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Published ahead of print on August 27, 2004, doi:10.1164/rccm.200402-146OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1239-1246, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200402-146OC

Original Article

Importance of Tumor Necrosis Factor-{alpha} Cleavage Process in Post-Transplantation Lung Injury in Rats

Taichiro Goto, Akitoshi Ishizaka, Fujio Kobayashi, Mitsutomo Kohno, Makoto Sawafuji, Sadatomo Tasaka, Eiji Ikeda, Yasunori Okada, Ikuro Maruyama and Koichi Kobayashi

Departments of Surgery, Medicine, and Pathology, School of Medicine, Keio University, Tokyo; Research & Development Division, Mitsubishi Pharma Corporation, Yokohama; and Department of Laboratory and Molecular Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan

Correspondence and requests for reprints should be addressed to Akitoshi Ishizaka, M.D., Department of Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan. E-mail: ishizaka{at}cpnet.med.keio.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) has two forms with apparently different biological activities: a membrane-associated form and a soluble form. TNF-{alpha}–converting enzyme (TACE) mediates a cleavage of membrane-associated TNF-{alpha} to induce its bioactive soluble form. We hypothesized that inhibition of TACE might prevent TNF-{alpha}–induced tissue injury while preserving the benefits of TNF-{alpha}. In this study, we evaluated the role of TACE in acute inflammation using an inhibitor of the enzyme in a rat model of lung transplantation. Inbred Lewis rats underwent left lung isotransplantation, and the donor lungs were kept in Euro-Collins solution with or without the inhibitor. After 6 hours of ischemia, the left lung was transplanted into the recipient rat and reperfused for 4 hours. Inhibition of TACE significantly attenuated endothelial and alveolar septal damage, as assessed by radiolabeled albumin leakage after transplantation. The inhibition also attenuated neutrophil accumulation in the alveolar space and other histopathologic findings, including intercellular adhesion molecule-1 expression. In addition, significantly lower levels of monocyte chemotactic protein-1, cytokine-induced neutrophil chemoattractant-1, high mobility group box-1, and soluble epithelial cadherin and decreased neutrophil elastase activity were observed in bronchoalveolar lavage fluid from the rats treated with the inhibitor. We conclude that TACE mediates a critical step in the development of post-transplantation lung injury.

Key Words: acute inflammation • epithelial cadherin • high mobility group box-1 • lung transplantation • TNF-{alpha}–converting enzyme

Although much progress has been made in transplantation immunology in recent years, reimplantation injury is still a serious problem. Indeed, severe acute injury has been reported to occur in 15 to 30% of patients after lung transplantation (1–4). It has also been reported that acute lung injury in the early stage after lung transplantation aggravates rejection via expression of various cytokines and adhesion molecules (5, 6). Despite the well-known proinflammatory effects of tumor necrosis factor-{alpha} (TNF-{alpha}), its role in the pathogenesis of lung injury after lung transplantation remains unclear. Much experimental work has addressed this issue with a view to develop procurement techniques, allowing such effective organ preservation as that achieved in liver and kidney transplantation. TNF-{alpha} plays a critical role in certain physiologic defensive response, but when produced in excess, it causes severe cellular and tissue damage in the host (7). TNF-{alpha} has two forms with apparently different biological activities: a membrane-associated form and a soluble form generated from the membrane-bound protein by proteolytic cleavage mediated by TNF-{alpha}–converting enzyme (TACE). TACE is present on the surface of macrophages, the major TNF-{alpha}–producing cells (8). TNF-{alpha} produced in response to various stimuli, such as bacterial challenge and tumor burden, is transported by the trans-Golgi network, expressed on the membrane surface, and transiently present as membranous TNF-{alpha} (memTNF-{alpha}). Within 1 hour, memTNF-{alpha} is processed by TACE and converted to soluble TNF-{alpha}, which is incorporated into tissues and plasma in the form generally called TNF-{alpha} (9–11). In the pathologic state of systemic inflammatory response syndrome, autotissue injury may be caused by excess expression of soluble TNF-{alpha} and other proinflammatory cytokines (12). When TACE is inhibited, a number of memTNF-{alpha} molecules remain on the macrophage surface because of blockade of memTNF-{alpha} processing. It has been suggested in recent years that these remaining memTNF-{alpha} molecules serve as a protection function such as defense against infection, tumor cell cytotoxicity, productive T cell–B cell interactions, and thymocyte proliferation (13–18). Thus, inhibition of TACE preserves beneficial memTNF-{alpha} and blocks the production of soluble TNF-{alpha}.

In this study, we performed orthotopic left lung transplantation in an inbred rat strain and investigated the involvement of soluble TNF-{alpha} in post-transplantation acute lung inflammation. We focused on chemokines and high mobility group box-1 (HMGB-1) as downstream mediators of TNF-{alpha} and examined their involvement in lung injury. To investigate the involvement of alveolar epithelial disorder in post-transplantation acute lung inflammation, we attempted detection of epithelial cadherin (E-cadherin)–soluble form in bronchoalveolar lavage fluid (BALF) of the injured lung.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A more detailed description of the methods is available in the online supplement.

Animal Model
Specific pathogen-free inbred Lewis rats were used as both donor and recipient animals. All procedures described in this report were approved by the institutional review board for animal studies.

To perform orthotopic left lung transplantations in the rats, we have modified surgical techniques described by other investigators (19, 20). Donor animals were injected with heparin (1,000 U/kg) intravenously. A 14-gauge catheter was inserted into the main pulmonary artery through the right ventricle. Immediately after the inferior vena cava was divided and the left and right atrial appendages were amputated, the pulmonary artery was flushed with 100 ml/kg of one of the cold (4°C) preservation solutions described later here at a pressure of 18 cm H2O. After the trachea was ligated and cut at an end-inspiratory phase during the ventilation, the donor heart and lungs were removed en bloc. Among five donor lung territories (right upper, middle and lower lobe, caudal lobe, and left lung), the right lower and caudal lobes immediately after excision were designated as "preischemic lung" and used for the evaluation of lung injury. Both the left lung and the right upper and middle lobes were wrapped in a bed of gauze soaked with 50 ml of preservation solution and stored at 4°C for a cold ischemic period. Then an orthotopic left lung transplantation was performed using a cuff technique for vessel and bronchial anastomoses, and blood flow and ventilation to the transplanted lung were reestablished after 6 hours of ischemia. The right upper and middle lobes of the donor were recovered from the preservation solution at the initiation of reperfusion and were designated as "postischemic lung" and used for the evaluation of lung injury. After the chest closure and awakening from the anesthesia, recipient animals were housed freely in room air. The rats were killed after 4 hours of reperfusion, and both the right and left lungs were evaluated for lung injury. Additional details are provided in the online supplement.

Experimental Protocol
Twenty-five Lewis rats were divided into three experimental groups (Figure 1). (1) In the transplantation group (n = 10), Euro-Collins solution was used as lung preservation solution, and physiologic saline was continuously infused via the tail vein at 0.75 ml/hour during 4 hours of reperfusion. (2) In the treatment group (n = 10), 1 mg/ml of TACE inhibitor (Y-41654) was added to the Euro-Collins solution. This modified Euro-Collins solution was used as lung preservation solution, and Y-41654 dissolved in physiologic saline was continuously infused via the tail vein at 3 mg/kg/hour (0.75 ml/hour) during 4 hours of reperfusion. (3) In the sham group (n = 5), after the same anesthesia, intubation, and artificial ventilation were given as in the previously mentioned two groups, only thoracotomy, dissection of the left hilum, and chest closure were performed. After dissection of the left hilum, physiologic saline was continuously infused via the tail vein at 0.75 ml/hour for 4 hours.



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  		Figure 1. Schematized experimental protocol. First, the heart and lungs were excised en bloc from the donor, immersed in the preservation solution, and kept in a refrigerator for a cold ischemic period. The recipient operation was started at an appropriate time. The recipient's left lung was excised, and the preserved left lung was transplanted orthotopically. Subsequently, the recipient's chest was closed, and after recovery from anesthesia, the recipient was housed freely in room air. After 4 hours of reperfusion, the recipient was killed and evaluated for right and left lung injury. Thus, we established a model of ischemia–reperfusion lung injury resulting from 6 hours of ischemia and 4 hours of reperfusion. At 3 hours after the start of reperfusion, 37 kBq of 125I-bovine serum albumin (BSA) were injected into the tail vein as a permeability tracer, and 10 kBq of 51Cr-labeled erythrocytes were injected as a pulmonary blood tracer 10 minutes before killing. RBC = red blood cell.

 
At 3 hours after transplantation, the recipient animals received an intravenous injection of 37-kBq 125I-labeled bovine serum albumin into the tail vein as a permeability tracer. Next, 10 minutes before killing, a 10-kBq 51Cr-labeled red blood cell was injected into the tail vein as a pulmonary blood tracer. Animals were killed by an intraperitoneal injection of 50 mg pentobarbital after 4 hours of reperfusion.

Blood samples were collected by cardiocentesis from each recipient at two points during the process: at the time of surgery (just before reperfusion) and at the time of killing. Bronchoalveolar lavage was performed in four specimens: (1) preischemic lung, (2) postischemic lung, (3) contralateral lung (recipient's right lung obtained at killing), and (4) graft lung (recipient's transplanted left lung obtained at killing). The BALF was centrifuged at 400 x g and 4°C for 10 minutes, and the supernatant was stored at –80°C until needed. Cell counts were done using a modified hemacytometer method. For differential counting of BALF cells, cell monolayers were prepared from BALF by cytocentrifugation. Differential counts were performed on 200 cells from smears stained with a modified Wright's stain. Additional details are provided in the online supplement.

Lung Water and 125I-labeled Albumin Index
Pulmonary edema was assessed by using a wet-to-dry weight ratio (W/D ratio). The isotope-specific radioactivity of excised lungs, blood, and BALF samples was measured. Transvascular flux of 125I-albumin was assessed by using the concentration ratio of lung tissue to plasma and that of BALF supernatant to plasma per unit weight, which were used as parameters of pulmonary endothelial and alveolar septal damage, respectively. Blood contamination in lung tissue and BALF was corrected using 51Cr counts. Additional details are provided in the online supplement.

Cytokine Determination
The cytokine concentration of TNF-{alpha}, monocyte chemotactic protein-1 (MCP-1), and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in each sample was determined using commercially available ELISA kits. Additional details are provided in the online supplement.

Measurement of Neutrophil Elastase Activity
Neutrophil elastase (NE) activity in BALF was determined with the synthetic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline, which is highly specific for NE, by the method described by Yoshimura and colleagues (21).

Histopathologic Examination
A portion of each lung was fixed with 4% paraformaldehyde for histopathologic and immunohistochemical examinations. After incubation with anti-rat TNF-{alpha} antibody, sections 5 µm thick were treated sequentially with rabbit anti-goat IgG conjugated to peroxidase. In the same manner, dewaxed paraffin sections were stained by the immunoperoxidase method using anti-rat intercellular adhesion molecule-1 (ICAM-1) antibody and goat anti-mouse IgG conjugated to peroxidase. Finally, color was developed with diaminobenzidine, and the sections were counterstained with hematoxylin.

Hematoxylin and eosin–stained sections of the graft lung tissue (n = 4 in each experimental group) were examined under light microscopy for a histologic scoring of lung injury. A pulmonary pathologist, who was blinded to the animals' group assignments, scored the histologic level of lung injury according to the following scoring system: grade 0, no abnormal findings; grade 1, patchy alveolar edema with widened interstitium with occasional erythrocytes in air spaces; grade 2, patchy hemorrhage and diffuse alveolar edema with widened interstitium; and grade 3, diffuse alveolar hemorrhage, massive hemorrhage, and necrosis of parenchyma.

Next, a quantitative morphometric analysis was performed on the findings in 10 randomly selected fields per slide of graft lung tissue subjected to TNF-{alpha} immunostaining or ICAM-1 immunostaining. The staining intensity of TNF-{alpha}– and ICAM-1–immunostained images in the lung tissue was measured as previously published (22–25). The total staining intensity was calculated as the summation of optical density of the positive area and was shown using the arbitrary units. Fields containing large vessels or bronchi were excluded. The total staining intensity was normalized to alveoli per field to control for inflation of the lung. The results were averaged from four rats in each experimental group. An observer, blinded to the experimental group, examined complete digitized images of specimens with Canvas 9J and Photoshop, version 7.0. Additional details are provided in the online supplement.

