Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0204110 on August 17, 2004

Published online before print August 17, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0204110v1
76/5/933    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuzzocrea, S.
Right arrow Articles by Riccardi, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cuzzocrea, S.
Right arrow Articles by Riccardi, C.
(Journal of Leukocyte Biology. 2004;76:933-940.)
© 2004 by Society for Leukocyte Biology

Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock

Salvatore Cuzzocrea*,1, Giuseppe Nocentini{dagger}, Rosanna Di Paola*, Emanuela Mazzon*, Simona Ronchetti{dagger}, Tiziana Genovese*, Carmelo Muià*, Achille P. Caputi* and Carlo Riccardi{dagger}

* Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, Messina, Italy; and
{dagger} Dipartimento di Medicina Clinica e Sperimentale, Università di Perugia, Italy

1 Correspondence: Institute of Pharmacology, School of Medicine, University of Messina, via C. Valeria, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail: salvator{at}unime.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we used glucocorticoid-induced tumor necrosis factor (TNF) receptor family gene knockout (GITR-KO) mice to evaluate a possible role of GITR on the pathogenesis of splanchnic artery occlusion (SAO) shock, which was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum of the SAO-shocked, GITR wild-type (WT) mice after reperfusion. The absence of GITR significantly reduced the lipid peroxidation in the intestine. SAO-shocked WT mice developed a significant increase of ileum tissue, TNF-{alpha}, and myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (5% survival at 24 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin, intercellular adhesion molecule 1 (ICAM-1), and E-selectin. The intensity and degree of P-selectin, E-selectin, and ICAM-1 were markedly reduced in tissue section from SAO-shocked, GITR-KO mice. SAO-shocked, GITR-KO mice also showed a significant reduction of the TNF-{alpha} production and neutrophil infiltration into the reperfused intestine, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that GITR plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of GITR expression may represent a novel and possible strategy.

Key Words: adhesion molecules • ischemia and reperfusion • neutrophil infiltration • lipid peroxidation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoid-induced tumor necrosis factor receptor (TNFR) family-related gene [GITR or TNFR superfamily 18 (TNFRSF18)] is a member of the TNFRSF, which was originally cloned in a glucocorticoid-treated hybridoma T cell line [1 ]. GITR is mainly expressed in lymphoid tissues including T lymphocytes [1 2 3 ]. In addition, like most of the members of the TNFRSF, GITR is overexpressed upon T lymphocyte activation, suggesting a role in the control of T cell activation [1 ]. Furthermore, GITR is able to protect T lymphocytes against T cell receptor (TCR)-activated apoptosis and acts as a coaccessory molecule in T lymphocyte activation [1 2 3 ].

T cell-mediated immunoregulation is one of the main mechanisms that is responsible for maintaining antigen-specific tolerance in vivo and for controlling T cell homeostasis [4 ]. Signals from TCR and coacessory molecules are required for full T cell activation [4 5 6 ]. Different mechanisms of energy and clonal selection are operative and determine the final outcome of immune response or tolerance [7 8 9 10 11 ]. In particular, deregulation of those mechanisms may break the immunological self-tolerance and gives rise to antigen-specific effector T lymphocyte activation and the consequent triggering of other autoimmune/inflammatory processes responsible for diseases [12 ]. Inflammation and tissue destruction are often the outcome of autoimmune disorders, and massive recruitment of leukocytes selects organs or tissues, the production of inflammatory cytokines, and intense cell death, leading to tissue destruction [13 ].

If GITR plays a role in immune response, it is plausible that it could play a role in the pathogenesis and development of acute/subacute and chronic autoimmune/inflammatory diseases.

Ischemia leads to hypoxia and is characterized by a series of events primarily related to activation of platelets and release of their vasoconstrictor mediators (e.g., thromboxane A2 and 5-hydroxytryptamine), which further restrict blood flow to the ischemic area. If the ischemia is severe enough, the rate of metabolism is diminished, and the generation of high-energy compounds subsequently declines (e.g., adenosine 5'-triphosphate). This degree of tissue injury is further enhanced and accelerated by reperfusion.

It is important to realize that reperfusion of an ischemic organ is not only associated with local changes. In some situations, reperfusion is also associated with systemic changes. For example, in a model of ischemia and reperfusion of the intestine, local, functional alterations include intestinal hyperpermeability as well as morphological changes, such as necrotic injury of the reperfused tissues [12 13 14 15 ]. Conversely, systemic alterations, seen post-reperfusion, include a progressive fall in the mean arterial blood pressure, release of proinflammatory mediators from the reperfused tissues into the systemic circulation, and ultimately, decreased survival [16 ].

Ischemia/reperfusion is also a stimulus for leukocyte-endothelial cell interaction and migration into tissues: Neutrophils provide another source of proinflammatory mediators [16 ]. Leukocyte-endothelial cell interaction involves a complex system of adhesion molecules including the selectins, ß2 integrins, and the immunoglobulin (Ig) superfamily [17 ]. Leukocyte interaction with the endothelium begins with leukocyte rolling, followed by adherence and transendothelial migration. P-selectin, a member of the selectin family of adhesion molecules, is believed to play a major role in the initial phase of leukocyte emigration, which is characterized by the rolling of leukocytes along the vascular endothelial surface. Although P-selectin is necessary for early neutrophil contact with the endothelium, P-selectin-mediated, leukocyte-endothelial interaction is not sufficient to allow neutrophil emigration from the vessel. E-selectin is expressed on inflamed endothelial cells in response to treatment with inflammatory cytokines [18 , 19 ]. In addition to mediating leukocyte rolling, E-selectin participates in the conversion of rolling to firm adhesion. E-selectin-deficient mice have a reduced number of firmly adherent leukocytes in response to local chemoattractant [20 ] or cytokine stimulation [21 ]. This defect may be related to the more rapid rolling velocities in the absence of E-selectin. Therefore, there is some evidence that E-selectin is of particular importance in skin inflammation, as it supports the recruitment of activated skin-specific effector T lymphocytes [22 ].

A more firm adherence of the neutrophil to the endothelial surface is required for transendothelial migration [23 ]. This firm adherence involves the interaction of ß2 integrins (i.e., CD11/CD18) on the polymorphonuclear neutrophil (PMN) surface and intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell surface [23 ]. Experimental studies have also shown that in vivo administration of antibodies raised against ICAM-1 reduced neutrophil infiltration into the inflamed lungs in the rabbit and protects the development of ischemia and reperfusion of the intestine [24 ].

In this study, we have investigated the role of GITR in a model of intestine ischemia and reperfusion using GITR-knockout (KO) mice. To characterize the role of GITR in this model of ischemia and reperfusion, we have determined the following endpoints of the inflammatory response: lipid peroxidation, PMN infiltration, expression of adhesion molecules (ICAM-1, P-selectin, E-selectin), TNF-{alpha} production, and intestinal injury and survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Mice (4–5 weeks old, 20–22 g) with a targeted disruption of the GITR-KO and littermate wild-type controls (GITR-WT) were purchased from C. Riccardi’s laboratories (Perugia, Italy). Mice were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations (D.M. 116192) regarding protection of animals used for experimental and other scientific purpose, as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 18/12/1986)

Surgical procedures
Male GITR-KO and GITR-WT mice were allowed access to food and water ad libitum. The mice were anaesthetized with chloral hydrate (380 mg/kg, intraperitoneally). After midline laparotomy, the celiac and superior mesenteric arteries were isolated near their aortic origins. During this procedure, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed 0.9% NaCl solution.

Mice (n=10 for each group) were observed for a 30-min stabilization period before splanchnic ischemia or sham ischemia. Splanchnic artery occlusion (SAO) shock was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 45 min. After this period of occlusion, the clamps were removed. In one study, the various groups of mice were killed at 60 min for histological examination of the ileum and for biochemical studies, as described below. In another sets of studies following reperfusion, the various groups of mice were observed for 24 h to determine survival differences.

Immunohistochemical localization of E-selectin, P-selectin, and ICAM-1
At 60 min after reperfusion, the ileum tissues were fixed in 10% buffered formaldehyde, and 8 µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin- or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with primary anti-E-selectin antibody (1:1000), anti-P-selectin antibody (1:500), anti-ICAM-1 antibody (1:500), or control solutions. Controls included buffer alone or nonspecific, purified rabbit IgG. Immunocytochemistry photographs (n=5) were assessed by densitometry. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). All the immunocytochemistry studies were performed in a blinded manner.

Light microscopy
Ileum biopsies were taken at 60 min after reperfusion. The biopsies were fixed for 1 week in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (thickness 7 µm) were deparaffinized with xylene, stained with haematoxylin/eosin, and studied using light microscopy (Dialux 22 Leitz). All the histological studies were performed in a blinded manner.

Terminal deoxynucleotidyltransferase (TdT)-mediated uridine 5'-triphosphate (UTP) end-labeling (TUNEL) assay
TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer’s instruction (Apotag, HRP kit DBA, Milan, Italy). Briefly, 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 immersed in terminal TdT buffer containing TdT and biotinylated deoxy-UTP in TdT 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 antifluorescein isothiocyanate horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.

