Figure 2.

MRs do not play a major role in the capture of HIV-1 by spermatozoa. (A) Spermatozoa were treated with 1,000 U/ml trypsin or 20 µg/ml pronase for 15 min at 37°C, washed thoroughly, and 1.5 × 10⁶/200 µl were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C. Cells were then washed, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (B) The expression of MRs in the spermatozoa was analyzed by measuring the binding of albumin bovine-α-d-mannopyranosylphenyl isothiocyanate-BMA revealed by streptavidin-FITC. Assays were performed by incubating spermatozoa for 30 min at 37°C with 100 µg/ml BMA in the absence or presence of 5 mg/ml mannan. The gray histogram represents spermatozoa incubated only with streptavidin-FITC (isotype). A representative experiment (n = 4) is shown. (C) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C in the absence or presence of mannan, mannose-BSA, or blocking antibodies directed to MR. Cells were washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of four to eight experiments performed in duplicate. (D) Human macrophages (10⁵/100 µl), obtained from monocytes cultured with GM-CSF for 5 d, were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C in the absence or presence of mannan, mannose-BSA, or blocking antibodies directed to MR. Cells were washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in duplicate. (E) B-THP-1-DC-SIGN⁺ cells (2.5 × 10⁵/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C in the absence or presence of mannan or mannose-BSA, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in duplicate. Asterisks in C–E represent statistical significance (P < 0.05 vs. controls). The capture mediated by THP-1 cells, which do not express DC-SIGN, is also shown (black bar).