Animal models of airway remodeling in asthma - Els Palmans…

GHENT UNIVERSITY HOSPITAL DEPARTMENT OF RESPIRATORY DISEASES

DIRECTOR: Prof. Dr. Romain A. Pauwels

Animal Models of Airway Remodeling in Asthma

Els Palmans

Promotor: Prof. Dr. Johan C. Kips Co-Promotor: Prof. Dr. Romain A. Pauwels

Thesis submitted as partial fulfillment of the requirements for the degree of Doctor in Medical Sciences

DANKWOORD

Merelbeke, 3 juli 2002

Mijn proefschrift is dan toch afgeraakt na een hele reeks experimenten en heel veel

schrijfwerk. Ik zou deze thesis echter nooit hebben kunnen presenteren zonder de hulp en

steun die ik heb gekregen van mijn promotoren, mijn collega’s, mijn vrienden en mijn familie

die ik bij aanvang van dit werk dan ook heel, héél hartelijk bedanken.

In de eerste plaats bedank ik mijn promotor Prof. Dr. Johan Kips voor de aangename

samenwerking gedurende deze 6 jaar. Hij heeft mij enorm geholpen om mij in te werken in dit

specifieke astma onderzoeksdomein en bij het leggen van verbanden tussen de resultaten uit

de dierenproeven en humaan astma. Met veel geduld heeft hij me geleerd mijn vaak ietwat

verwarde gedachtensprongen te ordenen. Ik dank hem ook voor de vele praktische tips en

inhoudelijke suggesties bij het schrijven van mijn manuscripten en dit proefschrift en omdat

hij, ondanks zijn eigen drukke agenda, veel tijd uittrok voor het nalezen van mijn teksten.

Mijn co-promotor Prof. Dr. Romain Pauwels dank ik voor de mogelijkheden die ik heb

gekregen op de dienst Longziekten, voor de vrijheid die ik had bij het uitvoeren van het

experimenteel werk en voor het feit dat ik kon deelnemen aan verschillende internationale

congressen en specifieke workshops. Ik dank hem voor de suggesties voor mijn onderzoek

en voor het uitschrijven van het proefschrift. Ik dank hem eveneens voor het nalezen van dit

proefschrift.

Een collega die ik in het bijzonder wil bedanken is Nele Vanacker. Een deel van het werk

dat ik hier vandaag presenteer, heb ik samen met haar uitgevoerd. Ik dank haar voor onze

leuke samenwerking en voor haar bemoedigende woorden op momenten dat het niet te best

lukte. Ik wens haar veel succes met haar thesisverdediging in het najaar, het allerbeste met haar

nieuwe job en de gezinsuitbreiding.

Alle laboranten die me geholpen hebben bij het vele praktische werk, wil ik ook extra in

de bloemen zetten. Eliane Castrique, Christelle Snauwaert, Kathleen De Saedeleer en Greet

Barbier dank ik voor de zorg van de proefdieren, voor de vele verstuivingen en voor het

‘prepareren’ van de ratten bij de longfunctiemetingen. Geert Van der Reysen hielp me bij de

‘buxco’-metingen en Marie-Rose Mouton met de ELISA-testen. Ann Neessen en Indra De

Borle zorgden voor de vele fijne weefselcoupes en voor de histologische en

immunohistochemische kleuringen.

Prof. Dr. Eric Veys wil ik bedanken voor het ter beschikking stellen van het LEICA

beeldverwerkingssysteem op de dienst Reumatologie zodat ik de vele analyses op de

rattenluchtwegen heb kunnen uitvoeren. Dominique Baeten dank ik voor de hulp bij het

gebruik van het systeem.

Alle ‘fellows’ met wie ik heb samengewerkt en heb samen gezeten in K12 en Blok B wil

ik bedanken, niet alleen voor hun interesse in het onderzoek, maar ook voor hun vriendschap

en voor de aanvoer van vaak veel te veel chocolade. Ik dank de ‘fellows-van-het-eerste-uur’

Veerle Van de Velde, Bart Lambrecht en Paul Germonpré; de ‘fellow-moedertjes’ Ines Carro-

Muino, Katy De Swert, Vanessa Schelfhout en Katia Mekeirele; de ‘computer-specialist-

helper-bij-figuren-fellow’ Karim Vermaelen; de rest van ‘Blok B-fellows’ Pieter Depuydt, Kurt

Tournoy, An D’hulst, Barbara Legiest en Ken Braecke; en de ‘nieuwe fellows’ Katrien

Moereloose, Tania Maes en Lander Robays.

De overige stafleden van het dienst Longziekten Prof. Dr. Guy Joos, Prof. Dr. Renaat

Peleman, Prof. Dr. Dirk Pevernagie en Prof. Dr. Eric Derom dank ik voor de interesse in mijn

werk. I thank Prof. Dr. Gillian Bullock to help me to get started with the histology and the

immunohistochemistry. Alle medewerkers van de receptie, het secretariaat, het

longfunctielabo, het computerlokaal en de verpleegkundigen dank ik voor de ontspannende

momenten en de praatjes tussendoor.

Verder zijn er een hele reeks vrienden die op de één of andere manier ook hun steentje

hebben bijgedragen waarvoor ik hen dan ook heel hartelijk bedank.

Mijn ouders dank ik voor hun steun, hun interesse, hun geduld en hun hulp, niet alleen

gedurende de periode dat ik aan dit proefschrift heb gewerkt, maar ook voordien tijdens mijn

studies. Naast mijn ouders wil ik ook mijn zus Anneke en mijn broer Hugo bedanken voor

hun steun en dank ik ook Denise, Hugo, Bert en Mila voor hun interesse en hun hulp

gedurende de laatste jaren.

Als laatste wil ik Steven bedanken voor al het geduld dat hij heeft gehad gedurende deze

lange periode waaraan maar geen einde leek te komen. Hij is er altijd geweest, hetzij thuis, hetzij

via de telefoon. Hij heeft me aangemoedigd om vol te houden. Hij heeft er vaak voor gezorgd

dat ik tijdens de weekends thuis ongestoord kon verder werken, zonder dat onze twee lieve

kindjes Mieke en Jan meehielpen aan de computer bij het ingeven van de teksten. Ons Mieke

zal ook blij zijn dat mama’s ‘doctoraat’ af is; deze zomer gaan we eindelijk naar Disneyland-

Parijs.

Els

TABLE OF CONTENTS

List of Abbreviations i Summary iii Samenvatting iv Résumé vii

Part I: Introduction 1

Review of the literature 3 Introduction 5 Histopathology of the asthmatic airway 6

Description of chronic structural alterations 6 Airway wall thickening 13

Pathogenensis of airway remodeling 15 The inflammatory basis of asthma 15 Inflammatory and structural cells involved in remodeling 16 Interdependence of inflammation and remodeling 20

Functional consequences of airway remodeling 21 Non-specific bronchial hyperresonsiveness 21 Irreversible component of the airway obstruction 26

In vivo experimental models of airway remodeling 27 Rat models of airway remodeling 28 Mouse models of airway remodeling 30

Conclusion 32 References 32

Aims of the studies 51

Part II: Publications 55

Details on methodology used in the experimental models 57 Respiratory measurements 59

Respiratory measurements in rats 59 Assessment of airway function in mice 61

Morphometric measurements 63 Nomenclature for quantifying subdivisions of the bronchial wall 64 Morphometric analysis of airways in rats and mice 65 Assessment of extracellular matrix deposition in the airway wall 65

References 69

List of publications 71 Prolonged allergen exposure induces structural airway changes in sensitized rats 73 Repeated allergen exposure changes collagen composition in airways of sensitized Brown Norway rats 101 The effect of repeated smooth muscle contractions on airway structure of Brown Norway rats 119 Allergen-induced inflammatory and structural airway changes in two inbred mouse strains 131 Effect of age on allergen-induced structural airway changes in Brown Norway rats 149 Is there an animal model of airway remodeling? 165

Part III: General Discussion 177

Summary 179 Future Perspectives 182

Abbreviations i

LIST OF ABBREVIATIONS Abm: area enclosed by the epithelial basement membrane AHR: airway hyperresponsiveness Amo: area enclosed by the outer muscle (outer border of airway smooth muscle) ANOVA: analysis of variance Ao: area enclosed by the outer border of the adventitia BAL: bronchoalveolar lavage BALF: bronchoalveolar lavage fluid BHR: bronchial hyperresponsiveness BM: basement membrane BN: Brown Norway BrdU: bromodeoxyuridine CD: cluster of differentiation CysLT: cysteinyl leukotriene DAB: 3,3’-diaminobenzidine ECM: extracellular matrix EDTA: ethylenediaminetetraacetic acid EGF: epidermal growth factor ELISA: enzyme-linked immunosorbent assay FEV1: forced expiratory volume in one second FP: fluticasone propionate GM-CSF : granulocyte-monocyte colony stimulating factor H&E: hematoxylin and eo sin HBSS: Hank’s balanced salt solution HRCT: high-resolution computed tomography i.p.: intraperitoneal ID: internal diameter Ig: immunoglobulin IGF: insulin-like growth factor IL: interleukin LSD: least significant difference LT: leukotriene MMPs: matrix metalloproteinases NRS: normal rabit serum NSS: normal sheep serum OA: ovalbumin PAF: platelet activating factor PAS: periodic acid shiff Pbm: perimeter basement membrane PBS: phosphate-buffered saline PC20FEV1: provocative concentration causing a 20% fall in FEV1 from baseline

Abbreviations ii

PC50RL: provocative concentration causing a 50% increase in baseline RL PC100RL: provocative concentration causing a 100% increase in baseline RL PC250Penh: provocative concentration causing a 250% increase in baseline Penh PD200RL: provocative dose required to double baseline resistance pCO2 : arterial carbon dioxide tension PDGF-ß: platelet derived growth factor-ß Penh: enhanced pause PEP: peak expiratory pressure PIP: peak inspiratory pressure Pmo: perimeter of te outer boundary of the smooth muscle Po: outer adventitial perimeter pO2: arterial oxygen tension Ppt : transpulmonary pressure RL: lung resistance rs : Spearman’s rank correlation coefficient SEM: standard error of the mean TBS: tris-buffered saline Te: expiratory time TGF-ß: transforming growth factor-ß Th: T helper TIMP: tissue inhibitor of metalloproteinase TMA: trimellitic anhydride TNF-a : tumor necrosis factor-a Tr: relaxation time vs: versus VT: tidal volume WAi: wall area of the inner airway wall between basement membrane and outer

boundary of the smooth muscle WAo: wall area of the outer airway wall between outer boundary of the smooth

muscle and the outer adventitia WAt: wall area of the whole airway wall between basement membrane and outer

adventitia WC: wall area occupied by collagen WF: wall area occupied by fibronectin wk: weeks

Summary iii

SUMMARY Asthmatic airways display characteristic morphological alterations, part of which are

structural changes, coined airway remodeling. These structural alterations include goblet-cell

hyperplasia, increased subepithelial deposition of collagen, changes in extracellular matrix

composition, neovascularization and smooth-muscle cell hyperplasia and/or hypertrophy.

The mechanisms that regulate the remodeling process as well as its functional consequences

remain to be fully elucidated. It has been postulated that airway remodeling results from an

inappropriate regeneration process in an attempt at tissue repair following repetitive bouts of

allergen-induced acute inflammatory changes in the airway wall. The functional consequences

of remodeling have mainly been related to lung function. In particular, mathematical models

predict that remodeling largely contributes to the development of bronchial

hyperresponsiveness, the functional hallmark of asthma. Acquiring further insight in the exact

pathophysiology of remodeling therefore seems important, for further optimizing asthma

treatment.

Human studies are often hampered by the limited accessibility to intrapulmonary airways.

Bronchial biopsies display only a part of the airway mucosa and do not allow a proper

assessment of the entire remodeling process throughout the airway wall. Relevant animal

models can contribute significantly to a better understanding of the underlying processes and

its functional consequences. Most animal models developed to date do not properly mimic

aspects of airway remodeling. In this study an in vivo model for allergen-induced structural

airway changes is described in rats and mice.

Sensitization of the animals to the allergen ovalbumin followed by a prolonged allergen

exposure period, resulted in both species in goblet cell hyperplasia, changes in extracellular

matrix proteins and increased airway wall thickness. These structural alterations bear

similarities to those observed in human asthma. Strikingly, for a same intensity of allergen

exposure, young animals develop more intense structural alterations when compared to adult

rats. An important observation, when relating these morphological alterations to lung function

indices, is that depending on the extent and distribution of the structural changes, the allergen

induced airway remodeling can oppose, rather than enhance bronchial responsiveness. These

findings underline the importance of linking morphological observations to functional

measurements, when evaluating the role of remodeling in asthma.

Summary iv

The models that we have developed should provide relevant data for a better

understanding of the pathogenesis of remodeling, in relation to airway behavior.

Summary v

SAMENVATTING Luchtwegen van astma patiënten vertonen een aantal karakteristieke morfologische

veranderingen waaronder de structurele veranderingen of ‘remodeling’. Deze omvatten onder

meer een verhoging van het aantal slijmbekercellen in het epitheel, een verhoging van collageen

depositie onder het epitheel en een verandering in de samenstelling van de extracellulaire matrix

van de luchtwegen, een toegenomen hoeveelheid gladde spiercelmassa en neovascularisatie.

Hoe deze structurele veranderingen in stand worden gehouden in de luchtwegen en wat de

functionele gevolgen ervan zijn, is echter nog niet volledig opgehelderd. Er wordt

verondersteld dat bij astmatici de luchtwegen veranderingen kunnen vertonen als gevolg van

herhaalde inflammatie na een allergeen blootstelling. Op basis van mathematische modellen

gaat men ervan uit dat remodeling de bronchiale hyperreactiviteit, de voornaamste

longfunctionele afwijking bij astma patiënten, in de hand werkt. De volledige pathofysiologie

van deze structurele veranderingen is echter onduidelijk. Nochtans zou een beter inzicht in de

relatie tussen remodeling en longfunctie kunnen leiden tot een betere therapeutische

behandeling voor astma.

Een belangrijk probleem bij humane studies is echter de beperkte toegankelijkheid tot

representatief luchtwegen weefsel. Endobronchiale biopten genomen in de centrale

luchtwegen zijn meestal beperkt tot mucosaal weefsel en het is niet duidelijk of de remodeling

hierin representatief is voor de ganse luchtwegwand. Representatieve in vivo dierenmodellen

kunnen bijgevolg een positieve bijdrage leveren aan het onderzoek van remodeling in

luchtwegen bij astma. Aangezien de meeste dierenmodellen in astma het aspect van de

structurele veranderingen onvoldoende benaderen, was het doel van dit werk om een model

voor allergeen geïnduceerde structurele luchtwegveranderingen te ontwikkelen in ratten en

muizen.

Het sensibiliseren van de proefdieren met het allergeen ovalbumine gevolgd door een

herhaalde allergeen blootstelling resulteerde in een toegenomen aantal slijmbekercellen, een

verhoogde afzet van extracellulaire matrix eiwitten in de luchtwegen en een verdikking van de

wand. Deze veranderingen vertonen gelijkenissen met remodeling van luchtwegen in humaan

astma. Bovendien bleek dat in vergelijking met volwassen dieren, jonge ratten voor een zelfde

graad van allergeen geïnduceerde inflammatie, een meer uitgesproken remodeling vertonen.

Opvallend was ook dat in ons model afhankelijk van de exacte locatie en uitgebreidheid van

de structuurveranderingen in de wand, deze kunnen beschermen tegen bronchiale

Summary vi

hyperreactiviteit in plaats van deze in de hand te werken. Hiermee werd aangetoond dat bij de

studie van luchtweg remodeling in astma, het noodzakelijk is om morfologische observaties te

koppelen aan functionele waarnemingen.

In deze studie werden twee modellen voorgesteld, waarmee verscheidene vragen rond de

pathogenese van remodeling en de functionele gevolgen ervan kunnen benaderd worden.

Summary vii

RÉSUMÉ

Les voies aériennes de patients asthmatiques démontrent certains changements

morphologiques caractéristiques, notamment des modifications d’ordre structurel, connues

sous le terme de « remodeling ». Sous ces changements on distingue entre autres une

augmentation du nombre de cellules « goblet » productrices de mucus, une augmentation de

la déposition sous-épithéliale de fibres collagènes ainsi qu’un changement dans la

composition de la matrice extracellulaire autour des voies aériennes. De surcroît, on observe

une augmentation de la masse musculaire lisse et des signes de néovascularisation font leur

apparition. Toutefois la manière dont ces changements structurels sont maintenus en place et

les conséquences fonctionnelles de ce phénomène n’ont pas été complètement élucidés. On

suppose que les voies aériennes des sujets asthmatiques manifestent de tels changements en

raison de tentatives inappropriées de régénération tissulaire suite à une inflammation chronique

due à l’exposition répétée à un allergène. Certains modèles mathématiques prédisent que l’

hyperréactivité bronchiale – l’anomalie fonctionnelle pulmonaire principale dans l’asthme –

est une conséquence du remodelage de la paroi bronchiale. Toujours est-il que la réelle

pathophysiologie de ces changements structurels reste encore à éclaircir, alors qu’une

meilleure compréhension de la relation entre le « remodeling » et la fonction respiratoire

pourrait mener à un traitement optimalisé de l’asthme.

Malheureusement les études humaines se heurtent aux limitations d’accès à des tissus

bronchiaux représentatifs. Des biopsies endobronchiales prélevées au niveau des voies

aériennes centrales sont le plus souvent limitées au tissu mucosal et il n’est pas démontré que

le remodelage que l’on y observe est représentatif pour toute la paroi bronchiale. Des modèles

animaux in vivo représentatifs peuvent par conséquent fournir une réelle contribution dans la

recherche concernant le remodelage des voies aériennes dans l’asthme. Cependant la plupart

des modèles expérimentaux animaux ne reflètent pas suffisamment les changements

structurels. C’est ainsi que le but du présent travail fût de développer dans le rat et la souris un

modèle adéquat de changements structurels bronchiaux dû à l’exposition à un allergène.

Dans les rongeurs cités, la sensibilisation à l’allergène ovalbumine, suivie d’une période

d’exposition répétée au même allergène résulte en une hyperplasie des « goblet cells », une

déposition accrue de protéines de matrice extracellulaire et un épaississement de la paroi

bronchiale. Toutes ces altérations structurelles se rapprochent des changements observés

dans l’asthme chez l’homme. Qui plus est, pour une même intensité d’exposition à un

Summary viii

allergène, les jeunes rats développent des altérations structurelles plus prononcées que leurs

congénères adultes. Lorsqu’on en vient à rapporter de tels changements histologiques à des

indices de fonction pulmonaire, il est surprenant d’observer qu’en fonction de l’étendue des

changements structurels, le remodelage dû à l’exposition chronique à un allergène peut

amortir plutôt qu’exacerber l’hyperréactivité bronchiale. Ces observations soulignent

l’importance de lier les paramètres morphologiques aux mesures de fonction lorsqu’il s’agit

d’évaluer le rôle du remodelage bronchial dans l’asthme.

Les modèles que nous avons développés devraient fournir des données relevantes en

vue d’une meilleure compréhension de la pathogenèse du remodelage en relation avec le

comportement des voies aériennes.

Introduction 1

PART I

INTRODUCTION

Review of the Literature 3

REVIEW OF THE LITERATURE


1. Introduction

As stated in its definition, asthma is a chronic inflammatory disease of the airways in

which many cell types play a role, in particular mast cells, eosinophils and T lymphocytes

(NHLBI/WHO workshop report, 1995). Persistent inflammation in asthma is considered to

be critical to the presence of symptoms and lung function abnormalities including increased

airflow obstruction and airway hyperresponsiveness. However it is increasingly recognized

that inflammation alone cannot explain all aspects of airway physiology in asthmatics (Haley et

al, 1998). Indeed, although airflow limitation in asthma has traditionally been thought to be

entirely reversible, some patients with asthma have evidence of irreversible airway obstruction

despite appropriate and aggressive anti-inflammatory treatment (Backman et al, 1997).

Similarly, treatment rarely restores bronchial hyperresponsiveness, another characteristic lung

function abnormality in asthma, to within normal limits. In addition it is shown that a number

of patients with asthma have an enhanced decline in lung function over time (Lange et al,

1998; Peat et al, 1987). Histological analysis of airway tissue from asthmatic patients reveals in

addition to acute inflammatory changes, structural abnormalities. These far less reversible

alterations coined airway remodeling are thought to be, at least in part, responsible for the

pulmonary function abnormalities.

At present, there are major gaps in our knowledge of the mechanisms regulating the

remodeling process. It has been postulated that airway remodeling results from a pathologic

regeneration process in an attempt at tissue repair following repetitive bouts of allergen-

induced acute inflammatory changes in the airway wall. Both inflammatory cells and resident

structural tissue cells within the airway wall are thought to contribute to this process. How

exactly airway remodeling is initiated and what causes perpetuation of the process is not

completely understood. Whether there is a mechanistic link between the altered airway

function observed in asthmatics and the structural changes throughout the airway wall also

remains largely unclear. As a result it remains to be established to what extent prevention or

reversal of remodeling should be an aim of asthma treatment. Hence the importance of gaining

more insight in the pathogenesis of this process.

Appropriate and relevant animal models of chronic airway inflammation and tissue

alterations, despite their own inherent shortcomings, can aid in understanding the underlying

processes that contribute to asthma. When developing an animal model it is necessary to

know what should be modeled and what has already been achieved. This review of the


literature attempts therefore to cover the descriptive pathology of airway remodeling in

asthma, describes the possible role of airway inflammation in the initiation and persistence of

this process, tries to link physiologic characteristics of asthmatics with airway remodeling and

briefly describes experimental models of airway remodeling.

2. Histopathology of the asthmatic airway

In the asthmatic airway it is important to differentiate between acute inflammatory and

chronic structural changes. Acute alterations such as cellular infiltration and accumulation of

edema fluid are usually reversible either spontaneously or by treatment. Chronic changes on

the contrary, resolve more slowly or may become irreversible. Airway remodeling can be

found in different compartments of the airway wall including airway epithelium, basement

membrane, the extracellular matrix in inner and outer airway wall, smooth muscle, blood

vessels and mucous glands (figure 1). The alterations might involve an increase in size and

number of the tissue elements, a long-term functional change of airway-resident cells or the

formation of new structures within the airway tissue.

Remodeling is distributed throughout the bronchial tree (Awadh et al, 1998; Okazawa et

al, 1996; Saetta et al, 1991) and is observed in different asthmatic subtypes (atopic, intrinsic

or occupational) (Paganin et al, 1996).

2.1. Description of chronic structural alterations

Epithelium

The surface of the intrapulmonary airways is covered by pseudostratified epithelium that

forms an active barrier between the external environment and the internal milieu. Shedding and

damage of the airway epithelium is a characteristic of asthma. Increased epithelial cell numbers

were found in sputum samples and bronchoalveolar lavage washings of asthmatic compared

to normal subject suggesting a more fragile epithelium in asthmatics (Taha et al, 2001; Chanez

et al, 1999; Fahy et al, 1995; Montefort et al, 1992; Beasley et al, 1989). Deranged and/or

damaged epithelial tight junctions might contribute to this increased epithelial fragility and

weaker cellular attachment of columnar epithelial cells to the basal layer in asthma (Laitinen and

Laitinen, 1994; Montefort et al, 1993; Montefort et al, 1992). Studies using bronchial biopsies


showed epithelial shedding in both fatal and mild asthma (Chanez et al, 1999; Jeffery et al,

1989; Beasley et al, 1989; Laitinen et al, 1985) although others did not find differences

between asthmatics and non-asthmatics (Boulet et al, 1997; Carroll et al, 1993; Lozewicz et al,

1990). In some studies a correlation was found between epithelial damage and in vivo airway

responsiveness (Jeffery et al, 1989; Beasley et al, 1989).

