ARTICLE IN PRESS

EXPERIMENTAL

ANDTOXICOLOGIC

PA THOLOGY

0940-2993/$ - se

doi:10.1016/j.et

�Correspond

fax: +49 6421 2

E-mail addr

Experimental and Toxicologic Pathology 56 (2005) 341–350

www.elsevier.de/etp

NO2-induced airway inflammation is associated with progressive airflow

limitation and development of emphysema-like lesions in C57BL/6 mice

Michael Wegmanna,�, Antonia Fehrenbachb, Simone Heimanna, Heinz Fehrenbachb,Harald Renza, Holger Garna, Udo Herza

aDepartment of Clinical Chemistry and Molecular Diagnostics, Hospital of the Philipps University Marburg, Baldingerstraße,

35033 Marburg, GermanybDepartment of Pneumology, Clinic of Internal Medicine, Germany

Received 25 October 2004; accepted 22 December 2004

Abstract

The major features of chronic obstructive pulmonary disease (COPD) comprise a not fully reversible airflowlimitation associated with an abnormal inflammatory response, increased mucus production and development ofemphysema-like lesions. Animal models that closely mimic these alterations represent an important issue for theinvestigation of pathophysiological mechanisms. Since most animal models in this area have focused on specific aspectsof the disease, we aimed to investigate whether exposure of C57BL/6 mice to nitrogen dioxide (NO2) may cause a morecomplex phenotype covering several of the characteristics of the human disease. Therefore, mice were exposed to NO2

for 14 h each day for up to 25 days. Initial dose response experiments revealed the induction of a significantinflammatory response at a dose of 20 ppm NO2. Mice developed progressive airway inflammation together with afocal inflammation of the lung parenchyma characterized by a predominant influx of neutrophils and macrophages. Inaddition, goblet cell hyperplasia was detected in the central airways and increased collagen deposition was found in thelung parenchyma. NO2-exposed mice developed emphysema-like lesions as indicated by a significantly increased meanlinear intercept as compared to control mice. Finally, the assessment of lung functional parameters revealed thedevelopment of progressive airway obstruction over time. In conclusion, our data provide evidence that theinflammatory response to NO2 exposure is associated with increased mucus production, development of airspaceenlargement and progressive airway obstruction. Thus, NO2-exposed mice may serve as a model to investigatepathophysiological mechanisms that contribute to the development of human COPD.r 2005 Elsevier GmbH. All rights reserved.

Keywords: Nitrogen dioxide; Airway inflammation; Fibrosis; Airflow limitation; Mouse model

e front matter r 2005 Elsevier GmbH. All rights reserved.

p.2004.12.004

ing author. Tel.: +49 6421 2866035;

866086.

ess: wegmann@med.uni-marburg.de (M. Wegmann).

Introduction

Chronic obstructive pulmonary disease (COPD) iscurrently the sixth leading cause of death worldwide andthe only one that is increasing in prevalence (Lodden-kemper et al., 2004; Stang et al., 2000). COPD is definedas a disease state characterized by airflow limitation that

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350342

is not fully reversible and associated with an abnormalinflammatory response to noxious gases or particles.Clinically, the loss of lung function measured as forcedexpiratory volume in 1 s (FEV1) is the most importantdiagnostic parameter combined with chronic symptomssuch as chronic cough and sputum production (Fabbriand Hurd, 2003; Gold, 2003; Hurd and Pauwels, 2002).Pathological changes comprise infiltrates of macro-phages, neutrophils and lymphocytes into airways, lungparenchyma, and pulmonary vasculature (Barnes, 2003;Di Stefano et al., 1996; Saetta et al., 1998). In addition,enlarged mucus secreting glands and increased numbersof goblet cells are observed that coincide with mucushypersecretion (Saetta et al., 2000). Structural remodel-ling is observed as a result of repair processes withincreasing collagen deposition within airway and bloodvessel tissues. Finally, destruction of lung parenchymaleads to the development of centrilobular emphysema.The most important aetiological factor is tobaccosmoking. Since only 15–20% of all smokers developclinically significant COPD a genetic predisposition issuggested to modify the individual risk (Stanescu et al.,1998). However, it remains still unclear which genetic orenvironmental factors account in smokers destined todevelop COPD compared to those who are resistant tothe development of the disease.

It is widely appreciated that appropriate animal modelsare needed in order to better understand initial steps andthe sequence of pathophysiological events finally leadingto the development of the disease (Barth and Mueller,1999; Croxton et al., 2003). Several approaches have beenundertaken to mimic the complex disease as closely aspossible. They have been recently reviewed by Mahadevaand Shapiro (Mahadeva and Shapiro, 2002). Severalspontaneous mutations have been identified that result inthe development of emphysema-like lesions such as thepallid (Martorana et al., 1993), blotchy (Fisk and Kuhn,1976) and tight-skin mice (Green et al., 1976). In the lastyears, genetically engineered mice over-expressing certaincytokines, e.g. IL-13 (Zheng et al., 2000) or IFN-g (Wanget al., 2000), or lacking other components, such as elastinor surfactant protein D (Wert et al., 2000), have beendescribed. In addition, long-term tobacco smoke expo-sure models have been applied (Cavarra et al., 2001;Wright and Churg, 1990). However, most models focusprimarily on the development of emphysema whereasother components of COPD, most importantly loss oflung function, are rarely investigated.

