Nef induces TNF-α release and anti-human TNF-α antibody neutralizes rhTNF-α-induced CD36 downregulation.

<p>TNF-α release was measured into supernatants collected from MDM cultures in presence or absence of Nef protein, (A) The column bar graph shows the amount of TNF-α released in the medium by untreated cells (Ctr) or Nef-treated cells (Nef) derived from PBMCs cultivated in HEMA condition w/o EPO for three days and in presence of rNef/myr for additional three days. (B) The column bar graph shows the amount of TNF-α released in the medium by MDMs differentiated in presence of M-CSF (10 ng/mL) for 5 days and treated with rNef/myr or infected with VSV-G pseudotyped HIV-1-expressing or not expressing the <i>nef</i> gene. (Ctr) untreated cells, (Nef(+)-HIV-1) infected MDMs with VSV-G pseudotyped HIV-1-expressing Nef, (ΔNef-HIV-1) infected MDMs with VSV-G pseudotyped HIV-1-not expressing Nef, (Nef) rNef/myr-treated cells. The levels of the cytokine are expressed as picograms/mL. The results (mean ± standard deviation) are representative of three independent experiments (*<i>p</i><0.05). (C) Cells isolated by using CD14 magnetic beads (Miltenyi Biotec) were cultured in presence of human M-CSF (10 ng/mL) for 5 days followed by additional three days in presence of different concentrations of rhTNF-α (10, 3, 1, 0.3 ng/mL). In the column bar graph are presented the MFI of untreated cells (Ctr CD36) and TNF-α-treated cells at different cytokine concentrations (TNFα 10, 3, 1, 0.3 ng/mL) stained with FITC-conjugated anti-CD36. Matched isotype (Ctr isotype) was used as control of non-specific fluorescence signals and SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of three independent experiments (*<i>p</i><0.05). (D) Measurement of cytotoxic activity of serial diluted concentrations of rhTNF-α on WEHI-164 cells by using a MTT assay. In the line graph the absorbance of multiwells containing cells pre-incubated in presence of actinomycin D (♦, 1 μg/mL) with addition of rhTNF-α alone (▪) or together with anti-human TNF-α antibody (▴) is reported. The data shown are representative of two independent experiments carried out in triplicate. (E) M-CSF-derived MDMs were treated for three days with different concentrations of rhTNF-α alone (10, 3, 1, 0.3 ng/mL) or together with anti-human TNF-α antibody (1 μg/mL). The column bar graph represent the MFI of untreated cells (Ctr CD36), TNF-α-treated cells at different cytokine concentrations (TNFα 10, 3, 1, 0.3 ng/mL) or cells incubate with both rhTNF-α and 1 μg/mL of anti-human TNF-α antibody (TNFα 10, 3, 1 ng/mL+anti TNFα) stained with FITC-conjugated anti-CD36. Matched isotype (Ctr isotype) was used as control of non-specific fluorescence signals and SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of three independent experiments (*<i>p</i><0.05).