SOD3 Derived Cell Migration Figure 4. Anti-inflammatory effect of dexamethasone. Open bars represent PBS treated animals and black bars represent dexamethasone animals (dosage 50 mg/kg). (a) Leukocyte infiltration was reduced by dexamethasone treatment from 51.56104 (64.7) to 36.56104 (67.1) cells/ milliliters of lavage. (b) Dexamethasone treatment had no effect on neutrophil migration, 16.66104 (62.9) and 21.56104 (65.2) cells/ml were found in PBS control group and dexamethasone treated animals, respectively. In contrast, accumulation of monocytes and lymphocytes were reduced from 21.46104 (62.7) to 8.86104 (62.2), and from 13.76104 (63.2) to 6.56104 (62.4), respectively. doi:10.1371/journal.pone.0005786.g004 As compared to neutrophils and macrophages, the role of CD3+ T-cells in recovery of ischemic tissue has remained uncertain. Despite relatively low level of infiltration, studies suggest an early role for T-cells in attracting neutrophils and macrophages to site of myocardial or peripheral ischemia/reperfusion injury [40,41]. The histological analysis at 3-day time point showed 114623 and 153646 (p = ns.) CD3+ T cells per ischemic tissue section in SOD3 and LacZ animals, respectively (Figure 2). At 10-day time point number of T cells had increased in LacZ animals by 60% to 247631 whereas in SOD3 animals T cell accumulation remained at similar level as compared to 3 day time point (125630, p,0.05). The analysis of leukocyte accumulation in the rat hind limb ischemia model shows selective inhibition of inflammatory cell migration and suggests SOD3 to have more prominent effect on macrophage infiltration as compared to lymphocytes. SOD3-mediated inhibition of leukocyte accumulation in peritonitis model To confirm the findings and to further analyze the SOD3-derived selective inhibition of cell migration we examined leukocyte migration in a mouse peritonitis model, which provides an efficient way to analyze leukocyte traffic in an acute inflammatory response. To induce peritoneal inflammation we used 5% solution of proteose peptone supplemented with IL-1b and counted the numbers of different leukocyte subtypes from the peritoneal lavage 18 hours after induction of inflammation. The analysis of SOD3 overexpres- sion derived inhibition of cell migration showed 20% (p = ns), 67% (p,0.001), and 33% (p,0.05) reduction in migrating neutrophils, monocytes/macrophages, and lymphocytes, respectively (Figure 3B). Moreover, the total number of infiltrated leukocytes was decreased by 30% in SOD3 animals as compared to LacZ controls (Figure 3A) (p,0.01), which is mostly caused by the effect of attenuated macrophage migration. The data effectively confirmed our findings in rat hind limb ischemia showing vastly stronger inhibition of macrophage infiltration as compared to other leukocyte subtypes. Dexamethasone, a corticosteroid that reduces swelling and inflammation is a potent anti-inflammatory drug used to treat many bacteria-free inflammatory conditions, including rheuma- toid arthritis and anaphylactic shock. Glucocorticoids exert their anti-inflammatory effect e.g. through repression of NF-kB mediated cytokine expression, which takes place after cytoplasmic glucocorticoid receptor translocates into the nucleus [5,42]. To compare SOD3 to clinically approved medication we gave an intra-peritoneal injection of dexamethasone (Oradexon) to animals 30 minutes before induction of peritoneal inflammation. Leuko- cyte traffic to the inflamed peritoneum was reduced by 20% (p,0.05) after treatment with 50 mg/kg dexamethasone (Figure 4A). Monocyte/macrophage migration was reduced by 60% (p,0.01), and that of lymphocytes by marked 50% indicating tendency, while no significant difference was seen in neutrophil accumulation in this setting (Figure 4B). PBS mock treated animals exhibited lower neutrophil and monocyte accumulation as compared to animals subjected to LacZ gene transfer, which may be result of the adenovirus vector used in the study. Intriguingly, SOD3 treatment reduced peritoneal monocyte and lymphocyte numbers to similar level as dexamethasone treatment although neutrophil numbers remained higher than what was observed in either PBS or dexamethasone treated animals. SOD3 inhibits NF-kB activation and suppresses the inflammatory cytokine and adhesion molecule expression Proinflammatory stimuli activate vascular endothelium leading to up-regulation of cell adhesion molecules and chemokines, NF- kB has been shown to be both necessary and sufficient for endothelial up-regulation of ICAM, VCAM, and MCP-1 [43]. Furthermore, ectopic expression of IkBa effectively abrogates expression of VCAM, IL-1, and IL-6 [44]. In vitro luciferase assay revealed 50% (p,0.01) decrease in NF-kB activity due to SOD3 transfection, which could at least partially be explained by increased IkBa expression (Figure 5A) suggesting that SOD3 promotes cytoplasmic localization of NF-kB rendering it incapable of binding DNA. NF-kB plays a central part in responses to inflammatory signaling by regulating the expression of cytokines suggesting that reduced NF-kB activity could lead to reduction in expression of inflammatory cytokines and chemokines. Therefore, we quantified cytokine and chemokine expression level in vivo from rat muscle three days after vessel ligation and SOD3 gene transfer. Quantitative RT-PCR showed significantly reduced expression of TNFa, IL1a, IL6, MIP2, and MCP1 (Figure 5B) in SOD3 animals suggesting reduced expression of several important inflammatory mediators. Specifically, MCP1 is an important macrophage PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5786