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Originally published online as doi:10.1189/jlb.0706480 on January 8, 2007

Published online before print January 8, 2007
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(Journal of Leukocyte Biology. 2007;81:1032-1043.)
© 2007 by Society for Leukocyte Biology

Splanchnic ischemia and reperfusion injury is reduced by genetic or pharmacological inhibition of TNF-{alpha}

Emanuela Esposito*,{ddagger},1, Emanuela Mazzon{dagger},{ddagger},1, Carmelo Muià{dagger}, Rosaria Meli*, Edoardo Sessa{ddagger} and Salvatore Cuzzocrea{dagger},{ddagger},2

* Department of Experimental Pharmacology, University of Naples "Federico II," Naples, Italy;
{dagger} Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy; and
{ddagger} IRCCS Centro Neurolesi "Bonino-Pulejo," Messina, Italy

2 Correspondence: Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. E-mail: salvator{at}unime.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we used TNF-{alpha} receptor 1 knockout (TNF-{alpha}R1KO) mice to evaluate a possible role of TNF-{alpha} on the pathogenesis of ischemia and reperfusion injury of the multivisceral organs. Ischemia and reperfusion injury was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by reperfusion. Sixty minutes after reperfusion, animals were killed for histological examination and biochemical studies. Injured wild-type (WT) mice developed a significant increase of ileum TNF-{alpha} levels, myeloperoxidase activity, and marked histological injury and apoptosis. Ischemia and reperfusion injury of the multivisceral organs was also associated with a significant mortality. Reperfused ileum sections from injured WT mice showed positive staining for P-selectin, VCAM, ICAM-1, and E-selectin. The intensity and degree of P-selectin, E-selectin, VCAM, and ICAM-1 were reduced markedly in tissue sections from injured TNF-{alpha}R1KO mice. Ischemia and reperfusion-injured TNF-{alpha}R1KO mice also showed a significant reduction of neutrophil infiltration into the intestine, a reduction of apoptosis, an improved histological status of the intestine, and survival. In addition, we investigated the effect of Etanercept, a TNF-{alpha} soluble receptor construct, on ischemia and reperfusion injury of the multivisceral organs. Etanercept (5 mg/kg administered i.p. 5 min prior to reperfusion) significantly reduced the inflammatory response and the ileum injury. Taken together, our results clearly demonstrate that TNF-{alpha} plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of TNF-{alpha} expression may represent a novel and possible strategy.

Key Words: adhesion molecules • Etanercept • neutrophil infiltration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Generally, intestinal ischemia is a phenomenon that can result from arterial occlusion by thrombi or embolisms and more frequently, from nonocclusive processes, such as in situations of low mesenteric flow, which occurs in cardiac insufficiency, sepsis, {alpha}-adrenergic agents, or digitalics administration [1 , 2 ]. The arteries most compromised by obstruction are the celiac trunk (or celiac artery), superior mesenteric (or cranial) artery (SMA), and inferior mesenteric (or caudal) artery [3 ]. The mid-gut receives arterial supply from the SMA, which arises from the aorta, close to the celiac artery (CA) at ~1 cm caudally. Branches include the middle, right, and ileocolic arteries as well as jejunal and ileal arteries and arterioles. Thus, the SMA and its branches are responsible for blood supply to the vast majority of the jejunum, ileum, and the ascending, transverse, and splenic flexure portions of the colon.

However, the sources of the collateral flow between the mesenteric blood vessels themselves or with adjacent circulation are numerous [4 ] and may compensate the blood flow to the tissues. Extensive collateralization between splanchnic vessels serves as a protective mechanism against ischaemia. As reviewed by Brandt and Boley [5 ], when a major intestinal artery is obstructed, a reduction in pressure in the distal arterial bed below systemic pressure triggers the opening of collaterals.

During chronic occlusive disease, an extensive collateral vascularization develops among the three main arteries. Therefore, in most instances, occlusion of at least two of the three major vessels is necessary to produce symptoms [6 , 7 ].

