Functional characterization of mouse granulocytes and macrophages produced in vitro from bone marrow progenitors stimulated with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF).
Bone marrow from C3H/ouj mice was depleted to < 1% of CD11b+ granulocytes and macrophages using paramagnetic beads coated with sheep anti-rat antibodies. CD11b- cells, enriched three- to fourfold in colony-forming cells, were stimulated in liquid culture with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Cultures stimulated with IL-3 or GM-CSF increased cell numbers fourfold at 7 days, with the CD11b+ population increasing to 63% +/- 9% (n = 5) with IL-3 or 96% +/- 1% (n = 4) cells with GM-CSF. Functional responsiveness of the granulocytes and macrophages was assessed by flow cytometry in an oxidative burst assay using dichlorofluorescein (DCF) and a quantitative phagocytosis assay using opsonized fluorescent beads. Granulocytes and macrophages, identified by light scatter characteristics and allophycocyanine staining of CD11b, were assayed simultaneously with granulocytes from fresh mouse bone marrow and peripheral blood. GM-CSF-generated CD11b+ cells had higher oxidative responses than similar populations produced in response to IL-3. The oxidative burst of these in vitro generated CD11b+ populations was similar to the equivalent fresh bone marrow population. Oxidative burst responses of peripheral blood phagocytic cells could not be adequately measured in this system. Peripheral blood CD11b+ cells were the most phagocytic, followed by GM-CSF-stimulated CD11b+ cells; IL-3-stimulated and bone marrow CD11b+ cells were the least phagocytic. These data demonstrate that functional granulocytes can be produced in vitro using growth factors and that GM-CSF produces a more responsive cell than IL-3.
Applied Sciences, Baxter Healthcare Corporation, Round Lake, IL 60073.
J G
JG
Bender
K L
KL
Unverzagt
P B
PB
Maples
Y
Y
Mehrotra
J
J
Mellon
D E
DE
Van Epps
C C
CC
Stewart
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells; D018845:CD11 Antigens; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006098:Granulocytes / Q000166:cytology / Q000502:physiology*; D006410:Hematopoiesis / Q000187:drug effects / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology
D015703:Antigens, CD; D018845:CD11 Antigens; D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006861:Hydrogen Peroxide; D010100:Oxygen