A Single Dose of Lipopolysaccharide into Mice withEmphysema Mimics Human Chronic ObstructivePulmonary Disease Exacerbation as Assessedby Micro-Computed Tomography

Satoshi Kobayashi1*, Reiko Fujinawa1*, Fumi Ota1, Shiho Kobayashi1, Takashi Angata2, Manabu Ueno3,Toshitaka Maeno3, Shinobu Kitazume1, Keiichi Yoshida1, Takeo Ishii4, Congxiao Gao1, Kazuaki Ohtsubo1,Yoshiki Yamaguchi5,6, Tomoko Betsuyaku7, Kozui Kida4, and Naoyuki Taniguchi1,6

1Disease Glycomics Team and 2Glycan Recognition Team, Systems Glycobiology Group, RIKEN Advanced Science Institute, Saitama, Japan;3Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Gunma, Japan; 4Respiratory Care Clinic, NipponMedical School, Tokyo, Japan; 5Structural Glycobiology Team, Systems Glycobiology Group, RIKEN Advanced Science Institute, Saitama, Japan;6RIKEN-Max Planck Institute Joint Research Center, Saitama, Japan; and 7Division of Pulmonary Medicine, Keio University School of Medicine,

Tokyo, Japan

Chronic obstructive pulmonary disease (COPD), manifested as em-physema and chronic airway obstruction, can be exacerbated bybacterial and viral infections. Although the frequency of exacerba-tions increases as the disease progresses, themechanisms underlyingthisphenomenonarelargelyunknown,andthereisaneedforasimplein vivoexacerbationmodel. In this study,we compared four groupsofmice treated with PBS alone, elastase alone, LPS alone, and elastaseplus LPS. A single intratracheal administration of LPS to mice withelastase-induced emphysema provoked infiltration of inflammatorycells, especiallyCD81Tcells, intoalveolar spacesand increasedmatrixmetalloproteinase-9, tissue inhibitor ofmetalloproteinase-1, andper-forin production in bronchoalveolar lavage fluid at the acute inflam-matoryphasecomparedwith theothergroups.Wealsomeasuredthepercentage of low-attenuation area (LAA%) in the above mice usingmicro-computed X-ray tomography. The LAA% was the most sensi-tiveparameter for quantitative assessments of emphysemaamongallthe parameters evaluated. Using the parameter of LAA%, we foundsignificantly more severe alveolar destruction in the group treatedwith elastase plus LPS compared with the other groups during long-term longitudinalobservations.Webuilt three-dimensional imagesofthe emphysema and confirmed that the lungs of elastase plus LPS–treatedmice contained larger emphysematous areas than mice trea-ted with elastase alone. Although human exacerbation of COPD isclinically and pathologically complicated, this simple mouse modelmimics human cases to some extent andwill be useful for elucidatingits mechanism and developing therapeutic strategies.

Keywords: emphysema; exacerbation; computed tomography; LPS; elastase

Chronic obstructive pulmonary disease (COPD), a major causeof chronic disease and death globally (1), is characterized bylargely irreversible expiratory airway obstruction and abnormal

immunoreactivity in the lungs. A mixture of small airway ob-struction and parenchymal destruction (emphysema) is thepathological hallmark of COPD (2, 3). The chronic lung inflam-mation in COPD largely results from invasion of the lung pa-renchyma by inflammatory cells, such as neutrophils, macrophages,and T cells (4).

Acute exacerbations of COPD can accelerate disease pro-gression and are a major cause of morbidity and mortality inpatients with COPD. Most exacerbations are precipitated byviral or bacterial infections (5), and the microorganisms mostcommonly isolated during these episodes are the Gram-negativebacteria nontypeable Haemophilus influenzae (6). Significantinflammatory responses occur in patients with COPD with exac-erbations compared with those without exacerbations, such asinfiltration of inflammatory cells into their sputum and produc-tion of proinflammatory cytokines (7, 8) and proteases. In par-ticular, previous studies have suggested that the level of matrixmetalloproteinase (MMP)-9 is significantly increased in thesputum and in the exhaled breath condensate, whereas the levelof tissue inhibitor of metalloproteinase (TIMP)-1, which inhib-its MMP-9 activity in vivo, is decreased in the sputum butincreased in the serum and expiration of patients with COPDwith exacerbation (9–11). The repeated exacerbations of COPDworsen lung function (12) and prognosis (13) and accelerate theprogression of emphysema (14) as COPD progresses. A betterunderstanding of the mechanisms and implications of this vi-cious circle of infections and inflammations in COPD has beenhampered by the pathological complexity of the underlying dis-ease and the limited availability of animal models.

