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Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università 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
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], 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-inflammatory properties of GITR-Fc.
).
Post-traumatic inflammation is determined by a number of cellular and molecular events (Carlson et al., 1998). Leukocytes 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 formation, and cell death (Kwon et al., 2004). Free radical production and lipid peroxidation is also important in the acute pathophysiology of SCI, both during the initial period of hypoperfusion and even more significantly during the period of reperfusion (Sakamoto et al., 1991).
Glucocorticoid-induced tumor necrosis factor (TNF) receptor-related (GITR) protein originally cloned in a glucocorticoid-treated hybridoma T cell line (Nocentini et al., 1997) is a protein belonging to TNF receptor superfamily. It is expressed in several cells and tissues, including T cells, where it acts as a costimulatory molecule (Tone et al., 2003; Ronchetti et al., 2004, 2007) after activation by its ligand (GITRL), mainly expressed on antigen-presenting cells (APC) and endothelial cells. GITRL/GITR system participates in the development of autoimmune diseases and potentiates response to infection and tumors (Nocentini et al., 2007b). Recent evidences suggest that physiological or pharmacological triggering of GITR exacerbates acute and chronic inflammatory response due not only to T cell modulation but also to modulation of extravasation and innate immunity (Krausz et al., 2007). Moreover, treating animals with GITR-Fc fusion protein ameliorates inflammation (Cuzzocrea et al., 2006, 2007). The main mechanism of action of GITR-Fc used as anti-inflammatory compound is still unknown, potentially being either inhibition of GITR triggering and/or stimulation of GITRL. In fact, GITRL can activate cytoplasmic signals upon GITR binding and privilege the development of tolerogenic dendritic cells (Agostini et al., 2005; Shin et al., 2005; Baltz et al., 2007; Grohmann et al., 2007). Establishing the main mechanism of action of GITR-Fc is crucial to plan treatment of diseases with different pathogenesis.
We here show that GITR plays a role in the modulation of secondary injury in an SCI model, demonstrating that GITR-deficient (GITR-/-) mice develop weaker SCI-induced injury and that GITR-Fc weakens SCI-induced injury. Moreover, GITR-Fc is active in GITR+/+ but not in GITR-/- mice, although GITR-/- mice develop a significantly reduced pathologic condition, suggesting that the main mechanism of action of GITR-Fc is the inhibition of GITR triggering.
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). Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and United States (Animal Welfare Assurance No A5594-01, Department of Health and Human Services). Spinal Cord Injury. Mice were anesthetized using chloral hydrate (400 mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away exposing T5-T8 vertebrae. The spinal cord was exposed via a four-level T5-T8 laminectomy, and SCI was produced by extradural compression of the spinal cord using an aneurysm clip with a closing force of 24 g. In all injured groups, the spinal cord was compressed for 1 min. Sham animals were only subjected to laminectomy. After surgery, 1.0 mL of saline was administered subcutaneously to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were individually housed in a temperature-controlled room at 27°C for a survival period of 10 days. Food and water were provided to the mice ad libitum. During this time period, the animals' bladders were manually voided twice a day until the mice were able to regain normal bladder function.
Mini-Osmotic Pump Implantation and Fusion Protein Delivery. Alzet pumps are precision drug administration tools that were used to deliver fusion proteins at a constant rate. In particular, we implanted Alzet model 2004 mini-osmotic pumps (Charles River Laboratories, Milan, Italy) 3 h after SCI, as described previously (Cuzzocrea et al., 2007). The pumping rate was 1 µl/h (± 0.15 µl/h), and the reservoir volume was 200 µl.
Experimental Groups. Mice were randomly allocated into the following groups: 1) SCI-GITR+/+ group: mice were subjected to SCI and received saline by mini-osmotic pump; 2) SCI-GITR-/- group: mice were subjected to SCI and received saline by mini-osmotic pump; 3) control GITR+/+ group (sham): GITR+/+ mice were subjected to the surgical procedures as the above except that the aneurysm clip was not applied and received saline by mini-osmotic pump; 4) control GITR-/- group (sham): GITR-/- mice were subjected to the surgical procedures as the above groups except that the aneurysm clip was not applied and received saline by mini-osmotic pump; 5) GITR-Fc-treated SCI-GITR+/+ group: GITR+/+ mice were subjected to SCI and received GITR-Fc (6.25 µg/mouse) by mini-osmotic pump. GITR-Fc was purchased from Alexis Corporation (Lausen, Switzerland) and is a dimer of a fusion protein formed by the extracellular domain of GITR and the Fc portion of human IgG1; 6) Fc-treated SCI-GITR+/+ group: GITR+/+ mice were subjected to SCI and received Fc control (6.25 µg/mouse) by mini-osmotic pump. Fc was purchased from Alexis and is a dimer of a fusion protein formed by the Fc portion of human IgG1; 7) GITR-Fc-treated SCI-GITR-/- group: GITR-/- mice were subjected to SCI and received GITR-Fc (6.25 µg/mouse) by mini-osmotic pump; 8) GITR-Fc-treated GITR+/+ group: GITR+/+ mice were subjected to the surgical procedures as the above groups, except that the aneurysm clip was not applied and received GITR-Fc (6.25 µg/mouse) by mini-osmotic pump; 9) Fct-reated GITR+/+ group: GITR+/+ mice were subjected to the surgical procedures as the above groups except that the aneurysm clip was not applied and received Fc control (6.25 µg/mouse) by mini-osmotic pump; and 10) GITR-Fc-treated GITR-/- group: GITR-/- mice were subjected to SCI and received GITR-Fc (6.25 µg/mouse) by miniosmotic pump.
Mice from each group were sacrificed 24 h after SCI to collect samples for the evaluation of the parameters as described below. In each experiment, five mice per group were treated, with the exception of groups 8, 9, and 10 (1-2 mice groups). Tree experiments were performed. No differences were observed in the histological results among groups 8, 9, and 10 and the respective sham-treated controls (groups 1-4) concerning each of the parameter studied. In the experiments investigating the motor score, the animals were observed for 10 days after SCI. In each experiment, eight mice per group were treated, and tree experiments were performed.
Grading of Motor Disturbance. The motor function of mice subjected to compression trauma was assessed once a day for 10 days after injury. Recovery from motor disturbance was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) (Basso et al., 1995) hind limb locomotor rating scale (Joshi and Fehlings, 2002a,b). The following criteria were considered: 0 = no hind limb movement; 1 = slight (<50% range of motion) movement of one to two joints; 2 = extensive (>50% range of motion) movement of one joint and slight movement of one other joint; 3 = extensive movement of two joints; 4 = slight movement in all three joints; 5 = slight movement of two joints and extensive movement of one joint; 6 = extensive movement of two joints and slight movement of 1 joint; 7 = extensive movement of all three joints; 8 = sweeping without weight support or plantar placement and no weight support; 9 = plantar placement with weight support in stance only or dorsal stepping with weight support; 10 = occasional (0-50% of the time) weight-supported plantar steps and no coordination (front/hind limb coordination); 11 = frequent (50-94% of the time) to consistent (95-100% of the time) weight-supported plantar steps and no coordination; 12 = frequent to consistent weight-supported plantar steps and occasional coordination; 13 = frequent to consistent weight-supported plantar steps and frequent coordination; 14 = consistent weight-supported plantar steps, consistent coordination and predominant paw position is rotated during locomotion (lift off and contact) or frequent plantar stepping, consistent coordination, and occasional dorsal stepping; 15 = consistent plantar stepping and coordination, no/occasional toe clearance, paw position is parallel at initial contact; 16 = consistent plantar stepping and coordination (Front/hind limb coordination) and frequent toe clearance and predominant paw position is parallel at initial contact and rotated at lift off; 17 = consistent plantar stepping and coordination and frequent toe clearance and predominant paw position is parallel at initial contact and lift off; 18 = consistent plantar stepping and coordination and consistent toe clearance and predominant paw position is parallel at initial contact and rotated at lift off; 19 = consistent plantar stepping and coordination and consistent toe clearance and predominant paw position is parallel at initial contact and lift off; 20 = consistent plantar stepping, coordinated gait, consistent toe clearance, predominant paw position is parallel at initial contact and lift off and trunk instability; 21 = consistent plantar stepping, coordinated gait, consistent toe clearance, predominant paw position is parallel at initial contact and lift off and trunk stability.
