Internalization of HIV-1 gp120 by immature MDDCs. The binding and entry of HIV-1 gp120 into immature MDDCs was examined using excess biotin-LC-hydrazide gp120. Original magnification, × 400. After a 30-minute pulse with gp120 (Ai) or no gp120 (Aii) or a subsequent 120-minute chase (Aiii), 5 × 104 cells were cytospun onto glass slides and fixed in 4°C acetone for 10 minutes. HIV-1 gp120 was detected using streptavidin Alexa Fluor 594 (red) and fluorescent colocalization examined with monoclonal antibodies to the lysosome marker LAMP-2 and the 2 CLRs, MR and DC-SIGN (green), by confocal microscopy. (Similar results were observed with alcian blue–adhered, paraformaldehyde-fixed, and saponin-permeabilized MDDCs stained with biotinylated CD4-IgG2.) (B) MDDCs were trypsinized, resulting in cleavage of the gp120 binding domains of functional CD4 (Leu3a) and DC-SIGN (AZN-D1) but not completely of MR. Solid gray histograms represent antibody (or streptavidin) staining for CD4, DC-SIGN, MR, and gp120 after trypsinization, and the open overlaid histograms represent their respective isotype or negative (no gp120) controls. The blue open overlaid histograms show respective staining in each panel before trypsinization. (C) HIV-1 gp120 uptake into different trypsinized MDDCs at 0 (Ci) and 120 (Cii) minutes after gp120 loading was detected as outlined for panel A. (D) Cell surface expression of MR on untreated (MR) or trypsinized MDDCs (MR+Tryp) and expression of DC-SIGN and CD4 (on untreated cells) was determined immediately before the gp120 pulse (before gp120; ▪), immediately after the 30-minute pulse (0 minutes; ▦) and after a 120-minute chase (120 minutes; □). Percent fluorescence intensity = MFI as a proportion of control.
