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Published ahead of print on June 5, 2008, doi:10.1164/rccm.200707-1069OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 356-362, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200707-1069OC

Original Article

Role of Soluble Receptor for Advanced Glycation End Products on Endotoxin-induced Lung Injury

Haiying Zhang1,2, Sadatomo Tasaka1, Yoshiki Shiraishi1, Koichi Fukunaga1, Wakako Yamada1, Hiroyuki Seki3, Yuko Ogawa1, Keisuke Miyamoto1, Yasushi Nakano1, Naoki Hasegawa1, Taku Miyasho4, Ikuro Maruyama5 and Akitoshi Ishizaka1

1 Division of Pulmonary Medicine, Keio University School of Medicine, Tokyo, Japan; 2 Emergency Department, The First Affiliated Hospital, China Medical University, Shenyang, China; 3 Department of Anesthesiology, Keio University School of Medicine, Tokyo, Japan; 4 Laboratory of Veterinary Biochemistry, Rakuno Gakuen University, Ebetsu, Japan; and 5 Department of Laboratory and Molecular Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan

Correspondence and requests for reprints should be addressed to Sadatomo Tasaka, M.D., Ph.D., Division of Pulmonary Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: tasaka{at}cpnet.med.keio.ac.jp


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The interaction of receptor for advanced glycation end products (RAGE) and its ligands often leads to inflammatory processes or tissue injury, although the effect of the blockade of RAGE signaling on lung injury remains to be investigated.

Objectives: Using a murine model of lung injury induced by intratracheal lipopolysaccharide (LPS), we evaluated RAGE expression in the airspace and the effect of recombinant soluble RAGE (sRAGE) on LPS-induced lung injury.

Methods: First, the expression of sRAGE in bronchoalveolar lavage (BAL) fluid was determined at 24 hours after intratracheal instillation of LPS or phosphate-buffered saline. Next, to evaluate the effect of sRAGE, BAL fluid was collected for cell counting and measurements of lung permeability and cytokine concentrations 24 hours after intratracheal LPS in the mice with or without intraperitoneal administration of sRAGE 1 hour after the instillation. In another series, lungs were sampled for histopathology and detection of apoptotic cells. The activation of nuclear factor (NF)-{kappa}B was analyzed 4 hours after LPS instillation.

Measurements and Main Results: In response to LPS challenge, a RAGE isoform of 48 kD was detected in the BAL fluid. Treatment with sRAGE significantly attenuated the increases in neutrophil infiltration, lung permeability, production of inflammatory cytokines, NF-{kappa}B activation, and apoptotic cells in the lung as well as development of pathologic changes after LPS instillation.

Conclusions: RAGE plays an important role in the pathogenesis of LPS-induced lung injury in mice. It was suggested that sRAGE should be tested as a treatment modality in other models of acute lung injury.

Key Words: RAGE • lung injury • apoptosis • chemokine • mouse model



   AT A GLANCE COMMENTARY
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 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The receptor for advanced glycation end products (RAGE) recognizes a variety of ligands, including high-mobility group box 1, but little is known concerning its role in the development of endotoxin-induced lung injury.

What This Study Adds to the Field
Soluble RAGE is up-regulated during LPS-induced lung injury, which was ameliorated by recombinant soluble RAGE. Soluble RAGE may be secreted as a decoy receptor and contribute to the suppression of excessive inflammatory response during acute lung injury/acute respiratory distress syndrome.

 
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are characterized by acute inflammation and disruption of the alveolar–capillary membranes, leading to alveolar flooding with protein-rich edema fluid (1). During ALI/ARDS, various proinflammatory cytokines and chemokines are up-regulated and contribute to the initiation and propagation of the inflammatory response (2). Despite various pharmacologic interventions developed in the last two decades that aim at specific targets, such as cytokines and adhesion molecules, the therapeutic strategy for this syndrome remains to be established (3).

The receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, is a multiligand-binding receptor, which can bind advanced glycation end products (AGE), amyloid β-peptide, and S100 proteins (4, 5). RAGE has also been implicated in mediating the cytokine activity of high-mobility group box 1 (HMGB1), which is a late inflammatory mediator of sepsis or lipopolysaccharide (LPS) lethality (4). Recent investigations using anti-HMGB1 antibody have shown that HMGB1 plays a critical role in the pathogenesis of lung injury by inducing neutrophil accumulation, lung edema, and cytokine release (6, 7). RAGE–ligand interaction results in rapid and sustained cellular activation and gene transcription, in most cases culminating in the activation of nuclear factor (NF)-{kappa}B (8–11). This cellular activation is related to inflammatory processes or tissue injury, such as diabetic microvascular injury, amyloidosis, and the immune-inflammatory process (4, 5, 12, 13).

Recently, several carboxyl-terminal truncated isoforms of RAGE, such as soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE), were identified in the lung of both humans and mice (14–17). Because these isoforms lack a transmembrane domain, they are secreted and act as decoy receptors (15). Indeed, sRAGE has been shown to prevent or reverse RAGE signal in experimental models of diabetic atherosclerosis, wound healing, amyloidosis, and colitis (10, 18–20). However, its putative protective roles on lung injury have not been previously characterized.

In this series of experiments, we aimed to evaluate the role of RAGE in the pathogenesis of LPS-induced lung injury in mice. First, the expression of RAGE isoform was measured in the bronchoalveolar lavage (BAL) fluid after intratracheal LPS challenge. Second, we tested the effect of blockade of ligand–RAGE interaction using recombinant sRAGE. We measured neutrophil accumulation into the alveolar space, pulmonary permeability, production of inflammatory mediators, activation of NF-{kappa}B, and apoptosis of the lung cells after LPS challenge, comparing the mice treated with intraperitoneal sRAGE with those treated with vehicle.


   METHODS
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An expanded description of methods can be found in the online supplement. Our experimental protocol was approved by the Council on Animal Care of Keio University and was in compliance with the guidelines of the National Institutes of Health.

Mice
Male C57BL/6J mice, 8–11 weeks of age and weighing 18–22 g, were purchased from CLEA Japan (Tokyo, Japan). Under anesthesia, the mice received 3.0 mg/kg Escherichia coli LPS as a solution of 1.2 mg LPS/ml phosphate-buffered saline (PBS) or an equal volume of PBS instilled into the left lung as previously described (21).

sRAGE Expression in the Alveolar Space
To determine the expression of sRAGE in the alveolar space, BAL fluid was collected at 6 or 24 hours after intratracheal instillation of LPS or PBS. Western blot analysis was performed using anti-mouse RAGE extracellular domain monoclonal antibody (R&D Systems, Minneapolis, MN). The images were converted into numerical data by quantitative scanning laser densitometry.

Effect of sRAGE on LPS-induced Lung Injury
To examine the effect of sRAGE on LPS-induced lung injury, mice were divided into three groups. The PBS group received intratracheal instillation of PBS (50 µl/mouse) followed by intraperitoneal injection of 100 µl PBS. The LPS group was injected intraperitoneally with PBS (100 µl/mouse) 1 hour after the intratracheal instillation of LPS. The sRAGE group was administered recombinant mouse sRAGE (100 µg/mouse diluted in 100 µl PBS) intraperitoneally 1 hour after the LPS instillation. The animals were killed at 4 or 24 hours after the instillation of LPS or PBS. The mice killed at 4 hours were subjected to lung sampling for NF-{kappa}B analysis. All other measurements were performed using the samples collected at 24 hours.

In the first series of the 24-hour experiments, the mice were subjected to BAL and blood sampling from the vena cava. The mice received an intravenous injection of human serum albumin (HSA) 1 hour before being killed, and HSA levels in plasma and BAL fluid were determined by ELISA. The permeability index (PI) was calculated as the BAL fluid-to-plasma ratio of the HSA concentration (22). The cytokine concentrations in plasma and BAL fluid were measured using a suspension array system. The HMGB1 level in BAL fluid was measured with ELISA. The sediments of BAL fluid were used for differential cell counts. White blood cell (WBC) counts in the blood samples were also examined.

In the second series, the lungs were sampled for pathology. The lung specimens were stained with hematoxylin–eosin (H&E) or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). In the H&E-stained slides, neutrophil emigration was quantitated by counting the number of neutrophils in 200 randomly selected alveoli and was expressed as the number of neutrophils per 100 alveoli. We also photographed in 30 randomly selected fields and quantified interstitial area/total lung area ratio (I/T ratio) by morphometric analysis. After TUNEL staining, the slides were visualized with a laser confocal microscopy to evaluate apoptotic cells.

Nuclear extracts were prepared from the lung tissue sampled at 4 hours after instillation. The DNA binding activity of NF- {kappa}B p65 in 1 µg nuclear extract was determined using ELISA.

Statistical Analysis
All data are expressed as the mean ± SE. The data were analyzed by one-way analysis of variance with the least squares differences test using SPSS Windows 14.0 statistical analysis software (SPSS, Inc., Chicago, IL). A value of P < 0.05 was considered to be statistically significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
sRAGE Expression in the Alveolar Space
To examine the potential impact of sRAGE on LPS-induced lung injury, we first explored the sRAGE expression in BAL fluid after LPS challenge. The specific antibody against RAGE extracellular domain recognized an isoform of RAGE that was approximately 48 kD in BAL fluid (Figure 1). On the basis of its molecular size, the isoform we detected was considered to be sRAGE (23). Although no significant expression was detected 24 hours after PBS instillation and 6 hours after LPS, significant up-regulation of sRAGE was observed 24 hours after intratracheal LPS instillation (P < 0.05).


Figure 1
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  		Figure 1. Expression of RAGE (receptor for advanced glycation end products) isoforms in bronchoalveolar lavage (BAL) fluid. In BAL fluid collected 6 or 24 hours after intratracheal instillation of LPS or 24 hours after phosphate-buffered saline (PBS), a single band of about 48 kD was observed. The band was stronger in the BAL fluid sampled 24 hours after LPS instillation than in other groups. The intensity data are expressed as the mean + SE (n = 4 in each group). *P < 0.05 was considered to be significantly different from the corresponding value of the PBS control.

 
sRAGE Ameliorates LPS-induced Lung Injury
To evaluate the effects of sRAGE on LPS-induced lung injury, we compared the severity of lung injury 24 hours after intratracheal instillation between the mice treated with sRAGE and those with PBS. In a preliminary experiment, prominent lung injury was observed 24 hours after an intratracheal challenge of 3.0 mg/kg LPS (data not shown).

Cell counts in BAL fluid were determined to assess the effects of sRAGE on neutrophil accumulation into the alveolar space. As shown in Figure 2, the mice instilled intratracheally with LPS revealed significantly increased numbers of both total cells and neutrophils compared with those administered PBS (P < 0.01). Mice treated with sRAGE 1 hour after LPS administration exhibited significantly lower numbers of both total cells and neutrophils in BAL fluid than those given PBS after LPS challenge (P < 0.05).


Figure 2
Figure 2
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  		Figure 2. Total and differential cell counts in bronchoalveolar lavage (BAL) fluid collected 24 hours after intratracheal instillation. The LPS + soluble RAGE (sRAGE) group received an intraperitoneal injection of sRAGE 1 hour after the intratracheal instillation of LPS. The LPS challenge caused significant increases in both total cell (A) and neutrophil (B) counts in BAL fluid, which was significantly attenuated by the treatment with sRAGE. All values are expressed as the mean + SE (phosphate-buffered saline [PBS], n = 8; LPS, n = 7; LPS + sRAGE, n = 8). *P < 0.05 and **P < 0.01 were considered to be significantly different from the corresponding value of the PBS group. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the LPS group.

 
Circulating WBC counts were significantly greater in the LPS group (4,830 ± 340 /mm3) than in the PBS group (2,240 ± 130 /mm3) at the time of killing (P < 0.05). There was no significant difference between the LPS and LPS + sRAGE (4,360 ± 550 /mm3) groups.

As a parameter of lung permeability, PI was calculated from the concentrations of HSA in BAL fluid and plasma (Figure 3). In the LPS group, the PI was significantly greater than in the PBS group (P < 0.01). The LPS + sRAGE group showed a significant decrease in the PI, compared with the LPS group (P < 0.05), which suggests that sRAGE might attenuate the LPS-induced increase in lung permeability.


Figure 3
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  		Figure 3. Permeability index (PI) calculated as the bronchoalveolar lavage (BAL) fluid-to-plasma ratio of human serum albumin (HSA) concentration. The LPS challenge caused a significant increase in the lung permeability, which was significantly mitigated by the treatment with soluble RAGE (sRAGE). All values are expressed as the mean + SE (phosphate-buffered saline [PBS], n = 8; LPS, n = 7; LPS + sRAGE, n = 8). *P < 0.05 and **P < 0.01 were considered to be significantly different from the corresponding value of the PBS group. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the LPS group.

 
Representative findings of the H&E-stained lung specimens are shown in Figure 4A. In the PBS group, no obvious neutrophil recruitment was observed at 24 hours after intratracheal instillation. The LPS group demonstrated numerous neutrophils in the alveolar space with thickening of the alveolar septa and areas of hemorrhage, which were markedly attenuated by the treatment with sRAGE. The result of morphometric quantitation of intraalveolar neutrophils is shown in Figure 4B. The LPS group had significantly increased neutrophil emigration compared with the PBS group (P < 0.01). The LPS + sRAGE group exhibited significantly decreased neutrophil emigration as compared with those given LPS followed by intraperitoneal injection of vehicle (P < 0.05). Using a microanalyzer, we determined the I/T ratio of each field for quantitative evaluation of edema formation in lung interstitium. An average I/T ratio was calculated for each animal and shown in Figure 4C. The I/T ratio in the LPS group was significantly greater than in the PBS group (P < 0.01). The I/T ratio in the LPS + sRAGE group was significantly decreased compared with the LPS group (P < 0.05).


Figure 4
Figure 4
Figure 4
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  		Figure 4. (A) Representative examples of lung pathology 24 hours after intratracheal instillation. No significant neutrophil emigration was observed in the phosphate-buffered saline (PBS) group. In the LPS group, marked neutrophil emigration, septal congestion, and exudate formation were observed and attenuated by the treatment with soluble RAGE (sRAGE). Original magnification, x100. (B) Neutrophil emigration was quantitated by counting the number of neutrophils in 200 randomly selected alveoli and was expressed as the number of neutrophils per 100 alveoli. Treatment with sRAGE significantly decreased the number of emigrated neutrophils after LPS instillation. (C) Interstitial area/total lung area (I/T) ratio 24 hours after intratracheal LPS instillation. The LPS group revealed increased I/T ratio (P < 0.01) compared with the control group. In the LPS + sRAGE group, I/T ratio was lower than LPS group (P < 0.05). All values are expressed as the mean + SE (n = 6 in each group). *P < 0.05 and **P < 0.01 were considered to be significantly different from the corresponding value of the PBS group. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the LPS group.

 
sRAGE Suppresses LPS-induced NF-{kappa}B Activation in the Lung
Because lung NF-{kappa}B is an important transcription factor modulating many proinflammatory cytokines and adhesion molecules involved in LPS-induced lung injury, we evaluated NF-{kappa}B activation in nuclear extracts from lung tissue sampled at 4 hours after the instillation of PBS or LPS (Figure 5). NF-{kappa}B p65 DNA binding activity was markedly increased in the LPS group compared with the PBS group (P < 0.01). Treatment with sRAGE 1 hour after LPS prevented the increase in NF-{kappa}B p65 DNA binding in LPS-challenged lungs (P < 0.05). Application of 500 ng of competitor oligo to nuclear extracts from the LPS group counteracted the activation caused by LPS instillation, which indicates the specific binding of NF-{kappa}B to NF-{kappa}B motif.


Figure 5
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  		Figure 5. Nuclear factor (NF)-{kappa}B p65 activity in nuclear extracts sampled 4 hours after intratracheal instillation of phosphate-buffered saline (PBS) or LPS. The DNA-binding activity is expressed in relative light units (RLU). Treatment with soluble RAGE (sRAGE) suppressed the increase of NF-{kappa}B p65 DNA binding in the LPS-challenged lungs. Application of 500 ng of a competitor oligo to nuclear extracts from LPS group counteracts the activation caused by LPS, which demonstrates the specific binding of NF-{kappa}B to NF-{kappa}B motif. All values are expressed as the mean + SE (n = 4 in each group). *P < 0.05 and **P < 0.01 were considered to be significantly different from the corresponding value of the PBS group. {dagger}P < 0.05 and {ddagger}P < 0.01 were considered to be significantly different from the corresponding value of the LPS group.

