Resveratrol in IL-2 Therapy against Melanoma Figure 6. Resveratrol inhibits IL-2-induced expansion of Treg. Splenocytes and infiltrating cells were stained with FITC-conjugated anti-CD4 mAb, fixed and permeabilized and incubated with PE-conjugated anti-FoxP3 mAb as described in Materials and Methods. The stained cells were analyzed by flow cytometry. The representative dot plots from spleen (A), lung (B), and liver (C) and the statistical analysis of the percentage mean 6SEM of Treg (D) are shown. doi:10.1371/journal.pone.0035650.g006 resveratrol can ameliorate IL-2 toxicity while promoting the effectiveness of IL-2 in metastatic melanoma therapy. Resveratrol does not affect infiltration of inflammatory cells but protects endothelial cell integrity and prevents the cells from undergoing apoptosis We previously found that HDIL-2-therapy caused endothelial cell (EC) damage by activating LAK cells [26,28,29]. Therefore, we investigated if resveratrol-mediated suppression of VLS resulted from its protective effect on ECs by pursuing ultrastruc- tural studies of the lungs. As shown in Fig. 3, ECs from PBS or resveratrol group showed normal morphological features. In contrast, ECs from IL-2-treatment group revealed extensive damage, including cell membrane folding or break and vacuola- tion of cytoplasmic particles. Some of the endothelial cells were severely damaged, with only extended cell membrane remnants remaining. Cellular debris from ECs was found in the blood capillary lumen [26]. Interestingly, IL2+resveratrol treatment exhibited morphologically normal ECs and the basal lamina, whereas we only found minor damage in few endothelial cells. We wondered whether suppression of IL-2 induced VLS by resveratrol resulted from inability of inflammatory cells to infiltrate into the organs. Therefore, the inflammatory cell extravasation was examined under microscope. The results showed that the IL-2 treatment induced significant perivascular infiltration both in lungs and liver. Notably, IL-2 + resveratrol co-treatment also exhibited similar levels of perivascular infiltration as the IL-2 treatment. The degree of infiltration was also measured by counting the layers of cell infiltration around each vessel and averaging the numbers for each group (Fig. 4). These data suggested that the suppression of VLS seen in IL-2 + resveratrol treated-mice was not due to the inability of infiltration of inflammatory cells. We previously reported that apoptosis could be one of the mechanisms of EC damage [26]. Hence, we applied TUNEL assay to examine apoptosis in lung sections. Our results showed that IL-2-treated mice exhibited a large number of ECs that had undergone apoptosis, which was determined by TUNEL-positive staining. In contrast, IL-2+resveratrol treated-mice did not show TUNEL-positive cells (Fig. 5). These data suggested that PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35650