Measurement of HMGB-1 Protein
HMGB-1 protein in BALF was quantified by ELISA with monoclonal antibodies, which do not cross-react with HMGB-2 by the method described by Yamada and colleagues (26).

Western Blot Analysis of Soluble E-cadherin Fragments
To detect the soluble fragments of E-cadherin in BALF, a Western blot analysis was performed using a rabbit polyclonal antiserum against the synthetic peptides for rat E-cadherin. Additional details are provided in the online supplement.

Statistical Analysis
All data were expressed as the mean ± SEM. One-way analysis of variance and a Tukey-Krammer multiple comparisons test were used to detect statistical significance between groups. Student's t test for paired data was used to detect significant changes in plasma TNF-{alpha} level within a group. A p value of less than 0.05 was used to determine significant differences between means.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} Level in Plasma and BALF
The plasma TNF-{alpha} level at the time of killing significantly increased as compared with at the time of surgery in the transplantation group, but not in the other two groups (Figure 2A). In BALF of the transplantation group, the TNF-{alpha} level increased in the bilateral lungs (graft and contralateral lungs) after the transplantation, particularly in the graft lung (Figure 2B). In the treatment group, however, the BALF TNF-{alpha} levels in the bilateral lungs after the surgery were not significantly different from those in the sham group and were significantly lower than those in the transplantation group.







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  		Figure 2. Tumor necrosis factor-{alpha} (TNF-{alpha}) level, lung water, and 125I-labeled albumin index. (A) In the transplantation group, plasma TNF-{alpha} significantly increased with reperfusion (p = 0.018) (closed triangle, sham-operated group; closed square, transplantation group; closed circle, treatment group). *p < 0.05. (B) In the transplantation group, bronchoalveolar lavage fluid (BALF) TNF-{alpha} increased in the bilateral lungs (graft lung in particular) after reperfusion. In contrast, in the treatment group, BALF TNF-{alpha} in the bilateral lungs after reperfusion was significantly lower than that in the transplantation group. (C) In the transplantation group, the wet-to-dry weight ratio (W/D ratio) was significantly higher in the bilateral lungs (graft lung in particular) after reperfusion. In the graft lung, the W/D ratio was significantly lower in the treatment group than in the transplantation group. (D and E) Both lung tissue to plasma and BALF supernatant to plasma ratios in the graft lung were significantly lower in the treatment group than in the transplantation group. (B–E) Gray bar, sham group; black bar, transplantation group; white bar, treatment group. *p < 0.05.

 
Lung Water and 125I-labeled Albumin Index
In the transplantation group, the W/D ratio significantly increased in the graft and contralateral lungs compared with those in the preischemic and postischemic lungs (Figure 2C). Furthermore, the W/D ratio was significantly higher in the graft lung than in the contralateral lung. In comparison of W/D ratios between the transplantation and treatment groups, the W/D ratio of the graft lung was significantly lower in the treatment group than in the transplantation group. However, no significant differences were observed in the W/D ratio of the preischemic, postischemic, or contralateral lungs between the transplantation and treatment groups.

Both lung tissue to plasma and BALF supernatant to plasma ratios in the graft lung were significantly lower in the treatment group than in the transplantation group (Figures 2D and 2E). In contrast, in the contralateral lung, there were no significant differences in the lung tissue to plasma or BALF supernatant to plasma ratio between the transplantation and treatment groups.

BALF Findings
In the transplantation group, no significant differences were observed in either the total or differential cell counts between the preischemic and postischemic lungs, and macrophages were predominant in the differential cell count in both lungs, which may have been the BALF feature of an almost normal lung (Table 1). The total cell count in BALF increased after reperfusion in the bilateral lungs, and the increase was marked in the graft lung. As for the differential counts, neutrophils increased after reperfusion in the bilateral lungs, particularly in the graft lung. In the treatment group, the total and differential cell counts were similar to those in the transplantation group in both preischemic and postischemic lungs, and increases with reperfusion in the total cell and neutrophil counts in the graft lung BALF were less than those in the transplantation group.


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  		TABLE 1. Bronchoalveolar lavage fluid cell percentage differential

 
In comparison of the BALF cell differential by the actual number of cells in the transplantation group graft lung and treatment group graft lung, the number of lymphocytes was similar, but the numbers of neutrophils and macrophages were significantly lower in the treatment group (Figure 3). As for differential cell count in BALF in the contralateral lung, the numbers of neutrophils and lymphocytes were similar in the transplantation and treatment groups, and only the number of macrophages was significantly lower in the treatment group.



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  		Figure 3. BALF cell count of the graft lung. In the treatment group, the number of lymphocytes was similar to that in the transplantation group, but the neutrophil and macrophage counts were significantly lower, resulting in significant decrease in the total cell count (gray bar, sham group; black bar, transplantation group; white bar, treatment group). *p < 0.05.

 
Histopathologic Findings
In the transplantation group, graft lung, alveolar hemorrhages, and interstitial thickening were marked, and growth of type II alveolar epithelial cells was observed (Figures 4B and 4D). In contrast, in the treatment group graft lung, only faint alveolar hemorrhages were present, and no obvious interstitial thickening was noted (Figures 4C and 4D).










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  		Figure 4. Histopathologic findings. (A–D) Hematoxylin and eosin staining of the graft lung tissue. (A) Sham group. (B) Transplantation group. (C) Treatment group. In the transplantation group, alveolar hemorrhages and interstitial thickening were marked, whereas only faint alveolar hemorrhages were observed in the treatment group. Scale bars, 150 µm (top row) or 25 µm (bottom row). (D) Histologic score for the graft lung injury. Data are shown as mean ± SEM (for grading, see the METHODS section). (E–H) TNF-{alpha} immunostaining of the graft lung tissue. (E) Sham group. (F) Transplantation group. (G) Treatment group. The top row presents immunostained lung tissues, and arrows indicate alveolar macrophages. The bottom row presents immunostained macrophages alone. In the sham group, lung tissue or macrophages were not immunostained. In the transplantation group, macrophage cytoplasm was mainly stained. In the treatment group, the entire macrophage, the alveolar epithelium, and the interstitium were prominently stained. Scale bars, 25 µm (top row) or 2 µm (bottom row). (H) Quantification of TNF-{alpha} immunostaining intensity in the graft lung tissue. In the graft lung, TNF-{alpha} staining was significantly increased in the treatment group. AU = arbitrary units. *p < 0.05. (D and H) Gray bar, sham group; black bar, transplantation group; white bar, treatment group.

 
In TNF-{alpha} immunostaining, no staining was observed in the lung tissue or macrophages in the sham group left lung, suggesting that TNF-{alpha} synthesis was not induced (Figures 4E and 4H). In the transplantation group graft lung, pale staining of the macrophage cytoplasm was mainly observed (Figures 4F and 4H). In contrast, macrophages were entirely stained deeply in the treatment group graft lung (Figure 4G). MemTNF-{alpha} on macrophages may have increased because of inhibition of TACE. We found also deeply stained TNF-{alpha} in the alveolar epithelium and interstitium, which may have been memTNF-{alpha} originating from alveolar epithelial cells, fibroblasts, and vascular endothelial cells (Figure 4H).

Chemokine, Adhesion Molecules, and NE Activity
MCP-1 and CINC-1 in graft lung BALF were significantly lower in the treatment group than in the transplantation group (Figures 5A and 5B). In the contralateral lung, no significant differences were observed in the BALF MCP-1 or CINC-1 level between the transplantation and treatment groups.









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  		Figure 5. Chemokine, adhesion molecule, and neutrophil elastase (NE) activity. (A and B) Both monocyte chemotactic protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in the graft lung BALF were significantly lower in the treatment group than in the transplantation group. (C–F) Intercellular adhesion molecule-1 (ICAM-1) immunostaining of the graft lung tissue. (C) Sham group. (D) Transplantation group. (E) Treatment group. In the transplantation group, vascular endothelium and alveolar epithelium were deeply stained, whereas these were only weakly stained in the treatment group. Scale bars = 25 µm. (F) Quantification of ICAM-1 immunostaining intensity in the graft lung tissue. In the graft lung, ICAM-1 staining was significantly increased in the transplantation group. AU = arbitrary units. (G) BALF NE activity increased in the transplantation group graft lung, but it was significantly decreased in the treatment group graft lung. N.D. = not detected; p-NA = p-nitroaniline. (A, B, F, and G) Gray bar, sham group; black bar, transplantation group; white bar, treatment group. *p < 0.05.

 
On ICAM-1 immunostaining, almost no staining was observed in the lung tissue in the sham group left lung (Figures 5C and 5F). The vascular endothelium and alveolar epithelium were deeply stained in the transplantation group graft lung but were only weakly stained in the treatment group graft lung (Figures 5D–5F).

The NE activity in BALF increased in the transplantation group graft lung, whereas it remained significantly lower in the treatment group graft lung (Figure 5G). In the contralateral lung, the NE activity in BALF of transplantation and treatment groups was similar.

HMGB-1 and E-cadherin Soluble Form
HMGB-1 in BALF was significantly higher in the transplantation group graft lung than in the sham group left lung (Figure 6A). In the treatment group, the HMGB-1 level in graft lung BALF was significantly reduced compared with that in the transplantation group.




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  		Figure 6. High mobility group box-1 (HMGB-1) protein and epithelial cadherin (E-cadherin)–soluble form. (A) The BALF HMGB-1 level was increased in the transplantation group graft lung, but it was significantly decreased in the treatment group graft lung (gray bar, sham group; black bar, transplantation group; white bar, treatment group). *p < 0.05. (B) Western blot analysis of E-cadherin–soluble form in the BALF from the bilateral lungs after the surgery. Molecular markers were applied in the extreme right lane (blue, 206 kD; magenta, 124 kD; green, 83 kD; violet, 42 kD; orange, 32 kD; red, 19 kD; and blue, 7 kD). A band consistent with the molecular weight of E-cadherin soluble form, approximately 85 kD, was detected in all samples. The BALF supernatant to plasma ratios were from the left, 0.0056, 0.0190, 0.0320, 0.1446, 0.0271, and 0.0683, and the band intensity changed in concordance with the BALF supernatant to plasma ratio. Contra = contralateral.

 
The soluble form of E-cadherin in BALF can be a parameter of lung epithelial damage. In this study, the BALF supernatants of the bilateral lungs after surgery in each experimental group were subjected to Western blotting described in the METHODS section (Figure 6B). A band corresponding to the molecular weight of E-cadherin soluble form, 85 kD, was detected in all samples. The variations in band intensity were concordant with the changes in BALF supernatant to plasma ratio, an isotopically derived index of alveolar septal damage, and the band intensity was macroscopically different between the transplantation group graft lung and treatment group graft lung.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this experiment, TACE inhibitor was used to inhibit cleavage of memTNF-{alpha} and release of soluble TNF-{alpha} in a rat lung transplantation model. Regional and systemic treatment with TACE inhibitor attenuated the severity of reimplantation injury of the graft lung with decreased lung local concentration of soluble TNF-{alpha}, suggesting the involvement of soluble TNF-{alpha} in post-transplantation acute lung inflammation. The decrease in soluble TNF-{alpha} inhibited chemokine production and ICAM-1 expression in the lung local region, reducing accumulation of inflammatory cells and tissue-injuring activity.

In this study, lung injury was evaluated 4 hours after reperfusion, whereas previous reports described lung injury evaluated within a relatively short time after reperfusion, such as 15 minutes to 2 hours (19, 27, 28). In an experiment tracing the time course of lung ischemia–reperfusion disorder in rats, the development of the disorder was biphasic (29). Neutrophil-independent mild lung injury occurred approximately 30 minutes after reperfusion, and neutrophil-dependent severe lung injury occurred approximately 4 hours after reperfusion (29). Based on investigations of myeloperoxidase activity in lung homogenates and histology (29, 30), accumulation of neutrophils in the lung is considered to require 2 or more hours after reperfusion. In this experiment, therefore, lung injury was evaluated 4 hours after reperfusion because our main objective was to investigate the role of TACE in neutrophil-dependent lung injury.

We used alveolar–septal permeability calculated from the leakage of radiolabeled albumin for the evaluation of lung injury. In previous lung transplantation experiments, acute lung injury was evaluated mostly based on oxygenation capability (e.g., arterial O2 tension), hemodynamics of the pulmonary circulation (pulmonary vascular resistance, pulmonary arterial flow), lung compliance, W/D ratio, or histologic examinations (19, 27, 28). We selected the isotopically derived indices because an accurate and quantitative evaluation of lung injury was anticipated (31, 32).

Inhibition of the release of soluble TNF-{alpha} decreased neutrophils and macrophages in the local graft lung. Thus, we investigated whether TACE inhibition affects recruitment of inflammatory cells, including chemokine induction and expression of adhesion molecules (33). In the treatment group, the levels of CINC-1, a C-X-C chemokine, and MCP-1, a C-C chemokine, in BALF recovered from the graft lung were significantly lower in cooperation with a decrease in the local concentration of soluble TNF-{alpha} compared with those in the transplantation group. In a preliminary in vitro study, we confirmed that the TACE inhibitor itself had no inhibitory action on CINC-1 or MCP-1 (data not shown). We inferred that the impaired release of TNF-{alpha} could be responsible for the decreased production of CINC-1 and MCP-1, which we observed. It was also revealed that the expression of ICAM-1, an adhesion molecule on endothelial cells mediating firm adhesion of neutrophils and endothelial cells, in the graft lung tended to be lower in the treatment group. Based on the findings mentioned previously here, we concluded that soluble TNF-{alpha} plays a critical role in the development of reimplantation lung injury by regulating accumulation of neutrophils and macrophages via induction of chemokines (CINC-1 and MCP-1) and upregulation of ICAM-1, which are consistent with previous reports (29, 30, 34, 35). Several investigators have shown that the administration of an antineutrophil antibody, a neutralizing monoclonal antibody against interleukin-8, or an anti–P-selectin antibody prevented neutrophil infiltration and tissue injury in the setting of lung transplantation (29, 30, 34). Also, the blocking antibody to MCP-1 was reported to be highly protective against lung reperfusion injury (35).

NE activity in the graft lung was decreased in the treatment group compared with the transplantation group. Because NE activity reflects the magnitude of neutrophil sequestration in the lung, decreased NE activity might be due to the decreased neutrophil recruitment by TACE inhibition, leading to attenuated lung injury.

In this study, we observed increased TNF-{alpha} levels in BALF after the transplantation, particularly in the graft lung, and in plasma 4 hours after transplantation, not during surgery, which suggests that the TNF-{alpha} production after reperfusion was mostly due to residential macrophages in the graft lung directly stimulated with ischemia–reperfusion and that plasma TNF-{alpha} may have been spilled over from the graft lung. It has been reported that during reperfusion oxidant stress activates macrophage nuclear factor-{kappa}B, leading to increased production of mRNA for TNF-{alpha} (36). Furthermore, soluble TNF-{alpha} in epithelial lining fluid, which is produced in the graft lung, may be more critical than TNF-{alpha} in plasma in the development of reimplantation injury. From this perspective, controlling the injurious potential of neutrophils and macrophages in the local lung may be effective as therapeutic strategy for reimplantation injury, and the addition of TACE inhibitor to the preservation solution may have significant clinical benefits. TACE inhibitor in the lung preservation solution could be distributed sufficiently in the lung tissue during 6 hours of preservation and may have formed the preparatory condition for inhibition of TNF-{alpha} release from residential macrophages after reperfusion (37). In addition, when the lung was stored in the preservation solution with TACE inhibitor, the total number and differential of BALF cells did not change after storage, which suggests exposure to the drug had no harmful effects.

In this experiment, TACE inhibitor did not significantly attenuate a slight increase in lung transvascular permeability in the contralateral lung, which could be induced by inflammatory mediators in systemic circulation (35). In the transplantation group, the TNF-{alpha} level in the BALF recovered from the contralateral lung was significantly higher than in other two groups. In contrast, no significant differences were observed in the BALF MCP-1 level, CINC-1 level, NE activity, or number of BALF neutrophils between the transplantation and treatment groups in the contralateral lung. Lung injury of the contralateral lung caused by indirect stimulation may not be mediated only by TNF-{alpha} but by several other mediators.

