Published online before print August 17, 2004
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* Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, Messina, Italy; and
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
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, 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- 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
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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- production, and intestinal injury and survival.
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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- 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.
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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.
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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.
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ileum levels were significantly increased in comparison with sham animals (Fig. 3
). This production was significantly reduced in the GITR-KO mice (Fig. 3)
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Figure 3. Reperfusion of the ischemic-splanchnic circulation leads to a profound increase in ileum TNF-, 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.
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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.
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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.
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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.
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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.
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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.
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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.
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GITR promoter region contains important binding sites such as the consensus element for nuclear factor (NF)-
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- 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-. 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- 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.
Received February 24, 2004; revised June 3, 2004; accepted July 7, 2004.
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