Effect of Ang-II on TNFα and IL-4 in human cells. Effect of TNFα and/or IL-4 on HPBMC recruitment by HUAECs. Time course of Ang-II-induced TNFα mRNA increase in HUAECs (A), HPBMCs (B), TNFα release in HPBMCs, (C) IL-4 mRNA expression in HPBMs (D), and recruitment of HPBMCs by TNFα and/or IL-4-stimulated HUAECs (E). HUAECs and HPBMCs (5 × 10⁶/mL) were stimulated with 1 μM Ang-II in the presence or absence of losartan (100 μM) for 1, 4, and 24 hours. Total mRNA was extracted. Relative quantification of the mRNA levels of TNFα, IL-4, and GAPDH was determined by using real-time quantitative RT-PCR by the comparative C_t method (ΔΔC_t method). Columns show the fold increase in expression of TNFα and IL-4 mRNA relative to control GAPDH values, calculated as mean ± SEM of the 2^{-ΔΔ Ct} values of 4 to 5 experiments. Protein content in the supernatant was determined by conventional sandwich ELISA and expressed as pM concentration of the cytokine of mean ± SEM from 4 to 5 experiments. *P < .05 or **P < .01 relative to values in the control group. + P < .05 relative to the Ang-II untreated group. HUAECs were incubated with medium, TNFα (0.2 ng/mL), IL-4 (20 ng/mL), or with a combination of both cytokines for 24 hours. Isolated HPBMCs (1 × 10⁶/mL) were perfused over the HUAECs for 5 minutes at 1 dyn/cm² and leukocyte accumulation quantified. Results are the mean (± SEM) for 4 experiments. **P < .01 relative to values in the control group; ++ P < .01 relative to the values in the TNFα or IL-4 groups.