A particular problem is the difficulty in identifying a suitablemodel of the exacerbation. Several groups have sought to estab-lish animal models of exacerbation in COPD (15, 16). Cigarettesmoke (17) and elastase/LPS treatment (18) exacerbate the in-flammatory responses to nontypeable H. influenzae in mice, andthis phenomenon was also observed after rhinovirus infection ofmice exposed to elastase/LPS (19).

(Received in original form February 18, 2013 and in final form June 21, 2013)

* These authors contributed equally to this work.

This work was supported by the Program for the Promotion of Fundamental

Studies in Health Science of the National Institute of Biomedical Innovation

(NIBIO) (N.T.), by the Global Center of Excellence (GCOE) program (N.T.),

and by a Grant-in-Aid for Scientific Research (A) no. 20249018 from the Ministry

of Education, Culture, Sports, Science and Technology, Japan (N.T.).

Correspondence and requests for reprints should be addressed to Naoyuki

Taniguchi, M.D., Ph.D., Disease Glycomics Team, RIKEN Advanced Science Institute,

2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: tani52@wd5.so-net.ne.jp

This article has an online supplement, which is accessible from this issue’s table of

contents at www.atsjournals.org

Am J Respir Cell Mol Biol Vol 49, Iss. 6, pp 971–977, Dec 2013

Copyright ª 2013 by the American Thoracic Society

Originally Published in Press as DOI: 10.1165/rcmb.2013-0074OC on July 3, 2013

Internet address: www.atsjournals.org

CLINICAL RELEVANCE

In this study, we tried to establish a simple in vivo exac-erbation model of chronic obstructive pulmonary disease(COPD) by a single administration of elastase followed byLPS. Although human exacerbation of COPD is clinicallyand pathologically complicated, this simple mouse modelmimics human cases and will be useful for elucidating itsmechanism and developing therapeutic strategies.

Recently, micro-computed X-ray tomography (micro-CT) forsmall animals has emerged as a powerful tool to track emphyse-matous changes over time in living mice. Several groups haveused micro-CT to evaluate the progression of alveolar destruc-tion (20–24), but different parameters were used to quantify theextent of the emphysematous disease, such as the mean CT den-sity, identification of low-density areas (i.e., low-attenuation area[LAA]) using a variety of thresholds, and total lung volume de-rived from CT data. Furthermore, micro-CT data enabled us toconstruct three-dimensional images, which helped us to furtherconfirm the area and progression of emphysematous changesvisually.

In this study, we report a simple experimental model of theexacerbation in COPD precipitated by administering a singledose of LPS into the trachea of mice with established emphyse-matous lung disease instigated by prior exposure to elastase. LPSis a toxic component of gram-negative bacteria that provokesprofound inflammation (25). Therefore, we hypothesized thatits administration would mimic bacterial infection by initiatingthe infiltration of inflammatory cells into the pulmonary alveoli,similar to patients with COPD with exacerbation. Moreover, toexamine whether our model of the exacerbation in COPDshowed progression of emphysema, similar to human cases,we performed micro-CT scanning to monitor the emphysema-tous changes by quantifying the percentages of LAA (LAA%).By evaluating these factors, we confirmed that our modelmimics human exacerbation in COPD. Our results indicatedthat the administration of elastase plus LPS augmented inflam-matory responses and MMP-9 and TIMP-1 production and pro-gressed the emphysematous changes, suggesting that our modelwould considerably mimic the clinical exacerbation in COPD.

MATERIALS AND METHODS

Mice

Male C57BL/6J mice were purchased from Japan SLC (Shizuoka,Japan). All experimental protocols and procedures were performedin compliance with the guidelines for animal experiments of RIKEN.

Porcine Pancreatic Elastase–Induced Emphysema

Mouse Model

Mice aged 10 to 14 weeks were anesthetized by intraperitoneal injectionof 1.25% (wt/vol) tribromoethanol, followed by spraying of 4.2 U of por-cine pancreatic elastase (PPE) (Wako Pure Chemical Industries Ltd.,Osaka, Japan) dissolved in 100 ml of sterile PBS or 100 ml of PBS alone(control animals) into the trachea using a MicroSprayer drug deliverydevice (Penn-Century Inc., Philadelphia, PA) as described previously(26).

