SOD3 Reduces Inflammatory Cell Migration by Regulating Adhesion Molecule and Cytokine Expression Juha P. Laurila1, Lilja E. Laatikainen1, Maria D. Castellone2, Mikko O. Laukkanen1* 1 Medicity Research Laboratory, University of Turku, Turku, Finland, 2 Institute of Experimental Endocrinology and Oncology (CNR), Department of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Naples, Italy Abstract Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNFa, IL1a, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E- selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders. Citation: Laurila JP, Laatikainen LE, Castellone MD, Laukkanen MO (2009) SOD3 Reduces Inflammatory Cell Migration by Regulating Adhesion Molecule and Cytokine Expression. PLoS ONE 4(6): e5786. doi:10.1371/journal.pone.0005786 Editor: Eshel Ben-Jacob, Tel Aviv University, Israel Received April 15, 2009; Accepted May 8, 2009; Published June 4, 2009 Copyright: 2009 Laurila et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Academy of Finland, Sigrid Juselius Foundation, Paolo Foundation, TUBS graduation school. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Mikko.Laukkanen@utu.fi Introduction The inflammatory process is initiated by endothelial cell (EC) activation comprising upregulation of chemokines and cell adhesion molecules, leukocyte activation and transmigration, and secretion of proinflammatory factors by leukocytes [1]. The inflammatory reaction is necessary for tissue recovery as it provides the correct cytokine signals and cell machinery to clear up the site for regeneration of the tissue [2]. However, uncontrolled inflammation has unfavorable effects on the course of tissue healing since the inflammatory cells are also capable of inducing tissue damage [2] and therefore many conditions involving inflammation, e.g. autoimmune diseases and tissue transplantations, are treated with immunosuppressants to reduce harmful leukocyte infiltration. Among the most potent drugs are glucocorticoids that downregulate the expression of numerous inflammatory chemokines, cytokines and adhesion molecules [3– 5], which, however, are not entirely without adverse effects such as delayed myocardial tissue healing, osteoporosis, and blood vessel calcification [6–9]. The most prominent outcome in the initial phase of inflammation is the enhanced production of cytokines, such as TNF-a and IL-1, and chemokines, such as monocyte chemoat- tractant protein-1 (MCP-1) [10,11], which further induce expression of a number of inflammatory cytokines [4]. Many of the stimulus-specific pathways converge in the production of superoxide (O22) and hydrogen peroxide (H2O2) as signal mediators, which in turn result in e.g. NFkB activation responsible for numerous stress-related functions [12–14]. Leukocytes are thus recruited by expression of various cell adhesion molecules, e.g. selectins, and intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1, respectively) [15,16]. They promote rolling and firm adhesion of leukocytes to endothelial wall, the necessary interactions preceding transmigration [17]. To aid leukocyte migration the vessel wall cells change their morphology by assuming cytoskeletal and cell-cell junction modifications in response to e.g. ligand binding to ICAM-1 and VCAM-1 [18–20], and when stimulated by O22 or TNF-a [21,22]. Previously, it has been shown that extracellular superoxide dismutase (SOD3) can attenuate tissue damage and inflammation but so far its mechanism of action has not been completely defined [23–27]. Since excess inflammation prevents the tissue injury recovery we investigated in the present study the effect of SOD3 overexpression on cell migration. We used two in vivo acute inflammation models to determine how SOD3 affects leukocyte extravasation, and compared the results to efficacy of the glucocorticoid immunosuppressant dexamethasone. The mouse peritonitis and rat hind limb ischemia models have been characterized previously: they induce rapid infiltration of leuko- cytes to the peritoneal cavity and large femoral muscles, PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5786