It is known that exposure to nitrogen dioxide (NO2)as an oxidative and nitrosative irritant may causehistomorphological changes in animals, particularly inrats and hamsters, that model certain aspects of COPD(Blank et al., 1998; Glasgow et al., 1987; Kleinermanand Ip, 1979; Stephens et al., 1974). However, analysesof lung function have not yet been reported. In thepresent study we exposed mice to NO2 and investigated

inflammatory response, histopathology including mu-cus-producing cells, parameters of emphysema develop-ment and, for the first time, influence on lung functionto cover the major features that are characteristic forhuman COPD. In addition, kinetic analyses wereperformed to analyze the association of severity of lunginflammation and lung function.

Material and methods

Animals

Pathogen free female C57BL/6 mice (Harlaan &Winkelmann, Borchen, Germany), 6–8 weeks of agewere used in all experiments. The animals were keptunder standard housing conditions. After delivery, micewere housed in our facility for at least 1 week beforestarting the exposure protocol. All animal studies werein accordance to German and international guidelines.

NO2 exposure protocol

C57BL/6-mice were exposed to 20 ppm NO2 for 25days, 14 h a day. For exposure, cages with a maximumof 12 C57BL/6-mice were placed into air-tight plexi-glaschambers with in- and outlet for the gas mixture and aventilator to ensure equal distribution of the gasatmosphere throughout the chamber. The volume ofthe exposure chambers was 60 l and continuous airflowwas adjusted to 12 l/min. The concentration of NO2

(Messer-Griesheim, Duisburg, Germany) was moni-tored several times a day using a fine-flow-meter (ECS102-1; MPS, Forschungswerkstaetten, Klinikum of thePhilipps-University, Marburg, Germany) (Barth andMueller, 1999).

Assessment of leukocyte distribution in BAL fluid

One hour after the last NO2-exposure, tracheas werecannulated and lungs were lavaged with 2� 0.9 ml of ice-cold PBS. The mean recovery volume was 1.470.2 ml.Total cell numbers were determined using a standardNeubauer chamber. BAL cells were cytocentrifuged at700 rpm and stained with Diff-Quicks (DADE Diag-nostics, Unterschleissheim, Germany) following fixation.Differentially stained BAL cell types were identified bylight microscopy using standard morphological criteria.Differential cell counts of 100 leukocytes were performedin duplicates (Herz et al., 1998).

Histology

To assess the extent of airway inflammation, lunghistology was analyzed. Therefore, lungs were fixed in

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350 343

situ with 4% formalin solution via the trachea, removedand stored in 10% formaldehyde in phosphate bufferedsaline at pH 7.2. Lung tissues were embedded intoparaffin, and cut into 3 mm thick sections that werestained with hematoxylin and eosin (HE) or periodicacid-Schiff staining (PAS) to identify mucus-producingcells (goblet cells) according to standard procedures(40). Collagen fibers were stained by sirius-red/fast-green staining (Malkusch et al., 1995).

Measurement of the specific lung volume

After 25 days of exposure to NO2 or room air,C57BL/6 mice (n ¼ 6) were sacrificed by cervicaldislocation. Heart–lung blocks were removed, and thelungs fixed by intratracheal instillation of 1.5% formalinand 1.5% glutaraldehyde in 0.1 M cacodylate buffer(vehicle osmolality: 300 mOsm/kg; pH 7.4) at a constantpressure of 20 cm H2O for 20 min. Tracheas were tightlyligated and lungs were stored in cold fixative overnight.Immediately prior to tissue sampling, left and rightlungs were separated, dissected free of any extrapul-monary components, and the fixed lung volumes weremeasured by fluid displacement (Scherle, 1970).

Tissue sampling and processing

Systematic random sampling of lung tissue wasperformed as described previously (Fehrenbach, 2002).Briefly, the whole lung was embedded in 2% agar-agardissolved in distilled water. By means of a tissue slicer,the organ was cut into 3 mm thick slices (3–5 per lung).A transparent point grid with 11� 11 points (distancebetween points, 10 mm) was superimposed over acollection of slices. Whenever a point hit the sectionface of a lung slice, a 3 mm3 tissue block was exciseddefining the point as the lower left corner of the tissueblock. Seven to nine blocks of tissue were obtained fromeach animal by this method.

After several buffer rinses, slices were post-osmicated(1% OsO4 in 0.1 M cacodylate buffer) and stained enbloc with half-saturated, aqueous uranyl acetate. Thetissue was dehydrated through a graded series of ethanoland embedded in araldite to minimize the degree of biasin the stereological analysis introduced by tissueshrinkage, which is regularly observed in paraffin-embedded material (Ladekarl, 1994). Semi-thin sections(1 mm) were stained with a mixture of methylene blue/Azur II (1:2).