The most-used method for producing ischemia was the arterial obstruction by vascular clamping, followed by stenosis or extrinsic vascular compression, surgical tapes to controlled release of blood flow, hypovolemic shock, and hypothermia. Most authors made the obstruction of the CA or SMA [8 , 9 ], but the obstruction of other arteries was necessary in studies performed to better understand the capacity of the collateral circulation [10 , 11 ], which varies according to the species, according to the experiment performed by Deune and Khouri [12 ]. Splanchnic artery occlusion/reperfusion (SAO/R) is characterized by a marked, systemic decrease in postreperfusion blood pressure and is associated with a high mortality rate [13 , 14 ]. It has been shown that endothelial dysfunction plays a key role in SAO/R, as it predisposes to vasospasm, platelet activation, and increased neutrophil adherence, which exacerbates the local bowel injury as well as the general cardiocirculatory failure [15 ].

Endothelial-leukocyte interaction is known to involve specific surface glycoproteins known as endothelial cell adhesion molecules [16 ]; they include early-phase molecules, such as P-selectin, involved in leukocyte tethering and rolling, which is rapidly translocated from the Weibel-Palade bodies to the endothelial cell surface upon stimulation by histamine, hypoxia, and reactive oxygen species [17 , 18 ], as well as late-phase molecules, such as E-selectin, VCAM-1, and ICAM-1, involved in leukocyte adhesion and extravasation into the tissue, whose expression is induced by most inflammatory mediators, including histamine and cytokines [16 , 19 20 21 ].

TNF-{alpha} is the prototypic member of a gene superfamily, which regulates essential biologic functions such as immune response, cell proliferation, survival, differentiation, and apoptosis, as well as host-damaging effects in sepsis, cachexia, autoimmune, and inflammatory diseases [22 ].

Various studies have implicated TNF-{alpha} in the pathogenesis of ischemia/reperfusion injury of the multivisceral organs [23 ]. In particular, an increase of serum and ileum TNF-{alpha} levels after reperfusion in an experimental model of ischemia/reperfusion injury of the multivisceral organs has been demonstrated [24 25 26 ]. Furthermore, the use of anti-TNF-{alpha} serum has been shown to reduce hepatic injury after liver ischemia [27 ]. Similarly, the use of cloricromene, an agent with known antithrombotic actions and with demonstrated anti-TNF-{alpha} activity, significantly reduced the ileum injury associated with ischemia/reperfusion injury [28 ]. Two distinct isoforms of the membrane receptors for TNF-{alpha} (TNF-R1 and TNF-R2) have been identified and molecularly cloned. Although most cell lines and primary tissues express both isoforms [29 ], most of the biological activities of TNF-{alpha} are mediated through TNF-R1 [30 31 32 ]. TNF-R2 is a poor inducer of apoptosis [33 ], and binding affinities of soluble TNF-{alpha} are significantly higher to TNF-R1 [31 ].

However, the biologic significance of TNFR shedding is unclear. It could represent a neutralizing mechanism to counteract excessive TNF-{alpha} activity, but conversely, it has been suggested that in relatively low concentrations, soluble TNFR (sTNFR) may serve as carriers to distant organs. Furthermore, sTNFR stabilize TNF-{alpha} trimeric structure, thereby prolonging its half-life and augmenting its biological effects [34 ]. Etanercept is a fully humanized, dimeric, soluble form of the p75 TNFR, which can bind to two TNF-{alpha} molecules, blocking their interaction with cell-surface TNFR and rendering TNF-{alpha} biologically inactive. TNF-{alpha} inactivation is 1000 times stronger than inactivation by p75 monomeric TNFR [35 ]. It inhibits the activity of TNF-{alpha} in vitro and has been examined in vivo for its effects in different animal model systems of inflammatory and autoimmune diseases [36 ].

In clinical settings, it has been tested in numerous trials and approved for the treatment of rheumatoid arthritis (RA), juvenile RA [37 , 38 ], ankylosing spondylitis [39 ], psoriatic arthritis [40 ], and plaque psoriasis [41 ].

In this study, we have investigated the role of TNF-{alpha} in a model of ischemia/reperfusion injury of the multivisceral organs using TNF-{alpha}R1 knockout (TNF-{alpha} R1KO) mice. Moreover, to our knowledge, the role of blockage through dimeric fusion protein Etanercept has not been investigated in ischemia/reperfusion injury of the multivisceral organs. Thus, we have also investigated whether Etanercept treatment attenuates the ischemia/reperfusion injury of the multivisceral tissues.


   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 TNF-{alpha}R1KO and wild-type controls (TNF-{alpha}WT), were purchased from Jackson Laboratories (Charles River, Italy). The University of Messina Review Board (Italy) approved the study for the care of animals, which were housed in a controlled environment and provided with standard rodent chow and water ad libitum. Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and the United States (Animal Welfare Assurance No. A5594-01, Department of Health and Human Services, Washington, DC, USA).

Surgical procedures
Male TNF-{alpha} R1KO and TNF-{alpha}WT mice were allowed access to food and water ad libitum. The mice were anesthetized with chloral hydrate (380 mg/kg i.p.). After midline laparotomy, the celiac and SMA 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, and ischemia and reperfusion injury of the mutlivisceral organs was induced by clamping the SMA 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 a separate set of experiments, splanchnic ischemia and reperfusion injury was induced in TNF-{alpha}WT treated with Etanercept (5 mg/kg i.p.) 5 min prior to reperfusion. 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.

Etanercept was given by i.p. injection after reconstitution with 1 mL sterile bacteriostatic water for injection (US Pharmacopeia, Rockville, MD, USA).

Immunohistochemical localization of E-selectin, P-selectin, VCAM, ICAM-1, Bax, Bcl-2, and Fas ligand (FasL)
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 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 (BD PharMingen, San Diego, CA, USA, 1:1000), anti-P-selectin antibody (BD PharMingen, CD62P, 1:500), anti-ICAM-1 antibody (BD PharMingen, CD54, 1:500), anti-VCAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500), and anti-Bax polyclonal antibody (pAb; Santa Cruz Biotechnology, 1:500 in PBS, v/v), along with anti-Bcl-2 pAb (Santa Cruz Biotechnology), anti-FasL pAb (Novocastra Laboratories, UK, 1:500 in PBS, v/v), or control solutions, which included buffer alone or nonspecific, purified rabbit IgG.

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, USA). Tissue sections (thickness, 7 µm) were deparaffinized with xylene, stained with H&E, and studied using light microscopy (Dialux 22 Leitz). The following morphological criteria were used for scoring: 0, No damage; 1 (mild), focal epithelial edema and necrosis; 2 (moderate), diffuse swelling and necrosis of the villi; 3 (severe), necrosis with evidence of neutrophil infiltration in the submucosa; 4 (major), widespread necrosis with massive neutrophil infiltration and evidence of hemorrhage. All the histological studies were performed in a blinded manner.

Terminal TUNEL assay
TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer’s instruction (Apoptag, 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 deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated deoxyuridine triphosphate 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 anti-FITC, HRP-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 [42 ], by measurement of the activity of MPO, an enzyme specific to granulocyte lysosomes and therefore, correlated directly 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 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 [43 ], in the intestinal tissues. Thiobarbituric acid-reactant substances were calculated by comparison with OD650 of standard solutions of 1,1,3,3-tetramethoxypropan, 99%, and malondialdehyde (MDA), 99% (Sigma-Aldrich Co., Milan, Italy). The absorbance of the supernatant was spectrophotometrically at 650 nm.

Measurement of cytokines
The levels of TNF-{alpha} were evaluated in the plasma and ileum, collected 60 min after reperfusion. The assay was carried out by using a commercial colorimetric kit (Diaclone Research Cell Science, Cedex, France), according to the manufacturer’s instructions. All TNF-{alpha} determinations were performed in duplicate serial dilutions. ELISA detection limit is less than 25 pg/ml.