Immunohistochemistry. Twenty-four hours after SCI, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8-mm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (Vector Laboratories, Burlingame, CA), respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit Ab, anti-iNOS Ab rat, anti-Fas-ligand monoclonal antibody, anti-Bax rabbit Ab, anti-Bcl-2 Ab, anti-TNF- Ab, anti-IL-2 Ab, anti-IL-12 Ab, anti-CD4 Ab, or anti-CD8 Ab. Dilution of Abs was 1:100 or 1:500 (v/v) in PBS as previously specified (Cuzzocrea et al., 2008). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (Vector Laboratories). To verify binding specificity, some sections were incubated with only the secondary antibody (no primary). Immunohistochemical photographs (n = 5 from each sample collected from each mouse in each experimental group) were assessed by densitometry by using Optilab software (Graftek, Austin, TX) as described previously (Shea, 1994).
Terminal Deoxynucleotidyltransferase-Mediated UTP Nick-End Labeling Assay. TUNEL assay is a technique revealing double strand breaks and used to detect apoptotic cells. It was conducted by using a TUNEL detection kit according to the manufacturer's instructions (Apotag HRP kit; DBA, Milano, Italy). In brief, sections were incubated with 15 µg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were dipped into terminal deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated dUTP in terminal deoxynucleotidyltransferase buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.
Light Microscopy. Spinal cord biopsies were taken 24 h after trauma. The biopsies were fixed for 24 h in paraformaldehyde solution (4% in PBS 0.1 M) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (thickness, 5 µm) were deparaffinized with xylene, stained with hematoxylin/eosin and Luxol Fast Blue (used to assess demyelination), and studied using light microscopy (Dialux 22; Leitz, Milan, Italy). Damaged neurons were counted, and the histopathologic changes of the gray matter were scored on a six-point scale (Sirin et al., 2002): 0 = no lesion observed, 1 = gray matter contained one to five eosinophilic neurons, 2 = gray matter contained 5 to 10 eosinophilic neurons, 3 = gray matter contained more than 10 eosinophilic neurons, 4 = small infarction (less than one third of the gray matter area), 5 = moderate infarction (one third to one half of the gray matter area), and 6 = large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for an individual mouse. All the histological studies were performed in a blinded fashion.
T-Cell Proliferation by CFSE Labeling. CFSE labeling technique reveals cells undergoing cell doublings during in vitro test. In fact, cells are labeled at time 0, cultured in vitro, and, at the end of the culture period, studied by flow cytometry to evaluate the amount of CFSE present. The more they have proliferated, the lower the amount of CFSE present in the cells. In brief, T lymphocytes were obtained from spleen or axillary, brachial, maxillary, and inguinal lymph node of GITR+/+ and GITR-/- mice. Cells were resuspended at 1 x 106 cells/ml in prewarmed (37°C) PBS with 0.1% bovine serum albumin. Freshly prepared CFSE (Invitrogen SRL, Milan, Italy) was added to a final concentration of 10 µM, and the cells were incubated for 10 min at 37°C. Excess CFSE was quenched by adding 5 volumes of ice-cold RPMI 1640 medium containing 10% fetal bovine serum and incubating the cells for 5 min on ice. CFSE-labeled cells were then washed three times with RPMI 1640 medium containing 10% fetal bovine serum and cultured with the indicated stimuli.
Cells (0.5 x 106 cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, streptomycin (100 µg/ml), 10 mM HEPES, 0.1% nonessential amino acids, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. Goat anti-CD3
monoclonal Ab (BD Biosciences Pharmingen, San Diego, CA) was added in soluble form, and the concentration was reported. GITR-Fc and Fc-isotype control (Alexis) were added at a final concentration of 2 µg/ml. Seventy-two hours later, the percentage of cells that have performed at least one cell cycle was evaluated through flow cytometry, conducted on a flow cytometer (EPICS XL-MCL; Beckman-Coulter, Fullerton, CA) running FCS Express 3.0 analysis software (De Novo Software, Los Angeles, CA).
Materials. Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Milan, Italy). GITR-Fc was purchased from Alexis. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter S.p.A., Milano, Italy) or 10% dimethyl sulfoxide.
Statistical Evaluation. All values in the figures and text are expressed as mean ± S.E.M. In the experiments involving histology or immunohistochemistry, figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. A p value of less than 0.05 was considered significant. BBB scale data were analyzed by the Mann-Whitney test and considered significant when p value was less than 0.05.