Sub-basement membrane thickening: extracellular matrix proteins in lamina reticularis

Asthmatic airways display an increase in collagen deposition just underneath the

basement membrane. This distinct collagenous layer is thickened in patients with asthma

measuring from 7 to 23 µm versus 4 to 5 µm in normal subject. Initially it was described as

basement membrane thickening (Dunnill, 1960). Now it is confirmed that the basement

membrane containing laminin and collagen type IV is not altered in asthma. In contrast, there

is a substantial deposition of interstitial collagens type I, III and V, fibronectin and tenascin in

the area below the true basement membrane, the lamina reticularis (Laitinen et al, 1997; Altraja

et al, 1996; Roche et al, 1989). Activated bronchial myofibroblasts lying close to the

epithelium are thought to be responsible for the production of these proteins in asthma. Their

numbers are increased and correlate with the extent of collagen deposition (Brewster et al,

1990).

Whether a correlation exists between the thickness of the subepithelial fibrosis and the

severity of asthma is debated. Although some authors found a correlation between the

thickness of the lamina reticularis and the severity of the disease (Niimi et al, 2000c; Chetta et

al, 1997; Boulet et al, 1997), this is not uniformly confirmed (Milanese et al, 2001; Chu et al,

1998; Saetta et al, 1992; Jeffery et al, 1989). Subepithelial-layer thickness does not correlate

with the disease duration nor etiology of asthma (Boulet et al, 2000; Niimi et al, 2000c; Jeffery

et al, 1989).


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Extracellular matrix components of the lamina propria

The extracellular matrix stabilizes the physical structure of tissues and plays a role in

regulating the behavior of cells. In the normal airway the extracellular matrix forms a complex

three-dimensional network which consists of fibrous proteins (collagen and elastin), structural

or adhesive proteins (fibronectin and laminin) embedded in a hydrated polysaccharide gel

containing several proteoglycans and glycosaminoglycans. The deposition of some of these

extracellular matrix proteins is altered in the airway wall of asthmatics.

Collagen is the major structural component of the extracellular matrix (Dunsmore and

Rannels, 1996). Increased amounts of collagen type III, V and VI have been found in the

deeper layer of the submucosa of asthmatic airways (Lee et al, 1999; Wilson and Li, 1997).

Total collagen was increased in the longitudinal bundles present in the submucosa of

conducting airways (Carroll et al, 2000). A higher amount of collagen was also observed

between the basement membrane and the smooth muscle layer in patients with asthma and the

increase was correlated with asthma severity (Minshall et al, 1997). Of note is that these

findings were not invariably confirmed, as others did not observe increased amounts of total

collagen nor increased deposition of collagen type I and III in asthmatic subjects (Chu et al,

1998; Godfrey et al, 1995; Roche et al, 1989).

Elastin is a cross-linked protein that gives tissues their elastic recoil. The network of

elastin fibers is fragmented in asthmatics (Bousquet et al, 1996) but the total amount of elastic

fiber appears to be unchanged (Mauad et al, 1999; Godfrey et al, 1995). The elastin content

of the longitudinal bundle was proportionally decreased in cases of asthma (Carroll et al,

2000). Higher levels of elastase in asthma might cause the increased elastic fiber disruption in

bronchi of asthmatics (Vignola et al, 1998a). Although the level of elastase in sputum was

correlated with the severity of the disease (Vignola et al, 1998a), the alterations in elastic fibers

in the submucosa observed in bronchial biopsies could not be linked to the severity and

duration of asthma (Bousquet et al, 1996).

Specific proteoglycans and glycosaminoglycans of the extracellular matrix influence

tissue biomechanics, fluid balance and cellular function. In the airway wall of patients with

mild atopic and severe asthma excess deposition of decorin, lumican and biglycan, members

of the family of small proteoglycans, and the large proteoglycan versican is observed (Huang

et al, 1999; Roberts, 1995). Hyaluronan is also overexpressed (Vignola et al, 1998b; Roberts,

1995). The levels of hyaluronan in bronchoalveolar lavage fluid were elevated in persistent

asthma compared to intermittent disease or control subjects (Vignola et al, 1998b).


Smooth muscle

The airway smooth muscle layer runs from the trachea to the smallest bronchioles (Ebina

et al, 1990b). Marked thickening of the airway smooth muscle layer is one of the best known

features of asthmatic remodeling (Kuwano et al, 1993; Ebina et al, 1993; Carroll et al, 1993;

Ebina et al, 1990a; James et al, 1989). Some degree of heterogeneity of the smooth muscle

thickening exists. The increased smooth muscle mass is either restricted to larger airways or

more uniformly present along the entire course of the bronchial tree (Ebina et al, 1990a).

When smooth muscle thickening is restricted to larger bronchi, hyperplasia of muscle cells

appeared responsible for the increase. Hypertrophy of individual smooth muscle cells causes

the thickening of smooth muscle over the entire tree (Ebina et al, 1993). Also extracellular

matrix and connective tissue surrounding smooth muscle bundles can contribute substantially

to the smooth muscle area (Thomson et al, 1996). In patients with fatal asthma the increase in

peripheral smooth muscle area was far greater than in asthma patients who died from other

causes (Carroll et al, 1993). Others did not find a correlation with the degree of asthma

severity (Cho et al, 1996).

Blood vessels

In the airway submucosa a dense microvasculature network is embedded in fibrous

tissue. Histological analysis of bronchial biopsies or resected tissue has revealed higher vessel

counts in asthmatics compared to normal controls (Li and Wilson, 1997; Carroll et al, 1997;

Kuwano et al, 1993). In bronchial biopsies the number of blood vessels per area tissue and

the percent biopsy occupied by vessels was increased in patients with mild allergic asthma

(Salvato, 2001; Li and Wilson, 1997). In cross sections of airways in autopsy specimens of

patients who died from or with asthma (Carroll et al, 1997; Kuwano et al, 1993) and in

broncial biopsies from mild-to-moderate asthmatic s (Chu et al, 2001) the vessels that were

present were larger suggesting vascular engorgement (Carroll et al, 1997). Vessels may

increase in number through proliferation of the vessel branches or elongation of the existing

vessels (Wilson and Stewart, 1999). Microvascular enlargement may result from proliferation

of endothelial cells resulting in new vessels that are larger than the original ones (Busse et al,

1999). The number of vessels was correlated with the severity of the disease (Salvato, 2001;

Orsida et al, 1999).


Mucous glands

Mucus secretions that coat the lining of the airway to protect the epithelium are produced

in submucosal glands and in secretory cells of the epithelium. In normal airways, mucous

glands are distributed throughout the cartilaginous airways and goblet cells are the most

prominent secretory cell type in the trachea and large airways. Excessive mucus production is

observed in fatal asthma (Aikawa et al, 1992; Dunnill, 1960). This hypersecretion of mucus

into the asthmatic airway lumen is caused by submucosal gland hypertrophy and/or goblet

cell hyperplasia. Morphometric analysis of airways in fatal asthma revealed an enlarged mass

of mucous gland compared to normal subjects as a result of hypertrophy of the gland cells

(Cho et al, 1996; Carroll et al, 1993; Aikawa et al, 1992). Whether the increase in number of

mucous glands is correlated with asthma severity is debated (Cho et al, 1996; Carroll et al,

1996; Carroll et al, 1993; Aikawa et al, 1992). In the epithelium a higher number of goblet cells

is observed in mild as well as in moderate and severe asthmatics (Ordonez et al, 2001;

Shimura et al, 1996; Laitinen et al, 1993; Aikawa et al, 1992). As a result of this increase in

goblet cell number, mucin stores in the airway epithelium were higher in asthmatic compared

to healthy control subjects (Ordonez et al, 2001).

2.2. Airway wall thickening

These various structural alterations can contribute to the overall thickening of the airway

wall as observed in asthmatics (Kuwano et al, 1993; Carroll et al, 1993; Saetta et al, 1991). In

fatal asthma a thickened airway wall was found in airways of all sizes and the difference was

most pronounced in small airways (Carroll et al, 1993). In non-fatal asthma only the overall

thickness of small airways is increased compared to controls. This increase is smaller

compared to fatal asthma (Kuwano et al, 1993; Carroll et al, 1993). Measurements of airway

wall thickness via high-resolution computed tomography (HRCT) scans confirm the

observations of autopsy studies (Kasahara et al, 2002; Awadh et al, 1998; Paganin et al, 1996;

Okazawa et al, 1996; Boulet et al, 1995). HRCT scanning revealed wall thickening in 16-92%

of the asthmatic patients (Little et al, 2002; Brown et al, 2001; Niimi et al, 2000b; Carr et al,

1998; Park et al, 1997; Paganin et al, 1996). HRCT showed that in mild asthmatics, increases

in airway size are limited to airways under 6 mm, while in severe asthmatics differences can be

seen in all airways except the largest (Awadh et al, 1998; Boulet et al, 1995). Also small

airways (2-4 mm diameter) showed the greatest difference between normal and asthmatics on

HRCT (Okazawa et al, 1996). Kasahara et al (2002) showed that sub-basement membrane


thickness measured in bronchial biopsy specimens correlated with whole airway wall

thickness assessed via HRCT.

Within the airway wall, three areas are defined: the inner wall, the outer wall and the

smooth muscle layer (figure 2). The inner wall comprises the area between the basement

membrane and the outer borders of the airway smooth muscle. The outer airway wall is

limited by the outer boundary of the smooth muscle and the outer adventitia (Bai et al, 1994).

Besides the above described increase in the smooth muscle area an increase in the thickness

of the inner and outer airway wall has been described in both fatal and non-fatal asthma

(Kuwano et al, 1993; Carroll et al, 1993; James et al, 1989).

Figure 2. Schematic representation of the different areas in the airway wall. A distinction is made between the inner airway wall, the outer airway wall and the smooth muscle layer. The inner wall comprises the basement membrane, the lamina propria and the airway smooth muscle. The outer airway wall consists of the adventitia.


3. Pathogenesis of airway remodeling

3.1. The inflammatory basis of asthma

Asthma is a disease that results from complex interactions between a variety of

environmental factors acting on a background of genetic predisposition and it is characterized

by a specific form of airway inflammation. The inflammation is orchestrated predominantly

by T helper-2 (Th-2) cells and involves a variety of cells such as eosinophils and mast cells.

Structural airway cells also contribute to the inflammatory events. A range of cytokines and

chemokines mediates interactions between the various cell types. Activated inflammatory cells

release a variety of pro-inflammatory mediators, resulting in morphologic features of

inflammation in the airway wall.

Evidence that inflammation is a component of asthma was initially derived from findings

in fatal asthma. Autopsy studies showed airway walls infiltrated with eosinophils, neutrophils

and mast cells in patients who died from asthma. More recently, biopsy studies showed that

even in mild asthmatics substantial inflammation is present in the wall. The inflammatory

changes occur in central as well as in peripheral airways and vary with the severity of the

disease (Moore et al, 2001; Hamid and Minshall, 2000; Kraft, 1999; Haley et al, 1998; Vignola

et al, 1998b; Hamid et al, 1997; Kraft et al, 1996; Ferguson and Wong, 1989). Further

evidence for an inflammatory response in asthma is the presence of cytokines and

chemotactic chemokines that mediate inflammation in bronchoalveolar-lavage fluid or

pulmonary secretions (Chung and Barnes, 1999).

The tissue infiltration of inflammatory cells may involve both the recruitment of cells

from the blood stream and proliferation of their progenitors in the submucosa. Increased

survival of these cells in asthma is influenced by the micro -environment within the airway wall

and might result from the interaction of inflammatory cells with other cell types and with

cytokines and growth factors (Chung and Barnes, 1999; Luster, 1998).

In asthmatics, allergen inhalation results in an acute inflammatory response characterized

by vascular leakage, mucus hypersecretion, epithelial shedding and widespread airway

narrowing. At the same time, through release of mediators, cytokines, chemokines and growth

factors, the inflammatory infiltrate persists in the airway wall and causes structural tissue

damage to it. Enhanced survival of cells and the resulting tissue damage can contribute to

airway wall remodeling (Bratton and Fadok, 1999).


3.2. Inflammatory and structural cells involved in remodeling

The inflammatory process underlying asthma results from the interaction of various cell

types (Busse and Lemanske, Jr., 2001). Inflammatory cells such as eosinophils, activated T-

cells, mast cells and macrophages and structural tissue cells in cluding epithelial cells,

fibroblasts and smooth muscle cells can play an important effector role in the remodeling

process in the asthmatic airway through the release of mediators, cytokines and chemokines

(Chung and Barnes, 1999).

Eosinophils

Eosinophils participate in the allergic inflammatory response in airways of mild-to

moderate asthmatics. The number of eosinophils are increased in peripheral blood, sputum,

BAL fluid and bronchial tissue of patients with asthma and their numbers have been claimed

to correlate positively with the severity of the disease (Louis et al, 2000; Chanez et al, 1999;

Vignola et al, 1998b; Bradley et al, 1991; Bousquet et al, 1990). The migration of eosinophils

from the circulation to the airway mucosa is stimulated by cytokines such as IL-5 and CC-

chemokines such as eotaxin and RANTES of which the local concentration is increased in

airways of asthmatic subjects(Hamid and Minshall, 2000; Wardlaw et al, 2000; Wardlaw,

1999; Luster, 1998; Berkman et al, 1996; Sedgwick et al, 1991). Eosinophils contribute to

their own survival by releasing several cytokines including GMCSF, IL-3 and IL-5

(Rothenberg, 1998).

Initially eosinophils were thought to be involved in the acute inflammatory process in the

asthmatic airway through the release of toxic granule proteins and other mediators such as

leukotrienes (LTC4 and LTD4) or platelet activating factor. During the late-phase inflammatory

reaction after allergen challenge eosinophils are recruited to the airway mucosa where they

become activated (Rossi et al, 1991; de Monchy et al, 1985). Once activated, products from

the eosinophils contract the airway smooth muscle, increase vascular permeability and induce

airway hyperresponsiveness (Rabe et al, 1994; Leff, 1994; Collins et al, 1993). The cytotoxic

eosinophil granule products such as major basic protein and eosinophilic cationic protein

seem to contribute to the denudation of the epithelium (Trautmann et al, 2002; Gleich et al,

1993).

Recent findings also indicate that eosinophils may be active in the airway remodeling

process in asthmatics. Possible mechanisms include the release of growth factors such as

transforming growth factor-ß (TGF-ß) and other profibrogenic cytokines such as IL-11.


TGF-ß possesses the ability to directly affect growth of different cell types and to regulate the

synthesis of matrix components (Cohen et al, 1997; Minshall et al, 1997). Transforming

growth factor-ß expression in asthmatic airways correlated with the degree of subepithelial

fibrosis (Ohno et al, 1996) and TGF-ß (Minshall et al, 1997) expression was located

predominantly in fibroblasts and eosinophils. Interleukin-11 which is able to induce structural

changes (Leng and Elias, 1997) is also expressed in the airway wall of asthmatics (Minshall et

al, 2000). Again, eosinophils are also considered the prime source of matrix

metalloproteinases (MMPs) that are involved in the degradation of extracellular matrix (Dahlen

et al, 1999; Ohno et al, 1997).

The selective action of IL-5 in several stages of eosinophil development supports an

important role for this cytokine in asthma (Chung and Barnes, 1999). Thus, indirectly IL-5

might also be important in the process of airway remodeling. In vivo models have shown that

selective suppression of eosinophils by anti IL-5 reduced subepithelial fibrosis in mice (Blyth

et al, 2000).

T-lymphocytes

T-cells have been linked with subepithelial fibrosis based on the association between

CD4/CD8 ratio in the airway wall and the thickness of the lamina reticularis in asthmatics

(Laprise et al, 1999). Whether T-lymphocytes can cause directly tissue damage and alter the

airway structure is debated. Indirectly they can contribute to the pathogenesis of airway

remodeling by the recruitment and activation of inflammatory cells in asthma. Th2-cells in the

airways are activated by inhaled allergen processed by antigen-presenting cells (Robinson et

al, 1992). In turn, the T-cells proliferate and produce a wide range of cytokines, such as IL-4,

IL-5, IL-9, IL-13 and GM-CSF which are relevant to asthma pathogenesis and play a central

role in the initiation and amplification of the inflammatory response (Ying et al, 1997;

Robinson et al, 1993a; Robinson et al, 1993b).

Mast cells

Mast cells have an important function in the regulation of allergic inflammatory processes

in asthmatic airways. Mast cell numbers are increased in the airway tissue of asthmatics

concomitantly with local recruitment and activation of Th2-cells (Amin et al, 2000; Laitinen et

al, 1993). Mast cells are activated in asthmatics predominantly via the allergen-specific IgE

receptor (Yssel et al, 1998). Upon activation they release cytokines and mediators involved in


recruitment and survival of inflammatory cells (Bingham, III and Austen, 2000; Kobayashi et

al, 1998; Bradding et al, 1994). In analogy to its role in other fibrotic diseases, the mast cell

may play an important role in the remodeling process of the lamina propria of asthmatic

airways by releasing proteases, proteoglycans, histamine and cytokines including tumor

necrosis factor-a (TNF-a) (Hart, 2001; Sommerhoff, 2001; Okuda, 1999; Cairns, 1998;

Inoue et al, 1996; Chanez et al, 1993). The positive correlation that has been found between

the thickness of the tenascin and laminin layer beneath the epithelium and mast cell numbers in

atopic asthmatics could be an indication for this role (Amin et al, 2000).

Macrophages

Macrophages have a central role in host defense against infection and in inflammatory

processes. Activated macrophages have the potential to secrete a wide variety of products,

many of which have the capacity to injure normal structures, leading to tissue repair and

remodeling. Macrophages are present in the airway inflammatory infiltrate of asthmatic patients

and a significant correlation is observed between their activation and the severity of asthma

(Kelly et al, 1988). Macrophages can regulate the airway inflammation in asthma through

release of enzymes, eicosanoids, PAF, oxygen free radicals and cytokines (Henderson, Jr.,

1991). In asthma, macrophages can also participate in tissue repair and remodeling by

releasing fibrogenic mediators (TGF-ß, PDGF-ß and endothelin) (Chanez et al, 1996; Vignola

et al, 1996; Taylor et al, 1994) and proteolytic enzymes (MMP-9) and their tissue inhibitors

(TIMP-1) involved in extracellular matrix turnover (Kelly et al, 2000; Mautino et al, 1999;

Vignola et al, 1998c; Mautino et al, 1997).

Epithelial cells

Epithelial cells are metabolically extremely active, and have been postulated to contribute

to a large extent to the remodeling process (Holgate et al, 1999). The epithelium in asthmatics

is injured via exogenous factors such as viruses, inhaled pollutants and allergens but also by

endogenous factors including proteolytic enzymes released by activated mast cells and

eosinophils (Holtzman et al, 2002). The respiratory epithelium is also a target of the

inflammatory attack by T-cells (Trautmann et al, 2002). Following injury, normal epithelium

releases a wide spectrum of molecules that participate in the repair process. It has been

hypothesized that in asthma these mechanisms have become disregulated resulting in


sustained production by the epithelial cells of cytokines and growth factors that are relevant to

the ongoing inflammatory response and to airway remodeling in asthma (Holgate, 1998).

Fibroblasts

Fibroblasts are involved in the deposition of collagen, reticular and elastic fibers,

proteoglycans and glycoproteins in the extracellular matrix within the airway wall. A range of

cytokines and growth factors regulates their biological activity. In asthmatic airways,

fibroblasts may also play a key role in the inflammatory and the remodeling processes.

A layer of myofibroblasts located immediately beneath the basement membrane has

attracted particular interest. After allergen challenge their number is increased and their number

correlates strongly with the depth of the collagen layer in the lamina reticularis of asthmatic

airways suggesting that these cells are responsible for the deposition of the increased amounts

of collagen beneath the epithelium (Gizycki et al, 1997; Brewster et al, 1990). These

myofibroblasts lie in close interaction with the epithelial cells, forming a functional unit (the so-

called epithelial-mesenchymal trophic unit). The close spatial relationship between the

epithelium and the myofibroblasts indicates a high level of organization between the two layers

(Holgate et al, 2000). After allergen challenge, epithelial cells stimulated collagen type I and III

synthesis by lung myofibroblasts (Hastie et al, 2002; Morishima et al, 2001). The injured and

repairing epithelium releases growth factors such as TGF-ß and epidermal growth factor. In

response to these mediators the fibroblasts in the asthmatic airway wall express a more

differentiated and responsive phenotype of fibroblasts (Zhang et al, 1999; Dube et al, 1998).

This bi-directional communication between repairing epithelium and the lining myofibroblasts

result in a chronic imbalance of matrix synthesis and remodeling (Hastie et al, 2002;

Morishima et al, 2001; Holgate et al, 2000).

Smooth muscle cells

Smooth muscle cells have recently been implicated as inflammatory cells in asthma

because they exhibit also synthetic and secretory potential besides their contractile and

mitogenic responses (Chung and Barnes, 1999; John et al, 1997). They can participate in the

chronic inflammation by releasing proinflammatory cytokines involved in the chemoattractant

activity for eosinophils, T-cells and macrophages (Chung and Barnes, 1999). Smooth muscle

also have the potential to produce matrix proteins and orchestrate key events in the process of


chronic airway remodeling (Coutts et al, 2001; Johnson et al, 2000; Hirst, 1996; Black et al,

1996).

3.3. Interdependence of inflammation and remodeling

Asthma is characterized by a persistent inflammation in the lower airways (Hamid and

Minshall, 2000; Vignola et al, 1998b; Laitinen et al, 1993). However a persistent inflammation

is not seen in all atopic individuals (Bradley et al, 1991). The mechanisms that account for this

persistence of inflammation in the lower airways remain largely unknown.

Remodeling in the airway wall might contribute in this respect. Components of the

extracellular matrix can interact profoundly with inflammatory cells and influence the

persistence and progression of the inflammatory response (Goldring and Warner, 1997;

Roman, 1996). Extracellular matrix proteins can affect migration, proliferation, differentiation

and activation state of inflammatory cells and influence their survival (Zhang and Phan, 1999;

Meerschaert et al, 1999; Vignola et al, 1999; Lavens et al, 1996; Neeley et al, 1994; Anwar et

al, 1993). Moreover some extracellular proteins can serve as a reservoir for growth factors

(Redington et al, 1998; Ruoslahti and Yamaguchi, 1991). Changes in the extracellular matrix

components could therefore influence the nature or persistence of the inflammatory process

in the airways.

Remodeling is usually considered to be a consequence of inflammation. Conversely, it

has also been claimed that airway remodeling as a result of epithelial injury might develop prior

to inflammatory changes in the airway wall. Injured and repairing bronchial epithelial cells

might contribute directly to the remodeling processes through increased production of

proliferative and fibrogenic growth factors (Holgate et al, 2000) which then activates the

myofibroblasts (Puddicombe et al, 2000). Upon activation myofibroblasts not only produce

extracellular matrix proteins but also mediators that can amplify the signals of structural

changes throughout the airway wall (Zhang et al, 1999). These inherent abnormalities result in

more pronounced structural alterations that subsequently can firmly anchor inflammation in

the lower airways (Holgate et al, 2000).

It appears that airway inflammation and remodeling are interdependent processes that

clearly influence the clinical long-term evolution of asthma. Modulation of the extracellular

matrix components by remodeling could therefore influence the nature or persistence of the

inflammatory process and contribute to its chronicity within the asthmatic airway wall. There

are however few hard data in this respect.


4. Functional consequences of airway remodeling

Airway remodeling could have important clinical impact in asthmatics. The functional

consequences of airway remodeling have mainly been related to the altered airway function

characteristic for asthmatics. Airway mechanics may be changed because of quantitative

changes in the airway wall compartments and/or by changes in the biochemical composition

or material properties of the various constituents of the airway wall. Airway remodeling may

contribute to bronchial hyperresponsiveness, loss of airway distensibility and accelerated

decline in FEV1 in asthmatics.

