Binding of live conidia of Aspergillus fumigatus activates in vitro-generated human Langerhans cells via a lectin of galactomannan specificity.

Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4 degrees C or 37 degrees C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.

Withdrawal of TNF-alpha after the fifth day of differentiation of CD34+ cord blood progenitors generates a homogeneous population of Langerhans cells and delays their maturation.

Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) generate a heterogeneous population of dendritic cells (DC), including Langerhans cells (LC). This combination of cytokines has been shown to be crucial for differentiation into LC. After day 5 of culture, TNF-alpha has been maintained in the medium in most studies despite the observation of spontaneous maturation of LC after day 12. Five-day samples of in vitro differentiated LC were cultured in parallel with or without TNF-alpha. The absence of TNF-alpha was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogate the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-alpha abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-alpha-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-alpha. These data indicate that the suppression of TNF-alpha after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.

Langerhans-like dendritic cells generated from cord blood progenitors internalize pollen allergens by macropinocytosis, and part of the molecules are processed and can activate autologous naive T lymphocytes.

BACKGROUND: The safety and efficacy of sublingual immunotherapy have been demonstrated in moderate allergic asthma and seasonal rhinitis. However, not much is known about the precise mechanism of action of the allergen when it crosses the oral mucosa. OBJECTIVE: To define this mechanism, we investigated the role of Langerhans' cells in the capture and internalization of allergens. METHODS: We generated dendritic cells in vitro with the phenotypic characteristics of Langerhans-like dendritic cells (LLDCs) from cord blood CD34(+) progenitors. We used two recombinant major allergens: Bet v 1 and Phl p 1 labeled with FITC. RESULTS: Internalization of allergens and control proteins was dose- and time-dependent and related to the immature state of the cells. LLDCs internalized allergens with a high efficiency in comparison with control molecules. Allergens were only internalized by macropinocytosis, as demonstrated by the use of various inhibitors. Addition of intracellular pH-modifying molecules indicated that only a part of the allergens was accumulated in acidic vesicles, whereas the majority remained in other cytoplasmic structures. Pulse-chase experiments calculated a half-life of 4 hours, suggesting that part of the molecules were not metabolized in the lysosome. Allergen internalization by LLDCs might be followed by processing in some experiments, as demonstrated by activation of autologous T lymphocytes in 4 of 9 experiments. CONCLUSION: These elements showed that Langerhans' cells present in mucosa might play an active role in immune responses to allergens.