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through the immunohistological staining. No staining resulted for either IL-2 (Figs. 6, a and b), IL-12 (Fig. 7a and Supplemental Fig. s1, a and b), and TNF- (Fig. 7b and Supplemental Fig. s2, a and b) in spinal cord obtained from the sham-operated mice. IL-2 (Fig. 6c) and IL-12 (Fig. 7a and Supplemental Fig. s1c) were found in inflammatory cells as well as in nuclei of Schwann cells in wm and gm of the spinal cord tissues of SCI-GITR+/+ mice. Levels of IL-2 (Fig. 6d) and IL-12 (Figs. 7a and Supplemental Fig. s1d) were significantly attenuated in GITR-/- mice subjected to SCI and in GITR-Fc-treated SCI-GITR+/+ mice (Fig. 6e 7a and Supplemental Fig. s1e). On the contrary, treatment of SCI-GITR-/- mice with GITR-Fc did not further decrease IL-2 and IL-12 expression (Figs. 6f and 7a and Supplemental Fig. s1f). Similar results were obtained with TNF- (Fig. 7b and Supplemental Fig. s2), suggesting that GITR-Fc decreases the production of pro-inflammatory cytokines that inhibit GITR triggering.
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Genetic and Pharmacological Inhibition of GITR Triggering Modulates Apoptosis, Fas Ligand Expression, and Regulation of Bcl-2 Family Members. To test whether inhibition of GITR triggering by GITR-Fc had antiapoptotic effects, we performed a TUNEL staining. Virtually no apoptotic cells were detected in the spinal cord from sham-operated mice (Fig. 9a and Supplemental Fig. s3, a and b). Tissues from SCI-GITR+/+ mice demonstrated dark brown apoptotic cells (approximately five cells per field) and intercellular apoptotic fragments (Fig. 9a and Supplemental Fig. s3c). In contrast, tissues obtained from SCI-GITR-/- mice showed a smaller number of apoptotic cells (approximately one per field) (Fig. 9a and Supplemental Fig. s3d). Likewise, very few apoptotic cells were observed in the spinal cord tissues collected from SCI-GITR+/+ mice treated with GITR-Fc fusion protein (Fig. 9a and Supplemental Fig. s3e). Neither treatment of GITR+/+ mice with control Fc nor treatment of GITR-/- mice with GITR-Fc reduced or increased the presence of apoptotic cells (Fig. 9a and Supplemental Fig. s3f).
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Because it has been demonstrated that apoptotic death of SCI is Fas-dependent (Casha et al., 2005), we evaluated the levels of Fas ligand (FasL) in our experimental system. There was no staining for FasL in spinal cord obtained from sham groups (Fig. 9b and Supplemental Fig. s4, a and b), but a significant presence was observed in the spinal cord tissues collected from SCI-GITR+/+ mice (Fig. 9b and Supplemental Fig. s4c). FasL levels were significantly decreased in SCI-GITR-/- mice (Fig. 9b and Supplemental Fig. s4d). Likewise, low levels of staining for FasL were observed in the spinal cord tissues collected from GITR-Fc-treated SCI-GITR+/+ mice (Fig. 9b and Supplemental Fig. s4e). The treatment of GITR+/+ mice with control Fc (Fig. 9b) and the treatment of GITR-/- mice with GITR-Fc (Figs. 9b and s4f) neither reduced nor increased the presence of FasL in the spinal cord compared with the respective control animals, further supporting the conclusion that GITR-Fc inhibits GITR triggering and that GITRL signaling has no role in the present inflammatory model.
A similar conclusion was reached studying the modulation of Bcl-2 family members that are involved in SCI-induced apoptosis (Dong et al., 2003; Seki et al., 2003). Sections of spinal cord from sham-operated mice did not stain for Bax (Fig. 9c and Supplemental Fig. s5, a and b) whereas spinal cord sections obtained from SCI-GITR+/+ mice exhibited a positive staining (Figs. 9c and s5c). Spinal cord levels of Bax were significantly attenuated in SCI-GITR-/- compared with SCI-GITR+/+ mice (Fig. 9c and Supplemental Fig. s5d). Likewise, lightly positive staining for Bax was observed in the spinal cord tissues collected from GITR-Fc-treated SCI-GITR+/+ mice (Fig. 9c and Supplemental Fig. s5e). Once again, the treatment of GITR-/- mice with control GITR-Fc (Fig. 9c and Supplemental Fig. s5f) and GITR+/+ mice with control Fc (Fig. 9c) did not modulate the levels of Bax.
