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Isolation of Human Skin Dendritic Cells by In Vitro Migration

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Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 64))

Abstract

In the human skin, various types of antigen-presenting cells (APC) are present. In the epidermis, they are identified ultrastructurally as Langerhans cells (LC) by the presence of Birbeck granules. LC are considered to belong to the family of dendritic cells (DC) that are important for the initiation of immune responses (1). In the dermis, macrophages and DC are present (2,3). The expression of CD1a molecules can be used to identify DC in the skin (4,5), because macrophages do not express this marker. In vivo, these skin DC are supposed to take up antigens penetrating in the skin. Thereafter, they migrate via the afferent lymphatics into the draining lymph nodes, where a T-cell response can be initiated (6,7). During migration, the DC mature into potent APC. Besides an increase in MHC class II expression, adhesion (8,9) and B7 co-stimulatory molecules (10) are up-regulated. Most research on skin DC has been carried out with cells isolated from enzyme digested skin (8-10). In this chapter, we describe a method to obtain DC from human skin without enzymes, by making use of their migratory capacities. The cells migrate “spontaneously” out of the skin during culture. Characterization of the cells shows that mature DC are obtained with a marker expression not influenced by enzymes.

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References

  1. Steinman, R. M. (1990) The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271–296.

    Article  Google Scholar 

  2. Richters, C. D., Hoekstra, M. J., van Baare, J., Du Pont, J. S., Hoefsmit, E. C., and M., Kamperdijk, E. W. A. (1994) Isolation and characterization of migratory human skin dendritic cells. Clin. Exp. Immunol. 98, 330–336.

    Article  PubMed  CAS  Google Scholar 

  3. Richters, C. D., Hoekstra, M. J., van Baare, J., Du Pont, J. S., Hoefsmit, E. C. M., Kamperdijk, E.W.A. (1995) Migratory properties and functional capacities of human skin dendritic cells. Br. J. Dermatol. 133, 721–727.

    Article  PubMed  CAS  Google Scholar 

  4. Murphy, G. F., Bhan, A. K. Sato, S., Mihm M. C., and Harrist, T. J. (1981) A new immunologic marker for human Langerhans cells. N. Engl. J. Med. 304, 791–792.

    PubMed  CAS  Google Scholar 

  5. Meunier, L., Gonzalez-Ramos, A., and Cooper K.D. (1993) Heterogeneous populations of class II MHC+cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogues to Langerhans cells. J. Immunol. 151, 4067–4080.

    PubMed  CAS  Google Scholar 

  6. Silberberg-Sinakin, I., Thorbecke, G. J., Baer, R. L., and Berezowsky, V. (1976) Antigen bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell Immunol. 25, 137–141.

    Article  PubMed  CAS  Google Scholar 

  7. Hoefsmit, E. C. M., Duijvestijn, A. M., and Kamperdijk, E. W. A. (1982) Relation between Langerhans cells, veiled cells and interdigitating cells. Immunobiol. 161, 225–265.

    Google Scholar 

  8. Teunissen, M. B. M., Wormmeester, J., Krieg, S. R.,et al. (1990) Human epidermal Langerhans cells undergo profound morphological and phenotypical changes during in vitro culture. J. Invest. Dermatol. 94, 166–173.

    Article  PubMed  CAS  Google Scholar 

  9. Romani, N., Lenz, A., Glessel, H., et al. (1989) Cultured Langerhans cells resemble lymphoid dendritic cells in phenotype and function. J. Invest. Dermatol., 93, 600–609.

    Article  PubMed  CAS  Google Scholar 

  10. Symington, F. W., Brady, W., and Linsley, P. S. (1993) Expression and function of B7 on human epidermal Langerhans cells. J. Immunol. 150, 1286–1295.

    PubMed  CAS  Google Scholar 

  11. Cordell, J. L., Faline, B., Erber, W. N., et al. (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem. 32, 219–229.

    PubMed  CAS  Google Scholar 

  12. Pease, D. C. (ed.) (1964) Histological techniques for electron microscopy. Academic Press, New York.

    Google Scholar 

  13. Liou, W., Geuze, H. J., and Slot, J. W. (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell. Biol. 106, 41–58.

    Article  PubMed  CAS  Google Scholar 

  14. Sallusto, F., Cella, M., Danielli, C., and Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the MHC class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182, 389–400.

    Article  PubMed  CAS  Google Scholar 

  15. Witmer-Pack, M. D., Olivier W., Valinsky, J., Schuler, G., and Steinman, R. M. (1987) Granulocyte/macrophage colony stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J. Exp. Med. 166, 1484–1498.

    Article  PubMed  CAS  Google Scholar 

  16. Heufler, C., Koch, F., and Schuler, G. (1988) Granulocyte/macrophage colonystimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J. Exp. Med. 167, 700–705.

    Article  PubMed  CAS  Google Scholar 

  17. Richters, C. D., Hoekstra, M. J., Hoefsmit, E. C. M., Kamperdijk, E. W. A. (1995) Phenotype of cells migrated from human skin explants. Adv. Exp. Med. Biol. 378, 247–251.

    PubMed  CAS  Google Scholar 

  18. Richters, C. D., Reits, E. A. J., van Pelt, A. M., et al. (1996) Effect of low dose UVB irradiation on the migratory properties and functional capacities of human skin dendritic cells. Clin. Exp. Immunol. 104, 191–197.

    Article  PubMed  CAS  Google Scholar 

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© 2001 Humana Press Inc.

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Richters, C.D., Hoekstra, M.J., du Pont, J.S., Kreis, R.W., Kamperdijk, E.W.A. (2001). Isolation of Human Skin Dendritic Cells by In Vitro Migration. In: Robinson, S.P., Stagg, A.J., Knight, S.C. (eds) Dendritic Cell Protocols. Methods in Molecular Medicine™, vol 64. Humana Press. https://doi.org/10.1385/1-59259-150-7:145

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  • DOI: https://doi.org/10.1385/1-59259-150-7:145

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-584-3

  • Online ISBN: 978-1-59259-150-3

  • eBook Packages: Springer Protocols

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