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Shimizu, Mie*; Hasegawa, Naoki*; Nishimura, Tomoyasu*; Endo, Yoshihiko*; Shiraishi, Yoshiki*; Yamasawa, Wakako*; Koh, Hidefumi*; Tasaka, Sadatomo*; Shimada, Hisato*; Nakano, Yasushi*; Fujishima, Seitaro; Yamaguchi, Kazuhiro*; Ishizaka, Akitoshi*

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doi: 10.1097/SHK.0b013e3181a2adb7
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Endotoxemia associated with septicemia is a major cause of acute lung injury (ALI) (1, 2). In experimental studies, endotoxemia has been observed to cause acute bronchoconstriction, production of TNF-α, protein leakage from the vascular to the alveolar space, and recruitment of neutrophils and alveolar macrophages, and several studies have found that TNF-α plays a central role in its development (1-5). It has been presented that in TNF-deficient mice, bronchoconstriction induced by LPS was prevented without changes in the recruitment of neutrophils into the lung (5). Therefore, TNF blockade has been proposed as therapeutic target against LPS-induced inflammatory responses.

Antibodies against TNF-α and its receptors and pharmacological inhibitors of TNF-α have been proposed since 1992 as a potential treatment of ALI/adult respiratory distress syndrome (6, 7), although no controlled clinical trial has confirmed their efficacy (8, 9). However, new information has since been gathered that has clarified the mechanisms behind the production, metabolism, and function of TNF-α (10-13).

Translated TNF-α is originally present on the cell surface as a 26 kd membrane-bound precursor (mTNF-α), which is enzymatically cleaved by the TNF-α-converting enzyme (TACE) into 17-kd soluble TNF-α (sTNF-α), before its release into the extracellular space (10, 11). During treatment with a TACE inhibitor, mTNF-α was increased on the LPS-activated cell surface, without an increase in sTNF-α in the medium (14-16). This TACE inhibitor-induced change in sTNF-α production was not observed after the administration of antibodies against TNF-α or its receptor (17-20).

Taken together, by controlling the overproduction of sTNF-α and by preserving mTNF-α on the cells, the blockade of TACE could modify acute inflammation. Y-41654 is a new low-molecular-weight TACE inhibitor with a short half-life. It was recently found to attenuate transplantation-related ALI in the rat (21). The present study, in a rat model, was designed to study the effect of Y-41654 on acute lung injury induced by intratracheal instillation of LPS.



The soluble TACE inhibitor Y-41654 was obtained from Mitsubishi Pharma Corporation (Osaka, Japan). It is succinate-based hydroxamate, of which molecular weight and half-life of the compound are approximately 700 d and 10 min, respectively. The serotype B:55 LPS derived from Escherichia coli was purchased from Sigma Chemicals (St. Louis, Mo).

Experimental animals

The experiments were performed in 55 specific, pathogen-free, inbred male Sprague-Dawley rats weighing 300 to 350 g (CLEA Japan Inc., Tokyo, Japan). All experimental procedures described in this report were reviewed and approved by the institutional board for animal studies of Keio University School of Medicine.

Effect of Y-41654 treatment on LPS-induced TNF-α production by isolated alveolar macrophages

Under anesthesia with pentobarbital (30 mg/kg body weight, i.p.), the animals were exsanguinated by section of the abdominal aorta. The chest was opened, and the lungs and trachea were removed. Bronchoalveolar lavage (BAL) was performed with 5 mL of saline via a 14-gauge tracheal cannula three times per animal to collect pulmonary alveolar macrophages. The cells contained in the BAL fluid, examined by Wrights-Giemsa stain, included 90% to 95% of alveolar macrophages and less than 2% of neutrophils, and their viability, measured by the trypan blue exclusion method, was greater than 90%. The cells were incubated with RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. The cells (8 - 9 × 104/200 μL) were seeded on a 96-well plate, placed in a 5% CO2 incubator at 37°C, and were divided among three groups, including control, LPS, and LPS + Y-41654. In two groups, the cells were cultured with LPS in a final concentration of 10 μg/mL in the presence (LPS) or absence (LPS+Y-41654) of 20 μL of 0.1-mM Y-41654 and were added to the medium 15 min before LPS stimulation in reference to the saline control (control) group. The supernatants of the cell cultures were collected at 0, 1, 2, and 4 h. Soluble TNF-α in the culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA; R&D Systems Minneapolis, Minn).

