| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, Messina, Italy;
Istituto di Ricovero e Cura a Carattere Scientifico Centro Neurolesi Bonino-Pulejo, Messina, Italy;
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, and Polo Scientifico e Didattico di Terni, Terni, Italy; and
Dipartimento di Farmacologia Sperimentale, Università di Napoli Federico II, Napoli, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
, IL-1
, myeloperoxidase, inducible NO synthase, and cyclooxygenase 2) in the pleural exudate and/or in the lung. Moreover, endothelial cells expressed lower levels of adhesion molecules. In lungs of GITR+/+ mice, GITR ligand expression was not modulated during pleurisy, while that of GITR increased, as a consequence of increased infiltration by GITR-expressing cells and of GITR up-regulation in macrophages and endothelial cells. Finally, cotreatment of GITR+/+ mice with carrageenan and Fc-GITR fusion protein decreased the number of inflammatory cells (pleural macrophages and lung neutrophils) as compared with carrageenan treatment alone, confirming that GITR plays a role in the modulation of pleurisy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
GITR is activated by its ligand (GITR ligand; GITRL), which is expressed in APCs, including macrophages, in endothelial cells, but not in T lymphocytes (5, 9, 10, 11, 12). Following GITR-GITRL interaction, GITRL also delivers signals to APC (13, 14, 15, 16). In vivo studies suggest that GITR triggering exacerbates autoimmune/inflammatory responses and potentiates antiviral and antitumoral immunity (5, 17, 18).
To investigate whether GITR also participates in the acute inflammatory response, GITR–/– and GITR+/+ mice were injected in the pleural cavity with carrageenan to obtain an acute lung inflammation, usually defined as carrageenan-induced pleurisy. Carrageenan-induced inflammation (paw edema or pleurisy) is a model of local acute inflammation commonly used to evaluate activity of anti-inflammatory drugs (19, 20, 21) and useful to assess the contribution of cells and mediators to the inflammatory process (22). The initial phase of carrageenan-induced pleurisy (0–1 h) has been attributed to the release of histamine, 5-hydroxytryptamine, and bradykinin, followed by a late phase (1–6 h) mainly sustained by PG release due to the induction of cyclooxygenase 2 (COX-2) in the tissues (23). PMNs moving out of the circulation into the inflamed tissue have a key function in the breakdown and remodeling of injured tissue (24, 25). Moreover, macrophages participate in the progression of experimental pleurisy producing proinflammatory cytokines such as TNF- and IL-1 (26).
In this context, a crucial role is played by the endothelium, which modulates extravasation and permeability in response to inflammatory products, further favoring the development of inflammation. In particular, migration and accumulation of PMNs and macrophages is a complex phenomenon involving endothelium-based adhesion molecules such as ICAM-1 and P-selectin (27). Moreover, tight junctions serve as a permeability barrier, and the expression of proteins involved in tight junction function, including occludin, claudin family members, and ZO-1, regulates its permeability (28, 29, 30, 31).
In our study, we demonstrate that GITR–/– mice exhibited a reduced degree of lung inflammation as evaluated by a number of parameters, including the following: leukocyte pleural recruitment and lung infiltration, expression of adhesion molecules, release of proinflammatory cytokines and NO, presence of oxidative stress, and other indices of inflammation and tissue damage. Moreover, a decreased inflammation level was obtained by injection with Fc-GITR fusion protein in carrageenan-treated GITR+/+ mice. Taken together, these data indicate that GITR participates in the carrageenan-induced acute inflammatory response in the lung.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Sv129 mice (8–9 wk old, 22–24 g) with a targeted disruption of the GITR gene (GITR–/–) and wild-type controls (GITR+/+) were used (7). Pleurisy was induced, as previously described (32). Animal care was in compliance with regulation in Italy (decreto ministeriale 116192), Europe (Official Journal of European Contract Law 358/1 12/18/1986), and U.S. (Animal Welfare Assurance No. A594-01, Department of Health and Human Services), and the study was approved by the Italian Ministero della Salute. At 2, 4, and 8 h after the injection of carrageenan, the animals were sacrificed and studied. In the experiments performed to study the modulation of GITR-GITRL system, Fc-GITR (Alexis) was injected in the pleural space (100 µl/mouse, 5 µg/mouse) 1 min before carrageenan injection, and animals were sacrificed and studied 4 h after injection.
