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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 microdomains on immature DCs bind virus-sized particles. Green fluorescent microbeads (40-nm diam) were coated with gp120 and added to the cells in a ratio of 20 beads/cell. The cells were incubated for 30 min at 37°C, washed, and fixed in PFA. DC-SIGN was stained with AZN-D1 and Alexa 647–conjugated goat anti–mouse Ab (red). Subsequently, the cells were mounted onto poly-l-lysine–coated glass coverslips and analyzed by confocal microscopy. The overview of binding to gp120 microbeads of (A) intermediate DCs and (D) immature DCs is shown. Two representative cells of (B) intermediate and (E) immature DCs are shown. Specific block with 100 μg/ml mannan was also performed by preincubating the cells at RT for 10 min before adding the microbeads (C and F); bars, 5 μm. Similar results were obtained with ICAM-3–coated microbeads (not depicted). (G) Binding of K-DC-SIGN, intermediate DCs, and immature DCs to soluble gp120 was also performed: 50,000 cells were incubated with 50 mM biotinylated gp120 for 30 min on ice, in presence or absence of 20 μg/ml anti–DC-SIGN blocking mAb (AZN-D1). A subsequent incubation with Alexa 488–conjugated streptavidin for 30 min on ice followed, and gp120 molecules bound to the cells were detected by flow cytometry. The values represent the mean of three independent experiments ±SD.
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fig6: DC-SIGN microdomains on immature DCs bind virus-sized particles. Green fluorescent microbeads (40-nm diam) were coated with gp120 and added to the cells in a ratio of 20 beads/cell. The cells were incubated for 30 min at 37°C, washed, and fixed in PFA. DC-SIGN was stained with AZN-D1 and Alexa 647–conjugated goat anti–mouse Ab (red). Subsequently, the cells were mounted onto poly-l-lysine–coated glass coverslips and analyzed by confocal microscopy. The overview of binding to gp120 microbeads of (A) intermediate DCs and (D) immature DCs is shown. Two representative cells of (B) intermediate and (E) immature DCs are shown. Specific block with 100 μg/ml mannan was also performed by preincubating the cells at RT for 10 min before adding the microbeads (C and F); bars, 5 μm. Similar results were obtained with ICAM-3–coated microbeads (not depicted). (G) Binding of K-DC-SIGN, intermediate DCs, and immature DCs to soluble gp120 was also performed: 50,000 cells were incubated with 50 mM biotinylated gp120 for 30 min on ice, in presence or absence of 20 μg/ml anti–DC-SIGN blocking mAb (AZN-D1). A subsequent incubation with Alexa 488–conjugated streptavidin for 30 min on ice followed, and gp120 molecules bound to the cells were detected by flow cytometry. The values represent the mean of three independent experiments ±SD.

Mentions: When comparing the capacity of intermediate and immature DCs to bind these virus-sized particles (Fig. 6), a very high binding was observed on immature DCs. Many virus-sized particles were bound to the plasma membrane and a significant amount was phagocytosed (Fig. 6, D and E). In contrast, intermediate DCs (Fig. 6, A and B) hardly bound any microbeads, comparable to background levels or when DCs were pretreated with the carbohydrate mannan to block DC-SIGN (Fig. 6, C and F). Similar results were obtained when microbeads coated with ICAM-3 were used (unpublished data). Moreover, DC-SIGN on intermediate as well as on immature DCs was found to be equally capable of binding to soluble recombinant gp120 (Fig. 6 G), suggesting that the intrinsic binding capacity of DC-SIGN molecule on the two different DC types is comparable. Similar results were obtained when soluble ICAM-3-Fc chimeras were used (unpublished data). All these observations support our hypothesis that DC-SIGN molecules acquire a higher avidity for multimeric ligands when organized in multimolecular assemblies.


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 microdomains on immature DCs bind virus-sized particles. Green fluorescent microbeads (40-nm diam) were coated with gp120 and added to the cells in a ratio of 20 beads/cell. The cells were incubated for 30 min at 37°C, washed, and fixed in PFA. DC-SIGN was stained with AZN-D1 and Alexa 647–conjugated goat anti–mouse Ab (red). Subsequently, the cells were mounted onto poly-l-lysine–coated glass coverslips and analyzed by confocal microscopy. The overview of binding to gp120 microbeads of (A) intermediate DCs and (D) immature DCs is shown. Two representative cells of (B) intermediate and (E) immature DCs are shown. Specific block with 100 μg/ml mannan was also performed by preincubating the cells at RT for 10 min before adding the microbeads (C and F); bars, 5 μm. Similar results were obtained with ICAM-3–coated microbeads (not depicted). (G) Binding of K-DC-SIGN, intermediate DCs, and immature DCs to soluble gp120 was also performed: 50,000 cells were incubated with 50 mM biotinylated gp120 for 30 min on ice, in presence or absence of 20 μg/ml anti–DC-SIGN blocking mAb (AZN-D1). A subsequent incubation with Alexa 488–conjugated streptavidin for 30 min on ice followed, and gp120 molecules bound to the cells were detected by flow cytometry. The values represent the mean of three independent experiments ±SD.
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Related In: Results  -  Collection

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fig6: DC-SIGN microdomains on immature DCs bind virus-sized particles. Green fluorescent microbeads (40-nm diam) were coated with gp120 and added to the cells in a ratio of 20 beads/cell. The cells were incubated for 30 min at 37°C, washed, and fixed in PFA. DC-SIGN was stained with AZN-D1 and Alexa 647–conjugated goat anti–mouse Ab (red). Subsequently, the cells were mounted onto poly-l-lysine–coated glass coverslips and analyzed by confocal microscopy. The overview of binding to gp120 microbeads of (A) intermediate DCs and (D) immature DCs is shown. Two representative cells of (B) intermediate and (E) immature DCs are shown. Specific block with 100 μg/ml mannan was also performed by preincubating the cells at RT for 10 min before adding the microbeads (C and F); bars, 5 μm. Similar results were obtained with ICAM-3–coated microbeads (not depicted). (G) Binding of K-DC-SIGN, intermediate DCs, and immature DCs to soluble gp120 was also performed: 50,000 cells were incubated with 50 mM biotinylated gp120 for 30 min on ice, in presence or absence of 20 μg/ml anti–DC-SIGN blocking mAb (AZN-D1). A subsequent incubation with Alexa 488–conjugated streptavidin for 30 min on ice followed, and gp120 molecules bound to the cells were detected by flow cytometry. The values represent the mean of three independent experiments ±SD.
Mentions: When comparing the capacity of intermediate and immature DCs to bind these virus-sized particles (Fig. 6), a very high binding was observed on immature DCs. Many virus-sized particles were bound to the plasma membrane and a significant amount was phagocytosed (Fig. 6, D and E). In contrast, intermediate DCs (Fig. 6, A and B) hardly bound any microbeads, comparable to background levels or when DCs were pretreated with the carbohydrate mannan to block DC-SIGN (Fig. 6, C and F). Similar results were obtained when microbeads coated with ICAM-3 were used (unpublished data). Moreover, DC-SIGN on intermediate as well as on immature DCs was found to be equally capable of binding to soluble recombinant gp120 (Fig. 6 G), suggesting that the intrinsic binding capacity of DC-SIGN molecule on the two different DC types is comparable. Similar results were obtained when soluble ICAM-3-Fc chimeras were used (unpublished data). All these observations support our hypothesis that DC-SIGN molecules acquire a higher avidity for multimeric ligands when organized in multimolecular assemblies.

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