4.1. Non-specific bronchial hyperresponsiveness

Airway hyperresponsiveness to non-specific stimuli (i.e. histamine, methacholine or cold

air) is one of the defining characteristics of asthma and is usually expressed as the dose of

bronchoconstrictor (e.g. methacholine) causing a 20% fall in FEV1 from baseline

(PC20FEV1). Non-specific airway hyperresponsiveness refers to an increase in the ease in

degree of airway narrowing in response to a wide range of bronchoconstrictor stimuli (Sterk

et al, 1993). In asthmatics this airway hyperresponsiveness is persistent even in periods of

symptomatic remission and after optimal therapy (Boulet et al, 2000). It is thought that

chronic airway inflammation and airway remodeling are critical factors in the development of

airway hyperresponsiveness (James et al, 1989). Non-specific airway hyperresponsiveness has

been correlated with inflammatory cells in the airways (Boulet et al, 2000; Laprise et al, 1999;

Chetta et al, 1996; Ohashi et al, 1992; Bradley et al, 1991), epithelial injury (Oddera et al, 1996;

Cho et al, 1996; Jeffery et al, 1989; Laitinen et al, 1985), and features of airway remodeling

(Boulet et al, 2000; Laprise et al, 1999; Boulet et al, 1997; Chetta et al, 1996; Bramley et al,

1994). However, not all studies have found an association between inflammation or structural

changes and the PC20FEV1 of asthmatic patients (Lozewicz et al, 1990) implying that altered

smooth muscle behavior might also be involved (Chu et al, 1998; Crimi et al, 1998).

In asthmatic subjects, mediators (histamine, LT, PAF or proteases) released by

inflammatory cells that are recruited to the airways after allergen inhalation can alter the

contractility of airway smooth muscle and induce airway hyperresponsiveness (Crimi et al,

2001; Oddera et al, 1998). The direct role of inflammatory cells however not fully accounts


for the chronicity of the altered airway mechanics in persistent asthma. Indeed, increased

airway responsiveness was observed in the absence of demonstrable inflammatory infiltrates in

the airway lumen or mucosa (Crimi et al, 1998). Hence, the hypothesis that airway

inflammation can affect airway hyperresponsiveness indirectly through its active role in the

restructuring of the airway wall.

How structural airway changes can result in increased airway narrowing and airway

hyperresponsiveness is debated. Different research groups have developed computer models

to predict the functional significance of altered airway dimensions and physical behavior on

airway resistance and airway hyperresponsiveness. Changes in thickness of airway wall

compartments could have profound effects on airway function (figure 3). When smooth

muscle shortens it has to deform the tissue between the smooth muscle layer and the lumen

into folds. Thickening of the inner wall in asthma has only little effect on baseline airway

resistance but can enhance the airway narrowing produced by smooth muscle shortening

resulting in exaggerated increase of airway resistance (Wiggs et al, 1992; James et al, 1989;

Moreno et al, 1986). It was also predicted that swelling of the inner airway wall after allergen

provocation in asthma could result in airway hyperresponsiveness (James et al, 1989). The

increased airway resistance can be further enhanced when intraluminal airway mucus

secretions are present (Postma and Kerstjens, 1998). Outer airway wall adventitial thickening

could also affect airway responsiveness. When airway smooth muscle contraction is initiated,

shortening occurs until the stress in the surrounding parenchyma prevents further shortening

and narrowing. An increase in the outer wall thickness could theoretically reduce the tethering

effect of the parenchyma on the airway and therefore reduce the afterload on the bronchial

smooth muscle (figure 4). As a result smooth muscle could shorten more for a given force

generation (Macklem, 1996; Pare and Bai, 1995; Lambert et al, 1994). The third compartment

of the asthmatic airway wall that could alter airway responsiveness is the amount of airway

smooth muscle. The increase in airway smooth muscle mass in asthma can have a simple

geometric effect on airway narrowing, much like the effect of thickening of the submucosal

region of the airway wall. It can also amplify the effect of smooth muscle shortening and

enhance airway narrowing. The increased area of airway smooth muscle can also increase the

force-generating ability and allow the smooth muscle to shorten more against the elastic loads

provided by the lung parenchyma and again can result in a larger airway narrowing (Postma

and Kerstjens, 1998; Pare et al, 1997). Maximal airway narrowing can be largely enhanced

both by an increase in the smooth muscle layer and changes in the adventitial structure,


leading to a loss of elastic parenchymal recoil on contracting airway smooth muscle

(Macklem, 1996; Lambert et al, 1993).

Changes in the stiffness of the airway wall areas due to biochemical changes of the tissue

can also affect airway behavior in asthmatics. The mechanical effects of collagen deposition

results from an altered size and composition of the collagen bundles in the subepithelial layer.

Increased collagen fibril density and thickening of this layer as observed in asthmatics (Carroll

et al, 2000) could increase the stiffness of the inner layer of the airway wall in comparison with

the surrounding submucosa. It was calculated that subepithelial thickening might reduce the

number of folds that forms when airway smooth muscle shortens. This then would favor

airway narrowing compared to a larger number of thin folds in contracting normal airways

(Wiggs et al, 1997). Also extracellular matrix protein changes in the submucosa may

contribute to airway hyperresponsiveness in asthmatics. The functional consequences of

proteoglycan deposition in the airway wall are unknown but hydrated proteoglycan may

contribute to an increased thickness of the submucosa in asthmatics and thus possibly also in

airway hyperresponsiveness (Kuwano et al, 1993). Degradation of elastin and matrix proteins

that form tethers between the submucosa and the airway smooth muscle may decrease the

load internal to airway smooth muscle and increase deformability of the airway (Bousquet et

al, 1996). This also has been postulated to induce increased smooth muscle contractility in

asthma (Bramley et al, 1994).

Figure 3. Increase in airway wall area enhances smooth muscle contraction. The normal airway has an inner wall area of 20%. When 30% airway smooth muscle (ASM) shortening occurs, the resistance (R) of the airway wall increases from 1 to 7.6. When inner wall area is 40%, baseline resistance is slightly


increased (R=1.8). However, with a thicker airway wall, 30% smooth muscle shortening increases resistance to 80 times baseline (Bosken et al, 1990).

Figure 4. Influence of outer airway wall thickening on smooth muscle contraction. The normal airway has a resistance (R) of 1. When airway smooth muscle (ASM) contraction is stimulated, shortening occurs until the stress of the surrounding parenchyma prevents further shortening. When the outer airway wall is thickened the stress in the surrounding parenchyma decreases and a greater smooth muscle shortening is possible (Pare and Bai, 1995).


Based on these theoretical assumptions it is clear that changes in compartments of the

airway wall have the capacity to influence airway function in asthma. However, it is not clear

whether they are indeed applicable in chronic asthma. Methodological problems hamper the

evaluation of correlations between parameters of remodeling and airway hyperresponsiveness.

Histological assessment of remodeling in asthmatic subjects is limited to the evaluation of the

thickness of the subepithelial fibrosis in bronchial biopsies from central airways. It is

unknown how well this correlates with other elements of the remodeling process. Results

from studies that have correlated PC20FEV1 with the thickness of subepithelial fibrosis are

conflicting. Although in some studies, a correlation was found between the PC20FEV1 and

the thickness of the subepithelial collagen layer (Niimi et al, 2000c; Laprise et al, 1999; Chetta

et al, 1997; Boulet et al, 1997) this was not confirmed by others (Boulet et al, 2000; Chu et al,

1998; Saetta et al, 1992). Similarly, studies using HRCT scans have not consistently shown a

correlation between the PC20FEV1 and the airway wall thickness at the level of the intermediary

bronchus (Niimi et al, 2000a; Carr et al, 1998; Boulet et al, 1995). In a human experimental

setting the allergen-induced increase of airway responsiveness that was observed in asthmatic

patients did not correlate with indices of airway edema (Peebles et al, 2001). These results

again contradict the theoretical models predicting that airway hyperresponsiveness in asthma

might be a result of airway wall edema.

Some have even observed a negative relationship between airway responsiveness and

subepithelial thickening (Milanese et al, 2001). This finding is in keeping with the theoretical

prediction that increased collagen deposition tends to increase airway wall stiffness, thus

providing additional internal load on contracting airway smooth muscle and protecting

against instead of enhancing airway narrowing (Seow et al, 2000; Lambert et al, 1994;

Lambert, 1991). Alterations in extracellular matrix proteins within the airway wall and more

specifically around the smooth muscle layer and between smooth muscle cells could have the

effect of banding and interfere with smooth muscle contractions, again resulting in limited

narrowing (Meiss, 1999; Bramley et al, 1995).

In addition, the above mentioned morphometric analysis fails to take into account airway

smooth muscle dynamics. The contractile properties of airway smooth muscle are also altered

in asthma. The maximal capacity and velocity of shortening is larger in airway smooth muscle

from patients with asthma than those from healthy subjects (Stephens et al, 1998). Moreover,

passive sensitization of airway smooth muscle by serum from atopic individuals resulted in


enhanced contractile responses in vitro (Wohlsen et al, 2001; Schmidt et al, 2000; Watson et

al, 1997).

These theories and observations illustrate that airway hyperresponsiveness in asthma is

not uniquely related to airway inflammation or structural airway changes but results from an

interaction between complex and multiple mechanisms that regulate narrowing of the airway

wall (Brusasco et al, 1998).

4.2. Irreversible component of the airways obstruction

It has long been recognized that reversible airflow limitation is one of the unique features

of asthma. A majority of asthmatic patients show a complete reversibility of airflow limitation

and become symptom-free, even when initially they had moderate to severe asthma

(Panhuysen et al, 1997). However, despite apparently optimal therapy a significant degree of

airflow obstruction may persist in some asthmatic patients, both children and adults (Ulrik

and Backer, 1999; Backman et al, 1997; Boulet et al, 1994). The irreversible airflow limitation

seem to develop more commonly in patients with severe asthma and in middle-aged and older

adults with asthma (Ulrik and Backer, 1999; Panhuysen et al, 1997). The phenomenon has

also been described in asymptomatic patients (Ferguson, 1988).

An indication of the irreversible changes in the histology of asthmatic airways is the

accelerated decline in FEV1. Longitudinal studies have shown that in patients with asthma the

decline in FEV1 is steeper compared to those without asthma (Lange et al, 1998; Ulrik and

Lange, 1994; Peat et al, 1987). The effect of asthma is variable and not all subjects with

asthma have accelerated rates of decline. The rate of decline has been reported to be larger in

patients with intrinsic asthma than in atopic asthma, (ten Brinke et al, 2001; Ulrik et al, 1992)

however this relation was not always found (Peat et al, 1987). The decline in lung function is

further accelerated by regular smoking (Lange et al, 1998; Peat et al, 1987). A relation was

observed between airway hyperresponsiveness and rate of decline in FEV1 (Peat et al, 1987)

but this also was not invariably confirmed (Ulrik and Backer, 1999).

Airway remodeling, especially the irreversible fibrotic change in the airway wall is thought

to contribute to the irreversible impairment of FEV1 in asthma (Ward et al, 2001; Boulet et al,

2000; Reed, 1999). Although somewhat debated, it was recently shown that longer duration

of asthma is associated with progressive remodeling of the airway wall. These changes

included a progressive increase in airway smooth muscle volume and a greater reduction in

airway lumen (Bai et al, 2000). This study thus provided a basis for understanding the


exaggerated decline in FEV1 reported in individuals with asthma, especially in the older

asthmatic patients. In asthma, studies of moderately severe asthmatic patients also have found

some loss of elastic recoil and lung elasticity (Zapletal et al, 1993; McCarthy and Sigurdson,

1980). These findings were thought to result from airway remodeling because increased

bronchial wall rigidity can reduce airway distensibility (Johns et al, 2000; Wilson et al, 1993).

Further evidence in this respect is given recently by showing a correlation between decreased

airway distensibility and the increase in sub-basement membrane thickening in asthmatic

subjects (Ward et al, 2001).

There are however no prospective studies showing that structural changes, pulmonary

function parameters and indices of inflammation are related longitudinally. Therefore the long-

term evolution of lung function in asthma and airway remodeling needs to be further

examined.

5. In vivo experimental models of airway remodeling

In vivo experimental models in which aspects of the asthma pathology are replicated are

necessary and useful to assess the contribution of particular mediators and cells to the

development of airway remodeling and its functional consequences. Further insight into the

pathogenic events leading to remodeled airways is clearly required. More information is

needed on which mediators, mediator-mediator interactions, cell-cell interactions, signal

transduction pathways and effector mechanisms are responsible for these biologic processes.

Also a further evaluation of the impact of these structural changes on airway function is

necessary. This implies that the animal model should display not only morphological

characteristics of remodeling but also a degree of airway hyperresponsiveness comparable to

human asthma.

The ideal animal model in which these morphological and physiological requirements are

combined does not yet exist. Animal models displaying naturally occurring asthma-like

syndromes are nearly inexistent. Two animal species, horses and cats can develop disease

profiles that resemble human asthma. Horses develop airway obstruction after exposure to

mold but the airway histology is different from asthma and resembles more bronchiolitis

(Derksen et al, 1985). The only species that seems to spontaneously develop an airway

disorder that bears similarities to asthma, both functionally and histologically, are cats.


Sensitization followed by repeated exposure to Ascaris has been shown to increase airway

responsiveness in conjunction with histological changes such as peribronchial eosinophil

infiltration, an extensive increase in smooth muscle mass and submucosal gland mass. The

overall airway wall thickness was not increased (Padrid et al, 1995). However the cat has not

gained widespread acceptance as an experimental animal.

The vast majority of animal models of allergic airway inflammation have been

experimentally induced in small rodents, mice and rats in particular. This choice of animal

species is in part dictated by economical and ecological considerations, but mainly

attributable to the huge range of immunological tools available in mice. The development of

monoclonal antibodies and more recently gene deletion technology allows to effectively

neutralize a given mediator or cell, thus allowing to establish its functional importance in the

disease process (Kips and Pauwels, 1997). To date, the majority of airway remodeling studies

in rat and mice have been conducted in previously characterized animal models of airway

hyperresponsiveness. The advantage of these models of airway hyperresponsiveness is that

they allow us to study the relationship between inflammatory and structural changes on one

hand and physiologic abnormalities on the other (Fernandes et al, 2001).

5.1. Rat models of airway remodeling

Rat models have received considerable attention during recent decades. Experimental

data suggest that some features of human asthma including pathological changes can be

duplicated in rats. Most studies on airway remodeling were conducted in the Brown Norway

species after chronic allergen challenge. Brown Norway rats produce IgE as the major

anaphylactic antibody and they exhibit both early and late responses to antigen. They are a

popular model of allergic bronchoconstriction. Sprague-Dawley rats were also used in a few

airway remodeling studies using especially non-allergic substances.

Chronic antigen challenge

In a series of studies, Brown Norway rats were sensitized and exposed to a number of

inhalation challenges with ovalbumin (OA) (Salmon et al, 1999; Du et al, 1996; Wang et al,

1993; Sapienza et al, 1991; Bellofiore and Martin, 1988). An increase in airway responsiveness

was observed in those animals that developed an early response to OA inhalation (Sapienza et

al, 1991; Bellofiore and Martin, 1988). The increase in airway responsiveness in some of the

animals persisted for up to 17 days after the last exposure (Bellofiore and Martin, 1988). The


altered airway behavior in these rats was not accompanied by a clear eosinophil influx into the

airways, nor altered airway wall thickness but with an increased thickness of the smooth

muscle layer attributed to smooth muscle cell hyperplasia (Panettieri, Jr. et al, 1998; Sapienza

et al, 1991). Others have reported increased numbers of replicating cells in airway epithelium

and in the airway smooth muscle layer after repeated OA exposure (Salmon et al, 1999). In

this report increased subepithelial collagen deposition and mucus secretion was observed

along with a significant recruitment of eosinophils and lymphocytes to the airways (Salmon et

al, 1999).

In addition to OA, the occupational allergen trimellitic anhydride (TMA) has also been

used to induce airway remodeling in Brown Norway rats (Cui et al, 1999). Sensitization and

repeated exposure to TMA conjugated to rat serum albumin resulted in enhanced bronchial

responsiveness that was accompanied by an increased thickness of smooth muscle, an

increase in narrowing of the small airways and goblet-cell hyperplasia but without a persistent

airway eosinophilia (Cui et al, 1999; Cui et al, 1997).

Non-antigenic inhaled substances

A number of models were able to induce aspects of remodeling in rats after exposure to

non-specific irritants. Chronic exposure of Sprague-Dawley rats to SO2 resulted in airway

hyperresponsiveness, accompanied by epithelial changes and increased thickness of the

airway smooth muscle layer (Long et al, 1997; Shore et al, 1995). In developing Sprague-

Dawley rats, chronic hyperoxia exposure resulted in an increased epithelial and airway smooth

muscle layer thickness and increased airway wall thickening that correlated with airway

hyperresponsiveness (Hershenson et al, 1992a; Hershenson et al, 1992b). These rats also

exhibited abnormally increased DNA synthesis in both the bronchiolar epithelium and smooth

muscle layer (Hershenson et al, 1994). Intratracheal instillation of vanadiumpentoxide induced

airway wall remodeling in Sprague-Dawley rats including airway smooth muscle thickening,

mucus cell metaplasia, and airway fibrosis. Myofibroblasts were the principal proliferating cell

type that contributed to the progression of the airway fibrosis (Bonner et al, 2000).

5.2. Mouse models of airway remodeling

The use of the mouse model of airway inflammation and airway hyperresponsiveness is

becoming more widespread. Mouse models present numerous advantages when compared

with other animal species as they offer the opportunity to explore mechanisms of allergic


reactions in view of the availability of numerous immunologic reagents and molecular

histological probes that far exceeds those of other species. Murine models have provided

fundamental information regarding the role of IgE and T lymphocyte cytokines in the

pathogenesis of airway inflammation that underlies asthma.

Antigen -induced structural airway changes

The majority of the currently developed murine models have focused on acute

inflammatory changes (Kips and Pauwels, 1997). However, in a few models, the effect of

more prolonged allergen exposure has been evaluated. Blyth and coworkers reported that

repeated intratracheal instillation of OA induced peribronchial eosinophilia and goblet cell

hyperplasia in BALB/c mice (Blyth et al, 1996). Repeated challenges also resulted in fibrosis

in the reticular layer beneath the basement membrane (Blyth et al, 1996). In another animal

experiment, prolonged allergen exposure of sensitized and challenged BALB/c mice resulted

in goblet cell hyperplasia and airway fibrosis but suppressed airway hyperresponsiveness and

reduced the number of peribronchial eosinophils (Sakai et al, 2001). Temelkovski and

coworkers reported that repeated exposure of BALB/c mice to low dose antigen over 6 to 8

weeks induced goblet cell hyperplasia as an early characteristic of airway remodeling followed

by epithelial thickening and subepithelial fibrosis. Allergen sensitization and exposure resulted

in a peribronchial accumulation of mainly mononuclear cells and in an increased sensitivity to

methacholine (Temelkovski et al, 1998). Interleukin-5 gene knockouts showed no

inflammatory changes or airway hyperresponsiveness while structural changes remained

present after OA exposure. It was therefore suggested that the structural alterations in these

BALB/c mice were not simply a consequence of the inflammation and did not correlate

directly with the airway hyperresponsiveness (Foster et al, 2000). Repeated intranasal OA

challenge of sensitized BALB/c mice during 12 weeks induced airway eosinophilia, airway

hyperresponsiveness and evidence of airway remodeling, including subepithelial collagen

deposition and an increase in α-smooth muscle actin positive cells in the airway wall. The

changes persisted for at least two weeks after the last exposure (Inman et al, 1999). Recently,

increased levels of cellular fibronectin in BAL and epithelial proliferation were observed after

repeated OA challenges in sensitized BALB/c and C57BL/6 mice (Trifilieff et al, 2001). These

parameters were used to indicate the presence of airway remodeling in these mice. The

changes were accompanied with inflammatory alterations and airway hyperreactivity (Trifilieff

et al, 2000). In contrast to the findings by Foster et al (2000), this model showed that


infiltration with eosinophils in the airways is a key event in the initiation of structural changes

that may lead to a further remodeling process. In this study it was also shown that IL-5 did

not affect the remodeling process directly but only via its mediator function on eosinophils

(Trifilieff et al, 2001).

More recently, a model of allergic airway remodelin g was described after sensitization

and repeated challenge with Aspergillus fumigatus. Intratracheal introduction of A. fumigatus

spores or conidia into the airways of sensitized mice resulted in a persistent airway

hyperresponsiveness, goblet cell hyperplasia and subepithelial fibrosis (Hogaboam et al,

2000).

Non antigen -induced models of airway remodeling in genetically manipulated mice

Overexpression of cytokines in murine airway epithelium, using transgene technology

has been reported to induce structural airway changes. Overexpression of IL-6 and IL-11,

two members of the IL-6-type cytokine family resulted in peribronchial fibrosis with the

accumulation of myofibroblasts (Tang et al, 1996; DiCosmo et al, 1995). Interleukin-6 did

not induce smooth muscle cell proliferation and did not induce airway hyperresponsiveness

(DiCosmo et al, 1995). Interleukin -11 overexpression was accompanied by an increase in true

airway smooth muscle mass. Despite similar histological changes IL-11 transgenic mice were

hyperresponsive, whereas IL-6 transgene animals were hyporesponsive (Tang et al, 1996). Of

note is that the airway caliber of IL-11 transgene mice was reduced, when compared to IL-6

transgenes (Kuhn, III et al, 2000). This difference in airway caliber probably accounts for the

airway hyperresponsiveness, and illustrates once more that histological analysis on its own is

insufficient to evaluate the full impact of airway remodeling in asthma.

Others have reported some degree of subepithelial fibrosis in addition to eosinophil

accumulation in transgenic animals expressing Th2-type cytokines. Overexpression of IL-13

generates a complex phenotype that includes eosinophilic and mononuclear inflammation,

subepithelial airway fibrosis, airway obstruction and airway hyperresponsiveness on

methacholine challenge (Zhu et al, 1999). Increased amounts of IL-5 and IL-9 has also been

demonstrated to cause eosinophilia and airway fibrosis (Temann et al, 1998; Lee et al, 1997).

These models obviously are of interest, illustrating the potential effect of high concentration of

a given cytokine in inducing structural airway changes. However, they do not address the role

of physiological concentrations of endogenously released cytokines in the pathogenesis of

the remodeling process.


6. Conclusion

In conclusion, the airway wall in asthma shows convincing signs of remodeling.

However, it remains largely unclear how the remodeling process is initiated and to what extent

these structural changes contribute to the abnormal airway physiology observed in

asthmatics. Moreover, methodological restrictions limit a full evaluation of all histological

changes in the airway wall and thus limit the investigation on the relation between the

remodeling process and the resulting airway physiology. Data derived from representative in

vivo animal models could therefore add valuable information. Most models developed to date

display only transient, acute inflammatory airway changes. To study the relation between

airway remodeling and physiology animal models need to be developed that also display the

chronicity of the inflammation and the structural changes in the airway wall, characteristic of

asthma.

7. References

Aikawa T, Shimura S, Sasaki H, Ebina M and Takishima T. Marked goblet cell hyperplasia with mucus accumulation in the airways of patients who died of severe acute asthma attack. Chest 1992, 101:916-921.

Altraja A, Laitinen A, Virtanen I, Kampe M, Simonsson BG, Karlsson SE, Hakansson L, Venge P, Sillastu H and Laitinen LA. Expression of laminins in the airways in various types of asthmatic patients: a morphometric study. Am J Respir Cell Mol Biol 1996, 15:482-488.