Numbers and phenotyping of cells infiltrating the pleural space
Two, 4, or 8 h after the injection of carrageenan, the animals were sacrificed and the pleural cavity was rinsed with 1 ml of saline solution containing heparin (5 U/ml) and indomethacin (10 µg/ml). The exudate and washing solution were removed by aspiration, and the total volume was measured. The amount of exudate was calculated by subtracting the volume injected (1 ml) from the total volume recovered. The leukocytes in the exudate were suspended in PBS and counted with an optical microscope in a Burker’s chamber after toluidine blue staining. Flow cytometry analysis was done using EPICS XL-MCL (Beckman Coulter). The following Abs (BD Pharmingen) were used: FITC anti-mouse CD3
-chain (clone 145-2C11), FITC anti-mouse CD11b (clone M1/70), and FITC anti-mouse Ly-6G (GR-1).
Histological examination
For histological examination, lung biopsies, taken at 4 h after injection of carrageenan, were fixed in 10% buffered Formalin phosphate, embedded in paraffin, sectioned, and stained with H&E.
TUNEL assay
TUNEL assay was conducted by using a kit according to the manufacturer’s instructions (ApopTag HRP kit, DBA). The number of TUNEL-positive cells/high-power field was counted in 5–10 fields for each coded slide, as previously described (17).
Immunohistochemistry
Tissue sections were prepared, as previously described (33). P-selectin (CD62P; BD Pharmingen), myeloperoxidase, ICAM-1 (CD54; BD Pharmingen), and nitrotyrosine Abs (Upstate Biotechnology) were used, as previously described (33). The polyclonal anti-GITR (R&D Systems) and anti-ZO-1 Abs were used 1/100 in the same experimental conditions as the others.
Measurement of inflammatory mediators
For TNF- and IL-1, ELISA tests were conducted, as previously described (33). PGE2 was measured by RIA without prior extraction or purification (34). Myeloperoxidase activity and nitrite/nitrate production were determined, as previously described (32).
Western blot analysis
Lung tissue and pleural infiltrate were homogenated in a buffer containing: 20 mM HEPES, 1.5 mM MgCl2, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 15 µg/ml trypsin inhibitor, 3 µg/ml pepstatin, 2 µg/ml leupeptin, 40 µM benzidamin, and 1% Nonidet P-40. Protein concentration was determinated by the Bio-Rad protein assay using BSA as standard. Equal amounts of protein (70 µg/lane) were dissolved in Laemmli’s sample buffer, boiled, and run on 8% SDS-PAGE gel, and then transferred to hybond polyvinylidene difluoride membrane. Membranes were blocked for 40 min in PBS and 5% (w/v) nonfat milk and subsequently probed overnight at 4°C with mouse monoclonal anti-inducible NO synthase (iNOS) (Upstate Biotechnology; 1/10,000), anti-COX-2 (Santa Cruz Biotechnology; 1/500), anti-I
B- (Santa Cruz Biotechnology; 1/1,000), or anti-phospho-NF-B p65 (Ser536) (Cell Signaling Technology; 1/1,000) Abs (in PBS, 5% w/v nonfat milk, and 0.1% Tween 20). Blots were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (Pierce; 1/5,000) for 1 h at room temperature. Immunoreactive bands were visualized using chemiluminescence assay detection system (LiteAblot; Euroclone), according to the manufacturer’s instructions, and exposed to Kodak X-OMAT film. Bands were quantified by densitometric analysis (Imaging Densitometer GS-700; Bio-Rad).