In addition to its role as a transcriptional regulatory factor, HMGB-1 protein has recently been identified as a late mediator of endotoxin lethality (38). Macrophages release HMGB-1 when exposed to early, acute cytokines, indicating that it is also positioned as a mediator of inflammatory conditions. Previous studies have demonstrated that TNF-{alpha} functions as an upstream regulator of HMGB-1 release (39). Our observations are consistent with the role of HMGB-1 as a distal inflammatory mediator and with its release induced primarily by soluble TNF-{alpha}. The role of HMGB-1 in the pathogenesis of acute lung injury seems distinct from any effects of earlier acting proinflammatory cytokines. It was reported that intratracheal administration of HMGB-1 causes acute lung injury, and antibodies against HMGB-1 attenuate lipopolysaccharide-induced pulmonary edema (40). In that study, anti–HMGB-1 antibody did not significantly reduce the concentrations of proinflammatory cytokines in lipopolysaccharide-induced lung injury, suggesting that HMGB-1 occupies a more distal position in the proinflammatory cascade (40). In our model as well, the delayed release of HMGB-1 may have participated in the downstream development of post-transplantation lung injury.

Soluble E-cadherin in BALF was measured as a direct index of alveolar epithelial injury. E-cadherin is a 120-kD transmembrane glycoprotein, predominantly localized to the lateral cell border and associated with the contractile cytoskeleton (41). Most epithelial cells express E-cadherin, and soluble E-cadherin may reflect loosened intercellular adhesion among epithelial cells. In this study, Western blot analysis showed soluble E-cadherin released into BALF, which might suggest the involvement of alveolar epithelial disorder in reimplantation lung injury.

In summary, TACE inhibition markedly attenuated reimplantation injury. A modest reduction in lung graft failure and early mortality rates caused by reimplantation injury would exert a significant effect on overall long-term survivals. The lung specimens were preserved for 6 hours in this study, but it is possible that organ preservation solution containing TACE inhibitor reduces reperfusion injury even though it was kept in ischemic condition for a prolonged period, and clinically, the ability to preserve donor lungs effectively for longer periods of time would increase the pool of potential donors.

Some studies have used anti–TNF-{alpha} antibodies for TNF-{alpha} inhibition in lung ischemia–reperfusion injury (42, 43) and noted improvement in lung injury. On the other hand, studies have reported that the complete blocking of the physiologic functions of TNF-{alpha} tends to result in pulmonary infections such as tuberculosis and fungal infection (44, 45). The administration of anti–TNF-{alpha} antibody in the perioperative period of lung transplantation might be risky and impractical. In general, the half-life of antibodies in the blood is relatively long up to 14 days, and anti–TNF-{alpha} antibody binds to soluble and memTNF-{alpha} in a specific and high-affinity manner, blocking all biological activities of TNF-{alpha}. Therefore, we speculate that anti–TNF-{alpha} antibodies might have disadvantages, such as potential for making the host vulnerable to infections. In contrast, TACE inhibitor inhibits only soluble TNF-{alpha}, which is distributed through the systemic circulation, and does not completely block the biological activities of TNF-{alpha}. Because TACE inhibitor with high water solubility used in the experiment has a very short half-life of approximately 20 minutes, we presume that the drug might have some advantage compared with anti–TNF-{alpha} antibodies because its blood concentration could be easily controlled, making it safer and more convenient compared with long-acting anti–TNF-{alpha} antibody.

In the graft lung after transplantation, TNF-{alpha} synthesis begins in alveolar macrophages in response to reperfusion stimulation after ischemia, but memTNF-{alpha} expressed on the cell surface does not cause pulmonary disorder. The lung injury cascade via soluble TNF-{alpha} does not start until memTNF-{alpha} is cleaved by TACE. TACE may be very important as a trigger of TNF-{alpha}–induced lung injury.


   Acknowledgments
 
The authors thank Mitsubishi Pharma Corporation (Osaka, Japan) for supplying the TACE inhibitor and pertinent information regarding the drug.


   FOOTNOTES
 
Supported in part by a grant-in-aid for Fundamental Scientific Research from the Education Ministry of Japan 13,770,743 (T.G.).

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

Conflict of Interest Statement: T.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; F.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Y.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 3, 2004; accepted in final form August 18, 2004


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