Effect of Y-41654 on the acute inflammatory responses induced by intratracheal instillation of LPS

Protocol of animal model

The four groups studied in vivo, each including 10 rats, were 1) a control group (control), 2) an LPS group (LPS), 3) a Y-41654 control group (Y-41654), and 4) a treatment group treated with Y-41654 after LPS instillation (LPS + Y-41654).

The rats were anesthetized with an injection of 40 mg/kg of pentobarbital (i.p.). After the placement of a 23-gauge catheter in a coccygeal vein, 1 mL of 0.04% human serum albumin (HSA), purchased from Sigma Chemicals, was injected through the catheter as previously described (22, 23). The trachea was exposed by a midline cervical incision and intubated with a 14-gauge catheter. The amount of LPS was determined based on preliminary study to develop acute inflammation.

Saline, or 300 μg/kg of LPS dissolved in 0.13 mL of saline, was instilled via the intratracheal catheter, followed by a flush of 0.3 mL of air. Based on its molecular size and half-life and previous report (21), Y-41654 (10 mg/kg in 0.7 mL of phosphate-buffered saline) or the equivalent amount of saline only was injected 15 min before and 0.5, 1.5, 2.5, and 3.5 h after the administration of LPS or saline (i.v.). A single bolus of 50 U of heparin, in a dilution of 1,000 U in 3 mL of saline, was injected (i.v.) 30 s before the end of the experiment to prevent coagulation. At 4 h after LPS or saline instillation, the animals were euthanized with pentobarbital, and blood samples were collected from the inferior vena cava. The lungs were removed, and the right upper lung was fixed by intratracheal instillation of 4% glutaraldehyde for pathological examinations. The peripheral white blood cells and their differential were counted using a hemocytometer and blood smears stained with Leukostat (Fisher Scientific, Pittsburgh, Pa). The remainder of the blood was centrifuged at 3,000 × g and 4°C for 15 min, and the supernatant was stored at −80°C until use. Bronchoalveolar lavage of the right lower lobe was performed three times using 5 mL of saline for each lavage. The fluid recovery rate was approximately 80% and was similar among animals. The BAL fluid was centrifuged at 1,500 × g and 4°C for 10 min, and the supernatant was stored at −80°C until use.

Evaluation of cell counts and differential in BAL fluid

The cell pellet of BAL fluid was resuspended in 1 mL of saline, and a cell count was done by a modified hemacytometer method (Unopet Microcollection System; Becton Dickinson, Rutherford, NJ). For differential count counting of white blood cells in BAL fluid, cell monolayers were prepared from BAL fluid by cytocentrifugation. Differential counts were performed on 200 cells from smear stained with a modified Wright stain (Diff-Quick; Baxter Healthcare Corp., McGraw Park, Ill).

Measurement of nuclear factor-κB in lung tissue

The protein was extracted from lung tissue using a protein extraction kit (PRO-PREP; INTRON Biotechnology, Gyeonggi-do, Korea). Briefly, 15 mg of lung tissue was homogenized in 600 μL of PRO-PREP protein extraction solution in the kit and incubated for 30 min on ice to induce cell lysis, then centrifuged at 15,000 × g for 5 min. The supernatant was saved and stored at −20°C to measure protein contents and nuclear factor (NF)-κB. Protein contents were measured by bicinchoninic acid assay using a commercial kit (Pierce Biotechnology, Rockford, Ill) according to the manufacturer's instructions. Nuclear factor-κB in the supernatant was measured by ELISA (Oxford Biomedical Research, Oxford, Mich), an assay that measures the amount of active NF-κB by detecting DNA-bound activated NF-κB using specific antibody against p50 and p105. The amount of activated NF-κB was standardized by the protein content of the sample.

Measurement of HSA concentration in BAL fluid

The concentration of HSA in BAL fluid was measured with an immunoenzymetric assay using a commercial kit (Cygnus Technologies, Southport, NC) that does not cross-react with rat serum proteins, including albumin. Therefore, HSA concentration in BAL fluid that was injected into intravascular space before LPS or saline injection reflected permeability of the alveolar septum (22, 23).

TNF-α, IL-1β, and cytokine-induced neutrophil chemoattractant 1 in saved plasma and BAL fluid

The concentrations of TNF-α, IL-1β, and cytokine-induced neutrophil chemoattractant (CINC) 1 in saved plasma and BAL fluid were measured by ELISA using commercially available kits (TNF-α and IL-1β, R&D Systems; CINC-1, Amersham Bioscience, Little Chalfont, Buckinghamshire, UK).

Histological and immunohistochemical examinations for assessment of inflammatory cellular infiltration and sTNF-α production

The lungs were fixed with 4% paraformaldehyde for histopathological and immunohistochemical examinations, and the tissues were embedded in paraffin. Sections 5 μm in thickness were cut from the paraffin blocks and stained with hematoxylin-eosin. The percentages of neutrophils in the lung tissue were evaluated among 200 cells on the stained smear under 400× magnification by an observer blinded to the animals' group. For immunochemistry, the tissues were deparaffinized. After incubation with antirat TNF-α antibody, the sections were treated sequentially with antigoat immunoglobulin G, the color was developed with diaminobenzidine, and the sections were counterstained with Mayer hematoxylin.

Statistical analysis

All data are expressed as means and SE indicated by error bars. ANOVA was used to examine the differences among groups. Between-groups differences were examined by Fisher test. A P value less than 0.05 was considered statistically different.


Effect of Y-41654 treatment on LPS-induced TNF-α production by isolated alveolar macrophages

Compared with the control group, stimulation with LPS increased significantly (P < 0.05) the production of sTNF-α in the cellular supernatant at 2 and 4 h (Table 1). However, when Y-41654 was added to the pretreatment, no increase in the production of sTNF-α was observed at any time point, suggesting LPS-induced sTNF-α production was inhibited by inhibitor of TNF-α-converting enzyme.

Effects of pretreatment of isolated rat alveolar macrophages with Y-41564 on the production of sTNF-α

Alveolar septum permeability

In the LPS group, the concentration of HSA in BAL fluid at 4 h was significantly higher than in the control group. In contrast, in the Y-41654 group, whether with saline (Y-41654) or with LPS instillation (LPS + Y-41654), the concentration of HSA in BAL fluid at 4 h was unchanged compared with the control group and significantly lower than in the LPS group (Fig. 1), suggesting Y-41654 decreased LPS-induced alveolar septum permeability.

Fig. 1:
Human serum albumin concentrations in BAL fluid supernatant. *P < 0.05 vs. LPS group.

Cytokine concentrations in serum and BAL fluid

The mean concentration of sTNF-α in serum at 4 in the LPS group was higher than in the control group. In the LPS + Y-41654 group, the mean serum sTNF-α concentration at 4 h was significantly lower (P < 0.05) than in the LPS group (Fig. 2A). The sTNF-α concentration in BAL fluid at 4 h in the LPS group was significantly higher than in the control group (P < 0.001). In the LPS + Y-41654 group, the sTNF-α concentration in BAL fluid was significantly lower than in the LPS group (P < 0.005; Fig. 2B). The mean IL-1β concentration in BAL fluid was higher in the LPS and the LPS + Y-41654 groups than in the control group, whereas the IL-1β concentrations in the LPS and the LPS + Y-41654 groups were similar (Fig. 2C). Likewise, the mean concentration of CINC-1 in BAL fluid was higher in both the LPS and the LPS + Y-41654 groups than in the control group and similar in the LPS and the LPS + Y-41654 groups (Fig. 2D).

Fig. 2:
Concentrations of sTNF-α in serum (A); sTNF-α in BAL fluid (B); IL-1β in BAL fluid (C); CINC-1 in BAL fluid (D). *P < 0.05 vs. LPS group in identical measurements (shown in A-C). **P < 0.01 vs. LPS group (D).

Neutrophil recruitment in lung tissue

The percentage of neutrophils among the 200 cells counted in the lung sections harvested from the LPS group was significantly higher than in the control group (P < 0.001) and was significantly decreased by treatment with Y-41654 (Fig. 3).

Fig. 3:
Percentage of neutrophils per 200 cells counted in lung sections. *P < 0.001 vs. LPS group.

Total cell and neutrophil counts in BAL fluid

In the BAL fluid, the total cell counts in the LPS and LPS + Y-41654 groups were similar. However, the mean number of neutrophils in the LPS group was significantly higher than in the LPS + Y-41654 group (Fig. 4), indicating Y-41654 decreased migration of neutrophil into alveolar space.

Fig. 4:
Overall cell counts and cell types contained in BAL fluid. *P < 0.001 vs. LPS group.

Activated NF-κB in lung tissue

In the LPS group, the concentration of activated NF-κB, standardized as picograms per microgram of total protein in the nuclear protein of lung tissue, was higher than in the control group and was lower in the LPS + Y-41654 than in the LPS group (Fig. 5).

Fig. 5:
Activated NF-κB (picograms per microgram protein) in lung tissue. *P < 0.005 vs. LPS group.

Histopathologic observations

Representative histopathologic view was demonstrated in Figure 6, whereas quantitative evaluation was not performed. Alveolar hemorrhages and interstitial thickening were more prominent in the LPS group than in the control group. In contrast, neither interstitial thickening nor alveolar hemorrhages were present in the LPS + Y-41654 group.

Fig. 6:
Histopathology of lung tissue stained with hematoxylin and eosin. In the LPS group, diffuse septal edema is present, with predominance of neutrophils, in contrast to the minimal ALI present in the tissue of the LPS + Y-41654 group.

TNF-α immunohistochemical staining of lung tissue

The cytoplasm and nucleus of the alveolar macrophages in the LPS group were deeply immunostained. In contrast, the entire alveolar macrophages in the LPS + Y-41654 group were less prominently immunostained except for marked staining of the cell membrane (Fig. 7).

Fig. 7:
TNF-α immunostaining of lung tissue. Alveolar macrophages in the LPS group were prominently immunostained. In the LPS + Y-41654 group, like in the control group, the macrophages are faintly immunostained.


Although a previous animal study had shown the prevention of endotoxemic death by TACE inhibition (15), this is the first study of its effects on acute inflammatory responses to intratracheal instillation of LPS. Advantages of Y-41654 include its small molecular size (700 d), short half-life (10 min), and its therapeutic specificity. Our observations pertaining to the concentrations of TNF-α in serum and BAL fluid suggest that the local inflammation induced by the instillation of LPS can spread over the entire organism by proinflammatory mediators released into the systemic circulation. Owing to its low molecular weight, a bolus injection of Y-41654 seems to be widely distributed, including to the air spaces, causing an increase in mTNF-α on the cell membrane and a decrease in the production of sTNF-α, as suggested in Figure 5.

It has been reported that mTNF-α might play important roles in the host defense against microorganism or tumor cells by the polyclonal activation of B cells and the generation by monocytes of anti-inflammatory cytokines such as IL-10 (12, 24, 25), whereas an excess of sTNF-α can be disseminated systemically via the circulation and contribute to the pathogenesis of ALI, cerebral malaria, rheumatoid arthritis, and Crohn disease (26-28). These findings suggest that mTNF-α and sTNF-α play separate and diverse, however coordinated, functional roles in acute and chronic inflammation (12, 24, 25). This balances the disadvantages of treatment with a monoclonal antibody against TNF-α or its receptor, which inhibits both types of TNF-α and suppresses mTNF-α-mediated host defense function against infection. In this regard, the short half-life of TACE inhibition allows a prompt and temporary suppression of prominent inflammatory reactions mainly induced by sTNF-α, obviating an undesirable prolonged immunosuppression concomitantly preserving anti-inflammatory beneficial roles by mTNF-α.

