Figure 5. SOD3-mediated reduction in activity of inflammatory mediators. Open bars represent LacZ tissue and black bars represent SOD3 animals. (a) Luciferase assay shows 50% reduction in NF-kB activity in vitro, and western blot analysis shows increased IkBa expression. (b) Quantitative RT-PCR analysis for cytokines and chemokines. SOD3 overexpression derived reduced expression of TNFa, IL-1a, IL-6, MCP-1, and MIP2 in injured tissue. (b) Analysis of ICAM, VCAM, P-selectin, and E-selectin expression. Expression of inflammatory adhesion molecules was significantly reduced in SOD3 muscle. doi:10.1371/journal.pone.0005786.g005 attractant [45,46], possibly explaining markedly reduced macro- phage accumulation. Furthermore, since TNFa, IL1a, and IL6 are important regulators of endothelial adhesion molecule expression we analyzed expression of ICAM, VCAM, E-selectin, and P-selectin from the tissue (Figure 5C). We found significant reduction in adhesion molecule expression, which further confirms the reduction in overall inflammation in the muscle of SOD3 recipient rats as compared to LacZ control animals. SOD3 Derived Cell Migration Discussion Tissue damage launches rapid recruitment of inflammatory leukocytes into injured tissue due to activation of endothelial cells. Inflammatory reaction promotes tissue healing by eliminating pathogens, clearing cellular debris, and promoting cell prolifera- tion. However, excessive inflammatory reaction promotes injury e.g. through neutrophil-derived superoxide production [47]. In fact, reactive oxygen species function as inflammatory mediators by activating expression of cytokines such as TNFa, IL-1, and IL-6 [48] and therefore ROS may contribute to tissue injury by not only directly damaging the tissue but also by enhancing further leukocyte accumulation. In the current work we showed that SOD3 is an important mediator of reduced CD68+ macrophage migration into the inflammatory area. Macrophages accumulate in high numbers to ischemic muscle forming the primary leukocyte population three days after injury [39]. CD68 staining showed significantly reduced inflammatory area and macrophage migration in SOD3 treated ischemic tissue as compared to LacZ control animals (Figure 1). The SOD3-mediated reduction in macrophage accumulation was evident in all of the studied time points. T-cells accumulate to ischemic muscle in vastly lower numbers as compared to macrophages. However, their presence is required for efficient neutrophil traffic, and they attract macrophages by secreting IL-16 [40]. In our studies, SOD3 did not prevent initial low level T-cell migration, but efficiently inhibited further increase at 10-day time point (Figure 2). Late effect on T-cell migration suggests an indirect mechanism for SOD3 mediated inhibition in T-cell traffic, which might be result of general decrease in inflammation. Inflammatory cytokines secreted by infiltrating macrophages attract other leukocytes to injured tissue [49]. Thereafter, inhibition of macrophage infiltration could lead to overall reduction in inflammatory reaction. Since the SOD3-derived reduction in inflammation showed selective inhibition of macrophage migration, we were prompted to confirm the finding and to better characterize the cell specific effect. The mouse peritonitis model supported the SOD3-derived reduction in the number of infiltrating leukocytes (Figure 3A), which was predominantly due to reduced macrophage numbers (Figure 3B). These results confirm the anti-inflammatory property of SOD3 and show a stronger inhibition of monocyte migration as compared to other analyzed leukocyte subtypes. The data suggest that reduced superoxide tissue concentrations caused by SOD3 overexpression may explain the anti-inflammatory effect of the enzyme. It has been previously shown that superoxide treatment of rat cerebral endothelial cells increases monocyte adhesion and migration, which, however, was not replicated by H2O2 treatment but was instead abrogated by superoxide scavengers suggesting superoxide as an inflammatory mediator [21]. We have shown in our previous works that SOD3 overexpression in vivo efficiently decreases the production of superoxide in cardiovascular injuries including our hind limb ischemia model [25–27]. Anti-inflammatory medications currently available for clinical use include glucorticoid drugs such as dexamethasone. Dexa- methasone binds the glucocorticoid receptor, which subsequently translocates to the nucleus and represses inflammatory gene expression by inhibiting e.g. NF-kB activity [4,5,42]. To compare the efficacy of SOD3 mediated anti-inflammatory effect to existing medication we determined the effect of Dexamethasone in mouse peritonitis. As a dose of 50 mg/kg, dexamethasone reduced leukocyte traffic in comparable levels to SOD3 gene transfer (Figure 4A). Dexamethasone-mediated effect was equally effective in monocyte/macrophage and lymphocyte lineages whereas no significant effect was seen in neutrophils (Figure 4B). Neutrophil PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e5786