error of the mean [SEM]; n = 4) compared with 10.0 +/- 0.7% on the control oxE layers. The peak plaque formation appeared to coincide with the increase in MFI of a large population of the cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, Dartmouth Medical School, Lebanon, NH.
authors.forename: S G
authors.initials: SG
authors.lastname: Ericson
authors.forename: N E
authors.initials: NE
authors.lastname: Benoit
authors.forename: L E
authors.initials: LE
authors.lastname: Mills
authors.forename: M W
authors.initials: MW
authors.lastname: Fanger
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000502:physiology*; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001853:Bone Marrow / Q000737:chemistry* / Q000502:physiology*; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006410:Hematopoiesis / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D010641:Phenotype; D017452:Receptors, IgG / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D007377:Interleukin-3; D017452:Receptors, IgG; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor