Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy.
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 immunofluorescence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37 degrees 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Rizova
H
H
Department of General Pharmacology, Sanofi Recherche, Montpellier, France.
Carayon
P
P
Michel
L
L
Barbier
A
A
Lacheretz
F
F
Dubertret
L
L
D002478:Cells, Cultured; D004705:Endocytosis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006684:HLA-DR Antigens / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007801:Langerhans Cells / Q000276:immunology / Q000378:metabolism*; D018613:Microscopy, Confocal; D011946:Receptors, Antigen / Q000378:metabolism; D012879:Skin Physiological Phenomena
D006684:HLA-DR Antigens; D011946:Receptors, Antigen