Real-time RT-PCR
Purified macrophages (obtained using CD11b (Mac-1) MicroBeads; Miltenyi Biotec), from pleural exudate, followed by a separation with Biomag sheep antifluorescein magnetic beads (Applied Biosystems) from pleural exudate, were homogenized in 1 ml of TRIzol reagent (Invitrogen Life Technologies). Lungs were immediately snap frozen on liquid nitrogen and homogenized in 1 ml of TRIzol reagent. Total RNA was isolated according to the manufacturer’s instructions. Reverse transcription of total RNA (1 µg) was performed with random primers and Superscript II (Invitrogen Life Technologies). PCR experiments were performed using specific primers: GITR forward (5'-AAGGTTCAGAACGGAAGTG-3'), GITR reverse (5'-GGGTCTCCACAGTGGTACT-3'); GITRL forward (5'-CGAGTCCTGCATGGTTAA-3'), and GITRL reverse (5'-TCAGCTTCCCATCAGATGTC-3'). PCR was done in the CHROMO 4 (MJ Research) using DyNAmo HS SYBR GREEN qPCR kit (Finnzymes). Gene expression was quantitated relatively to the expression of hypoxanthine phosphoribosyltransferase-1, evaluated in separate tubes.
Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich. Secondary and nonspecific IgG Abs for immunohistochemical analysis were from Vector Laboratories.
Data analysis
All values in the figures and text are expressed as mean ± SEM of the mean of n observations. For the in vivo studies, n represents the number of animals studied. Results were analyzed by one-way ANOVA, followed by a Bonferroni posthoc test for multiple comparisons. Value of p < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
To analyze the possible influence of GITR during acute inflammation in the lung, we examined the effect of GITR gene deletion on carrageenan-induced pleurisy. All GITR+/+ and GITR–/– mice that received carrageenan in the pleural space developed an acute pleurisy, as suggested by the production of turbid exudate (data not shown) and the increased number of cells collected from the pleural space following 2, 4, and 8 h of carrageenan administration (Fig. 1A). However, at every time point, the turbid exudate (data not shown) and the cells collected from the pleural space of carrageenan-treated GITR–/– mice were about half as compared with those from GITR+/+ mice (Fig. 1A, columns 4 vs 3). In fact, although the phenotype of infiltrating cells, as evaluated after 4 h of carrageenan administration, was similar (Fig. 1B), with macrophages far more represented in both groups, all the cell subpopulations, participating in the carrageenan-induced inflammation, were reduced in GITR–/– mice (Fig. 1C). Virtually no exudate and a low number of cells (resident macrophages) were detected in the pleural space of both GITR+/+ and GITR–/– sham-treated mice (Fig. 1A, columns 1 and 2).
|
|
Among infiltrating leukocytes, PMNs play a crucial role in tissue damage. Therefore, we evaluated the PMN infiltration of the lungs by using myeloperoxidase as a marker. In carrageenan-treated mice, the staining for myeloperoxidase was visibly lighter in GITR–/– (Fig. 3C) as compared with GITR+/+ mice (Fig. 3B). As expected, no positive staining was observed in lungs of sham-treated mice (Fig. 3A). To obtain more quantitative results, we measured myeloperoxidase activity of the lung tissue 2, 4, and 8 h after treatment. In GITR+/+ mice, it dramatically increased after carrageenan administration as compared with sham-treated controls (Fig. 3, D–F, columns 3 vs 1). In carrageenan-treated GITR–/– mice, myeloperoxidase activity was not increased or only slightly increased as compared with sham-treated controls (Fig. 3, D–F, columns 4 vs 2) and resulted significantly lower (p < 0.001) in comparison with that of carrageenan-treated GITR+/+ mice at all time points (Fig. 3, D–F, columns 4 vs 3).
|
GITR and its ligand are expressed in the lungs
To better understand the role of GITR in this process, we investigated the expression of GITR and GITRL in lungs of sham- and carrageenan-treated mice. Real-time PCR experiments demonstrated that GITRL is expressed at similar levels in GITR+/+ and GITR–/– lungs and that carrageenan treatment did not modulate its expression (Fig. 4A). On the contrary, GITR is up-regulated about 3-fold in the lungs of GITR+/+ mice following carrageenan treatment (Fig. 4B). Lungs of GITR–/– mice were used as negative control for the analysis of GITR expression (Fig. 4B, columns 2 and 4).
|
GITR is expressed in resident macrophages, as previously demonstrated also for peritoneal and splenic macrophages (3, 8, 9), and is up-regulated about 3-fold following carrageenan treatment (Fig. 4D, column 3 vs 1). Macrophages from GITR–/– mice were used as negative control for the analysis of GITR expression (Fig. 4D, columns 2 and 4).
It is well known that GITR is expressed in T cells and up-regulated following activation (5). GITR is also expressed in PMNs (35) and in pleural macrophages and is up-regulated during inflammation, as shown in Fig. 4D. Therefore, the increased expression of GITR mRNA in lungs of carrageenan-treated GITR+/+ mice (Fig. 4B) could be explained by leukocyte infiltration (as shown in Fig. 2) and by GITR up-regulation in activated cells. To verify that this was the case, we immunostained lung sections of sham- and carrageenan-treated mice with an anti-GITR Ab. In lung sections from sham-treated GITR+/+ mice, no positive staining for GITR was observed (Fig. 4E). Because real-time PCR indicated that GITR mRNA is expressed in the lung, this result probably means that the levels of expression are below the sensitivity level of the immunostaining. In lung sections from carrageenan-treated GITR+/+ mice, we obtained a positive staining for GITR in infiltrating macrophages, PMNs, and lymphocytes (see particles g1 of Fig. 4G). Surprisingly, we also observed a consistent staining of the vascular endothelium (see particle g2 of Fig. 4G). The absence of positive staining for GITR in both sham- and carrageenan-treated GITR–/– mice (Fig. 4, F and H) demonstrates the specificity of the staining.
Taken together, these results demonstrate that GITR is expressed in the lung, is up-regulated during the inflammatory response, and can be triggered by its ligand, thus suggesting a direct involvement of GITR in the carrageenan-induced pleurisy.
Lower levels of proinflammatory mediators in carrageenan-treated GITR–/– mice
In carrageenan-induced pleurisy, several proinflammatory factors are involved in the development of inflammatory response. Therefore, we evaluated the levels of the main mediators of the inflammation to verify whether the different response to carrageenan in GITR–/– mice was due to lack of one key player or to a general decrease in the proinflammatory factors.
First, we measured TNF- and IL-1 levels in pleural exudate of carrageenan-treated mice. Results of ELISA indicate that the levels of these cytokines were significantly lower in the exudate from GITR–/– as compared with that from GITR+/+ mice (Fig. 5, A and B, column 4 vs 3). Significantly higher levels of both cytokines were observed in the exudate of carrageenan-treated as compared with sham-treated GITR+/+ and GITR–/– mice.
|
|
Western blot analysis of lung homogenates obtained from carrageenan-treated GITR–/– mice revealed a COX-2 expression, which, however, was clearly weaker than that observed in lungs of GITR+/+ mice (Fig. 7A). As expected, COX-2 was undetectable in sham-treated mice (data not shown). To confirm these data, COX-2 activity was assessed by measuring the amount of PGE2 in the pleural exudate. Results indicate that the levels of PGE2 in carrageenan-treated GITR–/– mice were significantly lower as compared with those of carrageenan-treated GITR+/+ mice (Fig. 7B).
|
Lower levels of COX-2 in proinflammatory cells from carrageenan-treated GITR–/– mice
The lower expression of proinflammatory enzymes and the lower levels of their products in carrageenan-treated GITR–/– mice as compared with GITR+/+ mice might have been simply explained on the basis of the lower infiltration level of proinflammatory cells in the pleural space and in the lung. However, the possibility that these results were also due to a reduced activation level of proinflammatory cells in GITR–/– mice remained. Therefore, we evaluated the expression of COX-2, the main player of carrageenan-induced pleurisy (23), in the pleural infiltrate of carrageenan-treated mice. Western blot experiments demonstrated that COX-2 is expressed at a lower level in GITR–/– as compared with GITR+/+ cells (Fig. 8), suggesting that GITR–/– leukocytes are less activated as compared with GITR+/+ cells.
|
It is known that proinflammatory mediators modulate endothelial function, which, in turn, favors the extravasation and recruitment of leukocytes, further amplifying the inflammatory response. The decrease of all investigated inflammatory markers and the minor accumulation of leukocytes in GITR–/– mice as compared with GITR+/+ mice prompted us to investigate whether during acute inflammation the endothelial function of GITR–/– mice was modulated differently from that of GITR+/+ mice. In particular, we analyzed the expression of adhesion molecules, such as ICAM-1 and P-selectin, and the organization of tight junctions, which are both signs of the endothelial barrier response to proinflammatory signals, participating in the development of inflammatory response (28, 31, 36, 37).
Increased positive staining for ICAM-1 (Fig. 9, B and B1) and for P-selectin (Fig. 9, E and E1) was found along the bronchial epithelium and in vessels from carrageenan-treated GITR+/+ mice, as compared with sham-treated controls (Fig. 9A, ICAM-1; Fig. 9D, P-selectin). In carrageenan-treated GITR–/– lungs, the staining for ICAM-1 (Fig. 9C) and for P-selectin (Fig. 9F) was visibly reduced in comparison with carrageenan-treated GITR+/+ lungs (Fig. 9, B and E, respectively). Low, but detectable staining for ICAM-1, but not for P-selectin, was observed in lungs of sham-treated mice, both GITR+/+ (Fig. 9, A and D) and GITR–/– (data not shown), indicating that the basal expression of ICAM-1 is not differently regulated in GITR+/+ and GITR–/– mice.
|
|
B in lungs from carrageenan-treated GITR–/– mice
Most inflammatory mediators, including COX-2, iNOS, and ICAM-1, are controlled by NF-B transcription factor, which is kept inactive by IB while its trans activation potential is increased by phosphorylation of the p65 subunit (38, 39, 40, 41). Because the levels of proinflammatory markers evaluated are lower in lungs from carrageenan-treated GITR–/– as compared with GITR+/+ mice and it is known that GITR triggering activates TNFR-associated factor 2 and NF-B (5), we evaluated IB and phospho-p65 levels by Western blot experiments. Results in Fig. 11A indicate that while in carrageenan-treated GITR+/+ mice there was a decrease of IB levels as compared with sham control, no differences were detected between carrageenan- and sham-treated GITR–/– mice. Moreover, the phosphorylation of p65 subunit was highly increased in GITR+/+ mice upon carrageenan treatment, while it was not modulated in GITR–/– mice (Fig. 11B). These results confirm previous observation suggesting that GITR can induce NF-B activation, and are consistent with the role of NF-B in regulation of inflammatory mediators such as COX-2, iNOS, and ICAM-1 (5).
|
The above presented results demonstrate that GITR participates in the inflammatory response to carrageenan. To verify whether the inhibition of GITR-GITRL interaction and the consequent inhibition of GITR activation negatively modulate the development of the carrageenan-induced pleurisy, we cotreated GITR+/+ mice with carrageenan and an Fc-GITR fusion protein. Mice were injected in the pleural space with Fc-GITR (5 µg) and, 1 min later, with carrageenan. Four hours later, they were sacrificed, and two key markers of acute inflammation were evaluated: pleural infiltration of macrophages and lung infiltration of PMNs. Macrophage infiltration resulted significantly lower (p < 0.05) in the GITR+/+ mice cotreated with carrageenan and Fc-GITR as compared with GITR+/+ mice treated with carrageenan alone (Fig. 12A). Interestingly, macrophage infiltration of GITR+/+ mice cotreated with Fc-GITR and carrageenan was similar to that observed in carrageenan alone-treated GITR–/– mice (see Fig. 1 for comparison). To evaluate PMN infiltration of the lungs, we measured myeloperoxidase activity. It resulted significantly lower (p < 0.05) in Fc-GITR-cotreated GITR+/+ mice with respect to carrageenan alone-treated GITR+/+ mice (Fig. 12B), although higher than that observed in carrageenan alone-treated GITR–/– mice (see Fig. 3 for comparison). This latter effect may be due to the Fc-GITR administration route (intrapleural) that does not allow a sufficient plasmatic concentration of Fc-GITR.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B activation. The proinflammatory role of GITR in inflamed lung is not surprising, based on the expression of GITR and GITRL in lungs and leukocytes (including macrophages) and the up-regulation of GITR expression in macrophage during carrageenan-induced inflammation. We have demonstrated recently that GITR–/– mice have a higher survival rate than GITR+/+ mice in the splanchnic artery occlusion model (18). This observation, taken together with the differences seen in the expression of inflammatory markers, suggested that GITR could participate in life-threatening inflammatory response consequent to mechanical artery occlusion (18). The present finding indicates that GITR plays a role in the development of acute inflammation process triggered by an exogenous stimulus, and suggests that GITR-GITRL system may be a determinant player of the acute phase of inflammation and that the modulation of the GITR/GITRL system may be an approach to control inflammation development.
GITR and its ligand are expressed in macrophages, as demonstrated in this work and by other studies (3, 6, 8, 9). Moreover, we demonstrate in this study that GITR expression is up-regulated in macrophages upon carrageenan injection, suggesting that GITR participates in macrophage activation. In fact, in GITR+/+ mice, we found a higher level of macrophage-derived proinflammatory mediators (such as TNF-, IL-1, NO-derived products, and PGs) in the pleural space. Noteworthy is that the higher expression of proinflammatory molecules in the lungs (such as COX-2) of GITR+/+ as compared with GITR–/– mice is not only due to an increased infiltration of proinflammatory cells, including macrophages, but also to an increased production of mediators in proinflammatory cells (as shown for COX-2). Because macrophages express both GITR and GITRL, the increased expression of GITR may both positively modulate GITR-mediated pathways and trigger GITRL. In fact, recent studies demonstrate that GITRL can activate cytoplasmic signals following GITR binding (8, 13, 14, 16, 42). Future studies will clarify whether these mechanisms are both crucial in determining the decreased response of GITR–/– mice to carrageenan treatment.
In the lung, apoptosis is a common cellular reaction to an insult that can result in epithelial cell loss and fibrosis, as demonstrated in murine models of pulmonary fibrosis and LPS-induced lung injury (43, 44). The pleurisy model is also characterized by a substantial level of tissue injury and apoptosis (45), as seen in this study. The mechanisms responsible for epithelial cell apoptosis in acute lung injury are still unclear, but several lines of evidence point to the possible role of TNFRSF members, receptors deeply involved in the modulation of apoptosis (46, 47, 48, 49). In particular, it has been demonstrated that GITR binds and activates Siva, leading to cell death (46). Thus, a higher number of apoptotic cells in GITR+/+ as compared with GITR–/– lungs of carrageenan-treated mice might be due to a direct effect of GITR on pulmonary cells and/or on infiltrating cells. However, we cannot exclude that increased PMN infiltration and higher levels of proinflammatory and proapoptotic factors, such as, for example, TNF- and NO in GITR+/+ mice as compared with GITR–/– mice, may contribute to or directly cause the differences in apoptosis between carrageenan-treated GITR+/+ and GITR–/– mice.