Myeloperoxidase (MPO) activity
Assessment of neutrophil infiltration in the intestinal tissues was performed, as described previously [25 ], by measurement of the activity of MPO, an enzyme specific to granulocyte lysosomes and therefore, directly correlated to the number of neutrophils. MPO activity was defined as the quantity of enzyme degrading 1 µmol peroxide min at 37°C and was expressed in µ units per gram weight of wet tissue.

Thiobarbituric acid-reactant substance measurement
Thiobarbituric acid-reactant substance measurement, which is considered a good indicator of lipid peroxidation, was determined, as described previously [26 ], in the intestinal tissues. Thiobarbituric acid-reactant substances were calculated by comparison with an optical density at 650 nm of standard solutions of 1,1,3,3-tetramethoxypropan, 99%; malondialdehyde (MDA) bis(dymethyl acetal), 99% (Sigma-Aldrich Co., Milan, Italy). The absorbance of the supernatant was measured by spectrophotometry at 650 nm.

Measurement of cytokines
The levels of TNF-{alpha} were evaluated in the ileum collected 60 min after reperfusion. The assay was carried out by using a commercial colorimetric kit (Calbiochem-Novabiochem, Milan, Italy).

Reagents
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Co. Primary monoclonal P-selectin (CD62P) or ICAM-1 (CD54) for immunohistochemistry were purchases by PharMingen. Reagents (San Diego, CA) and secondary and nonspecific IgG antibody for immunohistochemical analysis were from Vector Laboratories (Burlingame, CA). Primary monoclonal anti-E-selectin antibody was purchased by Santa Cruz Biotechnolgoy. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl, Baxter Healthcare Ltd., Thetford, Norfolk, UK).

Data analysis
All values in the figures and text are expressed as means ± SEM of the mean of n observations, where n represents the number of animals studied. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. Nonparametric data were analyzed with the Fisher’s exact test. A Pvalue less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of GITR on mortality after splanchnic ischemia/reperfusion
To study the clinical situation of mesenteric infarction, mice were subjected to 45 min occlusion followed by reperfusion of the superior mesenteric artery and celiac trunk. Approximately 90% of the animals died at 24 h after reperfusion. The absence of GITR in mice (animals with the GITR-deficient phenotype) reduced the SAO-induced mortality (Fig. 1 ).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. SAO shock-induced mortality. Survival was monitored for 24 h after SAO shock. *, P < 0.01, versus Sham. °, P < 0.01, versus WT mice. I/R, Ischemia/reperfusion.

 
Splanchnic ischemia/reperfusion injury is reduced in GITR-KO mice
In sham GITR-WT and GITR-KO mice, the histological structure of the gastrointestinal tract was typical of a normal architecture (Fig. 2a ). In GITR-WT mice, splanchnic ischemia/reperfusion resulted in tissue injury mainly localized to the small intestine. Further histological examination of the tissue demonstrated damage localized to the villi and was associated with infiltration of the inflammatory cells in the mucosa as well as tissue hemorrhage (see Fig. 2b for representative section). The degree of the tissue injury (on an arbitrary score ranging from 0 to 4) was 3.14 ± 0.07. The damage score for GITR-KO mice was significantly lower (1.23±0.07) than that obtained from WT mice (P<0.001) as well as the histological observation (Fig. 2c) .



View larger version (124K):
[in this window]
[in a new window]
  		Figure 2. SAO shock-induced intestinal injury. (a) Distal ileum section from sham-operated mice demonstrating the normal architecture of the intestinal epithelium and wall. (b) Distal ileum section from SAO-shocked, GITR-WT mice showed inflammatory cell infiltration through the wall, concentrated below the epithelial layer, and demonstrating edema of the distal portion of the villi and necrosis of the epithelium at the villous tips. (c) Distal ileum from SAO-shocked, GITR-KO mice shows reduced SAO-induced inflammatory infiltration and villi damage. Original magnification, 125x. Figure is representative of at least three experiments performed on different experimental days.

 
GITR expression is required for the up-regulation of cytokine levels during splanchnic ischemia/reperfusion
Splanchnic ischemia/reperfusion results in the up-regulation of proinflammatory cascades in the intestine as well as in other organs [27 ]. The inflammatory response includes the expression of cytokines in the late phase of reperfusion. To determine whether the expression of GITR participates in the up-regulation of proinflammatory cytokine expression after splanchnic ischemia/reperfusion, WT mice and GITR-KO mice were subjected to splanchnic ischemia/reperfusion. TNF-{alpha} ileum levels were significantly increased in comparison with sham animals (Fig. 3 ). This production was significantly reduced in the GITR-KO mice (Fig. 3) .



View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Reperfusion of the ischemic-splanchnic circulation leads to a profound increase in ileum TNF-{alpha}, and this is inhibited in GITR-KO mice. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus Sham. °, P < 0.01, versus GITR-WT mice. I/R, Ischemia/reperfusion.

 
Expression of adhesion molecules (ICAM-1, P-selectin, and E-selectin) and neutrophil infiltration is reduced in GITR-KO mice
Assessment of neutrophil infiltration into the ileum was performed by measuring the activity of MPO, an enzyme that is contained in and specific for PMN lysosomes. MPO activity was significantly elevated after splanchnic ischemia/reperfusion in WT mice (Fig. 4 ). The elevation of the MPO activity was associated with the increase of imununohistochemical staining for ICAM-1 (Figs. 5b and 6 ), for P-selectin (Figs. 5e and 6) , and for E-selectin (Figs. 6 and 7b ) in the ileum section obtained from SAO-shocked, GITR-WT mice. In GITR-KO mice, tissue MPO activity (Fig. 4) was reduced markedly in comparison with those of WT control animals. In SAO-shocked, GITR-KO mice, the positive immunostaining in the intestine as well as in the lung for ICAM-1 (Figs. 5c and 6) , for P-selectin (Figs. 5f and 6) , and for E-selectin (Figs. 6 and 7c) was visibly and significantly reduced in comparison with the GITR-WT mice. Please note that staining of intestine tissue sections obtained from sham-operated mice with anti-ICAM-1 antibody showed a specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed (Fig. 5a) . No positive staining for P-selectin and E-selectin was found in intestine tissue section from sham-operated mice (Figs. 5d and 7a , respectively).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. MPO activity in the ileum from SAO-shocked mice. At 60 min of reperfusion, MPO activity was increased significantly in the ileum from SAO-shocked, GITR-WT mice in comparison with sham-operated mice. In contrast, the absence of GITR in mice significantly reduced the increase of MPO activity in the ileum. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus Sham. °, P < 0.01, versus GITR-WT mice. I/R, Ischemia/reperfusion.

 


View larger version (98K):
[in this window]
[in a new window]
  		Figure 5. (a) Staining of ileum tissue sections obtained from sham-operated mice with anti-ICAM-1 antibody showed a specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed. (b) Section obtained from SAO-shocked, GITR-WT mice showed intense positive staining for ICAM-1 on the vascular wall. (c) The degree of endothelial staining for ICAM-1 was markedly reduced in the tissue section obtained from GITR-KO mice. (d) No positive staining for P-selectin was observed in the ileum tissue sections obtained from sham-operated mice. (e) Section obtained from SAO-shocked, GITR-WT mice showed intense positive staining for P-selectin on the vascular wall. (f) The degree of endothelial staining for P-selectin was markedly reduced in the tissue section obtained from GITR-KO mice. Original magnification, 145x. Figure is representative of at least three experiments performed on different experimental days.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n=5) for ICAM-1, P-selectin, and E-selectin from ileum was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). *, P < 0.01, versus Sham. °, P < 0.01, versus GITR-WT mice. I/R, Ischemia/reperfusion.

 


View larger version (100K):
[in this window]
[in a new window]
  		Figure 7. (a) No positive staining for E-selectin was observed in the ileum tissue sections obtained from sham-operated mice. (b) Section obtained from SAO-shocked, GITR-WT mice showed intense positive staining for E-selectin on the vascular wall. (c) The degree of endothelial staining for E-selectin was markedly reduced in the tissue section obtained from GITR-KO mice. Original magnification, 145x. Figure is representative of at least three experiments performed on different experimental days.

 
Reduction of lipid peroxidation in GITR-KO mice
The release of free radicals and oxidant molecules during the early period of reperfusion has been suggested to contribute significantly to the tissue necrosis and mucosal dysfunction [6 7 8 ]. Splanchnic ischemia/reperfusion injury of WT mice was characterized by an increase in tissue MDA, indicative of lipid peroxidation (Fig. 8 ). In GITR-KO mice, tissue MDA level (Fig. 8) was markedly reduced in comparison with those of WT control animals.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 8. Thiobarbituric acid-reactant substances, a good indicator of lipid peroxidation, were determined in the ileum from SAO-shocked mice. At 60 min of reperfuion, MDA levels were significantly increased in the ileum from SAO-shocked, GITR-WT mice in comparison with sham mice. The absence of GITR in mice did not modify the lower increase of thiobarbituric acid-reactant substances in the ileum. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus Sham. °, P < 0.01, versus GITR-WT mice. I/R, Ischemia/reperfusion.

 
Reduction of apoptosis at villus tips in the intestine of GITR-KO mice
A few apoptotic cells were observed at the villus tips in the intestine from sham-operated, WT mice (Fig. 9b , see arrows). The number of apoptotic cells increased at 1 h after ischemia/reperfusion in intestine from WT mice (Fig. 9a , see particles a1 and a2). In contrast, only a few apoptotic cells were seen in the villus tips in the intestine from GITR-KO mice (Fig. 9c) .



View larger version (127K):
[in this window]
[in a new window]
  		Figure 9. A few apoptotic cells were observed at the villus tips in the intestine from sham-operated WT mice (b, see arrows). The number of apoptotic cells increased at 1 h after ischemia/reperfusion in intestine from WT mice (a, see particles a1 and a2). In contrast, only a few apoptotic cells were seen in the villus tips in the intestine from GITR-KO mice (c). The figure is representative of at least three experiments performed on different experimental days.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here that mice with a targeted deletion of the GITR gene (GITR-KO) are protected against the pathological changes caused by ischemia/reperfusion injury of the gut. Thus, we propose that GITR contributes to the pathophisiology of ischemia/reperfusion injury of the gut.

GITR promoter region contains important binding sites such as the consensus element for nuclear factor (NF)-{kappa}B, activated protein-1, and NF-interleukin-6 and other consensus sequences for transcription factors involved in T cell activation. Furthermore, GITR is able to protect T lymphocytes against TCR-activated apoptosis [1 ].

Suggestion of a possible role of GITR in T cell regulation came from the GITR-KO mice that we have generated, in which the GITR gene has been completely ablated [28 ]. T lymphocytes from GITR null mice show a disregulation of TCR-induced activation as compared with cells of WT mice. More recently, we have shown that GITR acts as a coactivating stimulus on effectors CD4+ and CD8+ single-positive (SP) lymphocytes and on CD4+CD25+ T regulatory (Treg) cells. Similar results have been reported with T helper lymphocytes [2 , 3 ]. GITR stimulus increases activation of effector SP lymphocytes and inhibits suppressor activity of Treg cells, thus suggesting that at least in certain conditions, lack of GITR expression may result in weaker effector T lymphocyte activation and higher Treg cell suppressor functions and consequently, in a decreased immune/inflammatory response.

There is evidence that the proinflammatory cytokine TNF-{alpha} help to propagate the extension of ischemia and reperfusion shock [29 ]. We confirm here that SAO shock leads to a substantial increase in the ileum levels of TNF-{alpha}. It is interesting that the levels of this proinflammatory cytokine are significantly lower in the ileum obtained from GITR-KO mice. These findings suggest that in the presence of GITR, the degree of SAO shock and hence, the formation of TNF-{alpha} are significantly enhanced. Using mice in which the gene for GITR was deleted (GITR-KO mice), we also demonstrate here that the activation of GITR mediates leukocyte-endothelial interactions by regulating the expression of P-selectin, E-selectin, and ICAM-1 during ischemia and reperfusion. In GITR-KO mice subjected to SAO shock, the up-regulation of P-selectin, E-selectin, and ICAM-1 in the intestine was largely attenuated. Endothelial cells appear to be major regulators of the neutrophil traffic, regulating the process of neutrophil chemoattraction, adhesion, and emigration from the vasculature to the tissue. P-selectin is rapidly recruited to the cell surface of platelets or endothelial cells from preformed storage pools after exposure to, e.g., hydrogen peroxide, thrombin, histamine, or complement and allows the leukocytes to roll along the endothelium [30 ]. E-selectin is expressed in skin microvessels under baseline conditions, and there is some evidence that E-selectin is of particular importance in skin inflammation also, as it supports the recruitment of activated, skin-specific effector T lymphocytes [31 ]. ICAM-1 is constitutively expressed on the surface of endothelial cells and is then involved in the neutrophil adhesion [32 ]. Hypoxic or injured endothelial cells synthesize proinflammatory cytokines, which can up-regulate endothelial expression of the constitutive adhesion molecule ICAM-1 in an autocrine manner [33 ]. Significant expression of ICAM-1 in microvessels of previously ischemic tissues occurs within 1 h after reperfusion [34 ]. The up-regulation expression of P-selectin, E-selectin, and ICAM-1 corresponds with the induction of neutrophil recruitment, which is maximal within the first hour of reperfusion, and persists at a lower rate in the late phase of reperfusion [35 ]. In accordance with these findings, we observed that SAO shock induced the appearance of P-selectin on the endothelial vascular wall and up-regulated the surface expression of E-selectin and ICAM-1 on endothelial cells in the lung and intestine section from GITR-WT mice. The genetic inhibition of GITR (KO mice) abolished the expression of P-selectin and the up-regulation of E-selectin and ICAM-1 but did not affect the constitutive expression of E-selectin and ICAM-1 on endothelial cells (data not shown). These results suggest that inhibition of GITR activity may interfere with the interaction of neutrophils and endothelial cells at the early rolling phase mediated by P-selectin and E-selectin and at the late firm-adhesion phase mediated by ICAM-1. The absence of an increased expression of the adhesion molecules in the intestine tissue of SAO-shocked, GITR-KO mice correlated with the reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPO and with the moderation of the tissue damage as evaluated by histological examination. It is noteworthy, however, that tissue MPO activity was not abolished completely. This result is consistent with previous studies demonstrating that constitutive levels of ICAM-1 appear to be sufficient to support a lower degree of CD11/CD18-dependent transendothelial migration of activated neutrophils [35 ]. In conclusion, the data presented here demonstrate that GITR is involved in the regulation of the expression of adhesion molecules and that consequently, GITR plays a role in the tissue infiltration of neutrophils.

Recent evidence has shown that ischemia and/or ischemia/reperfusion induce apoptosis in several tissues, such as brain [36 ], myocardium [37 ], intestine [38 ], and liver [39 ]. Previous studies using histological evaluations have described a spontaneous level of apoptosis as well as radiation-induced and chemically induced apoptosis in cells of small intestine [40 , 41 ]. Results described here confirm these observations.

TNFRSF members, including GITR and CD27, have been reported to regulate T lymphocyte development, as they can induce or counter apoptosis [1 , 2 , 42 , 43 ]. Recently, it has been demonstrated that GITR, like CD27, binds and activates murine Siva, which leads to cell death, suggesting that GITR may induce cell death [44 ]. Results described here indicate that GITR-KO mice have lower numbers of apoptotic cells, as evaluated by TUNEL, thus suggesting that GITR may regulate apoptosis after ischemia/reperfusion.

Taken together, the data presented in the present study demonstrate that GITR regulates the infiltration of neutrophils into the inflamed tissues via a number of distinct mechanisms. The discovery of the concept that GITR may regulate neutrophil trafficking, ischemia/reperfusion-induced inflammation, and injury may provide new insights in the interpretation of experimental models of ischemia/reperfusion injury and inflammation. Moreover, these data could be of interest for new therapeutic approaches aimed to modulate GITR expression.


   ACKNOWLEDGEMENTS
 
This work was supported by Ministero Pubblica Istruzione (Rome, Italy) and by Associazione Italiana Ricerca sul Cancro (AIRC; Milan, Italy).

Received February 24, 2004; revised June 3, 2004; accepted July 7, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nocentini, G., Giunchi, L., Ronchetti, S., Krausz, L. T., Bartoli, A., Moraca, R., Migliorati, G., Riccardi, C. (1997) A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis Proc. Natl. Acad. Sci. USA 94,6216-6221[Abstract/Free Full Text]
  2. Ronchetti, S., Zollo, O., Bruscoli, S., Agostini, M., Bianchini, R., Nocentini, G., Ayroldi, E., Riccardi, C. (2004) GITR, a member of TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations Eur. J. Immunol. 34,613-622[CrossRef][Medline]
  3. Tone, M., Tone, Y., Adams, E., Yates, S. F., Frewin, M. R., Cobbold, S. P., Waldman, H. (2003) Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells Proc. Natl. Acad. Sci. USA 100,15059-15064[Abstract/Free Full Text]
  4. Robey, E., Fowlkes, B. J. (1994) Selective events in T cell development Annu. Rev. Immunol. 12,675-705[CrossRef][Medline]
  5. Shevach, E. M. (2000) Regulatory T cells in autoimmunity Annu. Rev. Immunol. 18,423-449[CrossRef][Medline]
  6. Lutz, M. B., Schuler, G. (2002) Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. 23,445-449[CrossRef][Medline]
  7. Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russel, J. W., Loh, D. Y. (1988) Positive and negative selection of an antigen receptor on T cells in transgenic mice Nature 336,73-76[CrossRef][Medline]
  8. Kappler, J. W., Roehm, N., Marrack, P. (1987) T cell tolerance by clonal elimination in the thymus Cell 49,273-280[CrossRef][Medline]
  9. Nossal, G. J. V. (1983) Cellular mechanism of immunologic tolerance Annu. Rev. Immunol. 1,33-62[CrossRef][Medline]
  10. Kappler, J. W., Staerz, U., White, J., Marrack, P. (1988) Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex Nature 332,35-40[CrossRef][Medline]
  11. Hengartner, H., Odermatt, B., Schneider, R., Schreyer, M., Walle, G., MacDonald, H. R., Zinkernagel, R. M. (1988) Deletion of self-reactive T cells before entry into the thymus medulla Nature 336,388-390[CrossRef][Medline]
  12. Shigenatsu, T., Wolf, R. E., Granger, D. N. (2002) T-lymphocytes modulate the microvascular and inflammatory responses to intestinal ischemia reperfusion Microcirculation 9,99-109[CrossRef][Medline]
  13. Zimmermann, B. J., Arndt, H., Kubes, P., Kurtel, H. (1993) Reperfusion injury in the small intestine Schlag, G. Redl, H. eds. Pathophysiology of Shock, Sepsis and Organ Failure ,322-335 Springer-Verlag Berlin, Germany.
  14. Lefer, A. M., Lefer, D. J. (1993) Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock Annu. Rev. Pharmacol. Toxicol. 33,71-90[CrossRef][Medline]
  15. Zingarelli, B., Squadrito, F., Ioculano, M. P., Altavilla, D., Bussolino, F., Campo, G. M., Caputi, A. P. (1992) Platelet activating factor in splanchnic artery occlusion shock Eur. J. Pharmacol. 222,13-19[CrossRef][Medline]
  16. Granger, D. N., Hollwarth, M. A., McCord, J. M. (1986) Ischemia reperfusion injury: role of oxygen-derived free radicals Acta Physiol. Scand. Suppl. 548,47-63
  17. Butcher, E. C. (1993) Specificity of leukocyte-endothelial interactions and diapedesis: physiologic and therapeutic implications of an active decision process Res. Immunol. 144,695-698[CrossRef][Medline]
  18. Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A., Jr, Seed, B. (1989) Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins Science 243,1160-1165[Abstract/Free Full Text]
  19. Hickey, M. J., Kanwar, S., McCafferty, D. M., Granger, D. N., Eppihimer, M. J., Kubes, P. (1999) Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen J. Immunol. 162,1137-1143[Abstract/Free Full Text]
  20. Ley, K., Allietta, M., Bullard, D. C., Morgan, S. (1998) Importance of E-selectin for firm leukocyte adhesion in vivo Circ. Res. 83,287-294[Abstract/Free Full Text]
  21. Milstone, D. S., Fukumura, D., Padgett, R. C., O’Donnell, P. E., Davis, V. M., Benavidez, O. J., Monsky, W. L., Melder, R. J., Jain, R. K., Gimbrone, M. A., Jr (1998) Mice lacking E-selectin show normal numbers of rolling leukocytes but reduced leukocyte stable arrest on cytokine-activated microvascular endothelium Microcirculation 5,153-171[CrossRef][Medline]
  22. Picker, L. J., Kishimoto, T. K., Smith, C. W., Warnock, R. A., Butcher, E. C. (1991) ELAM-1 is an adhesion molecule for skin-homing T cells Nature 349,796-799[CrossRef][Medline]
  23. Butcher, E. C. (1992) Leukocyte-endothelial cell adhesion as an active, multi-step process: a combinatorial mechanism for specificity and diversity in leukocyte targeting Adv. Exp. Med. Biol. 323,181-194[Medline]
  24. Lawrence, M. B., Springer, T. A. (1991) Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins Cell 65,859-873[CrossRef][Medline]
  25. Mangino, M. J., Mangino, J. E., Murphy, M. K., Anderson, C. B. (1996) Arachidonic acid metabolism in intestinal hypotermic preservation injury in dogs Cryobiology 33,404-412[CrossRef][Medline]
  26. Mullane, K. M., Kraemer, R., Smith, B. (1985) Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium J. Pharmacol. Methods 14,157-167[CrossRef][Medline]
  27. Ohkawa, H., Ohishi, N., Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction Anal. Biochem. 95,351-358[CrossRef][Medline]
  28. Squadrito, F., Altavilla, D., Canale, P., Ioculano, P., Campo, G. M., Ammendolia, L., Squadrito, G., Saitta, A., Calapai, G., Caputi, A. P. (1994) Contribution of intercellular adhesion molecule 1 (ICAM-1) to the pathogenesis of splanchnic artery occlusion shock in the rat Br. J. Pharmacol. 113,912-916[Medline]
  29. Zingarelli, B., Cuzzocrea, S., Zsengeller, Z., Salzman, A. L., Szabó, C. (1997) Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase Cardiovasc. Res. 36,205-215[Abstract/Free Full Text]
  30. Hirano, T. (1992) Interleukin-6 and its relation to inflammation and disease Clin. Immunol. Immunopathol. 62,S60-S65[CrossRef][Medline]
  31. Szabó, C., Cuzzocrea, S., Zingarelli, B., O’Connor, M., Salzman, A. L. (1997) Endothelial dysfunction in endotoxic shock. Importance of the activation of poly (ADP ribose) synthetase by peroxynitrite J. Clin. Invest. 100,723-735[Medline]
  32. Az-ma, T., Fujii, K., Yuge, O. (1996) Self-limiting enhancement by nitric oxide of oxygen-free radical-induced endothelial cell injury: evidence against the dual action of nitric oxide as hydroxyl radical donor/scavenger Br. J. Pharmacol. 119,455-462[Medline]
  33. White, C. R., Brock, T. A., Chang, L. Y., Crapo, J., Briscoe, P., Ku, D., Bradley, W. A., Gianturco, S. H., Gore, J., Freeman, B. A. (1994) Superoxide and peroxynitrite in atherosclerosis Proc. Natl. Acad. Sci. USA 91,1044-1048[Abstract/Free Full Text]
  34. Zingarelli, B., Day, B. J., Crapo, J., Salzman, A. L., Szabó, C. (1997) The potential involvement of peroxynitrite in the pathogenesis of endotoxic shock Br. J. Pharmacol. 120,259-267[CrossRef][Medline]
  35. Granger, D. N. (1997) Cell adhesion and migration. II. Leukocyte-endothelial cell adhesion in the digestive system Am. J. Physiol. 273,G982-G986
  36. Hill, I. E., MacManus, J. P., Rasquinha, I., Tuor, U. I. (1995) DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat Brain Res. 676,398-403[CrossRef][Medline]
  37. Itoh, G., Tamura, J., Suzuki, M., Suzuki, Y., Ikeda, H., Koike, M., Nomura, M., Jie, T., Ito, K. (1995) DNA fragmentation of human-infarcted myocardial cells demonstrated by the nick end-labeling method and DNA agarose gel electrophoresis Am. J. Pathol. 146,1325-1331[Abstract]
  38. Noda, T., Iwakiri, R., Fujimoto, K., Matsuo, S., Aw, T. Y. (1998) Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa Am. J. Physiol. 274,G270-G276
  39. Fukuda, K., Kojiro, M., Chiu, J. F. (1993) Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis? Am. J. Pathol. 142,935-946[Abstract]
  40. Han, H., Iwanaga, T., Fujita, T. (1993) Species differences in the process of apoptosis in epithelial cells of the small intestine: an ultrastructural and cytochemical study of luminal cell elements Arch. Histol. Cytol. 56,83-90[Medline]
  41. Potten, C. S. (1992) The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice Cancer Metastasis Rev. 11,179-195[CrossRef][Medline]
  42. Wallach, D., Kovalenko, A. V., Varfolomeev, E. E., Boldin, M. P. (1998) Death-inducing functions of ligands of the tumor necrosis factor family: a Sanhedrin verdict Curr. Opin. Immunol. 10,279-288[CrossRef][Medline]
  43. Grammer, A. C., Lipsky, P. E. (2000) CD40-mediated regulation of immune responses by TRAF-dependent and TRAF-independent signaling mechanisms Adv. Immunol. 76,61-178[Medline]
  44. Spinicelli, S., Nocentini, G., Ronchetti, S., Krausz, L. T., Bianchini, R., Riccardi, C. (2002) GITR interacts with the pro-apoptotic protein Siva and induces apoptosis Cell Death Differ. 9,1382-1384[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
G. Nocentini, S. Cuzzocrea, T. Genovese, R. Bianchini, E. Mazzon, S. Ronchetti, E. Esposito, D. P. Rosanna, P. Bramanti, and C. Riccardi
Glucocorticoid-Induced Tumor Necrosis Factor Receptor-Related (GITR)-Fc Fusion Protein Inhibits GITR Triggering and Protects from the Inflammatory Response after Spinal Cord Injury
Mol. Pharmacol., June 1, 2008; 73(6): 1610 - 1621.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
L Santucci, M Agostini, S Bruscoli, A Mencarelli, S Ronchetti, E Ayroldi, A Morelli, M Baldoni, and C Riccardi
GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice
Gut, January 1, 2007; 56(1): 52 - 60.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. Cuzzocrea, S. Ronchetti, T. Genovese, E. Mazzon, M. Agostini, R. Di Paola, E. Esposito, C. Muia, G. Nocentini, and C. Riccardi
Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation
FASEB J, January 1, 2007; 21(1): 117 - 129.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Cuzzocrea, G. Nocentini, R. Di Paola, M. Agostini, E. Mazzon, S. Ronchetti, C. Crisafulli, E. Esposito, A. P. Caputi, and C. Riccardi
Proinflammatory Role of Glucocorticoid-Induced TNF Receptor-Related Gene in Acute Lung Inflammation
J. Immunol., July 1, 2006; 177(1): 631 - 641.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0204110v1
76/5/933    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuzzocrea, S.
Right arrow Articles by Riccardi, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cuzzocrea, S.
Right arrow Articles by Riccardi, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS