Glucocorticoid-Induced Tumor Necrosis Factor Receptor-Related (GITR)-Fc Fusion Protein Inhibits GITR Triggering and Protects from the Inflammatory Response after Spinal Cord Injury □ S Giuseppe Nocentini, Salvatore Cuzzocrea, Tiziana Genovese, Rodolfo Bianchini, Emanuela Mazzon, Simona Ronchetti, Emanuela Esposito, Di Paola Rosanna, Placido Bramanti, and Carlo Riccardi Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Universita ` di Perugia, Italy (G.N., R.B., C.R.); Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, Messina, Italy (S.C.); and Istituto di Ricovero e Cura a Carattere Scientifico Centro Neurolesi “Bonino-Pulejo,” Messina, Italy (T.G., E.M., E.E., D.P.R., P.B.) Received December 13, 2007; accepted March 5, 2008 ABSTRACT Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein is a costimulatory molecule that plays a role in inflammation so that GITR-Fc fusion protein can exert an anti- inflammatory effect. To investigate the mechanism by which GITR-Fc exerts its effects, we first used GITR knock-out (GITR -/- ) mice to verify whether GITR ligand (GITRL)/GITR system played a pro-inflammatory role in the spinal cord injury (SCI) model. It is noteworthy that less pronounced disease was induced in GITR -/- compared with GITR +/+ mice. We then evaluated the effect of GITR-Fc fusion protein against SCI-induced injuries in GITR -/- and wild-type (GITR +/+ ) mice. Administration of GITR-Fc amelio- rated SCI-induced inflammation in GITR +/+ mice as evaluated through: 1) histological damage and apoptosis, 2) modulation of apoptosis-related transduction factors (Bax and Bcl-2), 3) expres- sion of inflammatory markers [nitrotyrosine, inducible nitric-oxide synthase, interleukin (IL)-2, IL-12, and tumor necrosis factor-], and 4) T-lymphocyte infiltration. GITR-Fc was effective in GITR +/+ but not in GITR -/- , suggesting that in this experimental model, its anti-inflammatory action was due to inhibition of GITR triggering and not to GITRL activation. In conclusion, GITR plays a role in SCI, and administration of GITR-Fc results in amelioration of SCI severity, prompting further studies on the potential anti-inflamma- tory properties of GITR-Fc. The central nervous system is sensitive to mechanical in- juries, causing permanent functional deficits, as in patients who have spinal cord injury (SCI). The mechanical forces imparted to the spinal cord cause immediate tissue disrup- tion, with a direct axonal and neuronal injury, inducing the death of a number of neurons that can neither be recovered nor regenerated. Moreover, neurons continue to die for hours after SCI as a result of several mechanisms, including excito- toxicity, vascular abnormalities, and inflammatory response, that can contribute to evolution of spinal cord secondary injury (Kwon et al., 2004). Post-traumatic inflammation is determined by a number of cellular and molecular events (Carlson et al., 1998). Leuko- cytes are directly involved in the pathogenesis and extension of SCI (Carlson et al., 1998; Fleming et al., 2006). In fact, they infiltrate the marginal zone around the injured area, release inflammatory mediators, and activate endothelial cells, leading to increased vascular permeability, edema for- This study was supported by Associazione Italiana Ricerca sul Cancro (Milan, Italy) and by the network Fondo per gli Investimenti della Ricerca di Base grant, protocol RBPR05NWWC “CHEM-PROFARMA-NET” (Italy). G.N. and S.C. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.044354. □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: SCI, spinal cord injury; GITR, glucocorticoid-induced tumor necrosis factor receptor-related; GITR-Fc, fusion protein including the extracellular domain of GITR; TNF, tumor necrosis factor; APC, antigen-presenting cell; GITRL, GITR ligand; BBB, Basso, Beattie, and Bresnahan; PBS, phosphate-buffered saline; Ab, antibody; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CFSE, carboxyfluorescein diacetate succinimidyl ester; iNOS, inducible nitric-oxide synthase; wm, white matter; gm, gray matter; IL, interleukin; FasL, Fas ligand; TNF, tumor necrosis factor. 0026-895X/08/7306-1610 –1621$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 6 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 44354/3338428 Mol Pharmacol 73:1610–1621, 2008 Printed in U.S.A. 1610 by guest on May 14, 2012 molpharm.aspetjournals.org Downloaded from 4354.DC1.html http://molpharm.aspetjournals.org/content/suppl/2008/03/05/mol.107.04 Supplemental Material can be found at: