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Ann Thorac Surg 2006;82:2017-2023
© 2006 The Society of Thoracic Surgeons

Original Articles: General Thoracic

Toll-Like Receptor 4 Mediates Lung Ischemia-Reperfusion Injury

Akira Shimamoto, MD, PhDa,*, Timothy H. Pohlman, MDb, Shin Shomura, MDa, Tomohito Tarukawa, MDa, Motoshi Takao, MD, PhDa, Hideto Shimpo, MD, PhDa

a Department of Thoracic and Cardiovascular Surgery, Mie University Graduate School of Medicine, Tsu, Japan
b Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana

Accepted for publication June 19, 2006.

Abbreviations and Acronyms AP-1 = activator protein-1; BAL = bronchoalveolar lavage; BSA = bovine serum albumin; cpm = counts per minute; ERK = extracellular signaling-regulated kinase; HMGB1 = high-mobility group box 1; IL = interleukin; IR = ischemia-reperfusion; JNK = c-Jun NH2-terminal kinase; KC = keratinocyte chemoattractant; LIRI = lung ischemia-reperfusion injury; LPS = lipopolysaccharide; MAPK = mitogen-activated protein kinase; MCP-1 = monocyte chemoattractant protein-1; MIP = macrophage inflammatory protein; MPO = myeloperoxidase; NF-{kappa}B = nuclear factor-{kappa}B; TLR = Toll-like receptor; TNF-{alpha} = tumor necrosis factor-{alpha}; WT = wild-type

* Address correspondence to Dr Shimamoto, Department of Thoracic and Cardiovascular Surgery, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu, Mie 514-8507, Japan (Email: jj6jdv{at}clin.medic.mie-u.ac.jp).

Presented at the Poster Session of the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: We have previously reported that nuclear factor (NF)-{kappa}B activation and inflammatory cytokine expression were involved in the development of lung ischemia-reperfusion injury (LIRI). Because Toll-like receptor 4 (TLR4) activates NF-{kappa}B-dependent transcription of inflammatory cytokine genes during myocardial ischemia-reperfusion injury, we examined whether absence of TLR4 in TLR4-deficient mice protects against LIRI.

METHODS: Left lungs of wild-type (C57BL/6J) mice or TLR4-null (TLR4–/–) mice were made ischemic for 60 minutes and then reperfused for 180 minutes. Response to injury was quantified by tissue myeloperoxidase activity, vascular permeability ([125I]-bovine serum albumin extravasation), and leukocyte and inflammatory mediator accumulation in bronchoalveolar lavage expression. Lung homogenates were also analyzed for activation of mitogen-activated protein kinases and nuclear translocation of the transcription factors NF-{kappa}B and activator protein-1.

RESULTS: After LIRI, lungs from TLR4–/– mice demonstrated a 52.4% reduction in vascular permeability (p = 0.001), a 52.6% reduction in lung myeloperoxidase activity (p = 0.006), and a marked reduction in bronchoalveolar lavage leukocyte accumulation when compared with lungs from wild-type mice. The TLR4–/– mice lungs, subjected to LIRI, also demonstrated marked reductions in amounts of several proinflammatory cytokines/chemokines in bronchoalveolar lavage samples. Phosporylation of c-Jun NH2-terminal kinase, and activation of NF-{kappa}B and activator protein-1 were also significantly reduced in homogenates of lungs from TLR4–/– mice injured by ischemia and reperfusion (p < 0.05).

CONCLUSIONS: These data suggest that TLR4 plays a role in LIRI. Thus, TLR4 may be a potential therapeutic target to minimize ischemic-reperfusion–induced tissue damage and organ dysfunction.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Direct lung ischemia-reperfusion (IR) injury (LIRI) is a morbid complication of lung and heart–lung transplantation. It has been associated with more frequent episodes of acute cellular rejection [1], and predisposes lung recipients to an earlier onset of chronic rejection or obliterative bronchiolitis [2]. The development of LIRI is also associated with pulmonary thromboendarterectomy, open heart surgery, aortic surgery, severe hemorrhagic shock, and after resuscitation for circulatory arrest. Therefore, strategies aimed at amelioration of this form of acute lung dysfunction may have substantial effectiveness for patient care in general.

During IR, cell-surface inflammatory receptors of the innate immune system are stimulated, resulting in activation of intracellular signaling cascades, with subsequent upregulation of transcription of several proinflammatory mediators including cytokines, chemokines, and adhesion molecules, producing a robust inflammatory response [3]. Recently, we have focused on an innate immune receptor, Toll-like receptor 4 (TLR4), and the role that TLR4 may have in LIRI. Previous studies of TLR4 have focused on TLR4 activation during gram-negative sepsis, and activation of TLR4 by endotoxin. However, we [4] and others [5] have demonstrated a role for TLR4 in myocardial IR injury that does not involve interaction of TLR4 with an endotoxin ligand. In addition, a recent study has shown that TLR4 interacts with a protein ligand released from damaged hepatocytes to initiate an IR injury in the liver [6].

Two pathways of injury that may occur during ischemia and reperfusion of lung tissue are apoptosis, induced through activation of a transcriptional program controlled by nuclear factor (NF)-{kappa}B; and acute inflammation, promoted by activation of resident alveolar macrophages and expression of several proinflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, IL-8, and macrophage inflammatory protein (MIP)-2 [7]. We also have previously reported that TLR4 activates NF-{kappa}B-dependent transcription of inflammatory cytokine genes in myocardial IR injury [4, 8]. The TLR4-mediated injury appears to occur through activation of c-Jun NH2-terminal kinase (JNK) and translocation of NF-{kappa}B.

To examine the function of TLR4 in LIRI, we used a model of in situ single-lung ischemia and reperfusion in TLR4 knockout mice. We hypothesize that absence of TLR4 will reduce LIRI similar to the attenuation of myocardial IR injury we have observed in TLR4-null mice.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animals and Experimental Design
Male C57BL/6J mice (wild-type [WT]; CLEA Japan, Tokyo, Japan) or TLR4-null (TLR4–/–) mice (on the same genetic background as WT and each back-crossed with C57BL/6J mice at least eight times [9]), aged 7 to 14 weeks and weighing 20 to 25 g, were subjected to 60 minutes of warm in situ single-lung ischemia followed by 180 minutes of reperfusion by occlusion and release of the left hilum, modified as previously described [10]. Mice were intubated and placed under mechanical ventilation (tidal volume = 0.75 mL, respiratory rate = 120 breaths per minute) with an inspired oxygen content of 60% after undergoing general anesthesia with pentobarbital sodium (100 mg/kg, intrapercutaniously). After a left anterolateral thoracotomy through the fifth intercostal space, all animals were given 5 U heparin intrapercutaneously. Five minutes after heparin administration, the left pulmonary hilum, including the left main bronchus, artery, and vein, was occluded with a noncrushing microvascular clamp under the left lung in an inflated state. At the end of the 60-minute ischemic period, the clamp was removed and the lung was allowed to ventilate and reperfuse for as long as 180 minutes. Both TLR4–/– and WT mice underwent sham operations consisting of a thoracotomy without cross-clamping of the pulmonary hilum. All animals were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health and also with the "Guideline for Animal Experiments in Mie University Graduate School of Medicine."

Lung Vascular Permeability Index
To measure endothelial vascular injury secondary to ischemia and reperfusion, a lung permeability index was calculated. The 125I radiolabeled bovine serum albumin ([125I]-BSA; PerkinElmer Life and Analytical Sciences, Wellesley, Massachusetts), titrated to an activity of 8 x 105 counts per minute (cpm) per dose, was injected intravenously in a final volume of 50 µL of 1% BSA/phosphate-buffered saline solution 5 minutes before removal of the hilar clamp. At the end of the experiment, at 180 minutes of reperfusion, the radioactivity counts were quantitated in the left and right lung, and 1 mL blood was sampled from the inferior vena cava. The permeability index (PI) was calculated as:


Formula

This ratio corrects for any variation in systemic blood levels of radioactivity and provides a reproducible indicator of lung microvascular permeability secondary to acute oxidative lung injury.

Bronchoalveolar Lavage
Recovered left lung bronchoalveolar lavage (BAL) effluent afforded analysis of inflammatory cell accumulation in the alveolar spaces of ischemia-reperfused lungs. Left lungs were lavaged selectively through the tracheostomy tube with 300 µL 0.9% saline after a clamp was placed on the right hilum. At least 80% of instilled volume needed to be recovered for the sample to be considered adequate. This fluid was centrifuged (1,500g for 8 minutes at 4°C) to pellet the cells. The supernatant was snap frozen for cytokine analysis, and then stored at –80°C until subsequent analysis. Inflammatory cells were then counted using a hemacytometer.