LPS Administration

After 21 days, mice were given intracheal administration of LPS (1mg/kg) (Sigma-Aldrich Co., St. Louis, MO) dissolved in 100 ml ofsterile PBS into the tracheas of the control and PPE-exposed mice.

Collection and Analysis of Bronchoalveolar Lavage Fluid

At 1, 3, and 7 days after the LPS administration to the mice, lavage wasperformed by washing the lungs with 0.75 ml of PBS four times, and 3 mlof bronchoalveolar lavage fluid (BALF) was collected from eachmouse.The total number of cells in the BALF was determined with a hemocy-tometer after lysing the erythrocytes with 0.83% (wt/vol) Tris-bufferedammonium chloride (pH 7.2). Inflammatory cells were identified insmear preparations stained with Diff-Quik reagent (Sysmex Interna-tional Reagents, Kobe, Japan). The ratios of various inflammatory cellswere calculated by counting approximately 300 cells in each BALF sam-ple under a standard light microscope. Each ratio was multiplied by thetotal number of cells to estimate the number of each cell type.

FACS Analysis

The cells that had infiltrated into the BALF from elastase-treated micewith or without LPS administration were collected and washed threetimes with 0.2% (wt/vol) BSA dissolved in PBS. The washed cells werethen immunolabeled with antibodies against CD3e, CD4, and CD8a(eBioscience, SanDiego, CA) and analyzed using an LSR II flow cytom-eter (BD Bioscience, San Jose, CA) (19).

Western Blotting

Aliquots (50 ml) of BALF supernatants were acetone precipitated anddissolved in Laemmli sample buffer with 5% (vol/vol) 2-mercaptoethanol.The samples were electrophoresed in a 5 to 15% gradient polyacryl-amide gel and then tagged with a rat antimouse perforin antibody(eBioscience) followed by a horseradish peroxidase–conjugated antiratsecondary antibody (GE Healthcare UK Ltd., Buckinghamshire, UK)for enhanced chemiluminescence detection. The band intensities weremeasured using ImageQuant TL Software (GE Healthcare UK Ltd.).

Gelatin Zymography

Detailed methods are provided in the online supplement.

ELISA

BALF was analyzed for TIMP-1 levels using a RayBio Mouse TIMP-1ELISA Kit (RayBiotech Inc., Norcross, GA) in accordance with themanufacturer’s instructions.

Statistical Analysis

All data are expressed as means 6 SEM. Statistical analyses wereperformed by Student’s t test or with the Tukey-Kramer method asmultiple comparisons using Microsoft Office Excel 2010 (MicrosoftCorp., Seattle, WA). Values of P , 0.05 were considered to indicatestatistical significance.

RESULTS

Lung Inflammation Is Augmented in Elastase-Treated Mice

Challenged with LPS

To establish a practical animal model of lung inflammation inCOPD, we assessed the effects of enhanced lung inflammationin elastase-induced emphysema mice by intratracheal LPS ad-ministration, mimicking bacterial stimuli (Figure 1). We com-pared four groups of mice treated with PBS alone, elastasealone, LPS alone, and elastase plus LPS. On the first day ofthe experiment (Day 0), PPE or PBS was administered intra-tracheally once. At 3 weeks after the administration of elastase,the mice in the LPS alone and elastase plus LPS groups wereadditionally treated with LPS once. To evaluate whether themice treated with elastase plus LPS mimicked cases of patientswith COPD with exacerbations in terms of the inflammatoryresponses and proteinase production, we harvested BALF fromall groups at 1, 3, and 7 days after the LPS treatment. At 1 dayafter the LPS challenge, the total inflammatory cell count inBALF was highest in mice treated with LPS alone and wasalmost the same level in the groups treated with elastase plusLPS and elastase alone. However, at 3 days after the LPS chal-lenge, the total cell count in the elastase plus LPS group wassignificantly higher than that in the LPS alone group (Figure2A). Along with the total cell count, the number of neutrophilstended to be higher in the lungs of elastase plus LPS–treatedmice than in the lungs of LPS-treated mice (P ¼ 0.06, Student’st test) (Figure 2B). In the lungs of mice treated with LPS alone,the number of macrophages was higher at 1 day after LPStreatment than at 3 days, whereas the number was lower inthe lungs of mice treated with elastase plus LPS at 1 day after

972 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 49 2013

LPS treatment than at 3 days, indicating that the efflux of mac-rophages into the BALF was delayed in the mice exposed toboth elastase and LPS compared with the mice treated with LPSalone (Figure 2C). The accumulation of lymphocytes in theBALF was significantly higher in the group treated with elastaseplus LPS than in the other groups at 3 days after the LPStreatment (Figure 2D). At 7 days after the LPS challenge, mostof the inflammatory cells in the BALF had disappeared. Thesefindings suggested that our elastase plus LPS–treated miceshowed augmented inflammatory responses, similar to the casefor exacerbation in patients with COPD.

CD81 T Cells Are Significantly Accumulated in BALF

from Elastase Plus LPS–Treated Mice

We found a significant increase in lymphocytes in the BALFobtained from the elastase plus LPS–treated mice comparedwith the LPS-treated mice. Previously, Makris and colleagues(27) reported that the percentage of sputum CD81 T cellsamong lymphocytes was significantly increased at the onset ofCOPD exacerbations compared with stable condition. There-fore, we next characterized the T cells in their BALF afterthe LPS challenge. In this experiment, we could not evaluatethe groups of PBS- and elastase-treated mice because the num-ber of the total inflammatory cells accumulated into the lung ofthese two groups was not enough for flow cytometry analysis.The ratios of CD81 T cells to CD31 T cells (total T cells) weresignificantly higher in the BALF from the elastase plus LPS–treated mice compared with the LPS-treated mice at 3 daysafter the LPS challenge (Figures 3A and 3B). However, theratio of CD41 T cells showed no significant difference betweenthe elastase plus LPS–treated group and the LPS-treated group.These results are consistent with a previous study showing thatCD81 T cells are required for airspace enlargement in emphy-sema model mice (28). Perforin, a pore-forming protein, is re-leased from CD81 T cells and is associated with the progressionof emphysema in COPD (4). Therefore, we evaluated the protein

level of perforin in the BALF supernatants at 3 days after LPSadministration. Perforin production was significantly higher in thelungs of elastase plus LPS–treated mice than in LPS-treated mice(Figures 3C and 3D). These data also indicated an increase inCD81 T cells in the lungs of elastase plus LPS–treated mice.

MMP-9 and TIMP-1 Are Increased in BALF Supernatants

from Elastase Plus LPS–Treated Mice

Excessive inflammatory responses occur in patients with COPDwith exacerbations compared with those without exacerbations.

Figure 1. Flow chart of the animal experiment to establish the exacer-

bation model mice by single administrations of elastase and LPS. We

prepared four groups of mice treated with PBS alone (PBS), elastase alone

(Elastase), LPS alone (LPS), and elastase plus LPS (Elastase1 LPS). On thefirst day of the experiment (Day 0), 4.2 U of PPE or PBS was administered

intratracheally into mice. The mice were treated with 1 mg/kg of LPS

derived from Escherichia coli in the same way at 3 weeks after the elastaseor PBS administration. At Days 0 and 1 and at Weeks 3, 4, 5, 6, 8, 10, and

12 after the elastase or PBS administration, all groups of mice were

scanned by micro–computed tomography (CT). At 1, 3, and 7 days after

the LPS treatment, bronchoalveolar lavage fluid (BALF) was harvestedfrom each group. Each group consisted of at least 27 mice. Each BALF

analysis was performed for at least five mice per time point, and each

micro-CT analysis was performed on at least six mice per time point.

Figure 2. Elastase-treated mice challenged with LPS show an altered

cellular profile in the BALF. (A–D) BAL was performed for elastase-treatedand PBS-treated mice challenged with LPS or PBS. At 1, 3, and 7 days

after the LPS challenge, the BALF cells were assessed by microscopy to

count the numbers of total inflammatory cells (A), neutrophils (B), mac-

rophages (C), and lymphocytes (D) after preparation of slides and stain-ing with Diff-Quik reagent. The data are shown as means 6 SEM. All

experiments were performed for at least five mice per group. n.d., not

detected. Statistical analysis was performed with the Tukey-Kramer

method as multiple comparisons. *P , 0.05; **P , 0.01.

Kobayashi, Fujinawa, Ota, et al.: Simple Experimental Model for COPD Exacerbation 973

The inflammatory cells that infiltrate into their lungs releaseproinflammatory cytokines and proteinases. In particular, thebalance between the levels of MMP-9 and its inhibitor TIMP-1 is thought to be important in the pathogenesis of COPD withexacerbation. Previously, Kwiatkowska and colleagues (11)showed that MMP-9 and TIMP-1 were increased in the exhaledbreath condensate during COPD exacerbation and were nega-tively correlated with spirometric variables. To examine whetherour mouse model of the exacerbation also mimics patients withCOPD with exacerbations in terms of MMP-9 and TIMP-1 pro-duction, we evaluated the activity of MMP-9 by gelatin zymog-raphy and the production of TIMP-1 by ELISA at 3 daysafter the LPS administration. By gelatin zymography, we foundthat the MMP-9 activity tended to be higher in BALF fromelastase plus LPS–treated mice compared with LPS-treatedmice (P ¼ 0.05, Student’s t test) (Figures 4A and 4B). Further-more, we found that the production of TIMP-1 in the lungswas significantly higher in elastase plus LPS–treated mice com-pared with LPS-treated mice (Figure 4C). These results also

suggest that our model mimicked patients with COPD withexacerbations.

Progression of Emphysema Is Accelerated in Elastase Plus

LPS–Treated Mice Evaluated by Longitudinal Micro-CT

In addition to significant inflammatory responses, the progres-sion of emphysema is promoted in patients with COPD with ex-acerbations compared with those without exacerbations (14). Toconfirm whether our mouse model of exacerbations shows pro-gression of the emphysematous change seen in patients withCOPD with exacerbations, we evaluated the progression of em-physema using micro-CT, which allows us to perform quantita-tive assessments of emphysema in living mice for a long period.Figure E1 in the online supplement shows the density histo-grams of living mice lungs with or without emphysema. Refer-ring to the study about micro-CT analysis for emphysema modelof mice reported by Kawakami and colleagues (11), the LAAthresholds were set in the range –871 to –610 Hounsfield unitsin our analysis of the micro-CT data. By applying these thresh-olds, the LAA% was calculated as the ratio of LAA to the totallung area. We found a marked increase in the LAA% in theelastase-treated mice (31.6 6 1.6%), which was 15-fold higherthan that in the control mice (1.8 6 1.6%) (Figures E1 andE2A). In fact, the LAA% was the most sensitive parameteramong all the parameters we evaluated, including the meanCT density, CT-derived total lung volume, and mean linear

Figure 3. Elastase-treated mice treated with LPS show significant in-

creases in CD31 CD81 T cells and production of perforin in their BALF.

In this experiment, we could not evaluate the groups of PBS and elas-tase-treated mice because the number of the total inflammatory cells

accumulated into the lung of these two groups was not enough for

flow cytometry analysis. (A and B) BAL cells from elastase-treated and

PBS-treated mice were collected at 3 days after the LPS challenge andstained with anti-CD3, anti-CD4, and anti-CD8 antibodies. After incu-

bation with the antibodies, the cells in the BALF were analyzed by flow

cytometry, and the ratios of CD41 (A) and CD81 (B) T cells to CD31 T

cells, representing total T cells, were calculated. (C and D) The volumeof BALF taken from each mouse treated with LPS alone or elastase plus

LPS was 3 ml in total. To prepare the samples, 50 ml of BALF was

analyzed to examine the production of perforin by Western blotting

(WB) with normalization by the average intensity in LPS-treated mice.(C) The relative perforin immunoreactivity was calculated by the band

intensity using LPS-treated mice as a standard. (D) The data are shown

as means 6 SEM. All experiments were performed for at least five miceper group. Statistical analysis was performed with Student’s t test. *P ,0.01; **P , 0.005.

Figure 4. Elastase plus LPS–treated mice show a significant increase in

matrix metalloproteinase (MMP)-9 and tissue inhibitor of metallopro-

teinase (TIMP)-1 production in BALF. (A and B) BALF supernatants from

mice treated with PBS alone, elastase alone, LPS alone, or elastase plusLPS were collected at 3 days after the LPS challenge and analyzed by

gelatin zymography for MMP-9 activity. After staining of the gel and

washing, band densitometry was performed. Gel images were takenusing representative samples. AU, arbitrary unit; n.d., not detected. (C)

The production of TIMP-1 at 3 days after the LPS administration in BALF

supernatants from mice treated with PBS alone, elastase alone, LPS

alone, or elastase plus LPS was measured by ELISA. The data are shownas means 6 SEM. All experiments were performed for at least five mice

per group. Statistical analysis was performed with the Tukey-Kramer

method as multiple comparisons. *P , 0.01.

974 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 49 2013

intercept (Lm), representing the mean of the inner diameter ofpulmonary alveoli by lung morphometry (Figures E2A–E2D).Significant correlation was observed for LAA% with Lm (R2 ¼0.88) (Figure E3A) and with CT-derived lung volume (R2 ¼0.93) (Figure E3B). To verify the CT-derived lung volume, ac-tual lung volume was measured by immersing the lung in PBS.Actual lung volume was correlated with CT-derived lung vol-ume (R2 ¼ 0.57) (Figure E3C). We decided to use the LAA%,rather than the other parameters, to evaluate the degree ofemphysema.

To determine whether the elastase plus LPS–treated miceexhibited progression of emphysema, we monitored the lungconditions by micro-CT. After 12 weeks, micro-CT image anal-ysis showed that the mice treated with elastase plus LPS de-veloped a significantly higher LAA% than mice treated withelastase alone (Figure 5A). As an evaluation parameter of theprogression of emphysema, we also assessed, using Lm, a com-mon lung morphometric method. The Lm of elastase plus LPS–treated mice was not significantly different as compared withelastase-treated mice statistically, but it tended to be larger(Figures 5B and 5C). Moreover, to easily and spatially under-stand the emphysematous area in the lungs, we built stericimages of the emphysema at 3 and 12 weeks after the elastasetreatment using the micro-CT images. The three-dimensionalreconstructed images of the mouse lungs (Figure 6) confirmedthat the lungs of the elastase plus LPS–treated mice containedlarger emphysematous areas compared with the mice treatedwith elastase alone. These results indicated that elastase plusLPS–treated mice showed accelerated progression of emphy-sema during long-term follow-up, similar to the case for exac-erbation in patients with COPD.

DISCUSSION

We have established a simple mouse model of the exacerbationin COPD by a single administration of LPS into elastase-inducedemphysematous mice. Mice treated in this manner exhibiteda more robust inflammatory response than mice that had notbeen treated with elastase and thus did not have emphysematouslung disease, although the efflux of neutrophils and macrophagesinto the BALF was somewhat delayed and had almost subsidedat 7 days after the LPS administration. In patients with COPD,the exacerbation is normally of a longer duration, possibly owingto the presence and continued proliferation of living bacteria orviruses, whereas a single dose of a proinflammatorymediator wasused in our model. It commonly takes longer to recover frombacterial pneumonia, and this disease becomes severe in patientswith COPD, partly due to the prolonged proliferation of bacteriain the lungs. One way to mimic such a situation observed inhumans is to use multiple dosages of LPS.

At 3 days after LPS challenge, we found that the accumula-tion of total inflammatory cells in the lung was significantlyhigher in the group treated with elastase plus LPS than in allthe other treatment groups. Patients with COPD with exacerba-tions showed a higher number of inflammatory cells infiltrated intheir sputum (7, 8) and accelerated progression of emphysema(14) compared with those without exacerbations. These ob-servations suggested that pulmonary emphysematous changeswould accelerate the inflammatory responses induced by bacte-rial and viral infections. Among the inflammatory cells in theBALF, neutrophils showed the highest accumulation (5). Neu-trophils are known to recruit macrophages and lymphocytes andto play a key role in the progression of emphysema by therelease of elastase (29). Consistent with previous studies regard-ing models of the exacerbation in COPD (17, 18), neutrophilswere the major cells in the BALF at 3 days after the LPS

treatment as an early phase of exacerbation in the presentstudy. Furthermore, lymphocyte infiltration was significantlyhigher in mice treated with elastase plus LPS at 3 days afterLPS treatment, in agreement with a previous study reportingthat patients with severe COPD had increased numbers of Tcells in their airway (30). Some studies have suggested thatCD81 T cells play an important role in the development ofemphysema (4, 28), whereas others have shown links betweenthe presence of CD81 T cells and cigarette smoke in the airway(31, 32) and lung destruction (28, 33). A significant increaseof CD81 T cells in sputum was observed at the onset of

Figure 5. Elastase-treated mice challenged with LPS show a gradual

progression of emphysema. (A) The groups of mice treated with PBS

alone, elastase alone, LPS alone, or elastase plus LPS were scannedby micro-CT every week after the elastase or PBS treatment for up to