Amin K, Ludviksdottir D, Janson C, Nettelbladt O, Bjornsson E, Roomans GM, Boman G, Seveus L and Venge P. Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. Am J Respir Crit Care Med 2000, 162:2295-2301.

Anwar AR, Moqbel R, Walsh GM, Kay AB and Wardlaw AJ. Adhesion to fibronectin prolongs eosinophil survival. J Exp Med 1993, 177:839-843.

Awadh N, Muller NL, Park CS, Abboud RT and FitzGerald JM. Airway wall thickness in patients with near fatal asthma and control groups: assessment with high resolution computed tomographic scanning. Thorax 1998, 53:248-253.

Backman KS, Greenberger PA and Patterson R. Airways obstruction in patients with long-term asthma consistent with 'irreversible asthma'. Chest 1997, 112:1234-1240.


Bai A, Eidelman DH, Hogg JC, James AL, Lambert RK, Ludwig MS, Martin J, McDonald DM, Mitzner WA, Okazawa M, Pack RJ, Pare PD, Schellenberg RR, Tiddens HA, Wagner EM and Yager D. Proposed nomenclature for quantifying subdivisions of the bronchial wall. J Appl Physiol 1994, 77:1011-1014.

Bai TR, Cooper J, Koelmeyer T, Pare PD and Weir TD. The effect of age and duration of disease on airway structure in fatal asthma. Am J Respir Crit Care Med 2000, 162:663-669.

Beasley R, Roche WR, Roberts JA and Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989, 139:806-817.

Bellofiore S and Martin JG. Antigen challenge of sensitized rats increases airway responsiveness to methacholine. J Appl Physiol 1988, 65:1642-1646.

Berkman N, Krishnan VL, Gilbey T, Newton R, O'Connor B, Barnes P and Chung KF. Expression of RANTES mRNA and protein in airways of patients with mild asthma. Am J Respir Crit Care Med 1996, 154:1804-1811.

Bingham CO, III and Austen KF. Mast-cell responses in the development of asthma. J Allergy Clin Immunol 2000, 105:S527-S534.

Black PN, Young PG and Skinner SJ. Response of airway smooth muscle cells to TGF-beta 1: effects on growth and synthesis of glycosaminoglycans. Am J Physiol 1996, 271:L910-L917.

Blyth DI, Pedrick MS, Savage TJ, Hessel EM and Fattah D. Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Respir Cell Mol Biol 1996, 14:425-438.

Blyth DI, Wharton TF, Pedrick MS, Savage TJ and Sanjar S. Airway subepithelial fibrosis in a murine model of atopic asthma: suppression by dexamethasone or anti-interleukin-5 antibody. Am J Respir Cell Mol Biol 2000, 23:241-246.

Bonner JC, Rice AB, Moomaw CR and Morgan DL. Airway fibrosis in rats induced by vanadium pentoxide. Am J Physiol 2000, 278:L209-L216.

Bosken CH, Wiggs BR, Paré PD and Hogg JC. Small airway dimensions in smokers with obstruction to airflow. Am Rev Respir Dis 1990, 142:563-570.

Boulet L, Belanger M and Carrier G. Airway responsiveness and bronchial-wall thickness in asthma with or without fixed airflow obstruction. Am J Respir Crit Care Med 1995, 152:865-871.

Boulet LP, Laviolette M, Turcotte H, Cartier A, Dugas M, Malo JL and Boutet M. Bronchial subepithelial fibrosis correlates with airway responsiveness to methacholine. Chest 1997, 112:45-52.

Boulet LP, Turcotte H and Brochu A. Persistence of airway obstruction and hyperresponsiveness in subjects with asthma remission. Chest 1994, 105:1024-1031.

Boulet LP, Turcotte H, Laviolette M, Naud F, Bernier MC, Martel S and Chakir J. Airway hyperresponsiveness, inflammation, and subepithelial collagen deposition in recently


diagnosed versus long-standing mild asthma. Influence Of inhaled corticosteroids. Am J Respir Crit Care Med 2000, 162:1308-1313.

Bousquet J, Chanez P, Lacoste JY, Barneon G, Ghavanian N, Enander I, Venge P, Ahlstedt S, Simony-Lafontaine J, Godard P and . Eosinophilic inflammation in asthma. N Engl J Med 1990, 323:1033-1039.

Bousquet J, Lacoste JY, Chanez P, Vic P, Godard P and Michel FB. Bronchial elastic fibers in normal subjects and asthmatic patients. Am J Respir Crit Care Med 1996, 153:1648-1654.

Bradding P, Roberts JA, Britten KM, Montefort S, Djukanovic R, Mueller R, Heusser CH, Howarth PH and Holgate ST. Interleukin-4, -5, and -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am J Respir Cell Mol Biol 1994, 10:471-480.

Bradley BL, Azzawi M, Jacobson M, Assoufi B, Collins JV, Irani AM, Schwartz LB, Durham SR, Jeffery PK and Kay AB. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol 1991, 88:661-674.

Bramley AM, Roberts CR and Schellenberg RR. Collagenase increases shortening of human bronchial smooth muscle in vitro. Am J Respir Crit Care Med 1995, 152:1513-1517.

Bramley AM, Thomson RJ, Roberts CR and Schellenberg RR. Hypothesis: excessive bronchoconstriction in asthma is due to decreased airway elastance. Eur Respir J 1994, 7:337-341.

Bratton DL and Fadok VA. "Their's but to do and die": eosinophil longevity in asthma. J Allergy Clin Immunol 1999, 103:555-558.

Brewster CE, Howarth PH, Djukanovic R, Wilson JW, Holgate ST and Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990, 3:507-511.

Brown RH, Scichilone N, Mudge B, Diemer FB, Permutt S and Togias A. High-resolution computed tomographic evaluation of airway distensibility and the effects of lung inflation on airway caliber in healthy subjects and individuals with asthma. Am J Respir Crit Care Med 2001, 163:994-1001.

Brusasco V, Crimi E and Pellegrino R. Airway hyperresponsiveness in asthma: not just a matter of airway inflammation. Thorax 1998, 53:992-998.

Busse W, Elias J, Sheppard D and BanksSchlegel S. Airway remodeling and repair. Am J Respir Crit Care Med 1999, 160:1035-1042.

Busse WW and Lemanske RF, Jr. Asthma. N Engl J Med 2001, 344:350-362.

Cairns JA. Mast cell tryptase and its role in tissue remodelling. Clin Exp Allergy 1998, 28:1460-1463.


Carr DH, Hibon S, Rubens M and Chung KF. Peripheral airways obstruction on high-resolution computed tomography in chronic severe asthma. Respir Med 1998, 92:448-453.

Carroll N, Carello S, Cooke C and James A. Airway structure and inflammatory cells in fatal attacks of asthma. Eur Respir J 1996, 9:709-715.

Carroll N, Elliot J, Morton A and James A. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993, 147:405-410.

Carroll NG, Cooke C and James AL. Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 1997, 155:689-695.

Carroll NG, Perry S, Karkhanis A, Harji S, Butt J, James AL and Green FH. The airway longitudinal elastic fiber network and mucosal folding in patients with asthma. Am J Respir Crit Care Med 2000, 161:244-248.

Chanez P, Lacoste JY, Guillot B, Giron J, Barneon G, Enander I, Godard P, Michel FB and Bousquet J. Mast cells' contribution to the fibrosing alveolitis of the scleroderma lung. Am Rev Respir Dis 1993, 147:1497-1502.

Chanez P, Vignola AM, Albat B, Springall DR, Polak JM, Godard P and Bousquet J. Involvement of endothelin in mononuclear phagocyte inflammation in asthma. J Allergy Clin Immunol 1996, 98:412-420.

Chanez P, Vignola AM, Vic P, Guddo F, Bonsignore G, Godard P and Bousquet J. Comparison between nasal and bronchial inflammation in asthmatic and control subjects. Am J Respir Crit Care Med 1999, 159:588-595.

Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A and Olivieri D. Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest 1997, 111:852-857.

Chetta A, Foresi A, Del Donno M, Consigli GF, Bertorelli G, Pesci A, Barbee RA and Olivieri D. Bronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. Am J Respir Crit Care Med 1996, 153:910-917.

Cho SH, Seo JY, Choi DC, Yoon HJ, Cho YJ, Min KU, Lee GK, Seo JW and Kim YY. Pathological changes according to the severity of asthma. Clin Exp Allergy 1996, 26:1210-1219.

Chu HW, Halliday JL, Martin RJ, Leung DY, Szefler SJ and Wenzel SE. Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 1998, 158:1936-1944.

Chu HW, Kraft M, Rex MD and Martin RJ. Evaluation of blood vessels and edema in the airways of asthma patients: regulation with clarithromycin treatment. Chest 2001, 120:416-422.

Chung KF and Barnes PJ. Cytokines in asthma. Thorax 1999, 54:825-857.

Cohen MD, Ciocca V and Panettieri RA, Jr. TGF-beta 1 modulates human airway smooth-muscle cell proliferation induced by mitogens. Am J Respir Cell Mol Biol 1997, 16:85-90.


Collins DS, Dupuis R, Gleich GJ, Bartemes KR, Koh YY, Pollice M, Albertine KH, Fish JE and Peters SP. Immunoglobulin E-mediated increase in vascular permeability correlates with eosinophilic inflammation. Am Rev Respir Dis 1993, 147:677-683.

Coutts A, Chen G, Stephens N, Hirst S, Douglas D, Eichholtz T and Khalil N. Release of biologically active TGF-beta from airway smooth muscle cells induces autocrine synthesis of collagen. Am J Physiol Lung Cell Mol Physiol 2001, 280:L999-L1008.

Crimi E, Milanese M, Pingfang S and Brusasco V. Allergic inflammation and airway smooth muscle function. Sci Total Environ 2001, 270:57-61.

Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA and Brusasco V. Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med 1998, 157:4-9.

Cui ZH, Sjöstrand M, Pullerits T, Andius P, Skoogh BE and Lötvall J. Bronchial hyperresponsiveness, epithelial damage, and airway eosinophilia after single and repeated allergen exposure in a rat model of anhydride-induced asthma. Allergy 1997, 52:739-746.

Cui ZH, Skoogh BE, Pullerits T and Lotvall J. Bronchial hyperresponsiveness and airway wall remodelling induced by exposure to allergen for 9 weeks. Allergy 1999, 54:1074-1082.

Dahlen B, Shute J and Howarth P. Immunohistochemical localisation of the matrix metalloproteinases MMP-3 and MMP-9 within the airways in asthma. Thorax 1999, 54:590-596.

de Monchy JG, Kauffman HF, Venge P, Koeter GH, Jansen HM, Sluiter HJ and de Vries K. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 1985, 131:373-376.

Derksen FJ, Robinson NE, Armstrong PJ, Stick JA and Slocombe RF. Airway reactivity in ponies with recurrent airway obstruction (heaves). J Appl Physiol 1985, 58:598-604.

DiCosmo B, Geba G, Picarella D, Elias JA, Rankin JA, Stripp B, Whitsett JA and Flavell RA. Expression of interleukin-6 by airway epithelial cells. Effects on airway inflammation and hyperreactivity in transgenic mice. Chest 1995, 107:131S.

Du T, Sapienza S, Wang CG, Renzi PM, Pantano R, Rossi P and Martin J. Effect of nedocromil sodium on allergen-induced airway responses and changes in the quantity of airway smooth muscle in rats. J Allergy Clin Immunol 1996, 98:400-407.

Dube J, Chakir J, Laviolette M, Saint MS, Boutet M, Desrochers C, Auger F and Boulet LP. In vitro procollagen synthesis and proliferative phenotype of bronchial fibroblasts from normal and asthmatic subjects. Lab Invest 1998, 78:297-307.

Dunnill MS. The pathology of asthma with special reference to changes in the bronchial mucosa. J Clin Pathol 1960, 13:27.

Dunsmore SE and Rannels DE. Extracellular matrix biology in the lung. Am J Physiol 1996, 270:L3-L27.


Ebina M, Takahashi T, Chiba T and Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993, 148:720-726.

Ebina M, Yaegashi H, Chiba R, Takahashi T, Motomiya M and Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 1990a, 141:1327-1332.

Ebina M, Yaegashi H, Takahashi T, Motomiya M and Tanemura M. Distribution of smooth muscles along the bronchial tree. A morphometric study of ordinary autopsy lungs. Am Rev Respir Dis 1990b, 141:1322-1326.

Fahy JV, Wong H, Liu J and Boushey HA. Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects. Am J Respir Crit Care Med 1995, 152:53-58.

Ferguson AC. Persisting airway obstruction in asymptomatic children with asthma with normal peak expiratory flow rates. J Allergy Clin Immunol 1988, 82:19-22.

Ferguson AC and Wong FW. Bronchial hyperresponsiveness in asthmatic children. Correlation with macrophages and eosinophils in broncholavage fluid. Chest 1989, 96:988-991.

Fernandes DJ, Xu KF and Stewart AG. Anti-remodelling drugs for the treatment of asthma: requirement for animal models of airway wall remodelling. Clin Exp Pharmacol Physiol 2001, 28:619-629.

Foster PS, Ming Y, Matthei KI, Young IG, Temelkovski J and Kumar RK. Dissociation of inflammatory and epithelial responses in a murine model of chronic asthma. Lab Invest 2000, 80:655-662.

Gizycki MJ, Adelroth E, Rogers AV, O'Byrne PM and Jeffery PK. Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am J Respir Cell Mol Biol 1997, 16:664-673.

Gleich GJ, Adolphson CR and Leiferman KM. The biology of the eosinophilic leukocyte. Annu Rev Med 1993, 44:85-101.

Godfrey RW, Lorimer S, Majumdar S, Adelroth E, Johnston PW, Rogers AV, Johansson SA and Jeffery PK. Airway and lung elastic fibre is not reduced in asthma nor in asthmatics following corticosteroid treatment. Eur Respir J 1995, 8:922-927.

Goldring K and Warner JA. Cell matrix interactio ns in asthma. Clin Exp Allergy 1997, 27:22-27.

Haley KJ, Sunday ME, Wiggs BR, Kozakewich HP, Reilly JJ, Mentzer SJ, Sugarbaker DJ, Doerschuk CM and Drazen JM. Inflammatory cell distribution within and along asthmatic airways. Am J Respir Crit Care Med 1998, 158:565-572.

Hamid Q, Song Y, Kotsimbos TC, Minshall E, Bai TR, Hegele RG and Hogg JC. Inflammation of small airways in asthma. J Allergy Clin Immunol 1997, 100:44-51.


Hamid QA and Minshall EM. Molecular pathology of allergic disease: I: lower airway disease. J Allergy Clin Immunol 2000, 105:20-36.

Hart PH. Regulation of the inflammatory response in asthma by mast cell products. Immunol Cell Biol 2001, 79:149-153.

Hastie AT, Kraft WK, Nyce KB, Zangrilli JG, Musani AI, Fish JE and Peters SP. Asthmatic epithelial cell proliferation and stimulation of collagen production: human asthmatic epithelial cells stimulate collagen type III production by human lung myofibroblasts after segmental allergen challenge. Am J Respir Crit Care Med 2002, 165:266-272.

Henderson WR, Jr. Eicosanoids and platelet-activating factor in allergic respiratory diseases. Am Rev Respir Dis 1991, 143:S86-S90.

Hershenson MB, Aghili S, Punjabi N, Hernandez C, Ray DW, Garland A, Glagov S and Solway J. Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats. Am J Physiol 1992a, 262:L263-9.

Hershenson MB, Garland A, Kelleher MD, Zimmermann A, Hernandez C and Solway J. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness. Am Rev Respir Dis 1992b, 146:1294-1300.

Hershenson MB, Kelleher MD, Naureckas ET, Abe MK, Rubinstein VJ, Zimmermann A, Bendele AM, McNulty JA, Panettieri RA and Solway J. Hyperoxia increases airway cell S-phase traversal in immature rats in vivo. Am J Respir Cell Mol Biol 1994, 11:296-303.

Hirst SJ. Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma. Eur Respir J 1996, 9:808-820.

Hogaboam CM, Blease K, Mehrad B, Steinhauser ML, Standiford TJ, Kunkel SL and Lukacs NW. Chronic airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis during allergic airway disease induced by Aspergillus fumigatus. Am J Pathol 2000, 156:723-732.

Holgate ST. The inflammation-repair cycle in asthma: the piv otal role of the airway epithelium. Clin Exp Allergy 1998, 28 Suppl 5:97-103.

Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM and Lordan JL. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 2000, 105:193-204.

Holgate ST, Lackie PM, Davies DE, Roche WR and Walls AF. The bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma. Clin Exp Allergy 1999, 29 Suppl 2:90-95.

Holtzman MJ, Morton JD, Shornick LP, Tyner JW, O'Sullivan MP, Antao A, Lo M, Castro M and Walter MJ. Immunity, inflammation, and remodeling in the airway epithelial barrier: epithelial-viral-allergic paradigm. Physiol Rev 2002, 82:19-46.

Huang J, Olivenstein R, Taha R, Hamid Q and Ludwig M. Enhanced proteoglycan deposition in the airway wall of atopic asthmatics. Am J Respir Crit Care Med 1999, 160:725-729.


Inman MD, Ellis R, Wattie J, Gauldie J and O'Byrne PM. Airway remodelling and sustained dysfunction following chronic exposure of allergen to the mouse airway [Abstract]. Eur Respir J Suppl 1999, 14:A394.

Inoue Y, King TE, Jr., Tinkle SS, Dockstader K and Newman LS. Human mast cell basic fibroblast growth factor in pulmonary fibrotic disorders. Am J Pathol 1996, 149:2037-2054.

James AL, Pare PD and Hogg JC. The mechanisms of airway narrowing in asthma. Am Rev Respir Dis 1989, 139:242-246.

Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV and Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989, 140:1745-1753.

John M, Hirst SJ, Jose PJ, Robichaud A, Berkman N, Witt C, Twort CH, Barnes PJ and Chung KF. Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids. J Immunol 1997, 158:1841-1847.

Johns DP, Wilson J, Harding R and Walters EH. Airway distensibility in healthy and asthmatic subjects: effect of lung volume history. J Appl Physiol 2000, 88:1413-1420.

Johnson PR, Black JL, Carlin S, Ge Q and Anne UP. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture. The effect of beclomethasone. Am J Respir Crit Care Med 2000, 162:2145-2151.

Kasahara K, Shiba K, Ozawa T, Okuda K and Adachi M. Correlation between the bronchial subepithelial layer and whole airway wall thickness in patients with asthma. Thorax 2002, 57:242-246.

Kelly C, Ward C, Stenton CS, Bird G, Hendrick DJ and Walters EH. Number and activity of inflammatory cells in bronchoalveolar lavage fluid in asthma and their relation to airway responsiveness. Thorax 1988, 43:684-692.

Kelly EA, Busse WW and Jarjour NN. Increased matrix metalloproteinase-9 in the airway after allergen challenge. Am J Respir Crit Care Med 2000, 162:1157-1161.

Kips JC and Pauwels RA. Animal models of asthma. Clinical Asthma Reviews 1997, 1:45-53.

Kobayashi H, Okayama Y, Ishizuka T, Pawankar R, Ra C and Mori M. Production of IL-13 by human lung mast cells in response to Fcepsilon receptor cross-linkage. Clin Exp Allergy 1998, 28:1219-1227.

Kraft M. The distal airways: are they important in asthma? Eur Respir J 1999, 14:1403-1417.

Kraft M, Djukanovic R, Wilson S, Holgate ST and Martin RJ. Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1996, 154:1505-1510.

Kuhn C, III, Homer RJ, Zhu Z, Ward N, Flavell RA, Geba GP and Elias JA. Airway hyperresponsiveness and airway obstruction in transgenic mice. Morphologic correlates in


mice overexpressing interleukin (IL)-11 and IL-6 in the lung. Am J Respir Cell Mol Biol 2000, 22:289-295.

Kuwano K, Bosken CH, Pare PD, Bai TR, Wiggs BR and Hogg JC. Small airways dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1993, 148:1220-1225.

Laitinen A, Altraja A, Kampe M, Linden M, Virtanen I and Laitinen L. Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid. Am J Respir Crit Care Med 1997, 156:951-958.

Laitinen A and Laitinen LA. Airway morphology: epithelium/basement membrane. Am J Respir Crit Care Med 1994, 150:S14-S17.

Laitinen LA, Heino M, Laitinen A, Kava T and Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985, 131:599-606.

Laitinen LA, Laitinen A and Haahtela T. Airway mucosal inflammation even in patients with newly diagnosed asthma. Am Rev Respir Dis 1993, 147:697-704.

Lambert RK. Role of bronchial basement membrane in airway collapse. J Appl Physiol 1991, 71:666-673.

Lambert RK, Codd SL, Alley MR and Pack RJ. Physical determinants of bronchial mucosal folding. J Appl Physiol 1994, 77:1206-1216.

Lambert RK, Wiggs BR, Kuwano K, Hogg JC and Pare PD. Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 1993, 74:2771-2781.

Lange P, Parner J, Vestbo J, Schnohr P and Jensen G. A 15-year follow-up study of ventilatory function in adults with asthma. N Engl J Med 1998, 339:1194-1200.

Laprise C, Lavioette M, Boutet M and Boulet LP. Asymptomatic airway hyperresponsiveness: relationships with airway inflammation and remodelling. Eur Respir J 1999, 14:63-73.

Lavens SE, Goldring K, Thomas LH and Warner JA. Effects of integrin clustering on human lung mast cells and basophils. Am J Respir Cell Mol Biol 1996, 14:95-103.

Lee JJ, McGarry MP, Farmer SC, Denzler KL, Larson KA, Carrigan P, Brenneise IE, Horton MA, Haczku A, Gelfand EW, Leikauf GD and Lee NA. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997, 185:2143-2156.

Lee K, Li X, Carroll N, James A and Wilson JW. Subepithelial and submucosal collagen in fatal asthma [Abstract]. Am J Respir Crit Care Med 1999, 157:A840.

Leff AR. Inflammatory mediation of airway hyperresponsiveness by peripheral blood granulocytes. The case for the eosinophil. Chest 1994, 106:1202-1208.

Leng SX and Elias JA. Interleukin-11. Int J Biochem Cell Biol 1997, 29:1059-1062.


Li X and Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997, 156:229-233.

Little SA, Sproule MW, Cowan MD, Macleod KJ, Robertson M, Love JG, Chalmers GW, McSharry CP and Thomson NC. High resolution computed tomographic assessment of airway wall thickness in chronic asthma: reproducibility and relationship with lung function and severity. Thorax 2002, 57:247-253.

Long NC, Martin JG, Pantano R and Shore SA. Airway hyperresponsiveness in a rat model of chronic bronchitis: role of C fibers. Am J Respir Crit Care Med 1997, 155:1222-1229.

Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M and Djukanovic R. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med 2000, 161:9-16.

Lozewicz S, Wells C, Gomez E, Ferguson H, Richman P, Devalia J and Davies RJ. Morphological integrity of the bronchial epithelium in mild asthma. Thorax 1990, 45:12-15.

Luster AD. Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med 1998, 338:436-445.

Macklem PT. A theoretical analysis of the effect of airway smooth muscle load on airway narrowing. Am J Respir Crit Care Med 1996, 153:83-89.

Mauad T, Xavier AC, Saldiva PH and Dolhnikoff M. Elastosis and fragmentation of fibers of the elastic system in fatal asthma. Am J Respir Crit Care Med 1999, 160:968-975.

Mautino G, Henriquet C, Jaffuel D, Bousquet J and Capony F. Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects. Am J Respir Crit Care Med 1999, 160:324-330.

Mautino G, Oliver N, Chanez P, Bousquet J and Capony F. Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics. Am J Respir Cell Mol Biol 1997, 17:583-591.