Total protein extraction and Western blot analysis for Bax and Bcl-2
Tissues were disrupted by homogenization with a Ultra-turrax T8 homogenizer on ice in a buffer containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1.5 µg/ml trypsin inhibitor, 3 µg/ml pepstatin, 2 µg/ml leupeptin, 40 µM benzidamin, 1% Nonidet P-40, and 20% glycerol. The homogenates were centrifuged at 13,000 rpm for 15 min at 4°C; the supernatant was collected to evaluate contents of Bax and Bcl-2. Protein concentration was estimated by the Bio-Rad protein assay using BSA as standard (Bio-Rad Laboratories, Segrate, Milan, Italy). Equal amounts of protein (50 µg) were dissolved in Laemmli’s sample buffer (50 mM Tris, SDS, 10% glycerol, 10% 2-ME, 2 mg ml–1 bromophenol), boiled for 5 min, centrifuged at 10,000 rpm for a few seconds, and subjected to SDS-PAGE minigel (8% polyacrylamide). The blot was performed by transferring proteins from a slab gel to a nitrocellulose membrane at 240 mA for 40 min at room temperature. The filter was then blocked with 1x PBS and 5% (w/v) nonfat dried milk for 40 min at room temperature and subsequently probed with specific mAb against Bax (Santa Cruz Biotechnology, 1:1000) or Bcl-2 (Santa Cruz Biotechnology, 1:1000) in 1x PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20 at 4°C overnight. The secondary antibody (antirabbit IgG peroxidase-conjugated, Jackson ImmunoResearch, Laboratories, West Grove, PA, USA, 1:5000) was incubated for 1 h at room temperature. Subsequently, the blot was washed extensively with PBS, developed using the SuperSignal West Pico chemiluminescence substrate (Pierce, Milan, Italy) according to the manufacturer’s instructions, and exposed to Kodak X-Omat film. The protein bands of Bax (~29 kDa) and Bcl-2 (~26 kDa) on X-ray film were scanned and densitometrically analyzed with a model GS-700 imaging densitometer (Bio-Rad Laboratories).

Reagents
Etanercept was purchased by Policlinico Universitario Pharmacy (Messina, Italy). Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Co. Reagents and secondary and nonspecific IgG antibody for immunohistochemical analysis were from Vector Laboratories Inc. (Burlingame, CA, USA). 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 mean ± SEM of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. 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 P value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of TNF-{alpha} on mortality after ischemia/reperfusion injury of the multivisceral organs
To study the clinical situation of mesenteric infarction, mice were subjected to 30 min occlusion followed by reperfusion of the SMA and celiac trunk. Approximately 90% of the animals died at 24 h after reperfusion. The absence of TNF-{alpha}R1 in mice (animals with the TNF-{alpha}R1-deficient phenotype) reduced the ischemia/reperfusion injury of the multivisceral, organ-induced mortality (Fig. 1 ). Similarly, the treatment of TNF-{alpha}R1WT mice with Etanercept (5 mg/kg administered i.p. 5 min prior to reperfusion) reduced the ischemia/reperfusion injury-induced mortality (Fig. 1) .


Figure 1
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  		Figure 1. TNF-{alpha} gene deletion and Etanercept administration on ischemia/reperfusion (I/R) injury of the multivisceral organs reduced mortality. Mice (10 for each group) were subjected to 30 min occlusion followed by reperfusion of the SMA and celiac trunk. Survival was monitored for 24 h after ischemia/reperfusion injury. *, P < 0.01, versus WT mice.

 
Ischemia/reperfusion injury of the multivisceral organs is reduced in TNF-{alpha}R1KO mice
In sham TNF-{alpha}R1WT and TNF-{alpha}R1KO mice, the histological structure of the gastrointestinal tract was typical of a normal architecture (Fig. 2a ). In TNF-{alpha}R1WT mice, ischemia/reperfusion injury of the multivisceral organs 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 4.12 ± 0.05. The damage score for TNF-{alpha}R1KO mice was significantly lower (1.11±0.04) than that obtained from WT mice (P<0.001) as well as the histological observation (Fig. 2c) . Similarly, the treatment of TNF-{alpha}R1WT mice with Etanercept (5 mg/kg administered i.p. 5 min prior to reperfusion) reduced the villus tip alterations as well as the histological score at 60 min after reperfusion (Fig. 2d) .


Figure 2
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  		Figure 2. Effects of TNF-{alpha} gene deletion and Etanercept administration on ischemia/reperfusion injury of the multivisceral organs. Distal ileum section from sham-operated mice demonstrating the normal architecture of the intestinal epithelium and wall (a). Distal ileum section from ischemia/reperfusion-injured TNF-{alpha}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 (b). On the contrary, ileum section from mice lacking TNF-{alpha}R1 gene (TNF-{alpha} R1KO) (c) and TNF-{alpha}WT mice that had received Etanercept at the dose of 5 mg/kg (d) showed significantly less histological alterations. The figure is representative of at least three experiments performed on different experimental days.

 
TNF-{alpha} production is reduced in TNF-{alpha}R1KO mice
Ischemia/reperfusion injury of the multivisceral organs results in the up-regulation of proinflammatory cascades in the intestine as well as in other organs [44 ]. The inflammatory response includes the expression of cytokines in the late phase of reperfusion. To determine whether the expression of TNF-{alpha}R1 participates in the up-regulation of proinflammatory cytokine expression after splanchnic ischemia/reperfusion, WT mice and TNF-{alpha}R1KO mice were subjected to splanchnic ischemia/reperfusion. Ileum and plasma TNF-{alpha} levels were increased significantly in TNF-{alpha}R1WT mice in comparison with sham animals (Fig. 3A and 3B ). This production was reduced significantly in the TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 3A and 3B) .


Figure 3
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  		Figure 3. Effects of TNF-{alpha} gene deletion and Etanercept administration on ischemia/reperfusion injury of the multivisceral organs induced TNF-{alpha} production in the ileum (A) and plasma (B), MPO activity (C), and MDA levels (D). Reperfusion of the ischemic splanchnic circulation leads to a profound TNF-{alpha} increase in ileum (A) and plasma (B) at 60 min after reperfusion. TNF-{alpha} was not detected in the ileum, and plasma from ischemia/reperfusion-injured TNF-{alpha} R1KO as well as the treatment with Etanercept significantly reduced the ileum (A) and plasma (B) levels of TNF-{alpha}. In addition, at 60 min of reperfusion, MPO activity (C) and thiobarbituric acid-reactant substances (D), a good indicator of lipid peroxidation, were increased significantly in the ileum from ischemia/reperfusion-injured, TNF-{alpha}WT mice in comparison with sham-operated mice. In contrast, the absence of TNF-{alpha}R1 gene in mice as well as the treatment with Etanercept significantly reduced the increase of MPO activity and MDA levels in the ileum. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus sham. °, P < 0.01, versus TNF-{alpha}WT mice. ND, Not determined.

 
Expression of adhesion molecules (ICAM-1, VCAM, P-selectin, and E-selectin) and neutrophil infiltration are reduced in TNF-{alpha}R1KO 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 polymorphonuclear neutrophil lysosomes. MPO activity was elevated significantly after splanchnic ischemia/reperfusion in WT mice (Fig. 3C) . The elevation of the MPO activity was associated with the increase of imununohistochemical staining for ICAM-1 (Fig. 4b ), VCAM-1 (Fig. 4f) , P-selectin (Fig. 5b ), and E-selectin (Fig. 5f) in the ileum section obtained from ischemia- and reperfusion-injured, TNF-{alpha}R1WT mice. In TNF-{alpha}R1KO mice, tissue MPO activity (Fig. 3C) was reduced markedly in comparison with those of WT control animals. In ischemia- and reperfusion-injured, TNF-{alpha}R1KO mice, the positive immunostaining in the intestine for ICAM-1 (Fig. 4c) , VCAM-1 (Fig. 4g) , P-selectin (Fig. 5c) , and E-selectin (Fig. 5g) was reduced visibly and significantly in comparison with the TNF-{alpha}R1WT mice. Similarly, the treatment of TNF-{alpha}R1WT mice with Etanercept (5 mg/kg administered i.p. 5 min prior to reperfusion) reduced the neutrophil infiltration (Fig. 3C) as well as the positive immunostaining in the intestine for ICAM-1 (Fig. 4d) , VCAM-1 (Fig. 4h) , P-selectin (Fig. 5d) , and E-selectin (Fig. 5h) . Please note that no positive staining for ICAM-1, VCAM, P-selectin, and E-selectin was found in the intestine tissue section from sham-operated mice (Figs. 4a and 4e , and 5 , a and e, respectively).


Figure 4
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  		Figure 4. Immunohistochemical localization of ICAM-1 and VCAM-1 in the ileum tissues. Staining of ileum tissue sections obtained from sham-operated mice with anti-ICAM-1 (a) and anti-VCAM-1 (e) antibody showed no positive staining. Sections obtained from ischemia/reperfusion-injured, TNF-{alpha}WT mice showed intense positive staining for ICAM-1 (b) and VCAM-1 (f), mainly localized on the vascular wall. On the contrary, in tissue sections obtained from TNF-{alpha} R1KO and TNF-{alpha}WT mice, which had received Etanercept, the degree of endothelial staining for ICAM-1 (c and d, respectively) and VCAM-1 (g and h, respectively) was reduced markedly. Figures are representative of at least three experiments performed on different experimental days.