Stereological analysis

Mean linear intercept length (MLI), mean alveolarand airspace volumes as well as total alveolar surfacearea were quantified by light microscopy according to

standard stereological methods (Howard and Reed,1998) using a computer-based system (Cast-Grid 2.00,Olympus, Albertslund, Denmark). Because MLI doesnot allow to distinguish whether an observed effect waspresent at the level of alveoli or at the level of alveolarducts (Blanco et al., 1989), the volume-weighted meanvolumes ðn̄VÞ of alveoli and of the airspace comprisingalveoli and alveolar ducts were estimated using themethod of point sampled intercepts (Gundersen andJensen, 1985). As previously shown (Fehrenbach et al.,1999), a combined grid of points and intercepts wasprojected in random direction onto each field of viewsampled by meander scanning. Whenever a test-pointfell on gas-exchange region, the following measurementswere performed. The n̄V of airspaces was measured from‘‘wall to wall’’, which therefore comprised both alveoliand air-conducting paths distal to the terminal/transi-tional bronchioles. The measurement of n̄V of alveolihad to be modified in those instances in which part ofthe alveolar wall was not present (the mouth of thealveolus). According to Massaro and Massaro (1992),the alveolus was then interactively closed by drawing astraight imaginary line that connected the ends of thealveolar walls.

Non-invasive measurement of the midexpiratory

airflow (EF50)

The midexpiratory airflow (EF50) was measured usinghead-out body-plethysmography (Neuhaus-Steinmetz etal., 2000). Briefly, the system consists of a glass madebody plethysmograph to which a head exposurechamber was attached (Forschungswerkstaetten, Med-ical School Hannover, Germany). The mouse waspositioned in the body plethysmograph while the headof the animal protuded through a neck collar (9 mm ID,dental latex dam, Roeko, Langenau, Germany) into thehead exposure chamber, which was ventilated bycontinuous airflow of 200 ml/min. Small leaks, ifpresent, were not phasic with respiration and maytherefore be considered constant over a period of severalbreaths.

For airflow measurement, a calibrated pneumotacho-graph (PTM 378/1.2, Hugo Sachs Elektronic, March-Hugstetten, Germany) and a differential pressuretransducer (8T-2, Gaeltec, Dunvegan, Scotland) coupledto an amplifier (Gould Universal Amplifier, Dietzen-bach, Germany) were attached to the top port of eachplethysmograph. For each animal the amplified analogsignal from the pressure transducer was digitized via anA/D converter (DAS-16, Keithley, Germering, Ger-many) at a sampling rate of 2000/s. During a period of15 s, the signals of each breath were collected andcalculated.

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350344

Mid-expiratory airflow was measured before startingNO2 exposure and after 15, 20 and 25 days of NO2-exposure. The measurement was performed 1 h after endof the exposure-period.

Statistical analysis

Numerical data are presented as mean7SEM unlessstated otherwise. Differences between animal groupswere analyzed for significance using unpaired Student’st-test.

0

10

20

30

40

50

60

70

0 5 10 15 20

NO2 exposure [d]

Cel

l nu

mb

er [

104 /

BA

L lymphocytes

neutrophils

macrophages

leukocytes

********

*

*

*

25

Fig. 1. Kinetics of airway inflammation. Absolute numbers of

total leukocytes (black), macrophages (white), neutrophils

(light gray) and lymphocytes (dark gray) in BAL fluids after

exposure to 20 ppm NO2 (14 h/day) were determined at

indicated time points. Lavages of lungs were performed 1 h

after the last NO2 exposure as described in Materials and

methods. Mean7SEM is presented for each group with six

mice per group. Statistical significance is indicated by asterisks

with pp0:05 (*), pp0:01 (**), or pp0:001 (***), respectively.

Results

NO2 exposure causes inflammatory cell influx

C57BL/6 mice were exposed to NO2 at concentrationsof 10 or 20 ppm for 14 consecutive hours per day.Assessment of total cell counts in BAL fluids revealed amarked influx of inflammatory cells into the airwayslumen at a dose of 20 ppm by day 15, but there was nosignificant difference between animals exposed to10 ppm NO2 and untreated controls. Differentiation ofcell subpopulations revealed that the majority of BALcells of NO2-exposed mice consisted of macrophagesand neutrophils. However, a small but significant influxof lymphocytes was also detected (Table 1). Todetermine the kinetics of the inflammatory response toNO2 exposure, distribution of cell subpopulations wasfollowed up to 25 days (Fig. 1). An infiltration ofmacrophages was already detectable after 5 days ofexposure that further increased over time. Highestmacrophage levels were reached at day 15 followed bya decline, however, numbers remained markedly ele-vated when compared to controls. A similar pattern wasobserved for neutrophils. In contrast, lymphocyte influxstarted at day 5 of exposure, and lymphocyte numbersremained slightly elevated throughout the whole ex-posure period of 25 days. However, macrophages andneutrophils remained the dominating cell populations inthe BALs of NO2-exposed animals at each time pointinvestigated.

Table 1. Total leukocyte numbers and leukocyte differentiation in

Air

Leukocytes (� 104) 5.1170.47

Macrophages (� 104) 5.1170.47

Neutrophils (� 104) o0.01

Lymphocytes (� 104) o0.01

Leukocyte infiltration into the airway lumen was assessed in mice exposed to

methods. Results represent mean7SEM for each group (n ¼ 6). Statistical s

NO2 exposure causes inflammation of airway

mucosa and lung parenchyma associated with focal

fibrosis

Histological analysis of lung sections revealed a mildinflammation with moderate influx of mononuclear cellsinto the upper airway walls in animals exposed to10 ppm NO2 (data not shown). In contrast, exposure to20 ppm NO2 induced a marked peribronchial inflamma-tory cell infiltrate in the airway mucosa, again consistingmainly of macrophages and neutrophils (Fig. 2C). Inaddition, these animals developed goblet cell hyperpla-sia indicating increased mucus production in centralairways (Fig. 2D). Moreover, cellular infiltration couldalso be observed within the lung parenchyma (Fig. 3C)resulting in inflammatory lesions that were focallydistributed throughout all lobes. Especially at these

BAL fluids from C57BL/6 mice after NO2 exposure

NO2 (10 ppm) NO2 (20 ppm)

6.3970.46 51.6576.05***

6.2970.22 29.372.93***

0.1070.01 20.6173.35***

o0.01 1.7470.58***

10 or 20 ppm NO2 or room air for 25 days as described in Material and

ignificance is indicated by asterisks with pp0:001 (***).

ARTICLE IN PRESS

Air

NO2

200 µm

200 µm 50 µm

50 µm

A

C

B

D

Fig. 2. Peribronchial inflammation and development of goblet cell hyperplasia after NO2 exposure. The extent of lung inflammation

was assessed in sections of HE-stained (A and C) and PAS-stained (B and D) lung tissues from air- (A, B) or NO2-exposed mice (C,

D). Peribronchial inflammation was observed in the central airways of NO2-exposed mice (C) but not in sham-exposed animals (A).

In addition, hyperplasia of goblet cells was detectable in PAS-stained tissue slides of the central airways of NO2-exposed mice (D)

whereas almost no goblet cells were found in untreated controls (B). Lung tissues were prepared 1 h after the last NO2 exposure as

described in Materials and methods.

Air

NO2

20 µm

20 µm

20 µm

20 µm

A

C

B

D

Fig. 3. Parenchymal inflammation and increased collagen deposition in lung periphery after NO2-exposure. Inflammation of lung

parenchyma was assessed in sections of HE-stained lung tissues from air- (A) or NO2-exposed mice (C) and revealed a significant

inflammatory cell infiltration in the lungs of exposed animals. Furthermore, in these animals a deposition of collagen fibers could be

detected by staining with sirius red and fast green (D) that was not found in the lung parenchyma of untreated controls (B). Lung

tissues were prepared 1 h after the last NO2-exposure.

M. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350 345

ARTICLE IN PRESS

Table 2. Airspace enlargement in C57BL/6 mice after NO2

exposure

Air NO2

Mass-specific lung

volume (mm3/g bw)

52.974.4 64.674.5***

Mean airspace volumea

(104 mm3)

474764 566794

Mean alveolar volume

(104 mm3)

4.570.6 6.371.2**

Total alveolar surface

area (cm2)

533.4724.1 556.3753.5

Stereometrical analysis was performed on semi-thin sections of lungs

from mice exposed to 20 ppm NO2 or room air for 25 days as described

in Material and methods. Results represent mean7SEM for each

group (n ¼ 10). Statistical significance is indicated by asterisks with

pp0:01 (**) or pp0:001 (***).aComprises alveoli and alveolar ducts.

44

46

48

50 C

*

]

Air

NO2

100 µm

100 µm

A

B

M. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350346

sites, an increased deposition of collagen fibers wasdetectable (Fig. 3D).

Stereological analysis

For the investigation of structural changes lungs wereinflated ex vivo under constant pressure and analyzedstereologically. Lung volume referred to animal bodyweight (mass-specific) was significantly increased afterexposure to 20 ppm NO2 (Table 2). This was accom-panied by a significant increase in the MLI length of theairspaces (Fig. 4). Determination of the volume-weighted mean volume of alveoli revealed that airspaceenlargement was manifest at the level of the alveoli.Total alveolar surface area did not change in animalsexposed to 20 ppm NO2 (Table 2).

NO2-exposure causes development of progressive

airflow limitation

To assess the development of airway obstructionfollowing NO2 exposure, lung function was analyzed innon-anaesthetised, spontaneously breathing mice byhead-out body-plethysmography. Airflow at mid-tidalvolume during expiration (EF50) was measured for each

Fig. 4. Emphysema-like lesions in lung periphery after NO2-

exposure. Emphysema-like lesions are shown in the HE-

stained lung tissue section of a representative NO2-exposed

mouse (B) in comparison to an untreated animal (A). Lung

tissues were prepared 1 h after the last NO2-exposure as

described in Material and methods. Results of the analysis of

the mean linear intercept (MLI) of untreated (Nil) and NO2-

exposed (NO2) animals are shown in (C). Given are mean7SD

for each group with six animals per group. Statistical

significance is indicated by an asterisk with pp0:05:

breath, and a significant decrease was considered as anindex of airway obstruction. After the first 2 weeks ofNO2-exposure, the EF50 values decreased compared tobaseline levels, however, the difference did not reachstatistical significance. The effect was more pronounced

0

34

36

38

40

42

Air NO2

ML

I [µ

m

ARTICLE IN PRESS

**

2

3

4

5

6

0 5 10 15 20 25

NO2 exposure [d]

Mid

exp

irat

ory

airf

low

EF

50 [

ml/s

] AirNO2

Air

NO2

Time

B

A

Fig. 5. Decreased midexpiratory airflow (EF50) in NO2-

exposed mice. (A) Time course of the midexpiratory airflow

(EF50) demonstrates a continuous decrease in the EF50 value in

NO2-exposed mice (black squares) in comparison to air-

exposed controls (white squares). EF50 was measured by head-

out body-plethysmography at the indicated time points before

starting the daily NO2-exposure. Mean7SEM is presented for

both groups with 16 mice in each group. Statistical significance

is indicated by an asterisk with pp0:05 (*). (B) Representative

oscilloscopic tracings from plethysmograph airflow (VD)

signals (recorded at 40 mm/s) of an untreated control and an

NO2-exposed mouse demonstrate a flattening of the expiratory

airflow curve in the NO2-exposed animal.

Table 3. Breathing pattern of C57BL/6 mice after 25 days

NO2 exposure

Air NO2

Time of inspiration (ms) 6373 6572

Time of expiration (ms) 11973 14175*

Breathing frequency (bpm) 29475 26876*

Tidal volume (ml) 0.23270.017 0.19070.014*

Mid-Expiratory airflow (ml/s) 4.55970.303 3.32370.271**

Airflow was measured in mice exposed to 20 ppm NO2 or room air for

25 days as described in Material and methods. Results represent

mean7SEM for each group (n ¼ 16). Statistical significance is

indicated by asterisks with pp0:05 (*) or pp0:01 (**).

M. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350 347

over the following weeks reaching the maximal extentafter 4 weeks of exposure indicating a progressiveimpairment of airway function. At this time point, the20 ppm NO2-exposed group showed a 20% decrease ofthe EF50 value (Fig. 5A) and characteristic changes inthe breathing pattern (Fig. 5B) as compared to theuntreated control group. In accordance to this observa-tion, breathing frequencies and tidal volumes were also

significantly decreased, whereas the time of expirationwas significantly elevated in these animals (Table 3).

Discussion

It is widely accepted that cigarette smoking is the mostimportant risk factor for the development of COPD(Gold, 2003; Loddenkemper et al., 2004). Cigarettesmoke comprises a complex mixture of particular andgaseous compounds and so far it is unknown which of thecomponents are most important for the induction of thedisease. However, smoke derived oxidants are thought toplay a crucial role in this process (Pryor and Stone, 1983;Repine et al., 1997). It is known that the gas phase of onecigarette puff contains up to 1015 organic, short-lived andhighly reactive carbon- and/or nitrogen containingradicals. In addition, the concentration of nitrogen oxidesmay reach levels of 500 ppm (Repine et al., 1997). Thus,the exposure of laboratory animals to atmospherescontaining oxides of nitrogen such as NO2 may serve asa useful model to induce inflammatory reactions andsubsequent histomorphological and physiologicalchanges of the lung that show certain similarities to thesituation in COPD patients (Blank et al., 1998; Glasgowet al., 1987). There is no doubt that multiple interactingmechanisms and pathways are involved in the initiationand progression of pathological changes that finally leadto the complex phenotype of this disease. However, aclear relationship between persistent inflammation, em-physema and development of airflow obstruction stillrequires to be established. Therefore, animal models areneeded that may provide further information to increasethe understanding of the underlying pathomechanisms(Croxton et al., 2002; Fehrenbach, 2002). The presentstudy was carried out to determine if an extendedexposure to relatively high concentrations of NO2

(20 ppm) may induce such complex changes. The mainfindings of this study are (i) development of persistent

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350348

airway inflammation with a marked influx of neutrophilsand macrophages; (ii) goblet cell hyperplasia, which isindicative for mucus hypersecretion, in the upper airways;(iii) focal parenchymal inflammation associated withairspace enlargement; and (iv) progressive airflow ob-struction that all represent hallmarks of human COPD.

The present study was performed using C57BL/6 micefor several reasons. An important feature of thismouse strain seems to be its relative deficiency in a1-antitrypsin serum levels as compared to other strains(Gardi et al., 1994). Based on the concept that aproteinase–anti-proteinase imbalance is crucial for thedevelopment of emphysema in COPD (Barnes, 2003;Shapiro, 2000; Shapiro, 2002), this feature may result ina higher susceptibility of the C57BL/6 strain for thedevelopment of emphysema in response to oxidativeagents. In addition, there is a number of naturallyoccurring mutant mouse strains that develop airspaceenlargement and emphysema. These include tight skin,beige, blotchy and the pallid mouse (reviewed inMahadeva and Shapiro, 2002). All of these strains arebased on the C57BL/6 background. There are othermodels in which the deletion of specific genes resulted inthe development of airspace enlargement, mainly due toa failure in alveogenesis. These models include deletionof platelet derived growth factor A, FGF receptors 3and 4, fibulin 5, elastin, retinoid receptor gamma,surfactant protein B and many others (Bostrom et al.,1996; Weinstein et al., 1998; Wendel et al., 2000). Again,all of these strains were generated on the C57BL/6background. Finally, the model of NO2 exposure hasbeen extensively studied in rats (Barth et al., 1994, 1995;Barth and Mueller, 1999; Chitano et al., 1996; Glasgowet al., 1987; Mueller et al., 2001). However, mice presentmany advantages over rats including molecular geneticengineering and the availability of reagents.

A number of different agents have been used inanimals to induce inflammation and lung injury. Re-peated endotoxin administration leads to neutrophil andmacrophage recruitment into the airways associated withairspace enlargement (Wittels et al., 1974). Otheroxidants, e.g. ozone also cause lung injury. It has beenpreviously shown that long-term administration of NO2

results in mid focal emphysema, whereas ozone exposureresults in fibrosis (Chitano et al., 1995). Cigarette smokeinduced emphysema represents another important experi-mental model. Although this model is undoubtedly themost relevant to human disease, the extended timenecessary to induce changes in the airways and lungparenchyma, most importantly development of emphy-sema, limits its usefulness for a standardized evaluationof pathophysiological processes.

NO2 exposure for 25 days caused a marked influx ofinflammatory cells into the airway lumen and alveoli butalso airway mucosa and lung parenchyma. This infiltra-tion is dominated by macrophages and neutrophils,

which is a consistent finding not only in NO2-exposedmice (Ranga and Kleinerman, 1981; Suzuki et al., 1982),but also in rats and hamsters. Our findings of goblet cellhyperplasia and the focal parenchymal inflammationassociated with signs of developing fibrosis represent animportant extension of previous findings (Holroyd et al.,1997; Johnston et al., 2000; Richters and Damji, 1990). Itis important to note that airway and lung anatomy differsbetween mice and humans in several aspects. Althoughmice are obligatory nose breathers, they lack extensivecilia and occurrence of submucosal glands is limited tothe trachea. Furthermore, mice have less extensive airwaybranching and lack respiratory bronchioles. It is thereforenot surprising that epithelial metaplasia and mucushypersecretion was predominantly observed in the upperairways of mice following NO2 exposure.

Development of emphysema represents an importanthallmark in human COPD. Emphysema is defined byairspace enlargement accompanied by the destruction ofthe alveolar walls. After NO2 exposure, our micedemonstrated an airspace enlargement as demonstratedby the increased MLI in the NO2 exposure group. Thesechanges were accompanied by an elevation of thealveolar volumes in these mice. However, the alveolarsurface area, a parameter that gives reliable informationon the extent of alveolar wall destruction (Heemskerk-Gerritsen et al., 1996), was not altered. Taken together,these data indicate the onset of emphysema-likechanges, which together with a persistent inflammationmay ultimately result in pulmonary emphysema. Wehypothesize that NO2 exposure caused inflammatorycell recruitment into airways and lungs, associated withactivation and release of proteinases which lead to thedamage of retraction forces in the alveolar tissue.Further studies are required, however, to investigatethe link between inflammation and development ofairspace enlargement in this model.

So far, only a few physiological studies have beenconducted that explore the relationship between airflowobstruction and structural changes in the bronchioles. Inour approach we used the method of head-out body-plethysmography to analyze influences of the NO2

exposure on parameters of lung physiology. Thismethod has the advantage of continuous recording ofairflow signals in spontaneously breathing non-anesthe-tized mice (Braun et al., 2004; Hantos and Brusasco,2002). Furthermore, individual mice can be studied in aprospective fashion. We could demonstrate that mor-phological changes in lungs of NO2-exposed mice wereassociated with a progressive decline in expiratoryairflow. This resulted in a continuous decrease inbreathing frequency due to prolonged expiration time.It is likely that these changes in lung function areassociated with and related to the type and degree ofairway inflammation and the observed increase in mucusproduction.

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350 349

In conclusion, we can state that the model of NO2

exposure of mice resembles several features that areamong the most important characteristics of humanCOPD. These features comprise a significant inflamma-tory response in the airways that include goblet cellhyperplasia and increased mucus production. Thesechanges are indicative for the development of bronchitis,an important clinical feature of COPD. In addition, weobserved inflammatory changes in the lung parenchymafinally leading to the development of emphysema-likelesions, another important characteristic in humanCOPD. Finally, NO2-induced inflammatory and histo-pathological changes are associated with a progressiveloss in lung function. Actually, impaired lung functioncomprises the most important parameter for the diag-nosis of COPD. It is important to note that the NO2

exposure model remains a model with several advantagesand drawbacks (e.g. differences in airway and lunganatomy and physiology of mice and human). The mostimportant advantage of the model is, however, that asingle well-defined agent is able to induce importantfeatures of human COPD in a rather short time. Thus,the model seems to be suitable to investigate pathome-chanisms that are in involved in the onset and earlyperpetuation of the disease that finally lead to lungdamage and altered lung function. In addition, the modelmay be utilized to study the relationship between airwayinflammation, epithelial damage, goblet cell hyperplasiaas well as the relationship between development ofairspace enlargement and progressive airflow limitation.

Acknowledgments

The study was funded by the German Ministry forEducation and Research (BMBF), Grant number01GC0103.

References

Barnes PJ. New concepts in chronic obstructive pulmonary

disease. Annu Rev Med 2003;54:113–29.

Barth PJ, Mueller B. Effects of nitrogen dioxide exposure on

Clara cell proliferation and morphology. Pathol Res Pract

1999;195:487–93.

Barth PJ, Mueller B, Wagner U, et al. Quantitative analysis of

parenchymal and vascular alterations in NO2-induced lung

injury in rats. Eur Respir J 1995;8:1115–21.

Barth PJ, Uhlarik S, Bittinger A, et al. Diffuse alveolar

damage in the rat lung after short and long term exposure

to nitrogen dioxide. Pathol Res Pract 1994;190:33–41.

Blanco LN, Massaro GD, Massaro D. Alveolar dimensions

and number: developmental and hormonal regulation. Am

J Physiol 1989;257:L240–7.

Blank J, Glasgow JE, Pietra GG, et al. Nitrogen-dioxide-

induced emphysema in rats. Lack of worsening by beta-

aminopropionitrile treatment. Am Rev Respir Dis

1998;137:376–9.

Bostrom H, Willetts K, Pekny M, et al. PDGF—a signaling is

a critical event in lung alveolar myofibroblast development

and alveogenesis. Cell 1996;85:863–73.

Braun A, Lommatsch M, Neuhaus-Steinmetz U, et al. Brain-

derived neurotrophic factor (BDNF) contributes to neuro-

nal dysfunction in a model of allergic airway inflammation.

Br J Pharmacol 2004;141:431–40.

Cavarra E, Bartalesi B, Lucattelli M, et al. Effects of cigarette

smoke in mice with different levels of alpha(1)-proteinase

inhibitor and sensitivity to oxidants. Am J Respir Crit Care

Med 2001;164:886–90.

Chitano P, Hosselet JJ, Mapp CE, et al. Effect of oxidant air

pollutants on the respiratory system: insights from experi-

mental animal research. Eur Respir J 1995;8:1357–71.

Chitano P, Rado V, DI Stefano A, et al. Effect of subchronic

in vivo exposure to nitrogen dioxide on lung tissue

inflammation, airway microvascular leakage, and in vitro

bronchial muscle responsiveness in rats. Occup Environ

Med 1996;53:379–86.

Croxton TL, Weinmann GG, Senior RM, et al. Future

research directions in chronic obstructive pulmonary

disease. Am J Respir Crit Care Med 2002;165:838–44.

Croxton TL, Weinmann GG, Senior RM, et al. Clinical

research in chronic obstructive pulmonary disease: needs

and opportunities. Am J Respir Crit Care Med 2003;167:

1142–9.

Di Stefano A, Turato G, Maestrelli P, et al. Airflow limitation

in chronic bronchitis is associated with T-lymphocyte and

macrophage infiltration of the bronchial mucosa. Am J

Respir Crit Care Med 1996;153:629–32.

Fabbri LM, Hurd SS. Global strategy for the diagnosis,

management and prevention of COPD: 2003 update. Eur

Respir J 2003;22:1–2.

Fehrenbach A, Ochs M, Wittwer T, et al. Stereological

estimation of the volume weighted mean volumes of alveoli

and acinar pathways in the rat lung to characterise

alterations after ischaemia/reperfusion. J Anat 1999;194:

127–35.

Fehrenbach H. Animal models of chronic obstructive

pulmonary disease: some critical remarks. Pathobiology

2002;70:277–83.

Fisk DE, Kuhn C. Emphysema-like changes in the lungs of the

blotchy mouse. Am Rev Respir Dis 1976;113:787–97.

Gardi C, Cavarra E, Calzoni P, et al. Neutrophil lysosomal

dysfunctions in mutant C57 Bl/6J mice: interstrain varia-

tions in content of lysosomal elastase, cathepsin G and their

inhibitors. Biochem J 1994;299(Pt 1):237–45.

Glasgow JE, Pietra GG, Abrams WR, et al. Neutrophil

recruitment and degranulation during induction of emphy-

sema in the rat by nitrogen dioxide. Am Rev Respir Dis

1987;135:1129–36.

Global Initiative for Chronic Obstructive Pulmonary Disease

(GOLD). Global Strategy for the diagnosis, management,

and prevention of chronic obstructive pulmonary disease—

executive summary updated 2003.

Green MC, Sweet HO, Bunker LE. Tight-skin, a new mutation

of the mouse causing excessive growth of connective tissue

and skeleton. Am J Pathol 1976;82:493–512.

ARTICLE IN PRESSM. Wegmann et al. / Experimental and Toxicologic Pathology 56 (2005) 341–350350

Gundersen HJ, Jensen EB. Stereological estimation of the

volume-weighted mean volume of arbitrary particles

observed on random sections. J Microsc 1985;138:127–42.

Hantos Z, Brusasco V. Assessment of respiratory mechanics in

small animals: the simpler the better? J Appl Physiol

2002;93:1196–7.

Heemskerk-Gerritsen BA, Dijkman JH, et al. Stereological

methods: a new approach in the assessment of pulmonary

emphysema. Microsc Res Tech 1996;34:556–62.

Herz U, Braun A, Ruckert R, Renz H. Various immunological

phenotypes are associated with increased airway respon-

siveness. Clin Exp Allergy 1998;28:625–34.

Holroyd KJ, Eleff SM, Zhang LY, et al. Genetic modeling of

susceptibility to nitrogen dioxide-induced lung injury in

mice. Am J Physiol 1997;273:L595–602.

Howard CV, Reed MG. Unbiased stereology. Three-dimen-

sional measurement in microscopy. Oxford: BIOS Scientific

Publishers; 1998.

Hurd S, Pauwels R. Global initiative for chronic obstructive

lung diseases (GOLD). Pulm Pharmacol Ther 2002;15:

353–5.

Johnston CJ, Reed CK, Avissar NE, et al. Antioxidant and

inflammatory response after acute nitrogen dioxide and

ozone exposures in C57Bl/6 mice. Inhal Toxicol 2000;12:

187–203.

Kleinerman J, Ip MP. Effects of nitrogen dioxide on elastin

and collagen contents of lung. Arch Environ Health 1979;

34:228–32.

