Fig. 1.
Fig. 1. EBV infection induces apoptosis of monocytes cultured in GM-CSF and IL-4. / Monolayers of adherent PBMCs or purified CD14+monocytes were infected with supernatants from the EBV producer B95.8 cell line for 2 hours at 37°C and were cultured in medium with or without GM-CSF and IL-4. Each culture condition was tested in triplicate in every experiment. (A) Time kinetics of cell recovery in control (open symbols) and EBV-infected cultures (closed symbols) containing GM-CSF and IL-4. Mean ± SD of triplicates. Results are from 1 of 20 representative experiments. (B) Cytospins of cells cultured for 5 days in GM-CSF and IL-4 were examined by phase-contrast microscopy and Hoechst staining. From day 3, the percentage of apoptotic cells was significantly higher in EBV-infected cultures (gray bars) than in the uninfected control (white bars) and increased in parallel with the decrease of cell recovery. Results are from 1 of 2 representative experiments. (C) FACS analysis of FITC-conjugated Annexin V binding to EBV-infected and control cells collected at day 4 or day 6 of culture. Gated populations of propidium iodide-negative cells are shown. Values inside the histogram plots represent the percentage of gated cells in the M1 region. Results are from 1 of 2 representative experiments. (D) Caspase-3 cleavage in EBV-infected cells. Total cell lysates of EBV peptide-specific antigen-activated cytotoxic T-cell clone BK289 (lane 1), EBV-infected (lanes 2 and 4), and control (lanes 3 and 5) monocytes collected at day 4 (lanes 2 and 3) or day 6 (lanes 4 and 5) of culture with the lymphokines were analyzed by immunoblotting with caspase-3–specific rabbit polyclonal antibody. The 20-kd and 17-kd products, corresponding to enzymatically active forms of caspase-3 and generated by cleavage of 32-kd proenzyme, are easily detectable in the T-cell lysate because of Fas triggering.39 The same products are seen in the lysates of EBV-infected cells but not in control cells. The band of lower molecular weight most likely represents the enzymatically inactive 12-kd small subunit of caspase-3.4041 Identity of the fragment with the highest molecular weight remains uncertain. (E) Expression of Bim and Bcl-2 proteins in control (EBV−) or EBV-infected (EBV+) cultures was analyzed by immunoblotting after the indicated periods of time.

EBV infection induces apoptosis of monocytes cultured in GM-CSF and IL-4.

Monolayers of adherent PBMCs or purified CD14+monocytes were infected with supernatants from the EBV producer B95.8 cell line for 2 hours at 37°C and were cultured in medium with or without GM-CSF and IL-4. Each culture condition was tested in triplicate in every experiment. (A) Time kinetics of cell recovery in control (open symbols) and EBV-infected cultures (closed symbols) containing GM-CSF and IL-4. Mean ± SD of triplicates. Results are from 1 of 20 representative experiments. (B) Cytospins of cells cultured for 5 days in GM-CSF and IL-4 were examined by phase-contrast microscopy and Hoechst staining. From day 3, the percentage of apoptotic cells was significantly higher in EBV-infected cultures (gray bars) than in the uninfected control (white bars) and increased in parallel with the decrease of cell recovery. Results are from 1 of 2 representative experiments. (C) FACS analysis of FITC-conjugated Annexin V binding to EBV-infected and control cells collected at day 4 or day 6 of culture. Gated populations of propidium iodide-negative cells are shown. Values inside the histogram plots represent the percentage of gated cells in the M1 region. Results are from 1 of 2 representative experiments. (D) Caspase-3 cleavage in EBV-infected cells. Total cell lysates of EBV peptide-specific antigen-activated cytotoxic T-cell clone BK289 (lane 1), EBV-infected (lanes 2 and 4), and control (lanes 3 and 5) monocytes collected at day 4 (lanes 2 and 3) or day 6 (lanes 4 and 5) of culture with the lymphokines were analyzed by immunoblotting with caspase-3–specific rabbit polyclonal antibody. The 20-kd and 17-kd products, corresponding to enzymatically active forms of caspase-3 and generated by cleavage of 32-kd proenzyme, are easily detectable in the T-cell lysate because of Fas triggering.39 The same products are seen in the lysates of EBV-infected cells but not in control cells. The band of lower molecular weight most likely represents the enzymatically inactive 12-kd small subunit of caspase-3.40 41 Identity of the fragment with the highest molecular weight remains uncertain. (E) Expression of Bim and Bcl-2 proteins in control (EBV−) or EBV-infected (EBV+) cultures was analyzed by immunoblotting after the indicated periods of time.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal