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Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

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DC-SIGN cell surface distribution during monocyte-derived DC development. DC-SIGN binding activity was monitored during development of monocyte-derived DCs. As shown in the box, intermediate DCs indicate cells harvested after 3 d of monocytes differentiation. (A) The expression levels of DC-SIGN on monocytes, intermediate and immature DCs were assessed by FACS® analysis. The dotted line histogram represents the isotype control, and the thick line histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). Mean fluorescence intensity is indicated. One representative donor is shown. (B) The adhesion to ICAM-3 and gp120 was determined using 1 μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). One representative experiments out of three is shown. (C) Intermediate and immature DCs were let adhere onto fibronectin-coated formvar film, specifically labeled for DC-SIGN with 10-nm gold particles (Materials and methods), and analyzed by TEM. Results are representatives of multiple cells in several independent experiments. Bar, 200 nm.
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fig3: DC-SIGN cell surface distribution during monocyte-derived DC development. DC-SIGN binding activity was monitored during development of monocyte-derived DCs. As shown in the box, intermediate DCs indicate cells harvested after 3 d of monocytes differentiation. (A) The expression levels of DC-SIGN on monocytes, intermediate and immature DCs were assessed by FACS® analysis. The dotted line histogram represents the isotype control, and the thick line histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). Mean fluorescence intensity is indicated. One representative donor is shown. (B) The adhesion to ICAM-3 and gp120 was determined using 1 μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). One representative experiments out of three is shown. (C) Intermediate and immature DCs were let adhere onto fibronectin-coated formvar film, specifically labeled for DC-SIGN with 10-nm gold particles (Materials and methods), and analyzed by TEM. Results are representatives of multiple cells in several independent experiments. Bar, 200 nm.

Mentions: During the differentiation of DCs from monocyte precursors, the expression of DC-SIGN on the cell surface gradually increases (Geijtenbeek et al., 2000a). However, as shown by flow cytometry (Fig. 3 A), no significant increases in DC-SIGN expression levels are seen between cells harvested after 3 d of culture (designated intermediate DCs) and immature DCs. Maximum DC-SIGN–mediated adhesion to ICAM-3 as well as GP120 was observed on immature DCs, although already on intermediate DC DC-SIGN was capable of completely mediating the binding to ICAM-3 (Fig. 3 B, top), which on monocytes is LFA-1 dependent (unpublished data). Comparably, while on monocytes, binding to gp120 is mediated by CD4 (Kedzierska and Crowe, 2002; Kohler et al., 2003), on intermediate DCs, as well as on immature DCs, DC-SIGN is almost entirely responsible for binding to gp120 (Fig. 3 B, bottom). To examine DC-SIGN cell surface distribution, both intermediate and immature DCs were allowed to adhere to fibronectin, and DC-SIGN molecules were labeled with gold particles. Subsequently, the distribution on the plasma membrane was analyzed by TEM (Fig. 3 C). Given the high capacity of DCs to widely spread on the used substrates, very large membrane areas (often up to 60–70% of the whole visible plasma membrane) were available for gold particles analysis, ensuring that the areas used for quantitation were truly representative (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200306112/DC1). Surprisingly, we found that the distribution of DC-SIGN changes dramatically during DC development. Although on intermediate DCs, the gold particles are evenly distributed over the cell surface, on immature DCs, there is a clear organization of DC-SIGN in spatially well-defined microdomains. To exclude the possible influence of the fibronectin substrate on DC-SIGN distribution, TEM analysis was also performed on DCs that were gold labeled in suspension and mounted onto poly-l-lysine. No differences were seen between cells stretched on fibronectin or cells adhering to poly-l-lysine (unpublished data). Moreover, thin sections of resin-embedded immature DCs were gold labeled and analyzed by TEM. Clusters of DC-SIGN molecules could be observed exclusively at the plasma membrane (unpublished data). We also analyzed by TEM the cell surface distribution of other transmembrane receptors expressed on DCs, including LFA-1. Unlike DC-SIGN, LFA-1 did not show any changes in cell surface distribution pattern on intermediate and immature DCs (unpublished data).


Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

DC-SIGN cell surface distribution during monocyte-derived DC development. DC-SIGN binding activity was monitored during development of monocyte-derived DCs. As shown in the box, intermediate DCs indicate cells harvested after 3 d of monocytes differentiation. (A) The expression levels of DC-SIGN on monocytes, intermediate and immature DCs were assessed by FACS® analysis. The dotted line histogram represents the isotype control, and the thick line histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). Mean fluorescence intensity is indicated. One representative donor is shown. (B) The adhesion to ICAM-3 and gp120 was determined using 1 μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). One representative experiments out of three is shown. (C) Intermediate and immature DCs were let adhere onto fibronectin-coated formvar film, specifically labeled for DC-SIGN with 10-nm gold particles (Materials and methods), and analyzed by TEM. Results are representatives of multiple cells in several independent experiments. Bar, 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171967&req=5

fig3: DC-SIGN cell surface distribution during monocyte-derived DC development. DC-SIGN binding activity was monitored during development of monocyte-derived DCs. As shown in the box, intermediate DCs indicate cells harvested after 3 d of monocytes differentiation. (A) The expression levels of DC-SIGN on monocytes, intermediate and immature DCs were assessed by FACS® analysis. The dotted line histogram represents the isotype control, and the thick line histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). Mean fluorescence intensity is indicated. One representative donor is shown. (B) The adhesion to ICAM-3 and gp120 was determined using 1 μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). One representative experiments out of three is shown. (C) Intermediate and immature DCs were let adhere onto fibronectin-coated formvar film, specifically labeled for DC-SIGN with 10-nm gold particles (Materials and methods), and analyzed by TEM. Results are representatives of multiple cells in several independent experiments. Bar, 200 nm.
Mentions: During the differentiation of DCs from monocyte precursors, the expression of DC-SIGN on the cell surface gradually increases (Geijtenbeek et al., 2000a). However, as shown by flow cytometry (Fig. 3 A), no significant increases in DC-SIGN expression levels are seen between cells harvested after 3 d of culture (designated intermediate DCs) and immature DCs. Maximum DC-SIGN–mediated adhesion to ICAM-3 as well as GP120 was observed on immature DCs, although already on intermediate DC DC-SIGN was capable of completely mediating the binding to ICAM-3 (Fig. 3 B, top), which on monocytes is LFA-1 dependent (unpublished data). Comparably, while on monocytes, binding to gp120 is mediated by CD4 (Kedzierska and Crowe, 2002; Kohler et al., 2003), on intermediate DCs, as well as on immature DCs, DC-SIGN is almost entirely responsible for binding to gp120 (Fig. 3 B, bottom). To examine DC-SIGN cell surface distribution, both intermediate and immature DCs were allowed to adhere to fibronectin, and DC-SIGN molecules were labeled with gold particles. Subsequently, the distribution on the plasma membrane was analyzed by TEM (Fig. 3 C). Given the high capacity of DCs to widely spread on the used substrates, very large membrane areas (often up to 60–70% of the whole visible plasma membrane) were available for gold particles analysis, ensuring that the areas used for quantitation were truly representative (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200306112/DC1). Surprisingly, we found that the distribution of DC-SIGN changes dramatically during DC development. Although on intermediate DCs, the gold particles are evenly distributed over the cell surface, on immature DCs, there is a clear organization of DC-SIGN in spatially well-defined microdomains. To exclude the possible influence of the fibronectin substrate on DC-SIGN distribution, TEM analysis was also performed on DCs that were gold labeled in suspension and mounted onto poly-l-lysine. No differences were seen between cells stretched on fibronectin or cells adhering to poly-l-lysine (unpublished data). Moreover, thin sections of resin-embedded immature DCs were gold labeled and analyzed by TEM. Clusters of DC-SIGN molecules could be observed exclusively at the plasma membrane (unpublished data). We also analyzed by TEM the cell surface distribution of other transmembrane receptors expressed on DCs, including LFA-1. Unlike DC-SIGN, LFA-1 did not show any changes in cell surface distribution pattern on intermediate and immature DCs (unpublished data).

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Show MeSH
Related in: MedlinePlus