Ladekarl M. The influence of tissue processing on quantitative

histopathology in breast cancer. J Microsc 1994;174:

93–100.

Loddenkemper R, Gibson GJ, Sibille Y. Chronic obstructive

pulmonary disease. In: European lung white book. Lau-

sanne (CH): European Respiratory Society; 2004. p. 34–43.

Mahadeva R, Shapiro SD. Chronic obstructive pulmonary

disease � 3: Experimental animal models of pulmonary

emphysema. Thorax 2002 Oct;57(10):908–14 Review.

Malkusch W, Rehn B, Bruch J. Advantages of Sirius Red

staining for quantitative morphometric collagen measure-

ments in lungs. Exp Lung Res 1995;21:67–77.

Martorana PA, Brand T, Gardi C, et al. The pallid mouse. A

model of genetic alpha 1-antitrypsin deficiency. Lab Invest

1993;68:233–41.

Massaro GD, Massaro D. Formation of alveoli in rats:

postnatal effect of prenatal dexamethasone. Am J Physiol

1992;263:L37–41.

Mueller B, Oske M, Hochscheid R, et al. Effect of N-

acetylcysteine treatment on NO2-impaired type II pneumo-

cyte surfactant metabolism. Eur J Clin Invest 2001;31:

179–88.

Neuhaus-Steinmetz U, Glaab T, Daser A, et al. Sequential

development of airway hyperresponsiveness and acute

airway obstruction in a mouse model of allergic inflamma-

tion. Int Arch Allergy Immunol 2000;121:57–67.

Pryor WA, Stone K. Oxidants in cigarette smoke. Radicals,

hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann

N Y Acad Sci 1983;686:12–27.

Ranga V, Kleinerman J. Lung injury and repair in the blotchy

mouse. Effects of nitrogen dioxide inhalation. Am Rev

Respir Dis 1981;123:90–7.

Repine JE, Bast A, Lankhorst I. Oxidative stress in

chronic obstructive pulmonary disease. Oxidative Stress

Study Group. Am J Respir Crit Care Med 1997;156:

341–57.

Richters A, Damji KS. The relationship between inhalation of

nitrogen dioxide, the immune system, and progression of a

spontaneously occurring lymphoma in AKR mice. J

Environ Pathol Toxicol Oncol 1990;10:225–30.

Saetta M, Di Stefano A, Turato G, et al. CD8+ T-

lymphocytes in peripheral airways of smokers with chronic

obstructive pulmonary disease. Am J Respir Crit Care Med

1998;157:822–6.

Saetta M, Turato G, Baraldo S, et al. Goblet cell hyperplasia

and epithelial inflammation in peripheral airways of

smokers with both symptoms of chronic bronchitis and

chronic airflow limitation. Am J Respir Crit Care Med

2000;161:1016–21.

Scherle W. A simple method for volumetry of organs in

quantitative stereology. Mikroskopie 1970;26:57–60.

Shapiro SD. Evolving concepts in the pathogenesis of chronic

obstructive pulmonary disease. Clin Chest Med 2000;21:

621–32.

Shapiro SD. Proteinases in chronic obstructive pulmonary

disease. Biochem Soc Trans 2002;30:98–102.

Stanescu D, Sanna A, Veriter C, et al. Identification of

smokers susceptible to development of chronic airflow

limitation: a 13-year follow-up. Chest 1998;114:416–25.

Stang P, Lydick E, Silberman C, et al. The prevalence of

COPD: using smoking rates to estimate disease frequency

in the general population. Chest 2000;117:354S–9S.

Stephens RJ, Evans MJ, Sloan MF, et al. A comprehensive

ultrastructural study of pulmonary injury and repair in the

rat resulting from exposures to less than one PPM ozone.

Chest 1974;65(Suppl-13S).

Suzuki AK, Tsubone H, Sagai M, et al. Changes of gaseous

exchange in the lungs of mice exposed to nitrogen dioxide

and their recovery process. Toxicol Lett 1982;13:71–9.

Wang Z, Zheng T, Zhu Z, et al. Interferon gamma induction of

pulmonary emphysema in the adult murine lung. J Exp

Med 2000;192:1587–600.

Weinstein M, Xu X, Ohyama K, et al. FGFR-3 and FGFR-4

function cooperatively to direct alveogenesis in the murine

lung. Development 1998;125:3615–23.

Wendel DP, Taylor DG, Albertine KH, et al. Impaired distal

airway development in mice lacking elastin. Am J Respir

Cell Mol Biol 2000;23:320–6.

Wert SE, Yoshida M, Levine AM, et al. Increased metallo-

proteinase activity, oxidant production, and emphysema in

surfactant protein D gene-inactivated mice. Proc Natl Acad

Sci USA 2000;97:5972–7.

Wittels EH, Coalson JJ, Welch MH, et al. Pulmonary

intravascular leukocyte sequestration. A potential mechan-

ism of lung injury. Am Rev Respir Dis 1974;109:502–9.

Wright JL, Churg A. Cigarette smoke causes physiologic and

morphologic changes of emphysema in the guinea pig. Am

Rev Respir Dis 1990;142:1422–8.

Zheng T, Zhu Z, Wang Z, et al. Inducible targeting of IL-13 to

the adult lung causes matrix metalloproteinase- and

cathepsin-dependent emphysema. J Clin Invest 2000;106:

1081–93.