Figure 6

Activation of the cytokine signaling events by the Tat proteins. (A) Secretion of TNF-α from primary monocytes. Monocytes were isolated from peripheral blood by differential density gradient centrifugation and seeded in 96-well plates at 1 × 10⁴ cells/well. The culture medium was supplemented with 20 or 200 ng/ml of B-or C-Tat proteins or left without Tat and incubated for 30 or 60 min. Cells were washed 3 times to remove Tat, resuspended in complete medium and incubated for 24 h. The level of TNF-α secreted into the culture medium was assessed using a commericial kit following the manufacturer's instructions. The experiment was repeated three times and the data presented are from one of the representative experiments. The data are presented as the mean value of triplicate wells ± 1 S. D. Inset, purity of the monocytes evaluated by flow cytometry. The forward vs. side scatter profile of cells isolated is presented. Approximately 86% of the gated cells are monocytes. The differences between B- and C-Tat treatments under all the conditions were found to be statistically significant by Student's paired t-test. For instance, at 60 min time point, the p values were < 0.05* for 20 ng and < 0.002** for 200 ng of Tat (B) Upregulation of the IL-6 transcript in U373-MAGI cells. U373 MAGI cells were exposed to B- or C-Tat at a concentration of 0.2 or 2 μg/ml or left without Tat treatment. Twenty four hours after Tat treatment, cells were lysed by directly adding Trizol to the wells and the total cellular RNA was isolated using standard protocols. An RT-PCR (5 ng input RNA) was performed to estimate the levels of IL-6 or β-actin transcripts. Amplified DNA fragments were resolved on a 1% agarose gel and visualized by ethidium bromide staining. The relative densities of the PCR fragments were determined by scanning the gel on a Phosphor Imager (FLA5000, Fuji). The intensity values of IL-6 fragments were normalized against β-actin and the fold upregulation in IL-6 transcript as a result of Tat treatment was presented in the bar diagram. (C) Tat-induced upregulation of chemokine receptors on monocytes. Monocytes were stimulated with 100 ng/ml B-, C-Tat proteins or cells were left without Tat treatment. Cells were harvested after 72 h, incubated for 20 min with PE conjugated monoclonal antibodies to human CXCR4 (12G5; Pharmingen), or CCR5 (2D7; Pharmingen), fixed with 2% formaldehyde and analyzed for coreceptor expression by FACSCalibur flow cytometer. The live cells were gated on the basis of forward and side scatter. Unstained monocytes were used as control. Expression of the chemokine receptors was analyzed using histograms with FL-2 on the x-axis and percent positive cells on the y-axis. A total of 10,000 events were scored. The differences between controls and Tat treatments were evaluated using Student's paired t-test and found to be statistically significant. In the case of CXCR4 upregulation, No Tat vs. B-Tat, p < 0.00004; No Tat vs. C-Tat, p < 0.00001; B-Tat vs. C-Tat, *p < 0.184. In the case of CCR5 upregulation, No Tat vs. B-Tat, p < 0.00001; No Tat vs. C-Tat, p < 0.001; B-Tat vs. C-Tat, ** p < 0.019.