FIG 5.

HIV gp120-induced early activation of STAT3 is responsible for IL-6 production. (A and B) MDDCs were stimulated with gp120 (5 μg/ml) for the indicated time periods in the presence or absence of anti-IL-6 antibody (5 μg/ml, 1 h of pretreatment). Cell lysates were resolved by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis with antibodies specific for the total or phosphorylated forms of STAT3. Actin expression is shown as a gel loading control. Graphs in panels A and B show the ratio of phosphorylated to nonphosphorylated protein as determined by densitometry, where each sample was normalized to total STAT3. Fold phosphorylation was calculated relative to that of the untreated control, and the average fold change from four (A) and two (B) independent experiments was represented graphically, along with the SD. Statistical significance is indicated versus the untreated control. (C) MDDCs were pretreated for 1 h with the STAT3 inhibitor Stattic (10 μM) and stimulated with gp120 (5 μg/ml) for the indicated times. Total RNA was extracted and reverse transcribed, and real-time PCR was performed using primers specific for the human IL-6 gene and normalized with β actin. Data are expressed as means ± SE from three independent experiments performed. Results, analyzed by the relative quantification method (2^−ΔΔCT method), are shown as fold increase of IL-6 gene expression versus the untreated control. P values were calculated by Student's t test, and statistical significance is indicated (*, P < 0.05 for gp120 plus inhibitor versus gp120 alone). (D) MDDCs were pretreated for 1 h with the STAT3 inhibitor Stattic (10 μM) and stimulated with gp120 (5 μg/ml). After 18 h, supernatants were collected and IL-6 production was assessed by ELISA. Data are represented as means ± SE from five independent experiments. Statistical significance is indicated. **, P < 0.005 for gp120 plus inhibitor versus gp120 alone.