Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures.
Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11b(bright), CD8alpha(-)) and "lymphoid-related" (CD11b(dull), CD8alpha(+)), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)-dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c(+) population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro-derived DCs, when treated with interferon-alpha or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8alpha, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti-interleukin-6 (IL-6) antibody, but not anti-GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.
Brasel
K
K
Departments of Immunobiology and Discovery Research, Immunex Corporation, Seattle, WA 98101, USA. brasel@immunex.com
De Smedt
T
T
Smith
J L
JL
Maliszewski
C R
CR
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D051940:B7-2 Antigen; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D016167:Integrin alphaXbeta2 / Q000032:analysis; D018664:Interleukin-12 / Q000032:analysis; D015850:Interleukin-6 / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D019891:Leukopoiesis / Q000276:immunology*; D007959:Lymphocyte Culture Test, Mixed; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D051941:fms-Like Tyrosine Kinase 3
D015703:Antigens, CD; D051940:B7-2 Antigen; C497964:CD86 protein, human; C497965:Cd86 protein, mouse; D000949:Histocompatibility Antigens Class II; D016167:Integrin alphaXbeta2; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D018664:Interleukin-12; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3