Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells.
Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.
Peña-Cruz
V
V
Department of Immunology and Infectious Disease, Harvard School of Public Health, 651 Huntington Avenue, Boston, MA 02115, USA. vpenac@hsph.harvard.edu
Ito
S
S
Oukka
M
M
Yoneda
K
K
Dascher
C C
CC
Von Lichtenberg
F
F
Sugita
M
M
D015703:Antigens, CD; D018949:Antigens, CD1 / Q000032:analysis; D015820:Cadherins / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004817:Epidermis / Q000648:ultrastructure*; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D007801:Langerhans Cells / Q000166:cytology / Q000648:ultrastructure*; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology; D000097802:CD83 Antigen
D015703:Antigens, CD; D018949:Antigens, CD1; D015820:Cadherins; D000949:Histocompatibility Antigens Class II; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D000097802:CD83 Antigen; C419393:CD1a antigen