Analysis for Myeloperoxidase Activity
The myeloperoxidase (MPO) activity was used to quantitate lung parenchymal neutrophil accumulation. Frozen tissue samples were homogenized with ice-cold lysis buffer containing hexadecyltrimethylammonium bromide, and centrifuged, and supernatants were assayed for MPO activity using substrate buffer containing o-dianisidine dihydrochloride. The MPO activity was measured at an absorbance of 460 nm over 1 minute, and calculated as {Delta}OD460 nm/minute.

Western Immunoblotting Assay for Phosphorylation of Mitogen-Activated Protein Kinases
Whole-cell protein, extracted from frozen tissue samples with ice-cold lysis buffer (Cell Signaling Technology, Beverly, Massachusetts), were stored at –80°C until the time of assay. Whole-cell protein, 10 µg, was loaded onto 15% sodium dodecylsulfate polyacrylamide electrophoretic gels, and transferred to polyvinylidene difluoride membranes. The membranes were immunoblotted with primary antibodies contained in PhosphoPlus p38 mitogen-activated protein kinase (MAPK, Thr180/Tyr182), JNK (Thr183/Tyr185), and extracellular signal-regulated kinase (ERK, Thr202/Tyr204) Antibody Kits (1:1000; Cell Signaling Technology). Immunoreactivity was quantitated with enhanced chemiluminescence and determined with densitometry (ImageJ 1.34). The ratio of phospho- to total-MAPKs immunoreactivity was determined for each sample, and the results are expressed as fold increase over a control.

Electrophoretic Mobility Shift Assays for Transcription Factor Activities
Lung tissue nuclear proteins from frozen tissue samples were isolated as previously described. Each 10 µg of nuclear protein was incubated in a binding reaction with a 32P-end-labeled, double-stranded oligonucleotide containing the human and rodent consensus NF-{kappa}B binding, 5’-AGTTGAGGGGACTTTCCCAGGC-3’, and activator protein (AP)-1 binding 5’-CGCTTGATGAGTCAGCCGGAA-3’ (Promega, Madison, Wisconsin), subjected to electrophoresis in native 6% nondenaturing polyacrylamide gels. Densitometry was performed with ImageJ 1.34. The results were expressed as fold activation over a control.

Enzyme-Linked Immunosorbent Assay for Inflammatory Cytokine Expression in BAL
Cytokine expression in the supernatant of BAL stored at –80°C were measured with Quantikine colorimetric sandwich enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, Minnesota).

Statistical Analysis
All data were expressed as mean ± SE. The significance of the difference between group means was analyzed by a two-tailed Student’t t test after normality, confirmed by Kolmogorov-Smirnov test and Shapiro-Wilks test. All statistical analyses were done with SPSS 11.0 (SPSS, Chicago, Illinois), and a value of p less than 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
TLR4 Deficiency Reduces LIRI
To assess the role of TLR4 in LIRI, we used a murine model of complete, warm 60-minute lung ischemia achieved by cross-clamping one lung hilum in situ (with the lung inflated) followed by 180 minutes of reperfusion after the clamp was released. To ensure our mouse model was free from lipopolysaccharide (LPS [endotoxin]) contamination (an exogenous ligand for TLR4), levels of plasma endotoxin were measured with a chromogenic Limulus amebocyte lysate assay (Endotoxin Single Test; Wako Pure Chemical Industries, Osaka, Japan). Endotoxin was not detected in TLR4–/– and WT mice during IR or sham operation, or at the completion of each experiment (data not shown). The LIRI, as assessed by an increase in lung vascular permeability, was significantly increased in lungs from both WT and TLR4–/– mice after injury compared with sham-operated controls (WT, 0.06 ± 0.01 versus 0.82 ± 0.06, p = 0.00002; TLR4–/–, 0.05 ± 0.01 versus 0.39 ± 0.04, p = 0.0004). However, deficiency of TLR4 produced a significant reduction in LIRI-induced-lung vascular permeability (52.4%), when compared with lung vascular permeability in lungs from WT controls injured by ischemia and reperfusion (p = 0.001; Fig 1A). Similarly, LIRI significantly increased MPO activities in lung tissue from both TLR4–/– and WT mice compared with lung tissue from either TLR4–/– mice or WT mice undergoing sham operation only (WT, 0.22 ± 0.03 versus 0.76 ± 0.08 {Delta}OD460, p = 0.0001; TLR4–/–, 0.24 ± 0.02 versus 0.36 ± 0.16 {Delta}OD460, p = 0.024). In contrast to MPO activity in lungs injured by ischemia and reperfusion in WT mice, MPO activity in the TLR4–/– mouse lung subjected to LIRI was significantly lower (p = 0.006; Fig 1B). These findings were consistent with the observation that bronchioalveolar lavage cell counts that are elevated after LIRI were also significantly reduced in mice lungs deficient in TLR4 compared with lungs from WT mice (p = 0.00001; Fig 1C). These data suggest that LIRI induces damage to the alveolar-capillary barrier associated with an inflammatory cell infiltrate to the level of the alveolus, and that this damage after LIRI is diminished in the absence of the innate immune receptor, TLR4.


Figure 1
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  		Fig 1. Toll-like receptor 4 (TLR4) deficiency reduces lung ischemia-reperfusion injury. Lungs from either wild-type (WT) mice (open bars) or TLR4–/– (KO) mice (closed bars) were subjected to ischemia-reperfusion (IR) injury, as described in Methods. At the completion of lung ischemia-reperfusion injury, lung tissue was obtained to determine the following: (A) lung vascular permeability (values represent mean ± SE of 8 animals in each group; *p < 0.001 versus sham; {dagger}p = 0.001 versus WT); (B) tissue myeloperoxidase (MPO) activity as an indirect measure of neutrophil infiltration (values represent mean ± SE of 6 animals in each group; *p < 0.05 versus sham; {dagger}p = 0.006 versus WT); and (C) bronchoalveolar lavage (BAL) cell counts (values represent mean ± SE of 6 animals in each group; *p < 0.005 versus sham; {dagger}p = 0.00001 versus WT).

 
Activation of Intracellular Signaling Pathways During LIRI Is Attenuated in TLR4–/– Mice
The LPS stimulation of TLR4 activates several intracellular signaling pathways, which include the mitogen-activated protein kinase pathways, p38 MAPK, JNK, and ERK; these signaling pathways in turn activate transcription factors, including NF-{kappa}B (p50/p65) and AP-1 (c-Fos/c-Jun), which coordinate the transcription of many genes encoding inflammatory mediators [11]. To determine whether LIRI induces a comparable pattern of TLR4-mediated cellular signaling pathway activation, whole-cell proteins, immunoblotted for phosphorylated MAPKs, and nuclear proteins, analyzed by electrophoretic mobility shift assay for NF-{kappa}B and AP-1, were extracted from lungs in TLR4–/– and WT mice after LIRI. Phosphorylation of p38, JNK, and ERK were increased in both TLR4–/– mice and wild type mice in response to LIRI. The LIRI-induced p38 and ERK activation were similar in both TLR4–/– and WT mice; in contrast, JNK activation was significantly reduced in the TLR4-deficient mice when compared with WT. There were minimal to no activities of any of the MAPKs in sham-operated TLR4–/– or WT mice (Fig 2).


Figure 2
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  		Fig 2. The activity of c-Jun NH2-terminal kinase (JNK) during lung ischemia-reperfusion injury (LIRI) is diminished in Toll-like receptor 4 (TLR4) deficient mice. After LIRI, lung tissue from either wild-type mice (WT) or TLR4–/– mice (KO) were obtained and examined by Western immunoblotting analysis (A, B, C) for both phosphorylated and total amounts of mitogen-activated protein kinases (MAPKs): p38, JNK, and extracellular signal-regulated kinase (ERK). (D) Densitometry was used to quantify the ratio of phospho- to total-MAPK immunoreactivity for each sample from either WT mice (open bars) or KO mice (closed bars); the results are expressed as fold-increase of phospho-MAPK amount over total MAPK quantity. (Values represent mean ± SE of 4 animals in each group; *p < 0.005 versus sham; {dagger}p = 0.02 versus WT.)

 
TLR4 Deficiency Blocks Activation of NF-{kappa}B and AP-1 During LIRI
Compared with the sham group, there was a significant rise in transcription factor activation during lung ischemia and reperfusion in WT mice (NF-{kappa}B, 7.25 ± 0.60-fold increase, p = 0.000001; AP-1, 4.69 ± 0.43-fold increase, p = 0.0003). In contrast neither NF-{kappa}B nor AP-1 showed appreciable activation in TLR4–/– mice during LIRI (NF-{kappa}B, 2.70 ± 0.35-fold increase, p = 0.0005 versus WT; AP-1, 1.85 ± 0.56-fold increase, p = 0.0004 versus WT; Fig 3).


Figure 3
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  		Fig 3. Lung ischemia-reperfusion injury (LIRI)–induced nuclear factor (NF)-{kappa}B and activator protein (AP)-1 nuclear translocations are blocked in Toll-like receptor 4 (TLR4) deficient mice. (A, B) Electrophoretic mobility shift assays were performed on nuclear proteins isolated from tissue samples obtained from lungs subjected to LIRI in either wild-type mice (WT) or TLR4–/– mice (KO). (C) Shifted band strength was quantified by densitometry for each sample from either WT mice (open bars) or KO mice (closed bars). (Values represent mean ± SE of 4 animals in each group; *p < 0.0005 versus sham; {dagger}p < 0.001 versus WT.)

 
Inflammatory Cytokine Expression Is Reduced in TLR4–/– Mice During LIRI
Lung IR stress significantly increased keratinocyte chemoattractant (KC), TNF-{alpha}, MIP-1{alpha}, MIP-2, and monocyte chemoattractant protein (MCP)-1 in BAL in WT after LIRI (KC, 18.1 ± 1.96-fold increase, p = 0.0002 versus sham; TNF-{alpha}, 2.01 ± 0.30-fold increase, p = 0.0014 versus sham; MIP-1{alpha}, 13.6 ± 0.65-fold increase, p = 0.00004 versus sham; MIP-2, 10.7 ± 0.97-fold increase, p = 0.0075 versus sham; and MCP-1, 26.0 ± 3.11-fold increase, p = 0.00003 versus sham). However, the inflammatory cytokine response of LIRI was significantly reduced in TLR4–/– mice (KC, 8.85 ± 0.92-fold increase, p = 0.013 versus WT; TNF-{alpha}, 1.08 ± 0.49-fold increase, p = 0.042 versus WT; MIP-1{alpha}, 5.00 ± 0.33-fold increase, p = 0.022 versus WT; MIP-2, 1.16 ± 0.22-fold increase, p = 0.004 versus WT; MCP-1, 6.20 ± 1.11-fold increase, p = 0.0033 versus WT; Fig 4).


Figure 4
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  		Fig 4. Absence of Toll-like receptor 4 (TLR4) signal transduction is associated with decreased inflammatory cytokine expression in bronchoalveolar lavage during lung ischemia-reperfusion injury. Enzyme-linked immunosorbent assays were performed, as described in Methods, on lung samples from either wild-type (WT) mice (open bars) or TLR4–/– (KO) mice (closed bars) for keratinocyte chemoattractant (KC [a murine functional homologue of interleukin-8]) and other selected chemokines and cytokines identified on the x-axis. (Values represent mean ± SE of 8 animals in each group; *p < 0.05 versus sham; {dagger}p < 0.05 versus WT.) (MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; TNF = tumor necrosis factor.)

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
In a substantial number of patients undergoing lung transplantation, LIRI develops in the implanted graft, causing early graft dysfunction characterized by nonspecific alveolar damage, lung edema, increased pulmonary vascular resistance, and hypoxia [12]. Less well characterized forms of LIRI may also occur after cardiopulmonary bypass or after cardiopumonary resuscitation for cardiac arrest. A novel finding of the this study is that LIRI is ameliorated in TLR4-null mice, suggesting that the innate immune receptor, TLR4, mediates, in part, the response of the lung to ischemia and subsequent reperfusion. In addition, we have demonstrated that TLR4 activity during LIRI is associated with the activation of a MAPK, JNK, nuclear translocation of transcription factors NF-{kappa}B and AP-1, and subsequent expression of proinflammatory chemokines and cytokines.

An important consideration in this study is that warm ischemia rather than cold ischemia preceeds the reperfusion period. With respect to lung transplantation, our model more closely approximates retrieval of lungs from a non–heart-beating donor that has a necessary period of warm ischemia before the lungs are flushed with cold preservation fluid. Also, our model simulates the clinical state after circulatory arrest from any cause. Our results may not be generalized to lung retrieval from heart-beating donors that are not generally subjected to any significant period of warm ischemia.. Also, owing to technical limitations of the model, we could not assess lung function after LIRI that may be the result of inflammation-induced ventilation-perfusion mismatch and abnormalities in gas exchange, although these physiologic abnormalities are detected in larger animals after LIRI [13].

The molecular events leading to TLR-mediated activation of innate immunity during reperfusion of ischemic lung are not known. Recent studies have highlighted the role of the TLR4-mediated signaling pathway in the induction of innate immunity as an early response to infection by various micro-organisms [14]. Toll-like receptor 4 has been examined extensively as a receptor expressed on leukocytes that recognizes and binds LPS during the early course of infections with gram-negative pathogens [15]. Toll-like receptor 4 is also expressed under normal conditions in many tissues not generally thought to have a role in immunity [16, 17], suggesting that TLR4 may also function other than in innate immunity.

Toll-like receptor 4–initiated cell signaling in part activates transcription factors NF-{kappa}B, which has a critical role in regulating the transcription of genes involved in immune and inflammatory responses, programmed cell death and survival, and cell growth and the cell cycle [18–20]. Previous work has demonstrated that ischemia and reperfusion result in NF-{kappa}B activation [21]. The repertoire of genes regulated by NF-{kappa}B include those that encode cytokines (TNF-{alpha}, IL-1ß, IL-4, IL-5, IL-6, and granulocyte/macrophage-colony stimulating factor); chemokines (IL-8 [or the murine homologue of IL-8, KC], regulated upon activation normal T cells expressed and secreted, MIP-1{alpha}, and MCP-1); adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin); and enzymes (cycloxygenase-2, inducible nitric oxide synthase, phospholipase 2, and plasminogen activator inhibitor-1; reviewed by Bonizzi and Karin [22]), which initiate and propagate the innate immune response.

It is not known how TLR4 signaling is initiated during LIRI, although it is plausible that TLR4 recognizes an endogenous ligand released from cells damaged during ischemia and reperfusion. High-mobility group box 1 (HMGB1) is an intracellular protein present in many species that functions in regulation and modulation of transcription in the nucleus. High-mobility group box 1 is released readily from necrotic or damaged cells, which may signal through TLR4 the presence of advancing tissue injury, initiating an inflammatory response that further damages viable cells [23]. Previous studies have provided direct evidence that HMGB1 can interact with both TLR2 and TLR4 to induce an inflammatory response during liver IR injury similar to that initiated by LPS [24]. Whether HMGB1–TLR4 interactions mediate LIRI has yet to be proven. Furthermore, TLR4 has been shown to participate in the recognition of several other endogenous ligands, such as hyaluronic acid, heparin sulfate, fibrinogen, and perhaps, heat shock proteins [25–29]. The release of these ligands may stimulate inflammatory activity through TLR4 signaling. In this fashion, they may function as "danger signals" to innate immune cells, identifying ongoing tissue injury.

Glucan phosphate, a (1-3)-ß-D-linked polymer of glucose (isolated from fungal cell walls), is a ligand for TLR4 [30]. Glucan phosphate has been reported to decrease myocardial IR injury in an in situ rat model through an apparent interaction with TLR4, resulting in attenuation of NF-{kappa}B activation [21]. Although glucan phosphate blocked TLR4-mediated NF-{kappa}B activation during myocardial IR injury, glucan phosphate promoted tyrosine phosphorylation of TLR4, resulting in both inhibition of inflammation, regulated by NF-{kappa}B, and reduction in IR-induced cardiomyocyte apoptosis through activation of the phosphoinositide 3-kinase/Akt signaling pathway. The requirement for TLR4-mediated NF-{kappa}B activation in myocardial IR in a rat model [21] is consistent with our previous results demonstrating diminished myocardial IR injury in TLR4-null mice [4]. Our results may differ, however, in that TLR4-null mice would be expected to lack the protective effect of TLR4-induced antiapoptotic activity as well as lacking a TLR4-mediated proinflammatory response.

Eritoran is a second-generation structural analog of the lipid A portion of LPS. In vivo and in vitro models have shown Eritoran to be a potent antagonist of the biochemical and physiologic effects of LPS, inhibiting translocation of NF-{kappa}B and reducing expression of inflammatory cytokines [31]. Eritoran is safe in humans, and it is currently under clinical development as a possible therapeutic agent for the treatment of sepsis [32]. In our previous study, inhibition of TLR4 with Eritoran in an in situ murine model significantly reduced myocardial IR injury and markers of an inflammatory response [8]. We anticipate, based on the results of this study, that Eritoran will prove effective for treatment of LIRI.

In summary, we and others have shown previously that IR injury in the heart requires TLR4 expression [4, 21], and Tsung and colleagues [6] have reported that TLR4 is necessary for hepatic IR injury. We now extend these studies by demonstrating that TLR4 mediates LIRI as well, suggesting that TLR4 expression has a general role in the response to ischemia and reperfusion.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported in part by a Grant-in-Aid for Scientific Research (No. 14370408) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. We thank Dr Shizuo Akira (Department of Host Defense, Osaka University Research Institute for Microbial Diseases) for providing TLR4 deficient mice, as well as Oriental Bio Service (Kyoto, Japan) for the technical advice of these mice breeding.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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