respectively [28–30]. We then analyzed the proportions of the infiltrated leukocyte subtypes, and determined the effects on several mediators of the inflammatory reaction. Materials and Methods Ischemia model Fischer 344 rats (Harlan, Horst, Netherlands) and Balb/C mice (local colony) were maintained in specific pathogen-free conditions and had access to food and water ad libitum. All experimental procedures were approved by the Experimental Animal Commit- tee of University of Turku. Ischemic hind limb injury was induced to male Fischer 344 rats (5 to 6 weeks old, 86–115 g) by surgical closure of the distal femoral artery, lateral circumflex femoral artery, and the proximal femoral artery. The animals were anesthetized for the procedure by intra peritoneal administration of fentanyl fluanisone (Janssen Pharmaceutica, Beerse, Belgium) and midazolame (Roche, Basel, Switzerland). Gene transfer was done immediately after the ligation by intra muscular injection of 0.56109 pfu adenovirus SOD3 (AdSOD3) or LacZ (AdLacZ) in 50 ml PBS as described [27,31,32]. Uninjured muscle tissue was used as control. Peritonitis model Gene transfer was done to 8–10 week old female balb/c mice with intra peritoneal injection of 0.56109 pfu AdSOD3 or AdLacZ. Acute peritoneal inflammation was induced 72 hours later by i.p. injection of 1 ml PBS containing 5% proteose peptone (BD Difco, Sparks, MD, USA) and 10 ng of IL-1b (R&D Systems, Minneapolis, MN, USA). As a control treatment, animals were given 50 mg/kg of Dexamethasone (Oradexon, Organon, Oss, Holland) half an hour before proteose peptone injection. Cells from the peritoneal cavity were collected 18 hours after the induction of inflammation by washing with 10 ml of RPMI containing 5 U/ml heparin (Løvens Kemiske Fabrik, Ballerup, Denmark). Cells from peritoneal lavage were counted and cytocentrifuged at 1000 rpm for 5 minutes (Shandon cytospin 3, Shandon, Pittsburgh, PA, USA). Slides were stained with Reastain Diff-Quick (Reagena, Toivala, Finland) to analyze different leukocyte subtypes. Immunohistochemistry Rat muscle samples were frozen in liquid nitrogen and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Ten micrometer sections were fixed in acetone and stained with rabbit anti-rat CD3 and mouse anti-rat CD68 (Serotec, Oxford, UK). The sections were counterstained with hematoxylin/eosin (Sigma, Saint Louis, MI, USA). The number of CD3+ and CD68+ cells were analyzed from whole sections with Zeiss Axiovert 200 M (Carl Zeiss, Oberko- chen, Germany). Reporter assay HEK 293T cells were used for in vitro assay to provide a general cell model that we have previously used in our reporter, expression, and cell signalling studies allowing the comparison of the data with our previous results. HEK 293T cells were transfected with SOD3 expression vector together with pNFkB Luc reporter (Stratagene, Cedar Creek, TX, USA). Luciferase activity was quantified with Tecan Ultra XFluor4 Fluorescence Reader (Tekan, Mannedorf, Switzerlard). Western blot analysis HEK293 cells were homogenized in lysis buffer (50 mmol/l HEPES pH 7.5, 150 mmol/l NaCl, 10% glycerol, 1% Triton X- SOD3 Derived Cell Migration 100, 1 mmol/l MgCl, 10 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 1 mmol/l Na3VO4, 10 mg/ml approtinin, 10 mg/ ml leupeptin) (Sigma, Saint Louis, MI, USA). Mouse anti-human a- IkB (Santa Cruz, Santa Cruz, CA, USA) was used to detect IkB levels from tissues. Quantitative PCR Total RNA was extracted from a pool of four animals using Tri- reagent (Sigma, Saint Louis, MI, USA). The first strand synthesis was done with Revert-Aid M-MuLV (Fermentas, Burlington, Canada), and the following quantitative PCR with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Primers and cycling conditions are shown in the Table 1. Statistical Analysis All results are expressed as mean6SEM. A paired t-test was used to determine differences between groups. Results SOD3 inhibits leukocyte migration in acute ischemia Neutrophils, macrophages, and other inflammatory cells mediate a number of important cellular functions in injured tissue [33,34]. Phagocytotic macrophages clear cellular debris and secrete inflammatory cytokines such as MIP-2, a strong neutrophil attracting agent leading to further increase in inflammatory signaling [35]. However, excessive inflammatory reaction may also contribute to tissue damage by enhancing macrophage infiltration, which increases tissue free radical load leading to further tissue injury [36]. In addition, decreased neutrophil accumulation leads to reduced infarct size, reduced vascular permeability, and resistance in ischemia/reperfusion (I/R) injury [37,38]. Thus, it is suggested that the tissue recovery is dependent on the factors Table 1. Primers and cycling conditions. Gene TNFa for TNFa rev IL-1a for IL-1a rev IL-6 for IL-6 rev MCP-1 for MCP-1 rev MIP2 for MIP2 rev ICAM for ICAM rev VCAM for VCAM rev PSEL for PSEL rev ESEL for ESEL rev b-actin for b-actin rev Primer AGA TGT GGA ACT GGC AGA GG CCC ATT TGG GAA CTT CTC CT TCG GGA GGA GAC GAC TCT AA GAA AGC TGC GGA TGT GAA GT CCG GAG AGG AGA CTT CAC AG ACA GTG CAT CAT CGC TGT TC CTC ACC TGC TGC TAC TCA TTC ACT TGC TGC TGG TGA TTC TCT TGT AGT ATC CAG AGC TTG ACG GTG AC GGA CTT GCC GCT CTT CAG TA AGG TAT CCA TCC ATC CCA CA GCC ACA GTT CTC AAA GCA CA TGA CAT CTC CCC TGG ATC TC CTC CAG TTT CCT TCG CTG AC TTC CCA CAC TTC CTT CTG CT CAC GCT GTA GTC GGG GTA TT TTT TTG GCA CGG TAT GTG AA AGG TTG CTG CCA CAG AGA GT TCG TGC GTG ACA TTA AGG AG GTC AGG CAG CTC GTA GCT CT doi:10.1371/journal.pone.0005786.t001 Tm 60 58 55 55 55 55 55 57 57 55 PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5786