 

Figure 5
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  		Figure 5. Immunohistochemical localization of P-selectin in the ileum tissues. No positive staining for P-selectin (a) and E-selectin (e) was observed in the ileum tissue sections obtained from sham-operated mice. Sections obtained from ischemia/reperfusion-injured, TNF-{alpha}WT mice showed intense positive staining for P-selectin (b) and E-selectin (f) on the vascular wall. On the contrary, in tissue sections obtained from TNF-{alpha} R1KO and TNF-{alpha}WT mice, which had received Etanercept, the degree of endothelial staining for P-selectin (c and d, respectively) and E-selectin (g and h, respectively) was reduced markedly. Figures are representative of at least three experiments performed on different experimental days.

 
Reduction of lipid peroxidation in TNF-{alpha}R1KO 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 [45 ]. Ischemia/reperfusion injury of the multivisceral organs was characterized by an increase of tissue MDA, indicative of lipid peroxidation (Fig. 3D) . In TNF-{alpha}R1KO mice, as well as in TNF-{alpha}R1WT mice treated with Etanercept, tissue MDA level (Fig. 3D) was reduced markedly in comparison with those of WT control animals.

TNF-{alpha}R1 modulates expression of FasL after ischemia/reperfusion
Immunohistological staining for FasL in the ileum was also determined 60 min after reperfusion. Ileum sections from sham-operated mice did not stain for FasL (Fig. 6a ), whereas ileum tissue sections obtained from ischemia/reperfusion-injured TNF-{alpha}R1WT mice exhibited positive staining for FasL (Fig. 6b) . The degree of positive staining for FasL was reduced significantly in the ileum section obtained from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 6c and d , respectively).


Figure 6
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  		Figure 6. Immunohistochemical localization of FasL in the ileum tissues. No positive staining for FasL was observed in the ileum tissue sections obtained from sham-operated mice (a). Section obtained from ischemia/reperfusion-injured, TNF-{alpha}WT mice showed intense positive staining for FasL in inflammatory cells (b). The degree of endothelial staining for FasL was reduced markedly in tissue sections obtained from TNF-{alpha} R1KO (c) and TNF-{alpha}WT mice, which had received Etanercept at the dose of 5 mg/kg (d). Figures are representative of at least three experiments performed on different experimental days.

 
Reduction of apoptosis at villus tips in the intestine of TNF-{alpha}R1KO mice
No apoptotic cells were observed at the villus tips in the intestine from sham-operated WT mice (Fig. 7a ). The number of apoptotic cells increased at 1 h after reperfusion in intestine from WT mice (Fig. 7b , see Particle b1). In contrast, only a few apoptotic cells were seen in the villus tips in the intestine from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 7c and d , respectively).


Figure 7
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  		Figure 7. No apoptotic cells were observed at the villus tips in the intestine from sham-operated WT mice (a). The number of apoptotic cells increased at 60 min after ischemia/reperfusion in intestine from WT mice (b, see Particle b1). In contrast, only a few apoptotic cells were seen in the villus tips in the intestine from TNF-{alpha} R1KO (c) and TNF-{alpha}WT mice, which had received Etanercept at the dose of 5 mg/kg (d). Figure is representative of at least three experiments performed on different experimental days.

 
Western blot analysis and immunohistochemistry for Bax and Bcl-2
To detect Bax expression, whole extracts from ileum tissue of each mouse were also analyzed by Western blot analysis at 60 min after reperfusion. A basal expression of Bax was detectable in the ileum tissues from sham-operated animals (Fig. 8a 8a1 ). Bax expression was increased substantially in the ileum from ischemia/reperfusion-injured TNF-{alpha}R1WT mice (Fig. 8a 8a1) . A significant inhibition of Bax protein was observed in the ileum obtained from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 8a 8a1) .


