Limits...
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.

Show MeSH

Related in: MedlinePlus

Clustered DC-SIGN molecules efficiently bind HIV-1 particles and infect PBMC. DC-SIGN on immature DCs enhances HIV-1 infection as measured in a DC-PBMC coculture. Either intermediate or immature DCs (1.5 × 106) were preincubated for 20 min at RT with or without blocking mAb against 20 μg/ml DC-SIGN (AZN-D1 and AZN-D2). Preincubated intermediate or immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), and unbound virus particles and mAb were washed away. Subsequently, DCs were cocultured with activated PBMC (1.5 × 106) for 7 d. Coculture supernatants were collected, and p24 antigen levels were measured by ELISA. Black histogram represents PBMC infected in the absence of DCs. One representative experiment out of two is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171967&req=5

fig7: Clustered DC-SIGN molecules efficiently bind HIV-1 particles and infect PBMC. DC-SIGN on immature DCs enhances HIV-1 infection as measured in a DC-PBMC coculture. Either intermediate or immature DCs (1.5 × 106) were preincubated for 20 min at RT with or without blocking mAb against 20 μg/ml DC-SIGN (AZN-D1 and AZN-D2). Preincubated intermediate or immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), and unbound virus particles and mAb were washed away. Subsequently, DCs were cocultured with activated PBMC (1.5 × 106) for 7 d. Coculture supernatants were collected, and p24 antigen levels were measured by ELISA. Black histogram represents PBMC infected in the absence of DCs. One representative experiment out of two is shown.

Mentions: Having established that DC-SIGN microdomains bind virus-sized particles more efficiently in comparison to randomly distributed receptor molecules, we investigated whether the uptake of real virus was also enhanced by a clustered distribution of DC-SIGN, using p24 ELISA. Therefore, intermediate and immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), washed, and cultured in the presence of activated peripheral blood mononuclear cells (PBMC). As shown in Fig. 7, virus replication was significantly higher when PBMC were cocultured with either intermediate or immature DCs with respect to PBMC alone challenged with the same amount of infectious virus. However, infection of PBMC cultured with virus-pulsed immature DCs was much higher than the infection exhibited by PBMC cultured with intermediate DCs. In addition, the infection of PBMC with immature DCs was significantly blocked by anti–DC-SIGN mAbs that were added to DCs before incubation with HIV-1. In contrast, DC-SIGN contribution to PBMC infection was clearly much lower when intermediate DCs were used. The incomplete blocking observed in the presence of anti–DC-SIGN Abs suggests the involvement of other HIV receptors also expressed on DCs (Turville et al., 2002). The comparable expression levels of DC-SIGN, as shown in Fig. 3 A, as well as several costimulatory molecules (such as CD40 and CD80) on intermediate and immature DCs suggest that these two DC types have similar antigen presentation capacity (unpublished data). Altogether, our data demonstrate that DC-SIGN organized in microdomains rather than randomly distributed is more efficient in mediating binding and uptake of virus-sized microbeads as well as real HIV-1 particles.


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)

Clustered DC-SIGN molecules efficiently bind HIV-1 particles and infect PBMC. DC-SIGN on immature DCs enhances HIV-1 infection as measured in a DC-PBMC coculture. Either intermediate or immature DCs (1.5 × 106) were preincubated for 20 min at RT with or without blocking mAb against 20 μg/ml DC-SIGN (AZN-D1 and AZN-D2). Preincubated intermediate or immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), and unbound virus particles and mAb were washed away. Subsequently, DCs were cocultured with activated PBMC (1.5 × 106) for 7 d. Coculture supernatants were collected, and p24 antigen levels were measured by ELISA. Black histogram represents PBMC infected in the absence of DCs. One representative experiment out of two is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171967&req=5

fig7: Clustered DC-SIGN molecules efficiently bind HIV-1 particles and infect PBMC. DC-SIGN on immature DCs enhances HIV-1 infection as measured in a DC-PBMC coculture. Either intermediate or immature DCs (1.5 × 106) were preincubated for 20 min at RT with or without blocking mAb against 20 μg/ml DC-SIGN (AZN-D1 and AZN-D2). Preincubated intermediate or immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), and unbound virus particles and mAb were washed away. Subsequently, DCs were cocultured with activated PBMC (1.5 × 106) for 7 d. Coculture supernatants were collected, and p24 antigen levels were measured by ELISA. Black histogram represents PBMC infected in the absence of DCs. One representative experiment out of two is shown.
Mentions: Having established that DC-SIGN microdomains bind virus-sized particles more efficiently in comparison to randomly distributed receptor molecules, we investigated whether the uptake of real virus was also enhanced by a clustered distribution of DC-SIGN, using p24 ELISA. Therefore, intermediate and immature DCs were pulsed for 2 h with HIV-1 (M-tropic HIV-1Ba-L strain), washed, and cultured in the presence of activated peripheral blood mononuclear cells (PBMC). As shown in Fig. 7, virus replication was significantly higher when PBMC were cocultured with either intermediate or immature DCs with respect to PBMC alone challenged with the same amount of infectious virus. However, infection of PBMC cultured with virus-pulsed immature DCs was much higher than the infection exhibited by PBMC cultured with intermediate DCs. In addition, the infection of PBMC with immature DCs was significantly blocked by anti–DC-SIGN mAbs that were added to DCs before incubation with HIV-1. In contrast, DC-SIGN contribution to PBMC infection was clearly much lower when intermediate DCs were used. The incomplete blocking observed in the presence of anti–DC-SIGN Abs suggests the involvement of other HIV receptors also expressed on DCs (Turville et al., 2002). The comparable expression levels of DC-SIGN, as shown in Fig. 3 A, as well as several costimulatory molecules (such as CD40 and CD80) on intermediate and immature DCs suggest that these two DC types have similar antigen presentation capacity (unpublished data). Altogether, our data demonstrate that DC-SIGN organized in microdomains rather than randomly distributed is more efficient in mediating binding and uptake of virus-sized microbeads as well as real HIV-1 particles.

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