Intestinal ischemia-reperfusion (I/R) injury is a serious and common clinical entity and can result from different etiologic factors, including superior mesenteric artery (SMA) occlusion or hemorrhagic shock (1). This condition may result in severe local and extensive tissue injury and subsequent distant organ dysfunction, affecting mainly the lungs. Several studies (2, 3) point to the important role that intestinal I/R may play in the triggering of acute respiratory distress syndrome (ARDS), a syndrome characterized by hypoxemia, reduced lung compliance, fluffy diffuse infiltrates on the chest radiograph, and the presence of normal pulmonary capillary pressures (4). Although most patients survive the initial insult that precipitates ARDS, no therapeutic protocol has proven to modify the course of this condition and mortality remains greater than 40% (5).
Among the mediators that are altered during intestinal I/R, TNF-α is the most widely studied (6). TNF-α at high levels leads to the development of an inflammatory response that is the hallmark of many diseases because the mediator is implicated in acute lung injury and ARDS, and also more chronic lung diseases such as asthma, chronic bronchitis, and chronic obstructive pulmonary disease (7, 8).
TNF-α binds to two receptors, the 55-kd and 75-kd receptors, with similar affinity (6). The 55-kd receptor (TNFR1) mediates cytotoxic and proinflammatory effect, whereas the 75-kd receptor (TNFR2) mediates T-cell proliferation (8). Although there is a well-established link between TNF-α levels and sepsis, there is still a debate on the role of TNF-α in nonseptic acute inflammatory conditions. In fact, depending on the model used for the induction of the inflammatory insult, no significant increase of circulating TNF-α is detected. Kim et al. (9) showed that the levels of circulating TNF-α did not change compared with sham controls in a model of hemorrhage shock, whereas Grotz et al. (10) showed early TNF-α increases upon intestinal I/R in rats that are primarily caused by the laparotomy. Chen et al. (11) showed that the Toll-like receptor (TLR) ligand LPS decreased mesenteric I/R injury through TNF-α signaling.
The TLR family members recognize pathogen-associated molecular patterns derived from microbes as well as endogenous ligands from damaged/stressed cells (12). Toll-like receptor 2 and TLR4 are activated not only by LPS and lipoteichoic acid but also by extracellular matrix components (13) and possibly by heat shock proteins (14). Toll-like receptor 4 has also been implicated in the priming effect induced by hemorrhagic shock for lung inflammation (15).
Here, we tested the hypothesis that TNF-α is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-α antibodies and TNF ligand-deficient mice (KO). We report that after intestinal I/R in mice, several cytokines are upregulated, in particular of IL-6, granulocyte colony-stimulating factor (G-CSF), and KC, but TNF-α levels are very little affected. Importantly, neutralization of TNF-α by antibodies did not affect neutrophil recruitment to the lung, the cytokine response, and intestinal and pulmonary injury. Furthermore, the inflammatory response in the absence of TNF-α or of TNF receptors was not significantly different from wild-type controls upon intestinal I/R injury. Finally, we demonstrate that TLR2/TLR4 receptor signaling plays a critical role in the inflammatory process after intestinal I/R in mice.
MATERIALS AND METHODS
Male C57Bl/6 mice aged 8 to 10 weeks were purchased from Ace (Philadelphia, Pa) or obtained from the animal care facility at the Transgenose Institute (CNRS, Orléans, France). TNF KO and TNFR1/R2 KO (obtained from Jackson Laboratories, Bar Harbor, Me), and TLR2 and TLR4 KO (obtained from Dr Akira, crossed in-house to obtain TLR2/4 KO mice) (16) used in this study were bred under specific pathogen-free conditions at the Transgenose Institute (CNRS). Animals were housed, kept in sterile isolated ventilated cages, and used after an approved, reviewed, and monitored protocol from the Committee on Care and Use of Laboratory Animals of Centocor R&D and from the French Government's ethical and animal experiment regulations. All animals had access to water and food ad libitum before the experiments.
Animal model of acute lung injury
Intestinal I/R was performed as described elsewhere (17). In brief, laparotomy was performed under anesthesia with ketamine (100 mg/kg, intraperitoneally) and xylazine (20 mg/kg, intraperitoneally). In the ischemic group of mice, a microsurgical clip (Vascu-statt no. 1001-531; Scalan Internat, Minn) was applied to occlude the SMA. After 45 min occlusion, the clip was removed and intestinal perfusion was re-established. Animals were kept in anesthesia and killed 4 h after intestinal reperfusion by cervical dislocation. For the 24 h time point, the animals were awake after reperfusion and received acetaminophen-supplemented drinking water (3.2 mg/mL) to relieve pain. Blood samples were collected from the abdominal aorta of anesthetized mice. The aliquots were centrifuged (170 g, 10 min), and the sera obtained were stored at −80°C until further analyses. Sham-operated mice undergoing identical surgical intervention except for the occlusion of the SMA were used as control groups. An additional group of nonmanipulated mice (naive) was added to obtain basal values of the parameters studied.
Measurement of lactate dehydrogenase
Intestinal tissue damage caused by intestinal I/R releases lactate dehydrogenase (LDH) into the systemic circulation and was measured according to Cavriani et al. (17). Briefly, LDH activity was determined in the serum samples using a commercially available kit (Labtest Diagnostica, Belo Horizonte, Brazil) and expressed as units per microliter. Absorbance was read at 340 nm on a microplate reader spectrophotometrically (Bio-Tek Instruments).
Lung and intestine myeloperoxidase activity
Myeloperoxidase (MPO) activity was measured as an index of the presence of neutrophils. The lungs were perfused via the pulmonary artery with 3 mL of ice-cold pH 7.2 phosphate-buffered saline (PBS). Intestines were rinsed with ice-cold PBS, and the small intestine was excised and used in the analysis. This procedure was also used to obtain the samples for cytokine and histological analyses. Samples were prepared according to Goldblum et al. (18). In brief, to normalize the pulmonary and intestinal MPO activity among the groups, the whole tissue was homogenized with 3 mL/g of PBS containing 0.5% of hexadecyl-trimethylammonium bromide and 5 mM EDTA, pH 6.0. The homogenized samples were centrifuged at 37,000g for 15 min. Samples of lung and intestine homogenates (10 μL) were then incubated for 10 min with H2O2 and ortho-dianisidine, and the reaction was stopped by the addition of 1% NaNO3. The absorbance was determined at 450 nm using a microplate reader (Bio-Tek Instruments).
Intestine and lung were fixed in 10% buffered formalin. Tissues were dehydrated in ethanol and embedded in paraffin. Sections of 5 μm were stained with hematoxylin and eosin for evaluation of pathological changes. The microscopic slides were reviewed and assessed using a semiquantitative score by two investigators (F.R.C., B.R.) who were blinded to the groups and time points.
Cytokines levels were evaluated in serum and in lung and gut homogenates using a 22-cytokine Linco multiplex kit (LINCO Research, Inc, Mo) with a Luminex instrument (Luminex Corporation, Tex).
In vivo neutralization of TNF-α
Rat monoclonal antibody (mAb) with neutralizing activity for murine TNF-α was generated in house (Centocor R&D, Horsham, Pa) using standard hybridoma technology. The neutralizing property of this antibody has been tested in vitro and in vivo in response to TNF (data not shown). Prior (30 min) to intestinal I/R induction, mice were i.v. injected with 10 mL/kg of a solution containing 2.5 mg/mL of TNF-α antibody (final dose, 25 mg/kg of antibody). A group of mice i.v. injected with 10 mL/kg of PBS 30 min before the intestinal I/R induction was used as the control.
Expression of TLR2 and TLR4 mRNA in lung tissue
Samples of lungs were excised and frozen in liquid nitrogen after the experimental procedure. To harvest RNA, samples were lysed using Nucleic Acid Purification Lysis solution (Applied Biosystems, Foster City, Calif). RNA was prepared using ABI PRISM 6100 Nucleic Acid PrepStation (DNase step included). RNA quality was verified with the 2100 BioAnalyzer (Agilent Technologies, Palo Alto, Calif). Samples that demonstrated high quality (i.e., the ratio of 28S rRNA to 18S rRNA, >1.7) and had a minimum of 1 μg of RNA were submitted to real-time polymerase chain reaction (PCR) analysis. The expression of TLR2 and TLR4 mRNA was evaluated by real-time PCR using a TaqMan assay kit, following the manufacturer's recommendations.
Data are given as mean ± SEM. Comparisons between the groups were made by one-way ANOVA followed by unpaired Newman-Keuls post-test. A 4.0 version (2005) of the GraphPad Prism software was used for this purpose. Values of P < 0.05 were considered significant.
To study the inflammatory response upon intestinal I/R injury in the local (intestine) and in the remote organ (lung), the MPO activity was evaluated in tissue homogenates. Figure 1, A and B, shows that 45 min of ischemia followed by a 4-h reperfusion of the intestinal tissue caused significant neutrophil recruitment both in lung and in intestinal tissues. The MPO activity reached its peak at 4 h after intestinal reperfusion and returned almost to its basal levels after 24 h. Furthermore, intestinal I/R damage induces a significant increase of LDH activity in serum (Fig. 1C) and a decrease of LDH in the gut homogenates (Fig. 1D) as evidence of tissue injury caused by intestinal I/R.
The neutrophilic inflammation was confirmed by microscopic investigations of the lungs (Fig. 1F). We observe neutrophil recruitment in the interstitial space and capillaries of the lung upon intestinal I/R injury (Fig. 1H). There are also signs of edema and inflammatory cells in the perivascular space in the small vessels (Fig. 1F).
Cytokine profile in distinct compartments after intestinal I/R injury
To identify major changes in the pattern of inflammatory mediators expressed upon intestinal I/R injury, we evaluated the expression of 22 cytokines and chemokines using a Linco Multiplex kit in the homogenates of ileum and lung as well as in the serum 4 h after intestinal reperfusion.
In the ileum homogenate, the levels of IL-1α, IL-6, KC, G-CSF, monocyte chemotactic protein 1 (MCP-1), and TNF-α are increased (Fig. 2). The levels of the others cytokines and chemokines are summarized in supplemental Table 1 (see Supplemental Digital Content 1, https://links.lww.com/SHK/A36). In addition, macrophage inflammatory protein (FMIP) 1α is upregulated upon intestinal I/R injury in the ileum, whereas the other mediators such as IL-2, IL-4, IL-12, IL-15, IL-17, IFN-γ, and regulated upon activation, normal T cell expressed and secreted (RANTES) were below the detection levels.
In the serum, a similar profile of that in lung homogenates is observed, where the levels of IL-1α, IL-6, G-CSF, MCP-1, and KC were increased in the intestinal I/R group as compared with sham group (Fig. 3 and Table, Supplemental Digital Content 2, http://links.lww.com/SHK/A37). It is important to note that there was no significant increase of circulating TNF-α upon intestinal I/R injury.
In the lung, only IL-1α, IL-6, G-CSF, MCP-1, and KC were upregulated upon intestinal I/R injury compared with the sham-operated group, TNF-α concentration was increased less than a factor of 2 upon intestinal I/R injury (Fig. 4 and Table, Supplemental Digital Content 3, https://links.lww.com/SHK/A38).
Therefore, upon intestinal I/R, the TNF-α levels in intestine were slightly increased, whereas no increase was observed in serum. However other cytokines and chemokines must be important in this model.
Studies of TNF-α involvement in I/R-induced lung and gut inflammation
Although TNF-α levels were not markedly detectable in serum and lung, we decided to investigate the potential role of this cytokine in the inflammatory process induced by intestinal I/R injury by the use of neutralizing TNF-α mAb. The administration of a neutralizing TNF-α mAb 30 min before intestinal ischemia however did not reduce neutrophil recruitment as assessed by MPO activity in lung and intestinal tissues (Fig. 5, A and B). Moreover, the serum levels of cytokines and chemokines in mice subjected to intestinal I/R were not influenced by the administration of the TNF-α mAb (Fig. 5, C-H, and Table, Supplemental Digital Content 4, https://links.lww.com/SHK/A39).
To exclude a role of TNF-α in lung neutrophilic inflammation in this model of intestinal I/R, we further used TNF gene-deficient mice. However, the MPO activity in the lung from wild-type and TNF-deficient mice did not differ when submitted to intestinal I/R (Fig. 6A). Furthermore, neutrophilic inflammation was investigated in mice deficient for both TNF receptors 1 and 2 (TNFR1/R2 KO). In agreement with the data from both TNF-α mAb treatment and TNF KO experiments, MPO activity remains unchanged in the lung tissues of mice undergoing intestinal I/R injury in the absence of TNFR1/R2 signaling (Fig. 6B). Therefore, for this model of intestinal I/R, we can safely conclude that lung inflammation is TNF-α independent. We also analyzed mice at 8 h after intestinal I/R injury and did not find any consistent reduction of the global inflammatory response in TNF or TNFR1/R2 KO mice (data not shown).
TLRs mediate the lung inflammation induced by intestinal I/R
Intestinal ischemia causes substantial damage in the intestinal wall as shown before with leak of intestinal microbes and microbial products that activate the innate immunity through TLR ligation. Therefore, we asked whether ablation of the two major TLRs recognizing intestinal products, TLR2 and TLR4, would alter the inflammatory response after intestinal I/R injury. Indeed, pulmonary MPO activity was significantly decreased in TLR2/4 double-deficient mice when compared with their wild-type controls (Fig. 7A). We also investigated the expression of TLR2 and TLR4 in lung tissue in both naive and intestinal I/R mice and observed that TLR2 mRNA levels are significantly increased in wild-type mice subjected to intestinal I/R, whereas no significant upregulation of mRNA of TLR4 was detected (Fig. 7, B and C), which does not exclude signaling in response to intestinal I/R damage. Therefore, the data suggest that microbial products augment at least TLR2 expression and signal through TLR2/4 to induce the inflammatory response.
This study in the murine model of intestinal I/R injury provides comprehensive data on a wide range of cytokines and chemokines produced by the intestines and lung tissue and released in the serum. Despite a pivotal role in many acute inflammatory responses (19), we confirm our hypothesis that TNF-α plays no role in remote inflammation induced by intestinal I/R injury. Neither the neutralization of soluble TNF-α nor the absence of TNF-α or its receptors prevented lung inflammation upon intestinal I/R injury, supporting the view that TNF-α is not essential for the development of neutrophilic inflammation nor did it alter the pattern of other proinflammatory cytokines and chemokines in this model of intestinal I/R. However, we report that TLRs, notably TLR2/4, play a critical role for the induction of neutrophil recruitment into the lung as observed by increased lung MPO activity.
The intestinal I/R injury procedure as performed in this study is a simple and reproducible model of local and systemic inflammation with significant repercussions to lung and gut homeostasis (17, 20, 21). We used 45 min of intestinal ischemia followed by 4 h or 24 h of intestinal reperfusion, a condition considered as a mild model of injury with an inflammatory response characterized by neutrophil recruitment to gut and lung tissues that peaks at 4 h after with no significant mortality.
Although experimental data have been reported on the role of individual cytokines in lung injury after intestinal I/R and other forms of injury, no report so far exists on a broader panel of cytokines/chemokines. Here, we investigated a large number of cytokines and chemokines in ileum, lung, and serum of mice subjected to intestinal I/R injury using a multiplex platform (22). Such a comprehensive description of the changes of cytokines/chemokines in three different compartments might provide a better understanding of the role of the systemic inflammation induced by intestinal I/R injury on the multiple organ disease. We demonstrate here increased levels of IL-1α, IL-6, G-CSF, KC, MCP-1, macrophage inflammatory protein 1α, and TNF-α in the intestines. These factors are clearly relevant to mediate acute inflammation and intestinal tissue injury. Therefore, we extend previous data showing upregulation of platelet-activating factor (23), IL-1β (24), IL-6, and TNF-α (25) in the intestine. Furthermore, our data support the notion that cytokines/chemokines produced upon intestinal injury drive remote inflammation in the lung. IL-1α, IL-6, G-CSF, and KC were upregulated in lung and in serum after intestinal I/R injury. The gut, the initial site of ischemic injury with highest levels of cytokines/chemokines, may spill its inflammatory mediators causing remote inflammation as suggested for hemorrhagic shock by Deitch (26) and previously for intestinal I/R injury (17, 27).
Of great interest was the observation that KC level in lung homogenates from intestinal I/R mice were greater than 400 pg/mL upon intestinal I/R injury as compared with levels of 100 pg/mL in the sham-operated group. This elevated level of KC in lung tissue is even higher than that found in serum, suggesting that this chemokine is produced locally in the lung after intestinal I/R. In fact, Wiedermann et al. (28) reported that IL-8 (the human equivalent for KC) is increased in the bronchoalveolar lavage of patients with ARDS, and that this chemokine may be considered the main factor modulating the influx of neutrophils in the early stage of the disease.
Unexpectedly, there was no change in TNF-α levels in both serum and lung homogenates. Serum samples were taken also 1 h after the beginning of reperfusion to exclude a possible early peak of TNF-α, but no increased serum levels were observed (8.0 ± 1.5 for naive mice, 12.9 ± 1.3 for sham mice, and 10.2 ± 2.2 for mice upon intestinal I/R). Therefore, TNF-α seems to be unrelated to induction of lung inflammation in our model of intestinal I/R. Interestingly, Davidson et al. (29) did not implicate TNF-α in endothelial cell injury caused by hemorrhagic shock. Several studies suggested that TNF-α along with other cytokines may be important mediators of acute lung injury (30, 31). However, elevated TNF-α levels in serum and bronchoalveolar lavage fluid have not been consistently correlated with clinical outcomes in patients at risk of or with established acute lung injury (7).
Another important point in the discussion of the involvement of TNF-α in intestinal I/R is the different roles that this cytokine may play in the inflammatory process. Chen et al. (11) showed that the activation of TLR4 is capable of limiting the murine intestinal injury upon intestinal I/R, and that this protective effect is mediated by TNF-α. In addition, Teoh et al. (32) showed that a low dose of TNF-α administered before liver ischemia may have a protective effect. The discrepant results from our data may be explained by a different protocol used for intestinal I/R in the former study. Noteworthy, these studies did not preclude a pivotal role of TNF-α in acute inflammation, but suggest that TNF-α could have a more complex regulatory role. Our experimental data demonstrated that TNF-α does not participate in the modulation of some of the hallmarks of systemic inflammation caused by intestinal I/R injury. First, neutralization of TNF-α with antibody treatment did not diminish neutrophilic inflammation in lung and gut upon intestinal I/R injury. Second, the lack of TNF-α as investigated in TNF KO and in TNFR1/R2 double KO mice submitted to intestinal I/R injury did not abrogate inflammation. These data represent strong evidence that TNF-α is not associated with acute inflammation of the lung.
In contrast to the lack of involvement of TNF-α, TLR2 and TLR4 showed prominent roles in neutrophil recruitment after intestinal I/R injury. In fact, when we analyze lung homogenates, there was a significant reduction in MPO activity of TLR2/4 KO mice submitted to intestinal I/R injury when compared with their C57Bl/6 controls. Furthermore, quantification of mRNA by real-time PCR shows a small but significant increase of TLR2 mRNA expression in lung tissue upon intestinal I/R injury. These data demonstrate that TLR2 and TLR4 are important for neutrophil recruitment and suggest a possible bacterial translocation as the source of inflammation induced by intestinal I/R. However, in a rat model of intestinal I/R injury inflammation, inflammatory mediators preceded increased serum endotoxin levels (33). Furthermore, induction of oral tolerance to LPS or treatment with polymyxin B did not alter the inflammation and pulmonary edema induced by intestinal I/R injury (33). Clearly, more work is needed to understand the ligand-activating TLR2/4, which play a critical role in pathophysiology (34). Toll-like receptors may also recognize endogenous ligands such as heat shock proteins and extracellular matrix components that could act as "danger signals" (14, 34, 35).
In conclusion, intestinal I/R injury induced the upregulation of IL-6, G-CSF, and KC expression in local and remote organs, which seems to play a role in pulmonary neutrophil recruitment and inflammation, whereas TNF-α is not important for this response. Importantly, TLR2/4 signaling caused by microbial products accessing the body upon intestinal injury is critical to initiate the inflammatory response and remote inflammation in the lung.
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