Figure 8
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  		Figure 8. Representative Western blots showing the effects of TNF-{alpha} gene deletion and Etanercept administration on Bax (a, a1) and Bcl-2 (b, b1) expression at 60 min of reperfusion. A representative blot of lysates (a and b) obtained from five animals per group is shown, and densitometry analysis of all animals is reported. (a1 and b1) The results are expressed as mean ± SEM from n = 5/6 ileum tissues for each group. *, P < 0.01, versus Sham. °, P < 0.01, versus TNF-{alpha}WT mice.

 
A basal Bcl-2 expression was detected in ileum tissues from sham-operated mice (Fig. 8b 8b1) . Bcl-2 expression diminished significantly in whole extracts obtained from ischemia/reperfusion-injured TNF-{alpha}R1WT mice 60 min after reperfusion (Fig. 8b 8b1) . The loss of Bcl-2 expression was reduced significantly in the ileum tissues obtained from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 8b 8b1) . Moreover, ileum tissues were taken at 60 min after reperfusion to determine the immunohistological staining for Bax and Bcl-2. Sections of ileum from sham-operated mice did not stain for Bax (Fig. 9a ). Ileum tissue section obtained from ischemia/reperfusion-injured TNF-{alpha}R1WT mice exhibited positive staining for Bax (Fig. 9b) . The degree of positive staining for Bax was reduced significantly in the ileum section obtained from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 9c and d , respectively).


Figure 9
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  		Figure 9. Effect of TNF-{alpha} gene deletion and Etanercept administration on ischemia/reperfusion-induced Bax and Bcl-2 expression in the ileum. No positive staining for Bax was observed in the ileum section taken from sham-operated mice (a). Ileum sections taken from ischemia/reperfusion-injured, TNF-{alpha}WT mice showed positive staining for Bax, localized mainly in the inflammatory cells (b). The degree of positive staining for Bax was reduced markedly in the ileum section obtained from TNF-{alpha} R1KO (c) and TNF-{alpha}WT mice, which had received Etanercept at the dose of 5 mg/kg (d). In addition, positive staining for Bcl-2 was observed in the ileum sections taken from sham-operated mice (e). The degree of positive staining for Bcl-2 was reduced markedly in ileum sections obtained from ischemia/reperfusion-injured TNF-{alpha}WT (f). The reduction of Bcl-2 expression caused by SAO shock was attenuated significantly in the ileum section from TNF-{alpha} R1KO (g) and TNF-{alpha}WT mice, which had received Etanercept at the dose of 5 mg/kg (h). The figure is representative of at least three experiments performed on different experimental days.

 
In addition, sections of ileum tissues from sham-operated mice demonstrated positive staining for Bcl-2 (Fig. 9e) . Ileum tissue sections obtained from SAO-shocked TNF-{alpha}R1WT mice exhibited significantly less staining for Bcl-2 (Fig. 9f) . A significant inhibition of the loss of positive staining for Bcl-2 was observed in the ileum section obtained from TNF-{alpha}R1KO mice as well as in TNF-{alpha}R1WT mice treated with Etanercept (Fig. 9g and 9h , respectively).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here that the genetic (mice with a targeted deletion of the TNF-{alpha}R1 gene, TNF-{alpha}R1KO) or pharmacological (TNF-{alpha}R1WT mice treated with Etanercept) inhibition of TNF-{alpha} exerts a protective effect against the pathological changes caused by ischemia/reperfusion injury of the multivisceral organs. Thus, we propose that TNF-{alpha} contributes to the pathophysiology of ischemia/reperfusion injury. What is then the mechanism by which inhibition of TNF-{alpha} decreases the intestine inflammation caused by ischemia/reperfusion injury of the multivisceral organs?

There is evidence that the proinflammatory cytokine TNF-{alpha} helps to propagate the extension of ischemia and reperfusion injury [46 ]. We confirm here that ischemia/reperfusion injury of the multivisceral organs leads to a substantial increase of ileum TNF-{alpha} levels. It is interesting that the levels of this proinflammatory cytokine are significantly lower in the ileum obtained from TNF-{alpha}R1KO mice as well as in the TNF-{alpha}R1WT mice treated with Etanercept.

