Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor

doi:10.1084/jem.176.6.1693

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Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor

K Inaba, M Inaba, N Romani, H Aya, M Deguchi, S Ikehara, S Muramatsu and RM Steinman
Department of Zoology, Faculty of Science, Kyoto University, Japan.

Antigen-presenting, major histocompatibility complex (MHC) class II- rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II- negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma." At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.
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Li, C., Hirsch, M., Asokan, A., Zeithaml, B., Ma, H., Kafri, T., Samulski, R. J. (2007). Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo. J. Virol. 81: 7540-7547 [Abstract] [Full Text]
Inoue, Y., Kaifu, T., Sugahara-Tobinai, A., Nakamura, A., Miyazaki, J.-I., Takai, T. (2007). Activating Fc{gamma} Receptors Participate in the Development of Autoimmune Diabetes in NOD Mice. J. Immunol. 179: 764-774 [Abstract] [Full Text]
Figueiredo, R. T., Fernandez, P. L., Mourao-Sa, D. S., Porto, B. N., Dutra, F. F., Alves, L. S., Oliveira, M. F., Oliveira, P. L., Graca-Souza, A. V., Bozza, M. T. (2007). Characterization of Heme as Activator of Toll-like Receptor 4. J. Biol. Chem. 282: 20221-20229 [Abstract] [Full Text]
Tahk, S., Liu, B., Chernishof, V., Wong, K. A., Wu, H., Shuai, K. (2007). Control of specificity and magnitude of NF-{kappa}B and STAT1-mediated gene activation through PIASy and PIAS1 cooperation. Proc. Natl. Acad. Sci. USA 104: 11643-11648 [Abstract] [Full Text]
Izawa, T., Ishimaru, N., Moriyama, K., Kohashi, M., Arakaki, R., Hayashi, Y. (2007). Crosstalk between RANKL and Fas signaling in dendritic cells controls immune tolerance. Blood 110: 242-250 [Abstract] [Full Text]
Coutanceau, E., Decalf, J., Martino, A., Babon, A., Winter, N., Cole, S. T., Albert, M. L., Demangel, C. (2007). Selective suppression of dendritic cell functions by Mycobacterium ulcerans toxin mycolactone. JEM 204: 1395-1403 [Abstract] [Full Text]
Rogers, J., Hakki, A., Perkins, I., Newton, C., Widen, R., Burdash, N., Klein, T., Friedman, H. (2007). Legionella pneumophila Infection Up-Regulates Dendritic Cell Toll-Like Receptor 2 (TLR2)/TLR4 Expression and Key Maturation Markers. Infect. Immun. 75: 3205-3208 [Abstract] [Full Text]
Fang, R., Ismail, N., Soong, L., Popov, V. L., Whitworth, T., Bouyer, D. H., Walker, D. H. (2007). Differential Interaction of Dendritic Cells with Rickettsia conorii: Impact on Host Susceptibility to Murine Spotted Fever Rickettsiosis. Infect. Immun. 75: 3112-3123 [Abstract] [Full Text]
Majewski, M., Bose, T. O., Sille, F. C. M., Pollington, A. M., Fiebiger, E., Boes, M. (2007). Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells. Int Immunol 19: 719-732 [Abstract] [Full Text]
Nigg, A. P., Zahn, S., Ruckerl, D., Holscher, C., Yoshimoto, T., Ehrchen, J. M., Wolbing, F., Udey, M. C., von Stebut, E. (2007). Dendritic Cell-Derived IL-12p40 Homodimer Contributes to Susceptibility in Cutaneous Leishmaniasis in BALB/c Mice. J. Immunol. 178: 7251-7258 [Abstract] [Full Text]
Thomas-Rudolph, D., Du Clos, T. W., Snapper, C. M., Mold, C. (2007). C-Reactive Protein Enhances Immunity to Streptococcus pneumoniae by Targeting Uptake to Fc{gamma}R on Dendritic Cells. J. Immunol. 178: 7283-7291 [Abstract] [Full Text]
Wang, X.-Y., Facciponte, J., Chen, X., Subjeck, J. R., Repasky, E. A. (2007). Scavenger Receptor-A Negatively Regulates Antitumor Immunity. Cancer Res. 67: 4996-5002 [Abstract] [Full Text]