Our observations are concordant with the results of previous studies in vitro, which found that TACE inhibition decreased the release of sTNF-α from inflammatory cells induced by LPS, although it had little effects on the release of other cytokines and chemokines (12, 13, 15, 16). On the other hand, because mTNF-α and its membrane-associated receptors are dynamically recycled from the membrane to the Golgi fraction, (10, 11, 20), the functional effects induced by mTNF-α are expected to be transient, along with the short half-life of Y-41654, and limited to the local environment.

Although TACE is demonstrated to be essential for mammalian development, conditional temporal inactivation of TACE offered protection from lethality due to endotoxin-shock in mice by preventing increased TNF serum levels (29, 30). These reports suggest transient inhibition of TACE would be a principal target for the treatment of TNF-dependent pathologies.

As demonstrated in previous studies (15, 16), our in vivo and in vitro experiments showed that TACE inhibition did not significantly decrease the production of CINC-1, which corresponds to the human IL-8, suggesting a chemoattracting factor other than CINC-1 might be implicated in this model. Recently, a series of studies have demonstrated TACE-related mechanisms on neutrophil kinetics, including rolling, adhesion, tethering on the endothelium, and migration (31, 32). It is demonstrated that TACE expressed on neutrophil surface, induces shedding of adhesion molecules, including L-selectin and P-selectin in acute inflammation, which diminished by down-regulation of TACE. Schaff et al. (32) demonstrated that interaction of E-selectin, vascular adhesion molecule with L-selectin on the neutrophils induced dynamics of membrane redistribution of TACE and would affect circulating neutrophil kinetic processes such as rolling, activation, arrest, and transmigration.

The neutrophils accumulated in the lung tissue play an important role in the development of LPS and TNF-induced ALI (1, 4, 33). Therefore, the current study suggested the possibility that inhibition of TACE on neutrophils by Y-41654 decreased LPS-induced their accumulation in the lung, in part, leading to decreased degree of ALI assessed by protein leakage. Furthermore, considering that TNF-α can modify a variety of neutrophil functions, we hypothesize that TACE inhibition regulates their proinflammatory activities such as transmigration, oxygen radical production, or release of elastase. From such perspective, the functional modification of neutrophils induced by mTNF-α and sTNF-α separately, as well as together, warrant further evaluations in in vitro and in vivo studies.

In addition, TACE on neutrophil is reported to play an important role to induce their apoptosis (34). This suggests TACE on neutrophils has dual functions, both proinflammatory roles by promoting margination and migration of circulating neutrophils into inflammatory sites, and anti-inflammatory roles by inducing their apoptosis resulting in control of inflammation. Previous reports and current observation suggest that TACE exists on various cell membranes bearing individual responsibility for inflammatory responses beside generation of sTNF-α implying that TACE inhibitor could modify inflammatory responses through multiple mechanisms. In addition, recent reports have suggested that anti-inflammatory cytokine IL-10 induced by LPS could modify TACE dynamics on the monocytes through tissue inhibitor of metalloproteinase 3-dependent pathways in 24 h (35). Taken together, it has to be further studied to clarify a manner in which transient control of TACE during initial as short as 4 h of acute inflammatory phase could modify after course of inflammation. Furthermore, potential of Y-41654 when started after administration of LPS or other insults such as live bacteria should be evaluated before confirming usefulness of TACE inhibitor as a candidate of anti-inflammatory agents.

In conclusion, the inhibition of sTNF-α production by TACE inhibition, preserving mTNF-α on the cell surface, decreased LPS-induced proinflammatory responses with modification of neutrophil kinetics, suggesting that sTNF-α rather than mTNF-α, plays a key role in the development of lung injury during its very early phase.


The authors thank Dr. Fujio Kobayashi and Dr. Naruyasu Komorita for technical support offered to the study and Mitsubishi Pharma Corporation (Osaka, Japan) for supplying Y-41654.


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Soluble TNF; membrane-bound TNF; cytokine; LPS; neutrophil; lung inflammation; bronchoalveolar lavage

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