 
Cytokine and HMGB1 Levels in the Airspace after sRAGE Treatment
To evaluate the effects of sRAGE on LPS-induced cytokine production, we measured the levels of eight cytokines (IL-1β, IL-6, IL-10, TNF-{alpha}, macrophage inflammatory protein [MIP]-1{alpha}, MIP-1β, monocyte chemotactic protein [MCP]-1, and keratinocyte-derived chemokine [KC]) in BAL fluid 24 hours after the intratracheal instillation (Table 1). LPS instillation induced an increase in all eight cytokines as compared with the PBS-treated animals (P < 0.01). sRAGE treatment significantly attenuated the up-regulation of TNF-{alpha}, MIP-1{alpha}, and MIP-1β in LPS-challenged mice (P < 0.05), but no changes in the levels of other cytokines were observed between the LPS and LPS + sRAGE groups.


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  		TABLE 1. CYTOKINE PROFILES IN BRONCHOALVEOLAR LAVAGE FLUID

 
We also measured HMGB1 levels in BAL fluid with ELISA. HMGB1 levels in the control, LPS, and LPS + sRAGE groups were 23.2 ± 2.6, 50.6 ± 6.0, and 41.6 ± 3.8 ng/ml, respectively. Intratracheal LPS challenge significantly up-regulated HMGB1 in BAL fluid (P < 0.01), although sRAGE treatment made no difference in HMGB1 levels in BAL fluid.

Plasma Cytokine Levels after sRAGE Treatment
To evaluate the effects of sRAGE on LPS-induced cytokine production, we measured the levels of eight cytokines (IL-1β, IL-6, IL-10, TNF-{alpha}, MIP-1{alpha}, MIP-1β, MCP-1, and KC) in plasma 24 hours after the intratracheal instillation (Table 2). LPS instillation induced an increase in all eight cytokines as compared with the PBS-treated animals (P < 0.01). There was no significant difference in the cytokine levels between the LPS and the LPS + sRAGE groups. sRAGE treatment significantly attenuated the up-regulation of TNF-{alpha}, MIP-1{alpha}, and MIP-1β in LPS-challenged mice (P < 0.05), but no changes in the levels of other cytokines were observed between the LPS and LPS + sRAGE groups.


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  		TABLE 2. CYTOKINE PROFILES IN PLASMA

 
sRAGE Suppressed LPS-induced Apoptosis of Lung Cells
Apoptosis of the lung cells was evaluated by TUNEL assay using the lung samples obtained at 24 hours after intratracheal administration. A representative finding of the laser confocal microscopy and the numerical data are shown in Figures 6A and 6B, respectively. The PBS group showed few TUNEL-positive cells localized in the alveolar septa. Instillation of LPS resulted in significantly increased TUNEL-positive signals in the lung (P < 0.01), which were significantly diminished by sRAGE treatment (P < 0.01).


Figure 6
Figure 6
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  		Figure 6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed to detect apoptotic cells in the lung specimens sampled 24 hours after instillation. (A) Representative examples showing TUNEL-positive cells as bright green spots in the microphotographs (original magnification, x200). Few TUNEL-positive cells were observed in the phosphate-buffered saline (PBS) group. In the LPS group, a number of TUNEL-positive cells were observed, which was decreased by the treatment with soluble RAGE (sRAGE). The inset shows that the TUNEL-positive cells were epithelial cells. (B) Quantitative analysis of TUNEL-positive cells. Cells were counted in 10 random fields (original magnification, x100) and expressed as the number of apoptotic cells per millimeter squared. All values are expressed as the mean + SE (n = 6 in each group). *P < 0.05 and **P < 0.01 were considered to be significantly different from the corresponding value of the PBS group. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the LPS group.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study have revealed that sRAGE, which is up-regulated in the alveolar space in response to intratracheal LPS challenge, mitigates LPS-induced inflammatory events in the lung, including neutrophil infiltration, increased lung permeability, edema formation, production of inflammatory cytokines, and NF-{kappa}B activation, as well as apoptosis of the lung cells. These findings indicate that RAGE plays a critical role in the pathogenesis of LPS-induced lung injury and the blockade of RAGE signaling by sRAGE could be protective against the development of lung injury.

We observed significant up-regulation of a 48-kD isoform of RAGE in BAL fluid 24 hours after LPS instillation. It was previously reported that sRAGE was released into the BAL fluid and serum 6 hours after the intratracheal instillation of LPS as a single soluble isoform with a size of approximately 48 kD in a rat lung injury model (23). The authors also reported that the elevated levels of RAGE in the BAL fluid were correlated with the severity of the lung injury. We considered the possibility that the 48-kD isoform that was up-regulated was sRAGE.

In this study, sRAGE was administered only once at 1 hour after LPS challenge. It has been reported that recombinant sRAGE has an elimination half-life of 49.0 hours after intraperitoneal injection into normal rats (24). Considering the long half-life of RAGE, we believe that the effect of sRAGE administration could be sustained enough until the end of the observation. Because RAGE is a multiligand receptor, it remains unclear whether the effect of sRAGE observed was mediated by blockade of HMGB1, which is known to be up-regulated in serum from 8 to 32 hours after LPS exposure (6, 25). In response to LPS, HMGB1 is up-regulated in macrophages and causes an inflammatory response manifested by increased production of proinflammatory cytokines and neutrophil accumulation via activation of RAGE and other signaling pathways (6, 26). In this study, we observed up-regulation of HMGB1 in BAL fluid, which was not changed by sRAGE treatment. We speculated that, even after the binding with sRAGE, HMGB1 might not be degradated and still detected by its antibody. If the effect of sRAGE on LPS-induced lung injury is due to its interaction with HMGB1, the long half-life of sRAGE could be a therapeutic advantage.

During ALI/ARDS, various proinflammatory cytokines and chemokines are up-regulated and play a central role in the initiation and propagation of the inflammatory response (2). The activation of NF-{kappa}B is known to be a critical step for the expression of inflammatory mediators, including inflammatory cytokines, chemokines, and adhesion molecules (27). In the present study, sRAGE inhibited NF-{kappa}B activity as well as the expression of TNF-{alpha}, a major proinflammatory cytokine, and two CC chemokines, MIP-1{alpha} and MIP-1β, in the alveolar space after LPS challenge. Because these two CC chemokines induce activation and recruitment of neutrophils, we speculated that the suppression of these chemokines might contribute to the attenuation of neutrophil accumulation during lung injury (28, 29). KC is a recognized NF-{kappa}B–dependent CXC chemokine in rodents, which plays a major role in attracting neutrophils (30). It is noteworthy that, despite the suppression of NF-{kappa}B activation by sRAGE, no consequent decrease in the levels of KC and IL-6 was observed in our study. We speculate that, in addition to NF-{kappa}B, RAGE signaling might also activate a pathway hindering the production of IL-6 and KC, which thus neutralizes NF-{kappa}B–induced up-regulation of the mediators after the ligation of RAGE and its ligands. The reason for this discrepancy remains to be determined in future investigations.

In contrast to the cytokine profiles in BAL fluid, we found no difference in the plasma cytokine levels between the mice treated with sRAGE and those treated with vehicle. Although the detailed distribution of sRAGE in the body was not evaluated in the present study, it was previously reported that sRAGE is distributed in the various organs including the lungs (24). We speculated that, because HMGB1 was up-regulated in the alveolar space, the interaction between HMGB1 and sRAGE might be localized in the lung. It remains to be investigated whether sRAGE treatment could attenuate systemic inflammatory response and organ damage.

Other RAGE-related pathways for leukocyte recruitment were recently reported. Engagement of RAGE induces NF-{kappa}B–dependent up-regulation of the endothelial adhesion molecules VCAM-1 and ICAM-1 (5). RAGE also acts directly as a counter receptor for leukocytes by binding the β2-integrins Mac-1 and p150,95 (31), and thus promotes and perpetuates inflammatory cell recruitment. These findings indicate that a major action of sRAGE is to act as a powerful down-regulator of leukocyte trafficking in acute inflammation.

