Abstract
Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4+ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.
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Abbreviations
- Ab:
-
antibodies
- APC:
-
antigen-presenting cells
- BG:
-
Birbeck granules
- DNCB:
-
1-chloro-2,4-dinitrobenzene
- DNFB:
-
2,4-dinitrofluorobenzene
- DTAF:
-
dichlorotriazinylfluorescein
- FSC:
-
forward light scatter
- LC:
-
Langerhans cells
- LSCM:
-
laser scanning confocal microscopy
- MHC:
-
major histocompatibility complex
- MAb:
-
monoclonal antibodies
- PFA:
-
paraformaldehyde
- SSC:
-
side light scatter
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Rizova, H., Carayon, P., Michel, L. et al. Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy. Cell Biol Toxicol 10, 367–373 (1994). https://doi.org/10.1007/BF00755784
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DOI: https://doi.org/10.1007/BF00755784