The recruitment of cells in an area of inflammation is mediated by several proinflammatory factors promoting the activation and expression of adhesion molecules, such as P-selectin and ICAM-1, which play a crucial role in this process (36, 50). Another feature of lung inflammation is alveolar epithelium damage associated with severe injury of the alveolar capillary barrier and major increase in alveolar capillary permeability (51). Increased endothelial permeability results from several factors, including cytokine release (e.g., TNF-) (30, 52). We demonstrate in this study that during pleurisy, adhesion molecules are modulated at a lower level in GITR–/– mice as compared with GITR+/+ mice (Fig. 9), and that the disruption of ZO-1 pattern is less evident (Fig. 10). In our opinion, the differences seen are mainly due to the different levels of proinflammatory molecules observed in GITR–/– and GITR+/+ mice during pleurisy. The different modulation of the endothelial barrier further amplifies the inflammatory response of GITR+/+ mice and contributes to the lower inflammatory response of GITR–/– mice.
Even if the different response of endothelial barrier to carrageenan in GITR–/– and GITR+/+ mice seems secondary to the different amount of proinflammatory products, we cannot exclude a direct contribution of the GITR/GITRL system in extravasation process. In fact, it has been supposed that GITR plays a role in promoting extravasation of cells expressing GITR on their surface, due to the interaction with GITRL that is expressed on endothelial cells (10, 11, 12). Moreover, it is possible that GITR-expressing cells, including macrophages and PMNs, activate GITRL-dependent signals in endothelial cells, as demonstrated in APC (16), which leads to modulation of endothelial function. Finally, GITR, expressed on endothelial cells during inflammation (as shown in Fig. 4), may be triggered by GITRL-expressing cells, contributing to modulation of endothelial function as demonstrated for other TNFRSF members (53, 54).
Because our study strongly suggests the involvement of GITR in carrageenan-induced pleurisy, we wondered whether inhibition of GITR activation may modulate the response to carrageenan. Indeed, cotreatment of GITR+/+ mice with carrageenan and the fusion protein Fc-GITR decreased some of the key signs of carrageenan-induced inflammation. Interestingly, the number of macrophage collected from the pleural space of carrageenan-treated GITR+/+ mice cotreated with Fc-GITR was similar to that observed in carrageenan alone-treated GITR–/– mice. On the basis of the expression of GITR on both macrophages and PMNs, we speculate that Fc-GITR exerts its therapeutic anti-inflammatory activity by interfering with GITR/GITRL interaction and resembling GITR–/– mice model. As a consequence, GITR activation is inhibited. Moreover, if GITR/GITRL interaction participates in the extravasation process, Fc-GITR may hamper macrophages and PMN extravasation.
In conclusion, this study provides, for the first time, in vivo evidence that GITR is involved in the development of carrageenan-induced pleurisy, and suggests that the modulation of the GITR-GITRL system may help in the control of acute inflammation.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This study was supported by Associazione Italiana Ricerca sul Cancro (Milan, Italy) and a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (Rome, Italy). 
2 S.C. and G.N. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Salvatore Cuzzocrea, Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail address: salvator{at}unime.it
4 Abbreviations used in this paper: GITR, glucocorticoid-induced TNFR-related gene; COX-2, cyclooxygenase 2; GITRL, GITR ligand; iNOS, inducible NO synthase; PMN, polymorphonuclear leukocyte; TNFRSF, TNFR superfamily; Treg, regulatory T.
Received for publication January 18, 2006. Accepted for publication April 14, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B ligand (RANKL) in osteoclast cells. Bone 36: 832-839. [Medline] mediates morphine anti-inflammatory effect in carrageenan-induced paw oedema in mice. Cytokine 19: 102-105. [Medline] by lipopolysaccharide-activated J774 macrophages. Inflamm. Res. 48: 503-508. [Medline]-arrestins. J. Biol. Chem. 280: 31936-31948. B activation and IB dynamism involved in iNOS and chemokine induction in astroglial cells. Life Sci. 68: 1695-1701. [Medline]B and IB proteins: implications in cancer and inflammation. Trends Biochem. Sci. 30: 43-52. [Medline]
in murine macrophage. Exp. Mol. Med. 35: 175-180. [Medline]
(12,14)-prostaglandin J2 attenuates the development of acute and chronic inflammation. Mol. Pharmacol. 61: 997-1007. 
