Figure 5.

Spermatozoa strongly interact with DCs. (A) Spermatozoa (5 × 10⁶/400 µl) were incubated with 50 ng HIV-1 BAL for 60 min at 37°C. Cells were then washed thoroughly. Experiments were performed in 24-transwell plates with a polycarbonate filter (0.2-µm pore size). 5 × 10⁶ HIV-1–treated spermatozoa were in the upper compartment and 5 × 10⁵ DCs were seeded in the lower compartment. Controls were performed by incubating together HIV-1–treated spermatozoa and DCs in the lower compartment. Cells were cultured for 7 d and the infection of DCs was evaluated by measuring the amount of p24 in the supernatants of DC cultures. Results are the mean ± SEM of five experiments performed in triplicate. The asterisk represents statistical significance (P < 0.05 vs. controls). (B) CFSE-labeled spermatozoa (1.5 × 10⁶/200 µl) were incubated with or without HIV-1 BAL containing 25 ng p24 for 60 min at 37°C. Cells were then washed thoroughly and were incubated with unlabeled DCs at a spermatozoa/DC ratio of 10:1 during 1 h at 37°C. The interaction of spermatozoa and DCs were then analyzed by flow cytometry in the gate of DCs, which could be easily distinguished from spermatozoa because of their higher values of forward light scatter. Histograms from a representative experiment (n = 7) are shown. (C and D) CFSE-labeled spermatozoa (1.5 × 10⁶/200 µl) were incubated during 60 min at 37°C with DCs, previously labeled with PE–anti–HLA-DR antibodies, at a spermatozoa/DC ratio of 10:1. The interaction of spermatozoa and DCs were then analyzed by fluorescence microscopy (C) or laser confocal microscopy (D). Bars, 10 µm. (E) Spermatozoa (1.5 × 10⁶/200 µl) were incubated during 60 min at 37°C with DCs at a spermatozoa/DC ratio of 10:1. The interaction of spermatozoa and DCs were then analyzed by electron microscopy. Representative images are shown. Arrows in C–E indicate spermatozoa attached to or ingested by DCs. Bars: (top) 0.5 µm; (bottom) 1.5 µm. (F) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C and washed thoroughly. DCs were pretreated, or not, with blocking antibodies directed to either CD4 or DC-SIGN for 15 min at 4°C. Blocking antibodies were used at a concentration three- to fivefold higher than those needed to saturate all binding sites, as determined by FACS analysis. Spermatozoa and DCs were co-cultured during 7 d at a spermatozoa/DC ratio of 10:1, and the infection of DCs was then analyzed by measuring the levels of p24 antigen in cell supernatants. Also shown in the figure is the amount of p24 found in the supernatants of HIV-1–treated spermatozoa cultured for 7 d without DCs (gray bar). The results are expressed as the mean ± SEM of four experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. controls).