The humanized recombinant receptor construct Etanercept has been examined in vivo for its effects in different animal model systems of inflammation [36 , 37 ]. In various models of arthritis, it slowed down or retarded the onset and reduced the overall incidence and the severity of the joint disease. Etanercept was also evaluated as a TNF-{alpha} antagonist in several other preclinical models of disease such as septic shock, cachexia, allergic asthma, allograft rejection, response to vascular injury, and autoimmune encephalomyelitis. Recently, it has been reported that Etanercept blocks the acute inflammatory cell response in whole muscle autografts in mice in an vivo model [47 ], and in general, this new drug is now evaluated for treating many other clinical disorders. Etanercept has been approved for the treatment of autoimmune diseases such as RA, juvenile RA, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis in humans.

What are the secondary biochemical changes subsequent to TNF-{alpha} up-regulation that may contribute to experimental ischemia/reperfusion injury?

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. Hypoxic or injured endothelial cells synthesize proinflammatory cytokines, which can up-regulate the expression of P-selectin, E-selectin, VCAM, and ICAM-1, and correspond to the induction of neutrophil recruitment. This is maximal within the first hour of reperfusion, and persists, at a lower rate, in the late phase of reperfusion [48 ]. In accordance with these findings, we observed the expression of P-selectin, E-selectin, VCAM, and ICAM-1 on the endothelial vascular wall in the intestine section from SAO-shocked TNF-{alpha} R1WT mice. The genetic or pharmacological inhibition of TNF-{alpha} abolished the expression of P-selectin and the up-regulation of E-selectin, VCAM, and ICAM-1. These results suggest that inhibition of TNF-{alpha} 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 from ischemia/reperfusion-injured TNF-{alpha}R1KO as well as in Etanercept-treated TNF-{alpha}R1WT 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 [49 ]. In conclusion, the data presented here demonstrate that TNF-{alpha} is involved in the regulation of the expression of adhesion molecules, and that consequently, TNF-{alpha} plays a role in the tissue infiltration of neutrophils.

Neutrophils play a crucial role in the development and full manifestation of ischemia and reperfusion [50 ]. Their infiltration into inflamed tissue contributes to tissue destruction by the production of reactive oxygen metabolites [51 ], granule enzymes, and cytokines, which amplify further the inflammatory response by their effects on macrophages and lymphocytes [52 , 53 ]. In the present study, we confirm that ischemia/reperfusion injury of the multivisceral organs leads to a significant formation of lipid peroxidation in the ileum from TNF-{alpha}R1WT. Deletion of the TNF-{alpha}R1 gene (TNF-{alpha}R1KO) or treatment of TNF-{alpha}R1WT mice with Etanercept significantly reduced the lipid peroxidation in the ileum, probably, in part, related to the reduced neutrophil recruitment.

Recent evidence has shown that ischemia and/or ischemia and reperfusion induce apoptosis in several tissues, such as brain [54 ], myocardium [55 ], intestine [56 ], and liver [57 ]. Previous studies have suggested that the Fas/FasL interaction may regulate apoptosis [58 ]. Moreover, the Bcl-2 family, in addition to caspases, another one of the functional components of the apoptosis pathway. In our study, the presence of an increased apoptotic cell death was confirmed by TUNEL assay and by positive staining for FasL and Bax. Mice lacking the TNF-{alpha} R1 gene and animals that received Etanercept displayed diminished concentrations of FasL and Bax protein together with high expression of Bcl-2 protein in comparison with WT animals, thereby demonstrating that TNF-{alpha} regulates apoptosis in the ileum and that its inhibition exerts protective effects on cell death in ischemia/reperfusion injury of the multivisceral organs. These results are in agreement with recent evidence, which has demonstrated clearly that TNF-{alpha} contributes significantly to myocardial dysfunction and apoptosis in hind-limb ischemia/reperfusion [59 ]. In conclusion, we have demonstrated for the first time in vivo that the pharmacological inhibition of TNF-{alpha} by means of Etanercept attenuates the development of ischemia/reperfusion injury of the multivisceral organs. The effects of Etanercept are similar to those observed in TNFR1-KO mice. Our results evidence directly that TNF-{alpha} plays a pivotal role in ischemia-reperfusion injury and inflammation and that it may be a novel target by therapeutic applications for treating ischemia and reperfusion and shock.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received July 28, 2006; revised October 15, 2006; accepted November 9, 2006.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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