Langerhans-like dendritic cells generated from cord blood progenitors internalize pollen allergens by macropinocytosis, and part of the molecules are.

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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 Nadège Noirey, MSca, Nathalie Rougier, PhDa, Claude André, MDb, Daniel Schmitt, PhDa, Claude Vincent, PhDa Journal of Allergy and Clinical Immunology Volume 105, Issue 6, Pages 1194-1201 (June 2000) DOI: 10.1067/mai.2000.106545 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Internalization of allergens by LLDCs was dose- and time-dependent. A and B , LLDCs were incubated at 37°C in the presence of various concentrations of rBet v 1, rPhl p 1, FITC-dextran (FITC-DX) , and Lucifer yellow (LY) . C and D , LLDCs were incubated for various times at 37°C with 25 μg/mL rBet v 1 or rPhl p 1 and 1 mg/mL FITC-dextran or Lucifer yellow. One representative experiment from a set of 10 different experiments performed is shown here. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Internalization potencies reached a maximum in the premature state of LLDCs. A , Phenotypic evolution of DCs between day 6 and day 19. B , Modifications of the endocytic capacities expressed as a percentage of labeled cells. C , Modifications of the endocytic capacities expressed as molecules per cell. FITC-DX, FITC-dextran; LY, Lucifer yellow. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Internalized allergens had a 4-hour half-life in LLDCs. Nine-day-old LLDCs were pulsed for 15 minutes with rBet v 1 or rPhl p 1 at 2.5 μg/mL or with FITC-dextran (FITC-DX) or Lucifer yellow (LY) at 1 mg/mL, washed with cold medium, and then incubated in culture medium for various times. These data represent one experiment from a set of three. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Metabolic inhibitors indicated that protein allergen uptake was mediated by macropinocytosis. A , Inhibition of marker uptake by phenylarsine oxide (open bars) , monensin (shaded bars) , chloroquine (hatched bars) , or cytochalasin D (filled bars) . The drugs were added at 37°C for 1 hour with LLDCs, and then markers were added for 15 minutes at 37°C at the concentration shown in Fig 1, C . Results are expressed as a percentage of uptake without drug. B , Markers were internalized for 15 minutes at 37°C, and then LLDCs were washed and incubated with medium alone (open bars) or amiloride (filled bars) for 2 hours. Results are expressed as a percentage of labeling at the beginning of the 2-hour incubation. Data represent one experiment of a set of six. FITC-DX, FITC-dextran; LY, Lucifer yellow. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 The relative amount of allergen in acidic vesicles reached a maximum when LLDCs were in a premature state. Open circles represent direct measurement of fluorescent LLDCs after internalization of recombinant allergens tagged with FITC; filled circles represent fluorescent measurements of the same cell samples after an additional 30-minute incubation with monensin. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 6 Intracytoplasmic localization of rPhl p 1 (A and B ) and FITC-dextran (C and D ) analyzed by means of confocal microscopy. rPhl p 1 was used at 25 μg/mL, and FITC-dextran was used at 1 mg/mL. After 30 minutes of contact at 37°C, cells were analyzed directly (A and C ) or after 30 minutes of additional incubation at 4°C with monensin (B and D ). A phase-contrast picture is added as an inset , and immunofluorescence was observed at different levels of the cells from the top to the adherent side. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 7 LLDCs loaded with allergens and mixed with T lymphocytes induced a lymphoproliferative response in some of the experiments. LLDCs were incubated with medium alone (open bars) , rBet v 1 (shaded bars) , rPhl p 1 (hatched bars) , or BB, a chemical hapten used as positive proliferation control (filled bars) . *Significant difference (P < .05) of the corresponding mean in comparison with the control (C) incubated with medium alone. Journal of Allergy and Clinical Immunology 2000 105, 1194-1201DOI: (10.1067/mai.2000.106545) Copyright © 2000 Mosby, Inc. Terms and Conditions