McCarthy DS and Sigurdson M. Lung elastic recoil and reduced airflow in clinically stable asthma. Thorax 1980, 35:298-302.

Meerschaert J, Vrtis RF, Shikama Y, Sedgwick JB, Busse WW and Mosher DF. Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro. J Immunol 1999, 163:6217-6227.

Meiss RA. Influence of intercellular tissue connections on airway muscle mechanics. J Appl Physiol 1999, 86:5 -15.

Milanese M, Crimi E, Scordamaglia A, Riccio A, Pellegrino R, Canonica GW and Brusasco V. On the functional consequences of bronchial basement membrane thickening. J Appl Physiol 2001, 91:1035-1040.

Minshall E, Chakir J, Laviolette M, Molet S, Zhu Z, Olivenstein R, Elias JA and Hamid Q. IL-11 expression is increased in severe asthma: association with epithelial cells and eosinophils. J Allergy Clin Immunol 2000, 105:232-238.


Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P and Hamid Q. Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1997, 17:326-333.

Montefort S, Baker J, Roche WR and Holgate ST. The distribution of adhesive mechanisms in the normal bronchial epithelium. Eur Respir J 1993, 6:1257-1263.

Montefort S, Roberts JA, Beasley R, Holgate ST and Roche WR. The site of disruption of the bronchial epithelium in asthmatic and nonasthmatic subjects. Thorax 1992, 47:499-503.

Moore WC, Hasday JD, Meltzer SS, Wisnewski PL, White B and Bleecker ER. Subjects with mild and moderate asthma respond to segmental allergen challenge with similar, reproducible, allergen-specific inflammation. J Allergy Clin Immunol 2001, 108:908-914.

Moreno RH, Hogg JC and Pare PD. Mechanics of airway narrowing. Am Rev Respir Dis 1986, 133:1171-1180.

Morishima Y, Nomura A, Uchida Y, Noguchi Y, Sakamoto T, Ishii Y, Goto Y, Masuyama K, Zhang MJ, Hirano K, Mochizuki M, Ohtsuka M and Sekizawa K. Triggering the induction of myofibroblast and fibrogenesis by airway epithelial shedding. Am J Respir Cell Mol Biol 2001, 24:1-11.

Neeley SP, Hamann KJ, Dowling TL, McAllister KT, White SR and Leff AR. Augmentation of stimulated eosin ophil degranulation by VLA-4 (CD49d)- mediated adhesion to fibronectin. Am J Respir Cell Mol Biol 1994, 11:206-213.

NHLBI/WHO workshop report. Global Initiative for Asthma. Global strategy for asthma management and prevention. 1995, Publication no. 95-3659.

Niimi A, Matsumoto H, Amitani R, Nakano Y, Mishima M, Minakuchi M, Nishimura K, Itoh H and Izumi T. Airway wall thickness in asthma assessed by computed tomography. Relation to clinical indices. Am J Respir Crit Care Med 2000a, 162:1518-1523.

Niimi A, Matsumoto H, Amitani R, Nakano Y, Mishima M, Minakuchi M, Nishimura K, Itoh H and Izumi T. Airway wall thickness in asthma assessed by computed tomography. Relation to clinical indices. Am J Respir Crit Care Med 2000b, 162:1518-1523.

Niimi A, Matsumoto H, Minakuchi M, Kitaichi M and Amitani R. Airway remodelling in cough-variant asthma. Lancet 2000c, 356:564-565.

Oddera S, Silvestri M, Balbo A, Jovovich BO, Penna R, Crimi E and Rossi GA. Airway eosinophilic inflammation, epithelial damage, and bronchial hyperresponsiveness in patients with mild-moderate, stable asthma. Allergy 1996, 51:100-107.

Oddera S, Silvestri M, Penna R, Galeazzi G, Crimi E and Rossi GA. Airway eosinophilic inflammation and bronchial hyperresponsiveness after allergen inhalation challenge in asthma. Lung 1998, 176:237-247.

Ohashi Y, Motojima S, Fukuda T and Makino S. Airway hyperresponsiveness, increased intracellular spaces of bronchial epithelium, and increased infiltration of eosinophils and lymphocytes in bronchial mucosa in asthma. Am Rev Respir Dis 1992, 145:1469-1476.


Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, O' Byrne P, Tamura G, Jordana M and Shirato K. Transforming growth factor beta 1 (TGF beta 1) gene expression by eosinophils in asthmatic airway inflammation. Am J Respir Cell Mol Biol 1996, 15:404-409.

Ohno I, Ohtani H, Nitta Y, Suzuki J, Hoshi H, Honma M, Isoyama S, Tanno Y, Tamura G, Yamauchi K, Nagura H and Shirato K. Eosinophils as a source of matrix metalloproteinase-9 in asthmatic airway inflammation. Am J Respir Cell Mol Biol 1997, 16:212-219.

Okazawa M, Muller N, McNamara AE, Child S, Verburgt L and Pare PD. Human airway narrowing measured using high resolution computed tomography. Am J Respir Crit Care Med 1996, 154:1557-1562.

Okuda M. Functional heterogeneity of airway mast cells. Allergy 1999, 54 Suppl 57:50-62.

Ordonez CL, Khashayar R, Wong HH, Ferrando R, Wu R, Hyde DM, Hotchkiss JA, Zhang Y, Novikov A, Dolganov G and Fahy JV. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med 2001, 163:517-523.

Orsida BE, Li X, Hickey B, Thien F, Wilson JW and Walters EH. Vascularity in asthmatic airways: relation to inhaled steroid dose. Thorax 1999, 54:289-295.

Padrid P, Snook S, Funicane T, Shiue P, Cozzi P, Solway J and Leff AR. Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats. Am J Respir Crit Care Med 1995, 151:184-193.

Paganin F, Seneterre E, Chanez P, Daures JP, Bruel JM, Michel FB and Bousquet J. Computed tomography of the lungs in asthma: influence of disease severity and etiology. Am J Respir Crit Care Med 1996, 153:110-114.

Panettieri RA, Jr., Murray RK, Eszterhas AJ, Bilgen G and Martin JG. Repeated allergen inhalations induce DNA synthesis in airway smooth muscle and epithelial cells in vivo. Am J Physiol 1998, 274:L417-L424.

Panhuysen CI, Vonk JM, Koeter GH, Schouten JP, van Altena R, Bleecker ER and Postma DS. Adult patients may outgrow their asthma: a 25-year follow-up study. Am J Respir Crit Care Med 1997, 155:1267-1272.

Pare PD and Bai TR. The consequences of chronic allergic inflammation. Thorax 1995, 50:328-332.

Pare PD, Bai TR and Roberts CR. The structural and functional consequences of chronic allergic inflammation of the airways. In: Chadwick DJ and Cardew G (eds). The Rising Trends in Asthma: 1997, West Sussex, UK, John Wiley and Sons Ltd., p. 71-86.

Park CS, Muller NL, Worthy SA, Kim JS, Awadh N and Fitzgerald M. Airway obstruction in asthmatic and healthy individuals: inspiratory and expiratory thin-section CT findings. Radiology 1997, 203:361-367.

Peat JK, Woolcock AJ and Cullen K. Rate of decline of lung function in subjects with asthma. Eur J Respir Dis 1987, 70:171-179.


Peebles RS, Wagner EM, Liu MC, Proud D, Hamilton RG and Togias A. Allergen-induced changes in airway responsiveness are not related to indices of airway edema. J Allerg Clin Immunol 2001, 107:805-811.

Postma DS and Kerstjens HA. Characteristics of airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998, 158:S187-S192.

Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST and Davies DE. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J 2000, 14:1362-1374.

Rabe KF, Munoz NM, Vita AJ, Morton BE, Magnussen H and Leff AR. Contraction of human bronchial smooth muscle caused by activated human eosinophils. Am J Physiol 1994, 267:L326-L334.

Redington AE, Roche WR, Holgate ST and Howarth PH. Co-localization of immunoreactive transforming growth factor-beta 1 and decorin in bronchial biopsies from asthmatic and normal subjects. J Pathol 1998, 186:410-415.

Reed CE. The natural history of asthma in adults: the problem of irreversibility. J Allergy Clin Immunol 1999, 103:539-547.

Roberts CR. Is asthma a fibrotic disease? Chest 1995, 107:111S-117S.

Robinson DS, Bentley AM, Hartnell A, Kay AB and Durham SR. Activated memory T helper cells in bronchoalveolar lavage fluid from patients with atopic asthma: relation to asthma symptoms, lung function, and bronchial responsiveness. Thorax 1993a, 48:26-32.

Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM, Corrigan C, Durham SR and Kay AB. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992, 326:298-304.

Robinson DS, Ying S, Bentley AM, Meng Q, North J, Durham SR, Kay AB and Hamid Q. Relationships among numbers of bronchoalveolar lavage cells expressing messenger ribonucleic acid for cytokines, asthma symptoms, and airway methacholine responsiveness in atopic asthma. J Allergy Clin Immunol 1993b, 92:397-403.

Roche WR, Beasley R, Williams JH and Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989, 1:520-524.

Roman J. Extracellular matrix and lung inflammation. Immunol Res 1996, 15:163-178.

Rossi GA, Crimi E, Lantero S, Gianiorio P, Oddera S, Crimi P and Brusasco V. Late-phase asthmatic reaction to inhaled allergen is associated with early recruitment of eosinophils in the airways. Am Rev Respir Dis 1991, 144:379-383.

Rothenberg ME. Eosinophilia. N Engl J Med 1998, 338:1592-1600.

Ruoslahti E and Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 1991, 64:867-869.


Saetta M, Di Stefano A, Rosina C, Thiene G and Fabbri LM. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am Rev Respir Dis 1991, 143:138-143.

Saetta M, Maestrelli P, Di Stefano A, De Marzo N, Milani GF, Pivirotto F, Mapp CE and Fabbri LM. Effect of cessation of exposure to toluene diisocyanate (TDI) on bronchial mucosa of subjects with TDI-induced asthma. Am Rev Respir Dis 1992, 145:169-174.

Sakai K, Yokoyama A, Kohno N, Hamada H and Hiwada K. Prolonged antigen exposure ameliorates airway inflammation but not remodeling in a mouse model of bronchial asthma. Int Arch Allergy Immunol 2001, 126:126-134.

Salmon M, Walsh DA, Koto H, Barnes PJ and Chung KF. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling. Eur Respir J 1999, 14:633-641.

Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 2001, 56:902-906.

Sapienza S, Du T, Eidelman DH, Wang NS and Martin JG. Structural changes in the airways of sensitized brown Norway rats after antigen challenge. Am Rev Respir Dis 1991, 144:423-427.

Schmidt D, Watson N, Ruehlmann E, Magnussen H and Rabe KF. Serum immunoglobulin E levels predict human airway reactivity in vitro. Clin Exp Allergy 2000, 30:233-241.

Sedgwick JB, Calhoun WJ, Gleich GJ, Kita H, Abrams JS, Schwartz LB, Volovitz B, Ben Yaakov M and Busse WW. Immediate and late airway response of allergic rhinitis patients to segmental antigen challenge. Characterization of eosinophil and mast cell mediators. Am Rev Respir Dis 1991, 144:1274-1281.

Seow CY, Wang L and Pare PD. Airway narrowing and internal structural constraints. J Appl Physiol 2000, 88:527-533.

Shimura S, Andoh Y, Haraguchi M and Shirato K. Continuity of airway goblet cells and intraluminal mucus in the airways of patients with bronchial asthma. Eur Respir J 1996, 9:1395-1401.

Shore S, Kobzik L, Long NC, Skornik W, Van Staden CJ, Boulet L, Rodger IW and Pon DJ. Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis. Am J Respir Crit Care Med 1995, 151:1931-1938.

Sommerhoff CP. Mast cell tryptases and airway remodeling. Am J Respir Crit Care Med 2001, 164:S52-S58.

Stephens NL, Li W, Wang Y and Ma X. The contractile apparatus of airway smooth muscle. Biophysics and biochemistry. Am J Respir Crit Care Med 1998, 158:S80-S94.

Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O'Byrne PM, Anderson SD, Juniper EF and Malo JL. Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung


Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl 1993, 16:53-83.

Taha RA, Laberge S, Hamid Q and Olivenstein R. Increased expression of the chemoattractant cytokines eotaxin, monocyte chemotactic protein-4, and interleukin-16 in induced sputum in asthmatic patients. Chest 2001, 120:595-601.

Tang W, Geba GP, Zheng T, Ray P, Homer RJ, Kuhn C, Flavell R and Elias JA. Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest 1996, 98:2845-2853.

Taylor IK, Sorooshian M, Wangoo A, Haynes AR, Kotecha S, Mitchell DM and Shaw RJ. Platelet-derived growth factor-beta mRNA in human alveolar macrophages in vivo in asthma. Eur Respir J 1994, 7:1966-1972.

Temann UA, Geba GP, Rankin JA and Flavell RA. Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 1998, 188:1307-1320.

Temelkovski J, Hogan SP, Shepherd DP, Foster PS and Kumar RK. An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 1998, 53:849-856.

ten Brinke A, van Dissel JT, Sterk PJ, Zwinderman AH, Rabe KF and Bel EH. Persistent airflow limitation in adult-onset nonatopic asthma is associated with serologic evidence of Chlamydia pneumoniae infection. J Allergy Clin Immunol 2001, 107:449-454.

Thomson RJ, Bramley AM and Schellenberg RR. Airway muscle stereology: implications for increased shortening in asthma. Am J Respir Crit Care Med 1996, 154:749-757.

Trautmann A, Schmid-Grendelmeier P, Kruger K, Crameri R, Akdis M, Akkaya A, Brocker EB, Blaser K and Akdis CA. T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma. J Allergy Clin Immunol 2002, 109:329-337.

Trifilieff A, El Hashim A and Bertrand C. Time course of inflammatory and remodeling events in a murine model of asthma: effect of steroid treatment. Am J Physiol 2000, 279:L1120-L1128.

Trifilieff A, Fujitani Y, Coyle AJ, Kopf M and Bertrand C. IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation. Clin Exp Allergy 2001, 31:934-942.

Ulrik CS and Backer V. Nonreversible airflow obstruction in life-long nonsmokers with moderate to severe asthma. Eur Respir J 1999, 14:892-896.

Ulrik CS, Backer V and Dirksen A. A 10 year follow up of 180 adults with bronchial asthma: factors important for the decline in lung function. Thorax 1992, 47:14-18.

Ulrik CS and Lange P. Decline of lung function in adults with bronchial asthma. Am J Respir Crit Care Med 1994, 150:629-634.


Vignola AM, Bonanno A, Mirabella A, Riccobono L, Mirabella F, Profita M, Bellia V, Bousquet J and Bonsignore G. Increased levels of elastase and alpha1-antitrypsin in sputum of asthmatic patients. Am J Respir Crit Care Med 1998a, 157:505-511.

Vignola AM, Chanez P, Campbell AM, Souques F, Lebel B, Enander I and Bousquet J. Airway inflammation in mild intermittent and in persistent asthma. Am J Respir Crit Care Med 1998b, 157:403-409.

Vignola AM, Chanez P, Chiappara G, Merendino A, Zinnanti E, Bousquet J, Bellia V and Bonsignore G. Release of transforming growth factor-beta (TGF-beta) and fibronectin by alveolar macrophages in airway diseases. Clin Exp Immunol 1996, 106:114-119.

Vignola AM, Chanez P, Chiappara G, Siena L, Merendino A, Reina C, Gagliardo R, Profita M, Bousquet J and Bonsignore G. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J Allergy Clin Immunol 1999, 103:563-573.

Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D'accardi P, Bousquet J and Bonsignore G. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998c, 158:1945-1950.

Wang CG, Du T, Xu LJ and Martin JG. Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in the rat. Am Rev Respir Dis 1993, 148:413-417.

Ward C, Johns DP, Bish R, Pais M, Reid DW, Ingram C, Feltis B and Walters EH. Reduced airway distensibility, fixed airflow limitation, and airway wall remodeling in asthma. Am J Respir Crit Care Med 2001, 164:1718-1721.

Wardlaw AJ. Molecular basis for selective eosinophil trafficking in asthma: A multistep paradigm. J Allergy Clin Immunol 1999, 104:917-926.

Wardlaw AJ, Brightling C, Green R, Woltmann G and Pavord I. Eosinophils in asthma and other allergic diseases. Brit Med Bull 2000, 56:985-1003.

Watson N, Bodtke K, Coleman RA, Dent G, Morton BE, Ruhlmann E, Magnussen H and Rabe KF. Role of IgE in hyperresponsiveness induced by passive sensitization of human airways. Am J Respir Crit Care Med 1997, 155:839-844.

Wiggs BR, Bosken C, Pare PD, James A and Hogg JC. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992, 145:1251-1258.

Wiggs BR, Hrousis CA, Drazen JM and Kamm RD. On the mechanism of mucosal folding in normal and asthmatic airways. J Appl Physiol 1997, 83:1814-1821.

Wilson JW and Li X. The measurement of reticular basement membrane and submucosal collagen in the asthmatic airway. Clin Exp Allergy 1997, 27:363-371.

Wilson JW, Li X and Pain MC. The lack of distensibility of asthmatic airways. Am Rev Respir Dis 1993, 148:806-809.


Wilson JW and Stewart AG. Airway vascularity in asthma. Clin Exp Allergy 1999, 29:1295-1297.

Wohlsen A, Uhlig S and Martin C. Immediate allergic response in small airways. Am J Respir Crit Care Med 2001, 163:1462-1469.

Ying S, Humbert M, Barkans J, Corrigan CJ, Pfister R, Menz G, Larche M, Robinson DS, Durham SR and Kay AB. Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics. J Immunol 1997, 158:3539-3544.

Yssel H, Abbal C, Pene J and Bousquet J. The role of IgE in asthma. Clin Exp Allergy 1998, 28 Suppl 5:104-109.

Zapletal A, Desmond KJ, Demizio D and Coates AL. Lung recoil and the determination of airflow limitation in cystic fibrosis and asthma. Pediatr Pulmonol 1993, 15:13-18.

Zhang HY and Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am J Respir Cell Mol Biol 1999, 21:658-665.

Zhang S, Smartt H, Holgate ST and Roche WR. Growth factors secreted by bronchial epithelial cells control myofibroblast proliferation: an in vitro co-culture model of airway remodeling in asthma. Lab Invest 1999, 79:395-405.

Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y and Elias JA. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999, 103:779-788.

Aims of the Studies 49

AIMS OF THE STUDIES

Aims of the Studies 51

The overall aim of this study was to develop an in vivo animal model that displays

characteristics of allergen-induced airway remodeling. The rat model has several advantages.

Rats are easy to house and are readily available. They are of sufficient size to allow for easy

instrumentation and reliable lung function measurements. The strain chosen, Brown Norway

rats, are known to produce specific IgE and develop an acute airway inflammation in

response to active immunization and allergen challenge. A single allergen challenge in

immunized Brown Norway rats also causes an increase in bronchial hyperresponsiveness.

Mouse models are particularly interesting because of the wealth of immunologic tools and the

range of knockout mice that are available. Different inbred mouse strains develop

immunologic, inflammatory and smooth muscle responses when actively immunized and

exposed to allergen. Moreover, the existence of different inbred mouse strains allows

assessing the importance of genetic differences in the remodeling process.

The specific aims of our study were:

1. To develop an in vivo animal model in rat and in mice that mimics the chronic nature

of the asthmatic airway inflammation and the development of structural airway

changes.

2. To relate the allergen-induced structural changes to airway behavior.

3. To evaluate whether smooth muscle contractions, independent from allergen-induced

airway inflammation, can contribute to the remodeling process.

4. To evaluate whether the susceptibility of the airways to allergen-induced structural

changes is age dependent.

5. To evaluate if there is a difference in the remodeling process between species with

distinct genetically determined immunologic and smooth muscle responses to

allergen sensitization and exposure.

Publications 53

PART II

PUBLICATIONS

Methodology 55

DETAILS ON METHODOLOGY USED IN THE

EXPERIMENTAL MODELS

Methodology 57

1. Respiratory measurements

Overall, three techniques exist for the assessment of airway mechanics in rodents (Drazen

et al, 1999; Amdur and Mead, 1958). In the first, the physical properties of the lungs or

airways are assessed after their removal from the host. An example for this technique is the

measurement of contractions in isolated airway segments. The second set of techniques

requires anesthesia and instrumentation of the animal to allow the recording of airway

mechanics. We have used this approach to measure pulmonary resistance in the rat. In the

third technique neither anesthesia nor airway instrumentation is required. This has been used

to assess airway function in mice.

1.1. Respiratory measurements in rats

Lung resistance (RL) was used as a parameter of airway tone. As described by Amdur

and Mead (1958), the mechanical properties of the lung in rodents can be obtained by relating

tidal volume and flow rate to intrapleural pressure at specific time points during the respiratory

cycle. Intrapleural pressure can be divid ed into a flow-resistive and elastic component. The

flow-resistive component is related to resistance to flow of gas in lungs and airways and to

viscous resistance of lung tissue, whereas the elastic component is related to elastic recoil of

the lung. At points of equal volume during the respiratory cycle, the elastic forces must be

approximately equal and the pressure change between two such points must relate chiefly to

resistance to flow in lungs and airways (figure 1A). The pressure change is related to the

associated flow change to give a measure of the flow resistance of the lungs and airways. This

method gives a value that represents the average inspiratory and expiratory resistance near

peak inspiratory and expiratory flows. Therefore RL = ? P/?V (figure 1A). RL not only

measures airway resistance, but also the viscous resistance of lung tissue. Using a

plethysmographic technique in tracheostomized animals, it has however been shown that in

rats, RL is nearly identical to airway resistance (Raw) (Johanson and Pierce, 1971). In rats,

therefore RL is therefore considered to be a parameter of mainly airway resistance.

RL is derived from data of tidal volume (V), air flow (V) and transpulmonary pressure

(Ppt) measured at the outlet of an intratracheal cannula. Therefore rats are anaesthetized by the

intraperitoneal injection of pentobarbital (60 mg/kg; Sanofi, Libourne, France). The trachea is

cannulated with a Teflon cannula (35 mm long and 1.67 mm internal diameter [I.D.]). The

Methodology 58

femoral artery is cannulated with a polyethylene catheter (1.77 mm I.D., 2.80 mm outer

diameter; Intramedic; Clay Adams, Parsippany, NJ) to monitor blood pressure and heart rate

with a pressure transducer (Celesco Transducer Products, Canoga Park, CA) throughout the

experiment. A polyethylene catheter was inserted in the external jugular vein for administration

of intravenous drugs and fluids (figure 1B). Animals were placed on a heating pad (37°C),

were connected with a Palmer animal respirator (BioScience, Sheerness, UK), and were

ventilated with oxygen-enriched air at a rate of 75 strokes/min and a stroke volume of 2 to 2.5

ml. Animals were injected with pancuronium bromide (Organon Teknika, Boxtel, the

Netherlands), at 0.2 mg/kg body weight to interrupt spontaneous respiratory movements. The

animals’ arterial blood gases were measured after 10 min of ventilation, and the stroke volume

was adapted to keep pH, pO2 and pCO2 within the normal ranges.

A B

Figure 1. A. Method of measuring pulmonary flow resistance. At a point of equal volume during the respiratory cycle, the elastic forces are approximately equal and the pressure change between two such points relate chiefly to resistance to the flow sensitive component (Amdur and Mead, 1958). B. Schematic representation of the experimental setup used for the measurement of respiratory mechanics in rats. Ppt, transpulmonary pressure; V, airflow; V, volume; RL, lung resistance.

The animals’ transpulmonary pressure (Ppt) was measured with a differential pressure

transducer (Celesco Transducer Products), of which one end was attached to an air-filled

catheter inserted into the pleural cavity and the other end was attached to a catheter connected

Methodology 59

to a sideport of the outlet of the tracheal cannula. The airflow at the outlet of the intratracheal

cannula was measured with a pneumotachograph (Type 00000; Fleisch, Geneva,

Switzerland). The tidal volume (VT) was obtained by integration of the flow. RL was

continuously calculated from VT, airflow, and Ppt (PRS800; Mumed, London, UK).

