Detection of distinct subpopulations of langerhans cells by flow cytometry and sorting

Abstract

Flow cytometry was found to be a very appropriate tool for the study of Langerhans cells (LC), which represent a minor cell population (2-3%) of human epidermis, and allowed us to obtain new phenotypic, functional, and cell cycle data on these rare cells.The phenotypic analysis of cell surface antigens demonstrates the existence of two subpopulations of LC: the former is HLADR+and OKT 6 + (about 90% of total HLA-DR+ cells) and the latter is HLA-DR+ and OKT 6-(about 10% of total HLA-DR+ cells). These subpopulations of LC are both able to stimulate the proliferation of peripheral blood lymphocytes (PBL) in the presence of keratinocytes i.e., in mixed skin lymphocyte reaction (MSLR).Analysis of the cell cycle could be performed on OKT 6+ LC. Results show that they can be found in the various phases of the cell cycle, suggesting that the large majority of Langerhans cells are able to proliferate in situ in normal human epidermis.Key terms: Flow cytometry, Langerhans cells, double fluorescence, cell cycle, sterile sorting Langerhans cells (LC) are mononuclear, dendritic cells located among suprabasal keratinocytes in the epidermis of all mammalian species. Although they represent a minor population of epidermal cells (2-3%), great interest has been focused recently on them because their immunological role has become evident (23,261: 1) They express class 11 antigens and bear receptors for the Fc portion of IgG and several complement components (13,20,25); and 2) even more interestingly, LC are necessary for the induction by epidermal cells (EC) of autologous and allogeneic T-cell activation in the mixed skin cell lymphocyte reaction (MSLR) (3,8,26). Recently, human LC were also shown to be specifically stained with OKT 6 monoclonal antibody (MAb) (10). However, the functional importance of OKT 6 vs. HLA-DR determinants is still unclear. A serious hindrance to LC studies is their low number in epidermis. Their physical separation has been attempted by rosetting and monolayer techniques, with enrichment of 18 to 35% (8,26). Two other attempts have been made (19,211 with cell sorters, allowing purities higher than 80%. In the present study, we used the double immunofluorescence technique and sterile flow cytometry (FCM) sorting to analyze their antigenic determinants and their functional role.On the other hand, few data exist on the origin and renewal of LC in human epidermis. Katz et al. (12) have shown that LC originate in mice from bone marrow precursors, although some data suggest their possible renewal in the skin by mitosis (9, 14, 17). The FCM has proved to be a powerful tool in cell cycle studies (2) including those of murine and human keratinocytes (1,6). However, the low number of LC in the epidermis has limited until now the possibility of studying their kinetics accurately. To investigate whether LC go through the cell cycle, we chose three different approaches, using the cell sorter: 1) DNA staining of enriched LC suspensions, 2) simultaneous staining of LC membrane antigens and DNA, and 3) direct sorting ...

Comparative Cellular Morphology Suggesting the Existence of Resident Dendritic Cells Within Immune Organs of Salmonids

“…It may be possible in fish that, even when these cells are fully differentiated, they are still capable of proliferation. It has been shown that Langerhans cells in the normal epidermis undergo mitosis in situ to maintain their populations and during all phases of mitosis Birbeck granules remain within the cytoplasm (Miyauchi and Hashimoto, 1989; Oota, 1999; Vaigot et al, 2005).…”