12 weeks. At each time point, the percentage of low-attenuation area

(LAA%) was calculated using the data obtained from the CT scan. Black

and white arrowhead: time point of PBS or elastase administration; grayarrowhead: time point of treatment with or without LPS. (B) Represen-

tative lung sections at 12 weeks after PBS or elastase administration

stained with hematoxylin and eosin are shown. Original magnification:

3100. Scale bar ¼ 100 mm. (C) The mean linear intercept (Lm) valuesas an alternative parameter of emphysema are shown. The data are

shown as means 6 SEM. All experiments were performed for at least

six mice per group. Statistical analysis was performed with Student’s

t test. *P , 0.05; **P , 0.01 as compared with elastase alone group.

Kobayashi, Fujinawa, Ota, et al.: Simple Experimental Model for COPD Exacerbation 975

exacerbation in patients with COPD compared with stable con-dition (27). Our experimental mouse model showed significantincreases in CD81 T cells and enhanced production of perforinreleased from CD81 T cells in the BALF at 3 days after LPStreatment. These results resembled the condition of the patientswith COPD with exacerbation. Given recent evidence that thepathogenesis of COPD is dependent on T-cell activity (34, 35),the role of T cells in the progression of emphysema warrantsfurther study. Our results indicated that the increase in CD81 Tcells in the elastase plus LPS–treated mice might be partly cor-related with the progression of emphysema in the exacerbationthrough up-regulation of perforin. In addition to the accumula-tion of these inflammatory cells, we measured the levels ofMMP-9 activity and TIMP-1 production. MMP-9 and TIMP-1were increased in the BALF from the elastase plus LPS–treatedmice at 3 days after LPS treatment, compared with LPS alone–treated mice. These data are consistent with a previous studyon patients with COPD with exacerbations by measurementsof exhalation (11). These findings also implied that excessive

inflammatory responses led to the increase in MMP-9 and thatthe massive production of MMP-9 resulted in increased TIMP-1production at the early phase of exacerbation.

We found that the LAA% measured by micro-CT was themost sensitive parameter for quantifying the extent of emphy-sema, compared with the other parameters examined, includingLm, mean CT density, and CT-derived lung volume. Further-more, a significant correlation was observed for LAA% withLm. The elastase-treated mice exhibited a significantly higherLAA% than the control mice within 1 week of treatment. Lon-gitudinal data acquisition using micro-CT allowed us to nonin-vasively observe the progression of the emphysema provokedby LPS administration in the mice treated with elastase plusLPS comparedwith themice treatedwith elastase alone at 12weeksafter elastase treatment. In human cases, it was reported that theexacerbation in patients with COPD accelerated the progressionof emphysema (14). Our model shows the remarkable inflam-mation responses as compared with lung morphology change.This result suggests that the potential application of this modelmight be to investigate the inflammatory response in acute ex-acerbations of COPD. It is speculated from our data obtainedwith the elastase plus LPS–treated mouse model that the pro-gression of emphysema would be partly induced by excessiveinfiltration of inflammatory cells and enhancement of the produc-tion of proteases, such as perforin and MMP-9. The underlyingmechanisms for how the excessive accumulation of neutrophilsand CD81 T cells in the early phase of exacerbation can promotethe progression of emphysema need to be clarified. The presentfindings suggest that our model mimics the clinical exacerbationin COPD and could potentially be a robust means of studying theexacerbation of COPD and its influence on the disease progres-sion of emphysema.

Author disclosures are available with the text of this article at www.atsjournals.org.

Acknowledgments: The authors thank the Support Unit for Bio-material Analysis atthe RIKEN BSI Research Resources Center.

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Figure 6. Progression of emphysema visualized as three-dimensionalimages by integrating CT images. Elastase-treated mice with or without

the LPS challenge were scanned using micro-CT at 3 and 12 weeks after

the elastase treatment. (A) Three-dimensional lung images in which the

LAA (–871 to –610 Hounsfield units [HU]) is shown in yellow and thewhole lung field (–900 to –200 HU) is shown as a transparent shape.

The LAA% values shown are from representative mice. The three-

dimensional images were built to reflect the view from the back side.

(B) Images of lung cross-sections from an elastase-treated mouse andan elastase plus LPS–treated mice in which the LAA (–871 to –610 HU)

is colored in blue and the whole lung field (–900 to –200 HU) is colored

in gray. R, right side; L, left side.

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