Carbachol (carbamoylcholine hydrochloride; Merck, Darmstadt, Germany) was nebulized

with an ultrasonic nebulizer (Model 2511; Pulmo-Sonic, DeVilbiss Co., Somerset, PA). The

mean particle size of the aerosol was 3.8 µm and the output was 1.0 ml/min. The aerosol was

led into the inspiratory tube of the respiratory pump. Increasing concentrations of carbachol

were administered over a period of 30 s at 5 min intervals, allowing RL to return to baseline

between administrations of each concentration. Carbachol-induced bronchoconstriction was

measured as the percent increase in RL, comparing the peak of the reaction with baseline RL.

1.2. Assessment of airway function in mice

In mice, airway function is assessed in awake animals using a whole-body

plethysmograph system (Buxco; Buxco Electronics, Troy, NY). In barometric

plethysmography, unrestrained animals are placed in a chamber (figure 2) and the pressure

fluctuations that occur during a ventilatory cycle are monitored (figure 3) (Hamelmann et al,

1997). The major advantage of this technique is that this method does not require the use of

anesthetic agents or surgical preparation and therefore allows studying the same mouse on

different occasions.

The basic idea in barometric plethysmography is that during inspiration, tidal air is

warmed and heated up as it enters the thorax and the pressure change associated with this

change is mirrored in the pressure within the chamber. With few assumptions, the time of

inspiration and expiration can be accurately assessed. Using this approach an empiric

parameter ‘enhanced pause’ (Penh) is defined based on the respiratory pattern observed in

unrestrained mice (Hamelmann et al, 1997). The expiratory period is divided in two phases: an

early relaxation phase and a later phase (figure 3). A respiratory pause is defined by Pause =

(Te – Tr)/Tr where Te is time of expiration and Tr is time required for the area under the

chamber pressure-time curve during expiration to decrease to 36% of its total value.

Hamelmann and coworkers (1997) noted that when breathing was unobstructed, the

plethysmographic pressure signal during inspiration and expiration had equal deflections,

whereas when breathing was obstructed, the expiratory pressure deflection was greater than

was the inspiratory pressure deflection. Thus, they defined the parameter Penh as Penh =

Methodology 60

[(Te-Tr)/Tr] x (PEP/PIP) where PEP is peak expiratory pressure, and PIP is peak inspiratory

pressure (figure 3). Penh reflects changes in the waveform of the box pressure signal from

both inspiration and expiration (PIP, PEP) and combines it with the timing comparison of

early and late expiration (Pause). Hamelmann and coworkers (1997) demonstrated that the

changes in Penh reflected the changes in RL during bronchoconstriction induced by

aerosolized methacholine and an increase in Penh was an index of airway obstruction in mice

that were sensitized and challenged with allergen.

Figure 2. Schematic diagram of the whole-body plethysmograph. (A) Main chamber containing the mouse (A); (B) reference chamber; (C) pressure transducer connected to analyzer and (D) pneumotachograph. (1) Main inlet for aerosol closed by valve; (2) inlet for bias flow with four-way stopcock and (3) outlet for aerosol with four-way stopcock (Hamelmann et al, 1997).

Before performing readings, the system was calibrated by rapid injection of 1 ml air.

Pressure differences between the main chamber of the whole-body plethysmograph

containing the awake mouse and a reference chamber (this is the box pressure signal) were

recorded using the software BioSystem XA (version 157; Buxco). This pressure signal is

caused by volume and resultant pressure changes in the main chamber during the respiratory

cycle of the animal. A pneumotachograph with defined resistance in the wall of the main

chamber acts as a low-pass filter and allows thermal compensation (figure 2). For each

mouse, data of Penh were recorded at baseline, and after exposure to PBS or increasing

concentrations of methacholine (2 – 81 mg/ml). The solutions were nebulized through an inlet

of the main chamber during 1 min. Readings were taken during 5 min after each nebulization.

Between readings Penh value returned to baseline. Recordings of every 10 breaths are

extrapolated to define the respiratory rate in breaths per minute.

Methodology 61

Figure 3. Schematic figure of a box pressure wave in inspiration (down) and expiration (up) explaining the computation of the parameters by whole-body plethysmography. Ti, inspiratory time (s), time from start of inspiration to end of inspiration; Te, expiratory time (s), time from end of inspiration to start of next inspiration; PIP, peak inspiratory pressure (ml/s), maximal negative box pressure occurring in one breath; PEP, peak expiratory pressure (ml/s), maximal positive box pressure occurring in one breath; Tr, relaxation time (s), time of the pressure decay to 36% of the total box pressure during expiration (Hamelmann et al, 1997).

2. Morphometric measurements

In the airway wall the length of the basement membrane is relatively constant despite

passive changes in lung volume and changes in airway dimensions caused by smooth muscle

contraction (James et al, 1989; James et al, 1988; James et al, 1987). This observation

provides a mechanism by which airway size can be accurately assessed regardless of the state

of inflation of the lung and the degree of bronchoconstriction.

The airway can be divided into different layers that have a distinct composition.

Quantification of these layers is important because different pathophysiologic processes may

result in specific thickening of one of the compartments without thickening of the others.

Also the mechanisms by which each layer may be thickened could be different and the

functional consequences of thickening of individual layers are different. To quantify the

subdivisions in the bronchial wall the standardized nomenclature for the morphometric

measurements of the airway wall described by Bai et al (1994) was used in this study.

Methodology 62

2.1. Nomenclature for quantifying subdivisions of the bronchial wall

Figure 4 shows an idealized schematic cross section of a small cartilage-free airway in a

contracted state. The following abbreviations are used to define the different compartments of

the airway wall (Bai et al, 1994).

Pbm perimeter basement membrane Pmo perimeter outer muscle (outer border of smooth muscle) Po outer perimeter (outer border of the adventitia) Abm, Amo, Ao areas enclosed by the defined perimeters WA wall area WAi inner wall area (Amo – Abm) WAo outer wall area (Ao – Amo) WAt total wall area (Ao – Abm) WAm smooth muscle area

Areas denoted Abm, Ai and Ao include the luminal area, whereas wall areas denoted WAi,

WAo, WAt and WAm represent tissue area only.

Figure 4. Schematic diagram of an airway in a constricted state. Dl and Ds, long and short diameters, respectively of outer border of smooth muscle perimeter. WAi and WAo , inner and adventitial wall areas, respectively; Po and Ao, outer airway perimeter and area, respectively; Pmo and Amo, outer muscle perimeter and area respectively; Pbm and Abm, perimeter basement membrane respectively. The dashed line on the bronchus indicates the path to be traced in measuring Po. (Bai et al, 1994). 2.2. Morphometric analysis of airways in rats and mice

For the morphometric analysis of airways, lungs were fixed with 4% paraformaldehyde

via the trachea cannula. The lungs were removed and slices from different lobes were

embedded in paraffin and cut in 2 µm-thick sections. Measurements of morphometrical

Methodology 63

parameters are made on all airways observed in the stained lung sections. These airways are

cut in transverse section and free of branching. A transverse section is defined as one in

which there is an approximately constant thickness of the airway wall around the entire airway

and has a ratio of minimal I.D. to maximal I.D. greater than 0.5 (figure 4). The morphometric

measurements were made with a Zeiss Axiophot microscope or on the digital representation

of the airways via a computerized image analysis system (KS400; Zeiss, Oberkochen,

Germany).

In rats the following morphometrical parameters (Bai et al, 1994) were marked manually

on the airways: Pbm, Pmo and Po (figure 5A); the areas defined by these parameters (Abm,

Amo and Ao) and the length of Pbm are assessed via the computerized systems. The

following parameters were calculated based on the measured values: the total bronchial wall

area (WAt = Ao - Abm), the inner wall area (WAi = Amo - Abm) and the outer wall area (WAo

= Ao - Amo). The area of smooth muscle (WAm) was measured by specifically tracing

around the smooth muscle bundles. In mice, the following parameters were determined: Pbm,

the area defined by the basement membrane (Abm) and the area defined by the total adventitial

perimeter (Ao). The total bronchial wall area (WAt) was calculated (WAt = Ao - Abm) (figure

5B).

2.3. Assessment of extracellular matrix protein deposition in the airway wall

To assess the deposition of extracellular matrix proteins in the airway wall two

computerized image analysis systems were used in the experiments: the software system

KS400 (Zeiss) and the system LEICA Q500MC (Leic a Cambridge, Cambridge, UK). The

basic principle for the quantification of protein deposition in the airway wall is the same for

both image analysis systems.

Methodology 65

A

B

Figure 5. Morphological parameters assessed in rats (A) and mice (B). A. In rats the following parameters are shown Pbm (black line), WAi (red lines), WAo (blue lines) and WAt (red and blue lines) (hematoxylin and eosin stained section, magnification 200x). B. In mice the parameters Pbm (green line) and WAt (area between green and pink line) are shown (Congo Red stained section, magnification 400x). Pbm = perimeter basement membrane, WAi = inner wall area, WAo = outer wall area and WAt = total wall area.

Figure 6. Assessment of extracellular matrix protein deposition in an airway wall. The small insert shows the area covered by the red-stained collagen fibers for a part of the airway wall. This area was determined by the software KS400 (Zeiss) (Sirius Red stained section, magnification 200x).

Methodology 67

A camera sampled the image of the stained sections and generated an electronic signal

proportional to the intensity of illumination, which was then digitized into picture elements or

pixels. The digital representation of the airways cut in transverse sections was analyzed with

the software. Each pixel in a color image is divided into three color components (hue,

saturation, and intensity). The threshold for each color component for the specific stain of the

extracellular matrix protein (red or brown for total collagen stain with Sirius red or protein

staining via immunohistochemistry, respectively) was defined and kept constant throughout

the analysis. The area covered by the stain was determined by the software (figure 6). The

borders of the different compartments of the airway wall were marked manually and the

software analyzed the amount of protein deposited in these regions.

3. References

Amdur MO and Mead J. Mechanics of respiration in anaesthetized guinea pigs. Am J Physiol 1958, 192:364-368.


Drazen JM, Finn PW and De Sanctis GT. Mouse models of airway responsiveness: physiological basis of observed outcomes and analysis of selected examples using these outcome indicators. Annu Rev Physiol 1999, 61:593-625.

Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG and Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997, 156:766-775.

James AL, Hogg JC, Dunn LA and Pare PD. The use of the internal perimeter to compare airway size and to calculate smooth muscle shortening. Am Rev Respir Dis 1988, 138:136-139.


James AL, Pare PD, Moreno RH and Hogg JC. Quantitative measurement of smooth muscle shortening in isolated pig trachea. J Appl Physiol 1987, 63:1360-1365.

Johanson WG and Pierce AK. A noninvasive technique for measurement of airway conductance in small animals. J Appl Physiol 1971, 30:146-150.

List of Publications 69

LIST OF PUBLICATIONS

Prolonged Allergen Exposure in Sensitized Rats 71

CHAPTER I

PROLONGED ALLERGEN EXPOSURE INDUCES STRUCTURAL

AIRWAY CHANGES IN SENSITIZED RATS

Els Palmans, Johan C. Kips and Romain A. Pauwels

American Journal of Respiratory and Critical Care Medicine 2000, 161:627-635


1. Abstract

The pathogenesis and functional consequences of airway remodeling in asthma remain

to be fully established. In the present study we evaluated the effect of prolonged allergen

exposure on airway function and structure in rats. Sensitized Brown Norway rats were

repeatedly exposed for periods of 2, 4 or 12 wk to aerosolized ovalbumin (OA) or

phosphate-buffered saline (PBS). OA exposure induced a persistent increase in OA-specific

serum IgE and in the number of peribronchial eosinophils. After 2 wk of OA exposure,

airway histology revealed goblet-cell hyperplasia, an increase in bromodeoxyuridine-positive

cells in airway epithelium, increased fibronectin deposition, and a thickening of the airway

inner wall area. This coincided with airway hyperresponsiveness (AHR) to aerosolized

carbachol. After OA exposure for 12 wk increased fibronectin (p<0.05 versus PBS) and

collagen deposition (p<0.05 versus PBS) were observed in the submucosa. After 12 wk of

exposure, neither total nor inner wall area or airway responsiveness to carbachol were any

longer significantly different from those of PBS-exposed animals. In conclusion, prolonged

OA exposure in rats induces structural airway changes that bear similarities to airway

remodeling in asthma. The study further indicates that depending on the extent and

distribution of the remodeling, changes in the extracellular matrix can enhance or protect

against AHR.

2. Introduction

Airway morphology in asthma not only displays characteristics of an acute inflammatory

process, but also structural changes. These include goblet-cell hyperplasia; changes in

extracellular matrix (ECM) composition, with increased subepithelial deposition of collagens

I, III, and V, fibronectin, and tenascin; and neovascularisation and smooth-muscle-cell

hyperplasia and/or hypertrophy (Ebina et al, 1993; Jeffery et al, 1989; Laitinen et al, 1997; Li

and Wilson, 1997; Roche et al, 1989; Takizawa, 1972). These structural changes are

considered to play an important role in the pathophysiology of bronchial

hyperresponsiveness, the functional hallmark of asthma (James et al, 1989; Lambert et al,

1993; Macklem, 1996). The remodeling process that produces these changes is thought to

result from the combination of chronic, repetitive injury to the airway wall, and the ensuing


tissue repair process. A variety of proinflammatory mediators, enzymes, cytokines, and

growth factors have been implicated in the pathophysiology of airway remodeling (Cairns

and Walls, 1996; Cohen et al, 1997; Cohen et al, 1995; Glassberg et al, 1994; Hirst, 1996;

McKay et al, 1998; Panettieri et al, 1990; Stewart et al, 1995; Vignola et al, 1998); however,

both the pathogenesis and the functional consequences of such remodeling must be further

established.

In vivo animal models might provide interesting information in this respect. We and

others have previously shown that exposure to aerosolized allergen in Brown Norway (BN)

rats results in eosinophilic airway inflammation, IgE production, and an increase in airway

responsiveness (Bellofiore and Martin, 1988; Elwood et al, 1991; Haczku et al, 1994; Kips et

al, 1992; Sapienza et al, 1991). However, in these models, airway remodeling was either not

assessed or was revealed to be quite limited (Sapienza et al, 1991). The aim of the present

study was to evaluate whether longer exposure of sensitized animals to inhaled allergen could

result in more pronounced structural airway changes.

3. Methods

3.1. Animals

Specific pathogen-free, male BN rats were obtained from Harlan CPB (Zeist, the

Netherlands). They were housed in the animal research facilities of the Department of

Respiratory Diseases of the University Hospital, Gent, for 1 to 3 wk before testing, and

received food and water ad libidum. The rats were aged 2 to 5 months and weighed between

250 and 350 g at the time of testing.

3.2. Immunization procedures and exposure

All animals were actively sensitized by the intraperitoneal injection of 1 mg of ovalbumin

(OA) (Grade III; Sigma Chemical Co., Poole, UK) together with 200 µg Al(OH)3 in 0.5 ml

0.9% NaCl on the first day of the study (Day 0). Different groups of rats (each group

containing eight to 10 animals) were treated as follows: (1) Intraperitoneal injection of 1 mg

OA and 200 µg Al(OH)3 in 0.5 ml 0.9% NaCl on Day 7, followed from Day 14 to 28 by

thrice weekly exposure to aerosolized OA or PBS (2 wk). (2) Intraperitoneal injection of 1 mg

OA and 200 µg Al(OH)3 in 0.5 ml 0.9% NaCl on Day 7, followed from Day 14 to 42 by


thrice weekly exposure to aerosolized OA or PBS (4 wk). (3) Thrice weekly exposure to

aerosolized OA or PBS from Days 14 to 98 (12 wk).

The aerosol was generated with an ultrasonic nebulizer (Sirius Nova; Carl Heyer GmbH,

Bad Ems, Germany) and was drawn into an exposure chamber containing awake animals.

The output of the nebulizer was 3 ml/min and the median particle size was 3.2 µm according

to specifications by the manufacturer. The concentration of OA in the nebulizer was 1%

(wt/vol), and the duration of exposure was 30 min. Control animals were exposed to

aerosolized, sterile phosphate-buffered saline (PBS).

3.3. Measurement of bronchial responsiveness

At 24 h after the last exposure to OA or PBS, rats were anaesthetized by the

intraperitoneal injection of 60 mg/kg pentobarbital (Sanofi, Libourne, France). The trachea

was cannulated with a Teflon cannula (35 mm long and 1.67 mm internal diameter [I.D.]). The

femoral artery was cannulated with a polyethylene catheter (1.77 mm I.D., 2.80 mm outer

diameter; Intramedic; Clay Adams, Parsippany, NJ) to monitor blood pressure and heart rate

with a pressure transducer (Celesco Transducer Products Inc., Canoga Park, CA)

throughout the experiment. A polyethylene catheter was inserted in the external jugular vein for

administration of intravenous drugs and fluids.

Animals were placed on a heating pad (37°C), were connected with a Palmer animal

respirator (BioScience, Sheerness, UK), and were ventilated with oxygen-enriched air at a rate

of 75 strokes/min and a stroke volume of 2 to 2.5 ml. Animals were injected with

pancuronium bromide (Organon Teknika B.V., Boxtel, the Netherlands), at 0.2 mg/kg body

weight to interrupt spontaneous respiratory movements. The animals’ arterial blood gases

were measured after 10 min of ventilation, and the stroke volume was adapted to keep pH,

pO2 and pCO2 within the normal ranges.

The animals’ transpulmonary pressure (Ppt) was measured with a differential pressure

transducer (Celesco Transducer Products), of which one end was attached to an air-filled

catheter inserted into the pleural cavity and the other end was attached to a catheter connected

to a sideport of the outlet of the tracheal cannula. The airflow at the outlet of the intratracheal

cannula was measured with a pneumotachograph (Type 00000; Fleisch, Geneva,

Switzerland). The tidal volume (VT) was obtained by integration of the flow. Lung resistance

(RL) was continuously calculated from VT, air flow, and Ppt (PRS800; Mumed, London,

UK).


Carbachol (carbamoylcholine hydrochloride; Merck, Darmstadt, Germany) was

nebulized with an ultrasonic nebulizer (Model 2511; Pulmo-Sonic, DeVilbiss Co., Somerset,

PA). The mean particle size of the aerosol was 3.8 µm and the output was 1.0 ml/min. The

aerosol was led into the inspiratory tube of the respiratory pump. Increasing concentrations of

carbachol were administered over a period of 30 s at 5 min intervals, allowing RL to return to

baseline between administrations of each concentration. Carbachol-induced

bronchoconstriction was measured as the percent increase in RL, comparing the peak of the

reaction with baseline RL.

3.4. Bronchoalveolar lavage

After determination of the bronchial responsiveness, lungs were lavaged via the tracheal

cannula with six volumes of 6 ml each of Hank’s balanced salt solution (HBSS), free of

ionized calc ium and magnesium and supplemented with 0.05 mM sodium EDTA. Lavage

fluid was recovered by gentle manual aspiration with a syringe. The lavage fluid was

centrifuged and the cell pellet was washed in HBSS and incubated for 7 min in 1 ml of NH4Cl

to remove red blood cells. The cell pellet was then washed twice and resuspended in 1 ml

HBSS. Total numbers of leukocytes in the bronchoalveolar lavage fluid (BALF) were

determined with a Coulter counter (Coulter Electronics Ltd, Harpeneden, UK). A differential

cell count was performed on cytocentrifuged preparations (Cytospin 2; Shandon Ltd,

Runcorn, UK) stained with May-Grünwald-Giemsa, and was based on standard

morphological criteria of at least 300 cells.

3.5. Histological analysis of lung tissue

After being lavaged, lungs were fixed with 4% paraformaldehyde via the trachea cannula.

The lungs were removed and slices from different lobes were embedded in paraffin and cut in

2 µm-thick sections. Histological changes were evaluated on sections stained with

hematoxylin and eosin (H&E). Additionally, Congo Red and Sirius Red stains were

performed for specific assessment of eosinophil number and collagen deposition

respectively.

3.6. Immunohistochemical staining for fibronectin

The detection of fibronectin was done out as previo usly described (Casscells et al,

1990) with a slight modification. Sections were deparaffinized, rehydrated, and incubated in


3% H2O2 in methanol for 10 min at room temperature to block endogenous peroxidase

activity. The sections were then washed with 0.25% Brij 35 (Merck) in Tris -buffered saline

(TBS) (0.15M NaCl, 0.01M Tris-HCl; pH 7.4), incubated in 0.4% pepsin (Sigma Chemical)

in 0.01N HCl at 37°C for 15 min, and rinsed with TBS. Nonspecific binding sites were

blocked with 1% blocking reagent (Boehringer Mannheim GmbH, Mannheim, Germany) in

PBS for 30 min. Excess reagent was removed and the sections were incubated for 1 h with

goat antirat fibronectin antibody (Calbiochem-Novabiochem GmbH, Bad Soden, Germany)

at a dilution of 1:800 in TBS. After incubation with the primary antibody, the sections were

rinsed with 0.25% Brij 35 in TBS (pH 7.4) and incubated for 30 min with biotinylated rabbit

antigoat IgG (DAKO A/S, Glostrup, Denmark) diluted 1:200 in TBS (pH 7.4). The primary

antibody-secondary antibody complex was detected by incubating the sections for 30 min

with streptavidin -biotinylated horseradish peroxidase complex (Nycomed Amersham Plc.

Buckinghamshire, UK) diluted 1:200 in TBS (pH 7.4). The chromogenic substrate for the

peroxidase was 3,3’-diaminobenzidine (DAB) (DAKO), giving a brown reaction product.

Sections were counterstained with hematoxylin and mounted in Paramount. Two control

procedures were performed for fibronectin detection: (1) normal goat serum was substituted

for the primary antibody; and (2) goat antirat fibronectin antibody (diluted 1:800) was

adsorbed overnight at 4°C with 100 µg fibronectin, and the supernatant of the antibody-

fibronectin complex was substituted for the primary antibody.

3.7. Cell proliferation in the airways

To detect proliferating cells in lung tissue, animals were injected intraperitoneally twice

weekly from Day 14 onward with bromodeoxyuridine (BrdU) (20 mg/kg) (Nycomed

Amersham). BrdU is a thymidine analogue and is incorporated during the S phase of the cell

cycle. BrdU was detected immunohistochemically in 2 µm sections that were deparaffinized

and rehydrated. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for

10 min, after which the sections were incubated at 37°C for 15 min in 0.1% trypsin (Sigma

Chemical), rinsed in tap water, and incubated for a further 15 min at 37°C with 2M HCl.

Nonspecific binding sites were blocked with 5% normal sheep serum (NSS) for 30 min.

Excess reagent was removed and the sections were incubated for 2 h with mouse anti-BrdU

antibody (Nycomed Amersham) diluted 1:75 in 1% NSS. After incubation with the primary

antibody, the sections were rinsed with TBS (pH 7.5) and incubated for 30 min with

biotinylated sheep antimouse IgG (Nycomed Amersham) diluted 1:200 in TBS (pH 7.5). The


primary antibody-secondary antib ody complex was detected by incubating the sections for

30 min with streptavidin-biotinylated horseradish peroxidase complex (Nycomed Amersham)

diluted 1:200 in TBS (pH 7.5). The sections were then incubated for 10 min with DAB,

rinsed in tap water and counterstained with hematoxylin. As a negative control 1% NSS was

substituted for the primary antibody. Sections of the duodenum were used as a positive

control.