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Genetic and Pharmacological Inhibition of GITR-GITRL Interaction Reduced T-Cell Infiltration. The anti-inflammatory effect of GITR-Fc may also be due to inhibition of leukocyte extravasation, as suggested by previous data (Cuzzocrea et al., 2004, 2006; Nocentini et al., 2007a). To evaluate whether genetic and pharmacological inhibition of GITR triggering inhibits leukocyte extravasation in this inflammatory model, we looked for leukocyte infiltration by immunohistochemical staining for CD4+ and CD8+ T lymphocytes at the injury site. As expected, there was no staining for CD4+ (Fig. 10, a and b) and CD8+ (Fig. 11, a and b) T lymphocytes in spinal cord obtained from the sham groups. CD4+ (Fig. 10c) and CD8+ (Fig. 11c) cells were observed in the spinal cord tissues collected from SCI-GITR+/+ mice. CD4+ and CD8+ T lymphocytes infiltrates were significantly attenuated in SCI-GITR-/- mice (Figs. 10d and 11d, respectively) and in SCI-GITR+/+ mice treated with GITR-Fc fusion protein (Figs. 10e and 11e, respectively). Treatment of SCI-GITR+/+ mice with control Fc (Figs. 10g and 11g) and of SCI-GITR-/- mice with GITR-Fc (Figs. 10f and 11f) did not reduce the presence of CD4+ and CD8+ T lymphocytes compared with their respective control, suggesting that GITR-Fc inhibits extravasation by impeding GITR triggering.
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), and results here confirm that a lower level of inflammation correlates with decreased motor lesions as indicated by a higher motor score in SCI-GITR-/- mice. In addition, we demonstrate that antagonist GITR-Fc counters SCI-induced histological injuries and inflammation.
Development of SCI-induced lesions in GITR+/+ mice treated with GITR-Fc fusion protein was similar to that observed in GITR-/- mice concerning all the investigated parameters. This finding confirms the efficacy of GITR-Fc as an anti-inflammatory drug, as previously seen in other experimental models (Nocentini et al., 2007a), and suggests inhibition of GITR triggering results in disease reduction.
According to published data, GITR-Fc can function as 1) an antagonist that inhibits GITR triggering and 2) an agonist that stimulates GITRL. Based on different experimental models it is well known that GITR costimulates a number of cells, including T lymphocytes (Tone et al., 2003; Ronchetti et al., 2004; Cuzzocrea et al., 2005; Nocentini and Riccardi, 2005; Patel et al., 2005; Ramirez-Montagut et al., 2006; Nocentini et al., 2007b; Ronchetti et al., 2007). Moreover, GITR participates in the activation of neutrophils and other cells of innate immunity, playing a pro-inflammatory role in acute and chronic inflammation (Cuzzocrea et al., 2006, 2007; Krausz et al., 2007). Therefore, in GITR+/+ mice, inhibition of GITR activation by administration of GITR-Fc can account for the decreased inflammatory response. Moreover, whereas GITR-Fc worked efficiently in GITR+/+, it did not in GITR-/-, clearly indicating that it acts as a receptor antagonist.
An additional possibility is that GITR-Fc activates GITRL. In fact, evidence in other experimental models suggests that GITRL can activate cytoplasmic signals in a number of cells, including dendritic cells, where GITRL triggering by GITR inhibits IL-12 production and increases tryptophan catabolism, thus favoring a tolerogenic phenotype that could account for a decreased inflammation (Agostini et al., 2005; Grohmann et al., 2007). Moreover, results in other studies indicate that GITRL triggering on APC promotes inflammatory response (Shin et al., 2003; Kim et al., 2006). In the present experimental model we show that a milder inflammatory response was induced in GITR-/- compared with GITR+/+ mice, but also that GITR-Fc administration exerted no significative effect in SCI-GITR-/- mice but was effective in SCI-GITR+/+. In addition, in in vitro experiments, GITR-Fc exerted an antiproliferative activity by inhibiting GITR triggering and not acting as GITRL agonist (Fig. 9). These results clearly indicate that in SCI experimental model, GITRL triggering does not play a role in the modulation of inflammation and that the main mechanism of action of GITR-Fc is to antagonize GITR triggering.