Another intriguing finding of the present study is the role of sRAGE in abrogating pulmonary apoptosis induced by LPS. TUNEL assay demonstrated a significant apoptotic change, especially in alveolar epithelial cells after LPS challenge, which was remarkably diminished by blockade of RAGE signal by sRAGE. Excessive apoptosis resulting in alveolar epithelial injury is believed to be a causative factor for LPS-induced lung injury (32). It was recently reported that RAGE mediates apoptosis of osteoblasts via the mitogen-activated protein (MAP) kinase and cytosolic apoptosis pathways (33). We speculated that, in addition to its effect on neutrophil recruitment, RAGE signaling may contribute to the pathogenesis of LPS-induced lung injury by inducing apoptosis of lung cells. TUNEL assay, which was used in this study, identifies DNA breaks, but it is not completely specific for apoptosis (34). Although positive TUNEL is consistent with the occurrence of apoptosis, different overlapping methods, such as caspase assay, are needed to prove that apoptosis is involved in the mechanisms of the effectiveness of sRAGE. Further investigations should be conducted on the molecular mechanisms underlying the antiapoptotic effects of sRAGE.

The roles of endogenous sRAGE, which appeared in the alveolar space after LPS challenge, remain unclear. Uchida and colleagues reported that sRAGE is increased in plasma and pulmonary edema fluid of patients with ALI/ARDS and could be a marker of lung injury, in particular of alveolar type I cell damage (23). The results of the present study suggest that sRAGE may contribute to the resolution of lung injury as a decoy receptor. We speculate that sRAGE may be released from alveolar type I cells during ALI/ARDS and participates in the negative feedback after excessive inflammatory processes.

As described above, the major limitation of this study is that the ligand through which sRAGE showed its efficacy remains unclear, because RAGE is a multiligand receptor. sRAGE, due to its capacity to block RAGE signals, has been reported to reduce inflammatory responses in diverse models, such as delayed-type hypersensitivity, arthritis, colitis, and periodontitis (4, 5, 35). Because HMGB1 has both proinflammatory and apoptotic effects, it is likely to be a main target of sRAGE (6, 26, 36). In this study, however, we showed that sRAGE attenuates NF-{kappa}B activation as early as 4 hours after LPS challenge. Because HMGB1 is a late-phase mediator, we speculated that the effect of sRAGE might be mediated by its binding not only with HMGB1 but also with other known or unknown ligands. Wittkowski and coworkers described a role for neutrophil-derived S100A12 in subjects challenged with endotoxin (37). The interaction between RAGE and other ligands, especially in the early events in the inflammatory cascade, will be the subject of future investigation.

In clinical settings, patients with ALI/ARDS often have complications with infection. Neutrophils that accumulate into the lung are not only involved in tissue injury but also contribute to host defense. The benefit of blocking neutrophil-dominant inflammation for patients with ALI/ARDS is controversial, because it may impair the host defense. In this study, we showed that the treatment with sRAGE attenuates neutrophil accumulation, although its effect on host defense remains to be evaluated. However, because almost all patients with ALI/ARDS are treated with antibiotics, we think that the effect of sRAGE observed in this study could eventually become relevant for patients with ALI/ARDS. Because predisposing conditions of ALI/ARDS vary, the efficacy of sRAGE should be examined in further investigations using other models, such as the cecal ligation and puncture.

In summary, the results of the present study indicate that RAGE is involved in the pathogenesis of LPS-induced lung injury. sRAGE administered 1 hour after LPS challenge could sufficiently ameliorate lung injury. These findings suggest that sRAGE may be secreted as a decoy receptor and contribute to the suppression of excessive inflammatory response during ALI/ARDS. Although further investigation is necessary, sRAGE could be considered as a promising candidate of therapeutic modality for ALI/ARDS.


   Acknowledgments
 
The authors thank Ms. Miyuki Yamamoto of Keio University School of Medicine for her assistance. They also thank Dr. Nobuo Ida of Toray Corporation for helpful discussion.


   FOOTNOTES
 
Supported in part by a grant-in-aid for scientific research from the Ministry of Health, Labor, and Welfare of Japan (no. 18120101) (I.M.). H.Z. is a recipient of a fellowship from the Japan-China Sasakawa Medical Association.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200707-1069OC on June 5, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 20, 2007; accepted in final form May 30, 2008


   REFERENCES
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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