3.8. Morphometric analysis of the airways

H&E-stained tissue sections were examined light-microscopically, and morphometric

measurements were made with a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany)

at magnifications between x200 and x400. For each treatment group, four stained lung

sections from six to 10 rats each were analyzed. The following morphometric parameters (as

previously described by Bai et al 1994) were measured in all airways cut in reasonable cross-

sections (defined by a ratio of minimal I.D. to maximal I.D. greater than 0.5): the length of the

basement membrane of the epithelium (Pbm), the perimeter of the outer boundary of the

smooth muscle (Pmo) and the outer adventitial perimeter (Po); the areas defined by these

parameters (Abm, Amo and Ao) and the area of smooth muscle (WAm). The following

parameters were calculated based on the measured values: the total bronchial wall area (WAt =

Ao - Abm), the inner wall area (WAi = Amo - Abm) and the outer wall area (WAo = Ao - Amo).

Furthermore, the number of epithelial cells, the number of BrdU-positive cells, and the

number of goblet cells along the basement membrane were counted. The total number of

eosinophils and the number of BrdU-stained smooth muscle cells in the bronchial wall were

determined.

Deposition of collagen and fibronectin was examined by light microscopy, and

quantitative measurements of each were made with a computerized image analysis system

(LEICA Q500MC; Leica Cambridge Ltd., Cambridge, UK). A camera sampled the image of

the stained sections and generated an electronic signal proportional to the intensity of

illumination, which was then digitized into picture elements or pixels. The digital representation

of the airways was analyzed with Qwin software (Leica Cambridge). Each pixel in a color

image was divided into three color components (hue, saturation, and intensity). The threshold

for each color component for the red stain of collagen and the brown stain of fibronectin was

defined and kept constant throughout the analysis. In different fields around an airway, the

area covered by the stain was determined by the software, and its value was calculated. For


each experimental group, three lung sections from 10 rats each, stained for collagen, were

examined. Fibronectin deposition was measured in three lung sections from five rats exposed

to OA or PBS for 2 or 12 wk. The amount of protein deposited in the various components

of the total airway wall was measured for all airways cut in reasonable cross-sections.

3.9. Measurement of OA-specific IgE

OA-specific IgE was measured with an enzyme-linked immunosorbent assay (ELISA).

Blood was obtained by cardiac puncture at the end of the exposure period. Serum was

added to OA (Grade V; Sigma Chemical) -coated microwell plates, and OA-specific IgE was

detected with mouse antirat IgE antibody (H. Bazin; Experimental Immunology Unit,

Brussels, Belgium) followed by horseradish peroxidase-conjugated goat antimouse

immunoglobulins (DAKO). After addition of the substrate (o-phenylenediamine

dihydrochloride; Sigma Chemical) plates were read in a plate reader (Titertek Multiskan MCC;

Flow Laboratories, Irvine, Scotland) at 492 nm. The results are expressed in nanograms

ovalbumin bound to IgE per milliliter of serum.

3.10. Data analysis

Reported values are expressed as mean ± SEM. For measurements of bronchial

responsiveness, cumulative dose-response curves for the changes in RL with increasing doses

of carbachol were constructed. The changes in RL were expressed as percent increases in RL.

The concentration of carbachol causing a 50% increase in baseline RL (PC50RL) was

calculated by log-linear interpolation of the dose-response curve. The cumulative dose-

response curves were compared through analysis of variance (ANOVA). PC50RL values,

cellular composition of the BALF, OA-specific IgE, BrdU- positive cells and goblet cells in

the epithelium, and peribronchial eosinophils for the different experimental groups were

compared through the Kruskal-Wallis test for multiple comparisons. When significant

differences were observed, pairwise comparisons were made, using a Mann-Whitney U-test

with Bonferroni’s corrections.

For analysis of the morphometric measurements and the measurements of collagen and

fibronectin deposition, the data for the rats in a particular experimental group were pooled,

and the values of WAt, WAi, WAo, area of collagen deposition (WC), and area of fibronectin

deposition (WF) were normalized to Pbm. Thereafter, the airways were divided into three

categories according to the length of their basement membrane, as previously described by


Sapienza (Sapienza et al, 1991)): Pbm ≤ 1 mm (small), Pbm > 1 mm and ≤ 2 mm (medium)

and Pbm > 2 mm (large). To ensure that a similar range of airway sizes was being compared,

we compared the frequency distribution of Pbm for the PBS and OA experimental group for

each category of airways, using the Kolmogorov-Smirnov test. The mean values of WAt,

WAi, WAo, WC, and WF normalized to Pbm for each category of airways were compared

for the experimental groups through an unpaired t-test. Values of p<0.05 were regarded as

significant.

4. Results

4.1. Airway responsiveness

A 2-wk exposure to aerosolized OA induced an increase in airway responsiveness to

inhaled carbachol as compared with PBS-exposed animals. This was reflected by a

significant leftward shift of the dose-response curve (ANOVA, p<0.05) (Figure 1) and a

significant decrease in the PC50RL for carbachol (p<0.01) (Table 1). More prolonged

exposure to OA resulted in a loss of airway hyperresponsiveness. After 4 wk of OA

exposure, the PC50RL and dose-response curve for carbachol were no longer significantly

different from those of control animals (Table 1). The PC50RL for rats exposed to OA for 12

wk was significantly greater than that of control animals (Table 1), although the dose-response

curve for these animals was not significantly different from that of control animals (Figure 1).

The dose-response curves for carbachol of control animals exposed for 2, 4, or 12 wk to

PBS were not significantly different from one another. A leftward shift of the dose-response

curves for animals exposed to OA for 2 wk was observed as compared with the curves for

animals exposed to OA for 4 and 12 wk (ANOVA, p<0.05). No significant difference was

found in the dose-response curves for animals exposed to OA for 4 and 12 wk.

TABLE 1

EFFECT OF REPEATED OA EXPOSURE ON AIRWAY RESPONSIVENESS TO CARBACHOL AND ON OA-SPECIFIC IGE LEVELS IN THE SERUM*

PC50RL carbachol IgE serum

(mg/ml) (µg IgE/ml serum)


Treatment 2 wk 4 wk 12 wk 2 wk 4 wk 12 wk

PBS 1.73±0.25 1.86±0.45 0.86±0.13 1.48±0.60 1.66±0.80 1.42±0.53

OA 0.83±0.16‡ 2.75±0.89 ll 1.18±0.33† 9.24±1.70§ 15.87±1.4 § # 13.94±1.52§

¶ *n = 10 for all groups † p < 0.05, ‡ p < 0.01 and § p < 0.001, versus PBS ll p < 0.05, ¶ p < 0.01, OA versus OA 2 weeks # p < 0.001 OA versus OA 4 weeks

Figure 1. Bronchial responsiveness to aerosolized carbachol in sensitized BN rats exposed for (A) 2 wk,

(B) 4 wk, and (C) 12 wk to OA (closed squares) and PBS (open squares) (n = 10 for each group) (ANOVA for OA versus PBS, 2 wk, p<0.05).

4.2. OA-specific IgE

Serum levels of OA-specific IgE were consistently increased in sensitized rats exposed to

OA for 2, 4, and 12 wk as compared with those of control animals (Table 1). Peak levels of

OA-specific IgE were reached after 4 wk of OA exposure (Table 1).

4.3. BALF

Total cell counts were significantly increased in BALF obtained from OA-exposed

animals (Table 2). The to tal cell number increased at up to 4 wk and remained stable

afterwards. OA-exposed groups also had significantly greater numbers of eosinophils and

neutrophils in their BALF than did controls. The number of eosinophils was not significantly

different at 2, 4, or 12 wk, whereas the number of neutrophils increased further at 4 and 12 wk

(Table 2).

A. 2 weeks

0.01 0.1 1 10

0

50

100

150

200

concentration carbachol (mg/ml)

% i

ncr

ease

in

RL

B. 4 weeks

0.01 0.1 1 10

0

50

100

150

200


% i

ncr

ease

in

RL

C. 12 weeks

0.01 0.1 1 10

0

50

100

150

200


% i

ncr

ease

in

RL


4.4. Histology

Inflammatory cell infiltrates, including eosinophils, were observed in the peribronchial

area of sensitized and OA-exposed BN rats (Figure 2). After 2 wk of OA exposure, the

number of eosinophils around the airways was significantly increased compared to control

animals (Table 3). The number of infiltrating eosinophils remained increased after 4 and 12 wk

of OA exposure (Table 3).

Light-microscopic analysis did not reveal epithelial desquamation in any of the BN rats.

The ratio of goblet cells to epithelial cells was increased in all sensitized and antigen-exposed

rats (Table 3). The maximal increase in goblet cells was observed after 2 wk of exposure. The

number of BrdU-positive cells in the epithelium was measured in sensitized rats exposed to

PBS or OA during 2 or 4 wk. An increase in proliferating epithelial cells was observed after 2

wk (6.0±1.5 BrdU-positive cells/mm Pbm [mean±SEM] versus 1.9±0.6 BrdU-positive

cells/mm Pbm in PBS-exposed animals; p < 0.05) and 4 wk (11.1±0.9 BrdU-positive

cells/mm Pbm versus 2.3±0.9 BrdU-positive cells/mm Pbm in PBS-exposed animals; p <

0.001) of OA exposure. No BrdU-positive cells were found in the airway smooth muscle.


Lym

phoc

ytes

(x 1

05 )

1.57

±0.1

4

3.83

±0.0

8†

3.0

±1.1

5.50

±1.3

0

0.40

±0.0

1

7.64

±0.2

7§

N

eutr

ophi

ls

(x 1

05 )

1.02

±0.0

8

4.48

±0.1

4‡

1.40

±0.4

0

59.3

0±14

.00§

#

0.31

±0.0

1

69.9

9±1.

94§

#

Eos

inop

hils

(x 1

05 )

1.12

±0.0

6

7.59

±0.0

8§

0.20

±0.1

0

4.60

±1.4

0§

0.02

±0.0

1

3.69

±0.0

9§

Mon

ocyt

es

(x 1

05 )

42.6

7±0.

03

56.2

5±0.

23†

34.3

0±5.

60

88.4

0±5.

80‡

33.6

6±0.

01

89.9

8±1.

79§

Tot

al c

ells

(x 1

05 )

46.4

0±10

.70

72.2

0±7.

90†

38.8

2±6.

60

157.

80±1

7.70

§ ¶

34.4

0±4.

70

171.

3±3

0.00

§ ll

PBS

OA

PBS

OA

PBS

OA

TA

BL

E 2

TO

TA

L N

UM

BE

R O

F W

HIT

E B

LO

OD

CE

LL

S A

ND

CE

LL

UL

AR

DIS

TR

IBU

TIO

N

IN B

RO

NC

HIO

LA

R L

AV

AG

E F

LU

ID*

Tre

atm

ent

2 w

k

4 w

k

12 w

k

* n =

10

for a

ll g

roup

s

† p <

0.0

5, ‡ p

< 0

.01

and

§ p <

0.0

01 v

ersu

s PB

S

ll p <

0.0

5, ¶ p

< 0

.005

, # p <

0.0

01 O

A v

ersu

s 2

wee

ks O

A


A

B

Figure 2. Airway sections of sensitized BN rats exposed to PBS (A) and OA (B) for 2 wk (Congo Red; original magnification x400).


TABLE 3

NUMBER OF GOBLET CELLS IN EPITHELIUM AND NUMBER OF AIRWAY-WALL EOSINOPHILS AFTER REPEATED OVALBUMIN EXPOSURE

Peribronchial eosinophils Goblet cells

(cells/mm2 total airway wall) (% goblet cells/epithelial cells)

Treatment 2 wk 4 wk 12 wk 2 wk 4 wk 12 wk

PBS 254±49 125±33 124±57 4.8±2.2 1.7±0.6 1.5±0.3

OA 664±68‡ 728±84‡ 746±161‡ 11.3±1.4* 6.1±1.1† 9,6 ±1.1 ‡ * p < 0.05, † p < 0.01 and ‡ p < 0.001 versus PBS

4.5. Morphometric measurements

For the morphometric measurements, a mean of 54 airways (range: 45 to 68) per

experimental group were analyzed. The average number of airways analyzed within each

category (large, medium, and small) was 20 (range: 12 to 33). The total airway wall area of

small, medium, and large airways was increased in sensitized rats exposed to OA for 2 wk, as

compared with the areas in control animals (Figure 3). This increase disappeared upon

prolonged exposure. Similarly, inner wall area of small and large airways increased

significantly in rats exposed to OA for 2 wk, but not for 4 or 12 wk (Figure 3). As compared

with that of control animals, the outer wall area was increased in small (p<0.001), medium

(p<0.01), and large (p<0.05) airways after 2 wk of OA exposure, and in small (p<0.01), and

medium (p<0.05) airways after 4 wk of OA exposure (data not shown). The area of the

smooth-muscle layer around the airways did not change after OA exposure.


Figure 3. Effect of exposure to OA (solid bars) or PBS (open bars) for 2 wk (A and B), 4 wk (C and D),

and 12 wk (E and F) on WAt (A, C, and E) and WAi (B, D, and F) in small, medium, and large airways.

4.6. Fibronectin and collagen deposition

Fibronectin deposition was analyzed in a mean of 59 airways (range: 53 to 63) per

experimental group, and collagen deposition was analyzed in a mean of 43 airways (range: 39

to 51) per experimental group. The average number of airways studied for fibronectin

deposition in each category was 20 (range: 13 to 25), and the average number studied for

collagen was 21 (range: 11 to 20). At 2 wk, increased fibronectin deposition was observed in

large and medium airways of OA-exposed animals. This was further increased after 12 wk

exposure (Figures 4 and 5). Fibronectin was deposited in the outer airway wall adjacent to the

smooth-muscle layer (Figure 4). Large airways of rats exposed to OA for 12 wk also showed

increased fibronectin deposition in the inner airway wall (p<0.01) (data not shown). The

amount of collagen was increased in large airways of animals exposed to OA for 4 and 12 wk

(Figure 6). After 4 wk, an increased amount of collagen was deposited in the outer airway wall

(p<0.05) (data not shown); after 12 wk, an increase was observed in the inner (Figure 6) as

well as in the outer airway wall (p<0.01) (data not shown).

A B

A. 2 weeks

small medium large0

25

50

75

100

p<0.01

p<0.01

p<0.001

WA

t/Pbm

(µm

²/µm

)

C. 4 weeks

small medium large0

25

50

75

100

p<0.01

WA

t/Pbm

(µm

²/µm

)

E. 12 weeks

small medium large0

25

50

75

100

WA

t/Pbm

(µm

²/µm

)

B. 2 weeks

small medium large0

10

20

30

p<0.01

p<0.01

WA

i/Pbm

(µm

²/µm

)

D. 4 weeks

small medium large0

10

20

30

WA

i/Pbm

(µm

²/µm

)

F. 12 weeks

small medium large0

10

20

30

WA

i/Pbm

(µm

²/µm

)


C

D

Figure 4. Airway sections of sensitized BN rats exposed to PBS (A and C) and OA (B and D), stained

for fibronectin after an exposure period of 2 wk (A and B) and 12 wk (C and D) (hematoxylin counterstain; original magnification x400).


Figure 5. Effect of exposure to OA (solid bars) or PBS (open bars) for 2 wk (A and B) and 12 wk (C and D) on the amount of fibronectin deposited in the total airway wall (WFt) (A and C) and in the outer airway wall (WFo) (B and D) of small, medium, and large sized airways.

Figure 6. Effect of exposure to OA (solid bars) or PBS (open bars) for 2 wk (A and B), 4 wk (C and D),

and 12 wk (E and F) on the amount of collagen deposition in the total airway wall (WCt) (A, C and E) and in the inner airway wall (WCi) (B, D and F) of small, medium, and large airways.

A. 2 weeks

small medium large0

1

2

3

4

p<0.05

p<0.01WFt

/Pbm

(µm

²/µm

)

C. 12 weeks

small medium large0

1

2

3

4

p<0.01

p<0.01

p<0.001

WFt

/Pbm

(µm

²/µm

)

B. 2 weeks

small medium large0

1

2

3

p<0.01

p<0.05

WFo

/Pbm

(µm

²/µm

)

D. 12 weeks

small medium large0

1

2

3

p<0.01

p<0.01

p<0.001

WFo

/Pbm

(µm

²/µm

)

A. 2 weeks

small medium large0

10

20

30

40

WC

t/Pbm

(µm

²/µm

)

C. 4 weeks

small medium large0

10

20

30

40

p<0.05

WC

t/Pbm

(µm

²/µm

)

E. 12 weeks

small medium large0

10

20

30

40p<0.05

WC

t/Pbm

(µm

²/µm

)

B. 2 weeks

small medium large0

5

10

15

WC

i/Pbm

(µm

²/µm

)

D. 4 weeks

small medium large0

5

10

15

WC

i/Pbm

(µm

²/µm

)

F. 12 weeks

small medium large0

5

10

15

p<0.05

p<0.05

WC

i/Pbm

(µm

²/µm

)


5. Discussion

Airway remodeling encompasses the structural changes observed in asthmatic airways.

The pathophysiology of remodeling and its effect on airway physiology remain to be fully

established. In vivo animal models might provide useful information in this respect. However,

most of the currently developed animal models of allergic airway inflammation have been

restricted to acute inflammatory changes following relatively short periods of allergen

exposure. In the present study we investigated the effect of more prolonged OA exposure on

the airways of sensitized BN rats. In addition to producing an increase in OA-specific IgE and

persistent eosinophilic airway inflammation, repeated exposure to aerosolized OA over a

period of up to 12 wk also resulted in greater structural airway changes. These included

goblet-cell hyperplasia, increased mitogenic activity in the epithelium, and changes in ECM

components in the form of increased collagen and fibronectin deposition. The dynamics of

these structural changes are clearly different, since goblet-cell hyperplasia and fibronectin

deposition could be observed from 2 wk of exposure onward, whereas increased collagen

deposition occurred only after more prolonged exposure. Other models have also shown

goblet-cell hyperplasia after relative short exposure periods (Blyth et al, 1996; Temelkovski et

al, 1998). However, to the best of our knowledge, ours is the first model to show increased

mucosal collagen deposition following repeated allergen exposure, thus mimicking the

subepithelial fibrosis from increased deposition of collagen I, III and V observed in human

asthma (Laitinen et al, 1997; Roche et al, 1989).

Airway remodeling is thought to have a profound effect on airway responsiveness.

Different mathematical models describe the potential effect of structural changes on the

function of asthmatic airways (James et al, 1989; Lambert et al, 1993; Macklem, 1996).

Thickness of the inner airway wall, smooth-muscle mass, and adventitial structure are

considered to be the main determinants in the airway response to a bronchoconstrictor

stimulus (Moreno et al, 1986). Increased airway wall thickness has little effect on baseline

resistance, but enhances the effect of a given smooth-muscle contraction, following

Poiseuille’s law for laminar flow, thus contributing to increased airway sensitivity (Moreno et

al, 1986). It has been shown in rats that changes in pulmonary resistance after methacholine

inhalation are mainly due to changes at the large airway level (Sapienza et al, 1991). In our

model an increase in inner wall area at the large airway level was observed only in rats exposed


to aerosolized OA for 2 wk. The observed leftward shift of the dose-response curve of RL for

aerosolized carbachol could therefore result from this increase in inner airway wall thickness.

Increased smooth-muscle mass around the airways could also contribute to bronchial

hyperresponsiveness (Macklem, 1996). Our model showed no changes in the smooth muscle

layer, either by morphometric analysis or BrdU incorporation at 2 and 4 wk. This factor is

therefore unlikely to have contributed to the altered airway behavior in our model. This

contrasts with previous findings in BN rats of an increase in smooth-muscle thickness and

smooth-muscle proliferation following repeated allergen exposure (Panettieri, Jr. et al, 1998;

Sapienza et al, 1991). These divergent observations can at least be partly explained by

differences in the substrains of BN rats (BN/RyHsd versus BN/SSN Hsd) and in the

sensitization and exposure protocols used in these studies and our own.

Another element that could affect airway responsiveness are changes in the composition

of the ECM in the airways. It has been calculated that subepithelial collagen deposition in

asthmatic airways can influence the buckling pattern of the airway mucosa when the airway

narrows (Wiggs et al, 1997). A smaller number of folds result in an enhanced increase in

intraluminal pressure. In addition, changes in the ECM of the adventitial layer, in combination

with fluid fluxes during smooth-muscle contraction, can reduce elastic recoil exerted on the

airways by the lung parenchyma, thus contributing to AHR (Macklem, 1996). Conversely,

increased collagen deposition could increase airway wall stiffness, thus opposing against

narrowing of the airway wall (Lambert et al, 1994), and increased collagen deposition within

and around the smooth-muscle layer can interfere with smooth-muscle contraction, thus again

limiting airway narrowing (Bramley et al, 1995). In the present model, a small increase in

fibronectin deposition around the smooth-muscle layer was observed after a 2-wk exposure

period. At 12 wk this was far more pronounced. Increased deposition of collagen was

observed after 4 wk, but especially after 12 wk exposure. This coincided with a gradual

reduction in the airway wall thickness, whereas the AHR observed after 2 wk waned and

turned into hyporesponsiveness at 12 wk.

It has to be borne in mind that the ECM does not merely constitute a biologically

inactive framework within tissues, but consists of a highly regulated network of proteins and

proteoglycans that interact profoundly with inflammatory cells and other structural tissue

components (Raghow, 1994). Among its many effects, fibronectin binds to α5β1 integrins

on the surfaces of cells, thus not only influencing cell trafficking, but also affecting cell

function and survival (Zhang et al, 1995). During tissue regeneration, fibronectin production


precedes collagen synthesis (Welch et al, 1990). The ongoing inflammation observed in the

airways of rats after prolonged OA exposure might induce a repair process in which the ECM

is regenerated. Changes in the deposition of fibronectin starts after 2 wk, but the increase in

collagen content of the airway wall is observed only after a longer exposure period.

Fibronectin attaches collagen fibers to the elastin framework and to fibroblasts, mediating

contraction of collagen by fibroblasts (LeviSchaffer et al, 1999). The more pronounced

increase in fibronectin deposition around the airway smooth-muscle layer after 12 wk, in

combination with increased collagen deposition in the outer airway wall, could therefore result

in a tighter adhesion between lung parenchyma and the outer airway wall and prevent fluid

fluxes into the adventitium during smooth-muscle contraction. The changes of collagen

composition in the inner airway wall after 12 wk could also increase the stiffness of the airway

wall (Lambert et al, 1994). These observations suggest that increasing airway wall stiffness,

reducing airway wall thickness, enhancing elastic recoil, and changes in the ECM both within

and around the smooth-muscle layer might constitute an attempt to oppose against bronchial

hyperresponsiveness.

The present model also adds to previous rat models showing that the presence of

eosinophils per se in the airway tissue is insufficient to cause AHR (Haczku et al, 1994; Kips

et al, 1992). The influx of eosinophils in and around the airways persists with prolonged

allergen exposure, as does IgE synthesis, yet the increase in airway responsiveness

progressively declines. This illustrates that the loss of bronchial hyperresponsiveness is

probably not due to immunological tolerance, but is related to the development of

compensatory mechanisms, the loss of which could contribute to the persistent bronchial

hyperresponsiveness observed in human asthma.

In conclusion, we have described an in vivo rat model that displays characteristics of

airway remodeling. We postulate that the increase in airway responsiveness is due to increased

thickness of the inner airway wall of the large airways. Prolonged exposure results in scarring

of the airway wall, with a reduction in airway wall thickness and a loss of AHR. These

observations suggest that, depending on the extent and distribution of airway wall fibrosis,

airway remodeling might oppose, rather than enhance AHR.

6. References





Bramley AM, Roberts CR and Schellenberg RR. Collagenase increases shortening of human bronchial smooth muscle in vitro. Am J Respir Crit Care Med 1995, 152:1513-1517.

Cairns JA and Walls AF. Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J Immunol 1996, 156:275-283.