In the inflammatory response, extravasation of cells plays an important role. During inflammation, both GITR and GITRL are expressed on endothelial cells of vessels, and evidence has suggested that GITRL/GITR system modulates extravasation (Cuzzocrea et al., 2004, 2006). In the present experimental model, T cells are reduced in the perilesional area of SCI-GITR-/- and GITR-Fc-treated SCI-GITR+/+ mice compared with SCI-GITR+/+. These results are in agreement with our previous observation indicating a decreased extravasation of neutrophils in GITR-/- mice that correlates with a decreased expression of adhesion molecules such as intercellular adhesion molecule-1, P-selectin, and E-selectin (Cuzzocrea et al., 2004, 2006). The lack of differences of cell infiltration in SCI-GITR-/- and GITR-Fc treated SCI-GITR-/- mice, further suggest that a role is played by GITR activation, but not by GITRL activation, and that GITR-Fc inhibits extravasation by impeding GITR triggering. In our opinion, GITR, expressed on endothelial cells, is triggered by GITRL expressed on neutrophils, monocytes, and activated T lymphocytes (Krausz et al., 2007) and induces up-regulation of adhesion molecules. This hypothesis is in agreement with other studies demonstrating that GITR triggering increases expression of adhesion molecules, such as P-selectin and E-selectin, in activated T lymphocytes (Mahesh et al., 2006). Moreover, inhibition of inflammatory cell migration in the perilesional area contributes to a decreased concentration of pro-inflammatory cytokines that is also a consequence of GITR antagonism on pro-inflammatory cells as here demonstrated for TNF-, IL-2, and IL-12 (Figs. 6 and 7). The lower level of cytokines, in turn, contributes to a lower expression of adhesion molecules and lower levels of extravasation in a positive feedback process.
After SCI, apoptosis of neurons and oligodendrocytes is associated with axonal degeneration (Kwon et al., 2004). In fact, in SCI-GITR+/+ mice, we observed apoptotic death in the perilesional area that was less evident in SCI-GITR-/- and in GITR-Fc-treated SCI-GITR+/+ mice. The decreased apoptosis correlated with the modulation of transduction proteins belonging to Bcl-2 family. In particular, in SCI-GITR-/- mice, we observed lower expression levels of the proapoptotic protein Bax and higher expression levels of the antiapoptotic protein Bcl-2. These results are in accordance with previous observations showing that SCI-induced apoptosis is Bax-dependent (Martin and Liu, 2002; Dong et al., 2003) and that overexpression of Bcl-2 protects cells from SCI (Seki et al., 2003). However, considering that for signaling of Bcl-2 family members, cell localization is critical, the real contribution of these proteins to SCI-induced apoptosis has to be further investigated.
Apoptosis is favored by activation of Fas receptor, which is known to be involved in the SCI lesions (Demjen et al., 2004; Casha et al., 2005). We found higher levels of FasL in SCI-GITR+/+ mice compared with GITR-/- and GITR-Fc-treated SCI-GITR+/+ mice. Thus, GITR triggering up-regulates FasL in GITR positive cells, including activated T cells that are present in the perilesional area (Figs. 10 and 11), and induces higher apoptosis level in SCI-GITR+/+ mice. Similar conclusions were reached by Muriglan et al. (2004), working on a graft-vs-host experimental model, showing that GITR stimulation increases FasL expression in CD4+ effector T cells, and by Ramirez-Montagut et al. (2006), showing that tumor rejection elicited by GITR triggering depends on augmentation of FasL expression. We also demonstrated that GITR triggering inhibition by GITR-Fc inhibited SCI-induced Bax and FasL up-regulation as well Bcl-2 down-regulation.
In conclusion, GITRL/GITR system plays a role in the inflammatory neuronal lesion secondary to SCI that can be antagonized by treatment with GITR-Fc. Moreover, those results, considering the different role of GITR in different steps of inflammatory process, including cell activation, cytokine production, and cell migration, prompt to further studies aimed at better defining the potential use of GITR-Fc as anti-inflammatory drug and the mechanisms responsible for GITR-Fc therapeutic effect.
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G.N. and S.C. contributed equally to this work.
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.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Salvatore Cuzzocrea, Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail: salvator{at}unime.it
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