Abstract
Objective: This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. Methods: PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFα) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFα or lipopolysaccharide (LPS) stimulations for 24 h. Results: After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFα treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. Conclusion: This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.
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References
Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature, 392(6673):245–252. doi:10.1038/32588.
Bender, A., Sapp, M., Schuler, G., Steinman, R.M., Bhardwaj, N., 1996. Improved methods for the generation of dendritic cells from nonproliferation progenitors in human blood. J. Immunol. Methods, 196(2):121–135. doi:10.1016/0022-1759(96)00079-8.
Bhardwaj, N., Bender, A., Gonzalez, N., Bui, L.K., Garrett, M.C., Steinman, R.M., 1994. Influenza virus-infected dendritic cells stimulate strong proliferate and cytolytic responses from human CD8+ T cells. J. Clin. Invest., 94(2):797–807.
Cella, M., Sallusto, F., Lanzavecchia, A., 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol., 9(1):396–404. doi:10.1016/S0952-7915(97)80153-7.
Duperrier, K., Eljaafari, A., Dezutter-Dambuyant, C., Bardin, C., Jacquet, C., Yoneda, K., Schmitt, D., Gebuhrer, L., Rigal, D., 2000. Distinct subsets of dendritic cells resembling dermal DCs can be generated in vitro from monocytes in the presence of different serum supplements. J. Immunol. Methods, 238(1–2):119–131. doi:10.1016/S0022-1759(00)00147-2.
Ebner, S., Ratzinger, G., Krosbacher, B., Schmuth, M., Weiss, A., Reider, D., Kroczek, R.A., Herold, M., Heufler, C., Fritsch, P., Romani, N., 2001. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4. J. Immunol., 166(1):633–641.
Grassi, F., Dezutter-Dambuyant, C., McIlroy, D., Jacquet, C., Yoneda, K., Imamura, S., Boumsell, L., Schmitt, D., Autran, B., Debre, P., Hosmalin, A., 1998. Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules. J. Leukoc. Biol., 64(4):484–493.
Hashimoto, S., Suzuki, T., Dong, H.Y., Nagai, S., Yamazaki, N., Matsushima, K., 1999. Serial analysis of gene expression in human monocyte-derived dendritic cells. Blood, 94(3):845–852.
Jenkins, D.E., Yasukawa, L.L., Benike, C.J., Engleman, E.G., Arvin, A.M., 1998. Isolation and utilization of human dendritic cells from peripheral blood to assay an in vitro primary immune response to varicella-zoster virus peptides. J. Infect. Dis., 178(Suppl 1):39–42.
Johansson, E., Domeika, K., Berg, M., Alm, G.V., Fossum, C., 2003. Characterizations of porcine monocyte-derived dendritic cells according to their cytokine profile. Vet. Immunol. Immunopathol., 91(3–4):183–197. doi:10.1016/S0165-2427(02)00310-0.
Kunitani, H., Shimizu, Y., Murata, H., Higuchi, K., Watanabe, A., 2002. Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases. J. Hepatol., 36(6):734–741. doi:10.1016/S0168-8278(02)00062-4.
Labeur, M.S., Roters, B., Pers, B., Mehling, A., Luger, T.A., Schwarz, T., Grabbe, S., 1999. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage. J. Immunol., 162(1):168–175.
Matteo Rigolin, G., Howard, J., Buggins, A., Sneddon, C., Castoldi, G., Hirst, W.J., Mufti, G.J., 1999. Phenotypic and functional characteristics of monocyte-derived dendritic cells from patients with myelodysplastic syndromes. Br. J. Haematol., 107(4):844–850. doi:10.1046/j.1365-2141.1999.01781.x.
Morse, M.A., Lyerly, H.K., Gilboa, E., Thomas, E., Nair, S.K., 1998. Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells. Cancer Res., 58(14):2965–2968.
Paillot, R., Laval, F., Audonnet, J.C., Andreoni, C., Juillard, V., 2001. Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes. Immunology, 102(4):396–404. doi:10.1046/j.1365-2567.2001.01200.x.
Rieser, C., Bock, G., Klocker, H., Bartsch, G., Thurnher, M., 1997. Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. J. Exp. Med., 186(9):1603–1608. doi:10.1084/jem.186.9.1603.
Romani, N., Reider, D., Heuer, M., Ebner, S., Kampgen, E., Eibl, B., Niederwieser, D., Schuler, G., 1996. Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability. J. Immunol. Methods, 196(2):137–151. doi:10.1016/0022-1759(96)00078-6.
Summerfield, A., Guzylack-Piriou, L., Schaub, A., Carrasco, C.P., Tache, V., Charley, B., McCullough, K.C., 2003. Porcine peripheral blood dendritic cells and natural interferon-producing cells. Immunology, 110(4):440–449. doi:10.1111/j.1365-2567.2003.01755.x.
Zhou, L.J., Tedder, T.F., 1996. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc. Natl. Acad. Sci. USA, 93(6):2588–2592.
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Project (No. 398441) supported by the Natural Science Foundation of Zhejiang Province, China
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Tang, Ll., Zhang, Z., Zheng, Js. et al. Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes. J. Zhejiang Univ. - Sci. B 6, 1176–1181 (2005). https://doi.org/10.1631/jzus.2005.B1176
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DOI: https://doi.org/10.1631/jzus.2005.B1176