Casscells W, Kimura H, Sanchez JA, Yu Z-X and Ferrans VJ. Immunohistochemical study of fibronectin in experimental myocardial infarction. Am J Pathology 1990, 137:801-810.

Cohen MD, Ciocca V and Panettieri RA, Jr. TGF-beta 1 modulates human airway smooth-muscle cell proliferation induced by mitogens. Am J Respir Cell Mol Biol 1997, 16:85-90.

Cohen P, Noveral JP, Bhala A, Nunn SE, Herrick DJ and Grunstein MM. Leukotriene D4 facilitates airway smooth muscle cell proliferation via modulation of the IGF axis. Am J Physiol 1995, 269:L151-L157.


Elwood W, Lotvall JO, Barnes PJ and Chung KF. Characterization of allergen-induced bronchial hyperresponsiveness and airway inflammation in actively sensitized brown-Norway rats. J Allergy Clin Immunol 1991, 88:951-960.

Glassberg MK, Ergul A, Wanner A and Puett D. Endothelin -1 promotes mitogenesis in airway smooth muscle cells. Am J Respir Cell Mol Biol 1994, 10:316-321.

Haczku A, Moqbel R, Elwood W, Sun J, Kay AB, Barnes PJ and Chung KF. Effects of prolonged repeated exposure to ovalbumin in sensitized brown Norway rats. Am J Respir Crit Care Med 1994, 150:23-27.





Kips JC, Cuvelier CA and Pauwels RA. Effect of acute and chronic antigen inhalation on airway morphology and responsiveness in actively sensitized rats. Am Rev Respir Dis 1992, 145:1306-1310.




LeviSchaffer F, Garbuzenko E, Rubin A, Reich R, Pickholz D, Gillery P, Emonard H, Nagler A and Maquart FAX. Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro: A role for transforming growth factor beta (TGF-beta). Proc Natl Acad Sci U S A 1999, 96:9660-9665.



McKay S, de Jongste JC, Saxena PR and Sharma HS. Angiotensin II induces hypertrophy of human airway smooth muscle cells: expression of transcription factors and transforming growth factor-beta1. Am J Respir Cell Mol Biol 1998, 18:823-833.

Moreno RH, Hogg JC and Pare PD. Mechanics of airway narrowing. Am Rev Respir Dis 1986, 133:1171-1180.

Panettieri RA, Yadvish PA, Kelly AM, Rubinstein NA and Kotlikoff MI. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am J Physiol 1990, 259:L365-L371.



Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J 1994, 8:823-831.



Stewart AG, Tomlinson PR, Fernandes DJ, Wilson JW and Harris T. Tumor necrosis factor alpha modulates mitogenic responses of human cultured airway smooth muscle. Am J Respir Cell Mol Biol 1995, 12:110-119.

Takizawa T. Muscle and mucous gland size in the major bronchi of patients with chronic bronchitis , asthma and asthmatic bronchitis. Am Rev Respir Dis 1972, 104:331-336.


Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D'accardi P, Bousquet J and Bonsignore G. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998, 158:1945-1950.

Welch MP, Odland GF and Clark RA. Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction. J Cell Biol 1990, 110:133-145.


Zhang Z, Vuori K, Reed JC and Ruoslahti E. The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci U S A 1995, 92:6161-6165.

Collagen Deposition in Rat Airway Wall 99

CHAPTER II

REPEATED ALLERGEN EXPOSURE CHANGES COLLAGEN

COMPOSITION IN AIRWAYS OF SENSITIZED BROWN NORWAY

RATS

Els Palmans, Romain A. Pauwels and Johan C. Kips

European Respiratory Journal, In Press


1. Abstract

Increased or altered collagen deposition in the airway wall is one of the characteristics of

airway remodeling in asthma. The mechanisms underlying this increase, and its functional

consequences remain to be further established. Representative in vivo animal models might be

useful in this respect. In the present study we characterized collagen deposition after

prolonged allergen exposure in the airway wall of Brown Norway rats. Sensitized rats were

repeatedly exposed to ovalbumin (OA) or phosphate-buffered saline (PBS) during 2 and 12

weeks. The deposition of collagen type I, III, IV, V and VI was not altered in animals

exposed to OA for 2 weeks. After 12 weeks of OA exposure, more collagen type I was

deposited in the inner and outer airway wall (p < 0.01 versus PBS) and more type V and VI

collagen were observed in the outer airway wall (p < 0.05 versus PBS). At 12 weeks the

number of vessels, identified via type IV collagen staining was not increased, but the total

vessel area was (p < 0.05 versus PBS). In conclusion, prolonged allergen exposure in

sensitized rats is associated with enhanced deposition of type I, V and VI collagens and

increased vascularity.

2. Introduction

Different extracellular matrix (ECM) proteins have been identified throughout the airway

wall. The basement membrane (BM) underneath the airway epithelium consists of type IV

collagen, proteoglycans, laminin, and fibronectin (Roche et al, 1989). Beneath this BM the

ECM of normal airways is composed of collagens type I, III and V, elastins, proteoglycans,

fibronectin and laminins. The major fibrillar components of the matrix, type I and III collagen

and elastin, are the primary determinants of the mechanical properties of airways (Campa et al,

1993).

The asthmatic airway wall displays structural changes including sub-basement membrane

thickening, altered submucosal ECM deposition and neovascularization (Bousquet et al,

1996; Lee et al, 1999; Li and Wilson, 1997; Roche et al, 1989; Wilson and Li, 1997). The

thic kened layer beneath the BM comprises predominantly types I, III, and V collagen together

with fibronectin and tenascin (Laitinen et al, 1997; Roche et al, 1989). Moreover, increased

deposition of collagen type III, V and VI has also been observed in the bronchial submucosa


in chronic asthmatics (Lee et al, 1999; Wilson and Li, 1997). Others have observed the

presence of increased amounts of fragmented elastin fibers under the epithelium (Bousquet et

al, 1996). Both the pathogenesis and the functional consequences of these ECM changes in

asthmatic airways remain uncertain.

An appropriate in vivo animal model could provide useful information in this respect.

We have previously described that prolonged allergen exposure induced structural changes in

the airways of sensitized Brown Norway (BN) rats (Palmans et al, 2000). In these animals

exposure to allergen for up to 3 months resulted in an increased amount of collagen and

fibronectin in the large airways (Palmans et al, 2000). We now further characterized the

increased collagen deposition by evaluating the amount of different collagen types in the

airway wall of sensitized rats exposed to allergen for 14 days and 3 months.

3. Methods

3.1. Sensitization and exposure of animals

Male BN rats (Harlan CPB; Zeist, the Netherlands) were sensitized and exposed to

allergen as previously described (Palmans et al, 2000). In brief, all animals were sensitized by

the intraperitoneal (ip) injection of 1 mg ovalbumin (OA) (Sigma Chemical Co., Poole, UK)

and 200 µg Al(OH)3 on day 0. One group of animals (n=5) was boosted with 1 mg OA and

200 µg Al(OH)3 ip on day 7 and exposed from day 14 to day 28 to aerosolized 1% OA

thrice weekly (2 weeks). A second group of animals (n=5) was similarly exposed to

aerosolized 1% OA from days 14 to 98 (12 weeks). Control animals (n=5 per group) were

exposed to sterile phosphate-buffered saline (PBS).

3.2. Lung histology

Twenty-four hours after the last aerosol exposure rats were anaesthetized (60 mg/kg

pentobarbital [Sanofi, Libourne, France], ip) and the trachea was cannulated. Following

lavage, lungs were fixed with 4% paraformaldehyde via the tracheal canulla. Lung sections

were processed for immunohistochemistry and for Congo Red staining. The total number of

peribronchial eosinophils was determined on Congo Red stained sections (Palmans et al,

2000).


3.3. Immunohistochemical staining of type I, III, IV, V and VI collagen

Type I collagen was stained with a polyclonal goat anti-type I collagen in lung tissue

embedded in Technovit 8100 (Heraeus Kulzer GmbH, Wehrheim, Germany) according to

the manufacturers instructions. The other collagen types were detected in paraffin embedded

tissue. Sections of 2 µm were pre-treated with H2O2 to block endogenous peroxidase activity

and 0.1% trypsin (Sigma Chemical) in 0.1% CaCl2 (pH 7.8) at 37°C. The sections were

rinsed in distilled water and subsequently incubated with normal rabbit serum (NRS) for 1h

and with the primary polyclonal antibody (Southern Biotechnology Associates Inc.,

Birmingham, AL) diluted in 1% NRS as follows: goat anti-type I collagen (1:20), goat anti-

type III collagen (1:100), goat anti-type IV collagen (1:50), goat anti-type V collagen (1:50)

and goat anti-type VI collagen (1:300). The sections were washed and incubated with

biotinylated rabbit anti-goat IgG (DAKO A/S, Glostrup, Denmark) diluted 1:200. The

primary antibody-secondary antibody complex was detected with the streptavidin-

biotinylated horseradish peroxidase complex (Nycomed Amersham Plc., Buckinghamshire,

UK) diluted 1:200. Peroxidase activity was developed in diaminobenzidine (DAB) (DAKO)

giving a brown reaction product. Sections were counterstained with hematoxylin. Rat skin

was used as a positive control for the different collagen types. The primary antibody was

substituted with normal goat serum as a negative control.

3.4. Quantification of collagen deposition

The deposition of the different types of collagen was examined by light microscopy and

quantitative measurements were performed using a computerized image analysis system (Leica

Q500MC; Leica Cambridge Ltd., Cambridge, UK) as previously described (Palmans et al,

2000). For each experimental group three lung sections per animal were examined. The

amount of the specific collagen deposited in the different compartments of the airway wall

was measured in all large airways (length of BM [Pbm] greater than 2 mm) cut in reasonable

cross sections (defined by a ratio of minimal to maximal internal diameter greater than 0.5).

The different compartments of the airway wall are the total wall defined by the BM and the

outer adventitia; the inner wall defined by the BM and the outer boundary of the smooth

muscle; and the outer wall defined by the outer boundary of the smooth muscle and the outer

adventitia. The borders of these compartments were marked manually and the software

analyzed the amount of specific collagen deposited in these regions.


3.5. Measurement of vessel area

Vessels were identified by the staining of their BM with type IV collagen. With the image

analysis system (Leica Q500MC) the vessel area in the airway wall of large airways was

measured on the sections used for type IV collagen quantification. The vessel area is defined

by all the structures internal to the vessel BM and was calculated by the software Qwin. The

total amount of vessels in the total airway wall was counted.

3.6. Data analysis

Reported values are expressed as mean ± standard error of the mean (SEM). The data of

collagen deposition, vessel area and vessel number of all sections in one experimental group

were pooled. Each quantification was performed in a mean of 16 large airways (range 12 to

25) per experimental group. Values of the area of collagen deposition in total, inner and outer

airway wall (WCt, WCi and WCo respectively) were normalized to Pbm. The vessel area was

normalized to the total wall area (WAt) and the number of vessels was divided by WAt to

obtain the amount of vessels per square mm. The frequency distribution of Pbm for the PBS

and OA experimental group was compared using the Kolmogorov-Smirnov test to ensure the

comparison of a similar range of airway sizes. Mean values of WCt, WCi and WCo, the vessel

area and the amount of vessels per square mm were compared between the OA- and PBS-

exposed animals in one experiment by an unpaired t-test. Spearman’s rank correlation

coefficient was used to examine the correlation between the amount of collagen deposited in

the airway wall and the number of peribronchial eosinophils. P-values less than 0.05 were

regarded as significant.


4. Results

4.1. Distribution of different collagen types

Type I collagen was clearly present in the total airway wall and formed bundles parallel

with the airway epithelium (figure 1A and B). Type III and type VI collagen were ubiquitous

in the airway mucosa and adventitia and their deposition pattern was similar (figure 1C, G and

H). Type V collagen was predominantly deposited in the outer airway wall close to the airway

smooth muscle cells and the blood vessels (figure 1F). Type IV collagen was found in the

basement membrane beneath the epithelium and surrounding blood vessels, and within

smooth muscle bundles (figure 1E).

The distribution pattern of the different types of collagen was similar in control animals

and allergen exposed animals independent of the duration of the exposure (figure 1).

4.2. Quantification of type I, III, V and VI collagen deposition

The amount of the collagen subtypes deposited in the total, inner and outer airway wall

of large airways did not differ between sensitized BN rats exposed to PBS or OA during 2

weeks (data not shown).

Quantitative changes in collagen types were observed in rats exposed to allergen during

12 weeks. Type I and type VI collagen were increased in the total airway wall (p<0.001 versus

PBS-exposed animals) (figure 2A and C). The increase in type I collagen was observed in the

inner (p<0.01) as well as in the outer airway wall (p<0.001) (figure 2A) and the increase in

type VI collagen was found mainly in the outer airway wall (p<0.001) (figure 2C). Although

the amount of type V collagen in the total airway wall of OA- and PBS-exposed rats did not

differ, more type V collagen was deposited in the outer airway wall of allergen exposed

animals (p<0.05 versus PBS) (figure 2B). No difference was observed in the deposition of

type III collagen between OA- and PBS-exposed rats (12.56±1.24 µm2/µm BM versus

13.93±2.33).


A

B

C

D

E

F

G

H

Figure 1. Photomicrographs of type-specific collagen staining in large airways of BN rats exposed during 12 weeks (counterstain hematoxilyn, magnification x400). Type I collagen in airways of rats exposed to OA (A) or PBS (B). Type III collagen (C), type IV collagen (E) and type V collagen (F) in OA-exposed rats. Type VI collagen in animals exposed to OA (G) or PBS (H). A representative negative control for the collagen staining (D).


0.0 2.5 5.0 7.5 10.0 12.5 15.00

250

500

750

1000

1250

rS = 0.721p < 0.05

type I collagen/Pbm (µm²/µm)

eosi

noph

ils (

cells

/mm

² W

At)

hier plaats laten

A. type I collagen

total inner outer0

5

10

15

p<0.001

p<0.001

p<0.01ar

ea c

olla

gen/

Pbm

B. type V collagen

total inner outer0

5

10

15

p<0.05

area

col

lage

n/P

bm

C. type VI collagen

total inner outer0

5

10

15

p<0.001

p<0.001

area

col

lage

n/P

bm

Figure 2. Deposition of (A) type I, (B) V and (C) VI collagen in the total, inner and outer airway wall of sensitized rats exposed to ovalbumin (solid bars) or PBS (open bars) during 12 weeks.

The amount of type I collagen present in the airway wall correlated with the number of

peribronchial eosinophils in rats exposed during 12 weeks (rs = 0.72, p<0.05) (figure 3).

Deposition of type VI collagen was also related to the number of eosinophils in the airway

wall (rs = 0.86, p<0.01) (data not shown). The amount of type V collagen in the airway wall

did not correlate with the number of eosinophils.

Figure 3. Correlation between the amount of type I collagen deposited in the airway wall and the number of peribronchial eosinophils in BN rats exposed during 12 weeks. Open circles represents data from rats exposed to PBS; closed circles represent data from OA-exposed animals.

4.3. Basement membrane type IV collagen

No differences were observed in the amount of type IV collagen deposited in the airway

wall of rats exposed to OA during 2 and 12 weeks (1.51±0.21 µm2/µm BM versus 1.33±0.4

in PBS-exposed animals). Also no difference was seen in the amount of vessels per area of

the bronchial wall. After 2 weeks the airways of OA-exposed animals contained 265 ± 27

vessels per mm2 compared to 332 ± 20 vessels per mm2 in PBS exposed rats. After 12 weeks


318 ± 13 vessels per mm2 were counted in the OA group compared to 288 ± 31 vessels per

mm2 in the PBS group. The vessel area normalized to WAt was increased in animals exposed

to OA during 12 weeks (figure 4) compared to PBS-exposed animals (p<0.05).

Figure 4. The effect of ovalbumin (solid bars) or PBS (open bars) exposure during 2 weeks and 12 weeks on the vascularity in large airways.

5. Discussion

The purpose of this study was to characterize changes in collagen deposition in the large

airway walls of sensitized BN rats repeatedly exposed to allergen (Palmans et al, 2000). The

results indicate that these airways contained increased amounts of collagen types I, V and VI

after 12 weeks of allergen exposure. An increase of all three collagen types was observed in

the outer airway wall; collagen type I was also deposited in the inner wall. No differences were

observed in the deposition of type III and type IV collagen. Vascular perfusion was increased

after 12 weeks of OA exposure.

Two aspects of changes in the collagen composition in the airway wall that have been

linked to chronic asthma are the thickened lamina reticularis and increased deposition of

collagen in the submucosa (Lee et al, 1999; Wilson and Li, 1997). The information obtained

from endobronchial biopsy studies relates mainly to the composition of the area beneath the

basal lamina. In the asthmatic airway the reticular layer represents an area of irregularly

distributed fibrosis. This collagenous matrix band beneath the BM contains increased

amounts of the fibrillar collagens type I, III and V but also of fibronectin and tenascin

(Laitinen et al, 1997; Roche et al, 1989; Wilson and Li, 1997). In the present model the

increase in type I collagen in this area is most prominent. Although the thickness of the lamina

2 weeks 12 weeks0

5

10

15p<0.05

vess

el a

rea

(mm

²/mm

²W

At)

hier plaats laten


reticularis is increased in bronchial biopsies from asthmatic when compared to normal

subjects (Jeffery et al, 1989; Roche et al, 1989) the significance of this subepithelial fibrosis

remains unclear. In some studies no relation was found between the thickening and the

severity, duration or etiology of asthma (Chu et al, 1998; Jeffery et al, 1989; Milanese et al,

2001). Others confirmed thickening of the matrix band beneath the BM in atopic, but not in

non-atopic asthma (Amin et al, 2000), and reported a correlation with the severity but not with

the duration of asthma (Boulet et al, 1997; Chetta et al, 1997).

Far less data are available on the distribution of collagen in the submucosa and the

adventitial area. Wilson and colleagues (Wilson and Li, 1997) observed increased amounts of

type III and type V collagen in the submucosa of asthmatic airways and Lee and colleagues

(Lee et al, 1999) found an increase in type VI collagen in the submucosa of asthmatics. These

findings have not been invariably confirmed, as others did not observe increased amounts of

total collagen nor increased deposition of type I and III collagen in asthmatic subjects (Chu et

al, 1998). In the present study repeated allergen challenge was shown to induce increased

deposition of collagen I, V and VI in the outer airway wall. This area corresponds to the

adventitial tissue between the cartilage and/or the smooth muscle layer. In normal airways, the

adventitia consists mainly of fibrous tis sue and blood vessels but to the best of our

knowledge data on collagen deposition in the adventitia of the asthmatic airways are not

available. It is therefore unclear whether subepithelial fibrosis serves as a marker of changes

such as ECM protein deposition throughout the submucosa and the adventitia, as has been

suggested (Bradding et al, 1997). Therefore we cannot ascertain that the changes observed in

this study are consistent with those in human asthma.

Data on vascular changes within the airway wall of asthmatics are also sparse. Recent

reports illustrate an increase of vessels and vascularity in the airway wall of asthmatics (Kuhn

et al, 1989; Orsida et al, 2001; Skalli et al, 1986). In patients with fatal asthma the number of

large blood vessels was increased compared to control subjects in large but not in smaller

airways (Carroll et al, 1997). Similarly, the total area occupied by the blood vessels in the

airway submucosa was not different between asthmatics and control subjects (Carroll et al,

1997). In the present model, blood vessels were identified by immunostaining for collagen

type IV which is the major collagen subtype present in the vascular BM. Using this approach,

we could not identify an increase in the total amount of vessels per square mm. However, in

line with the data by Li and Wilson an increase in the total area of the mucosa occupied by

blood vessels was found.


The mechanisms underlying the allergen induced increased collagen deposition in this

model remain to be further elucidated. Collagen formation is part of tissue repair response to

injury, in an attempt at tissue regeneration. When this repair process is dysregulated and

prolonged, it may result in progressive fibrosis with its specific changes (Busse et al, 1999).

Different types of collagen seem to be produced at various stages in the regeneration process.

The production of ECM in airways of rats repeatedly exposed to allergen resembles the

process of tissue regeneration in which fibronectin precedes collagen deposition forming a

provisional ECM (Welch et al, 1990). Previously we have shown that changes in the

fibronectin deposition start after 2 weeks, but the increase in collagen content and

composition of the airway wall is observed only after a longer exposure period (Palmans et al,

2000). Moreover collagen type I and V are usually considered to reflect fully established

fibrotic lesions, occurring at a later stage during the regeneration process (Martin et al, 1985;

McAnulty and Laurent, 1995). The main sources of collagen probably are fibroblasts and/or

myofibroblasts responding to a wide range of stimuli including various growth factors such

as transforming growth factor-β , insulin like growth factor or platelet derived growth factor

(Hoshino et al, 1998; Zhang et al, 1999). These factors can in turn be released from different

cell types, epithelial cells and eosinophils in particular (Ohno et al, 1995; Vignola et al, 1997;

Zhang et al, 1999). Of note is the positive correlation between the amount of collagen type I

and VI and the number of eosinophils in the airway wall of BN rats exposed during 12 weeks

found in the present study. Whether the eosinophils express these growth factors is yet

unknown.

The increased and altered collagen deposition in the airway wall, observed in the current

study could have important functional consequences. Mathematical models link airway

remodeling to airway hyperresponsiveness (AHR) (James et al, 1989). Changes in the

composition of the airway connective tissue can influence smooth muscle shortening

(McFawn and Mitchell, 1997) and depending on the extent and location of the altered ECM

deposition these might protect against instead of enhancing airway responsiveness to

bronchoconstrictor stimuli (Lambert et al, 1994; Milanese et al, 2001; Seow et al, 2000). In

the initial study we observed that the loss of AHR in rats exposed to OA during 12 weeks

was associated with an increase in total collagen in the total airway wall and fibronectin in the

outer airway wall (Palmans et al, 2000). We suggested that tighter adhesion between lung

parenchyma and the outer airway wall could influence the mechanical properties of the

airways (Palmans et al, 2000). Type VI collagen microfibrils are known to form fibrillar


connections between other ECM components (Davidson, 1990). These fibers might thus

function as a mechanical link between interstitial collagen fibrils and other matrix structures

including basement membranes and elastin microfibrils (Keene et al, 1988). Type I collagen

forms a relatively rigid linear fibrillar network and could also increase the stiffness of the airway

wall (Raghow, 1994). The increase of both collagen type I and VI in the OA-exposed rats

might therefore increase rigidity of the airway wall and thus oppose against instead of induce

AHR.

These observations stress the importance of including all structural changes that occur

throughout the airway wall, when evaluating the functional role of remodeling. To date, in

man, this analysis has been limited to subepithelial collagen deposition, relating the extent of

collagen type I and III deposition to an increase as opposed to a decrease in airway

responsiveness. The causal relationship between both however is not known. The present

model cautions against relating one single aspect of the structural alterations to airway

function, without taking into account the potential contribution of co-existing alterations.

In conclusion, the increased collagen type I, V and VI deposition in the airway wall of

sensitized rats exposed to allergen during 12 weeks might result from repeatedly induced

tissue repair processes in response to the persistent presence of inflammatory cells. The

different collagens may increase the rigidity and stiffness of the airway wall and thus prevent

AHR.

6. References



Bousquet J, Lacoste JY, Chanez P, Vic P, Godard P and Michel FB. Bronchial elastic fibers in normal subjects and asthmatic patients. Am J Respir Crit Care Med 1996, 153:1648-1654.

Bradding P, Redington AE and Holgate ST. Airway wall remodelling in the pathogenesis of asthma: cytokine expression in the airways. In: Steward AG (ed). Airway wall remodelling in asthma: 1997, Boca Raton, USA, CRC Press, Inc., p. 29-63.


Busse W, Elias J, Sheppard D and BanksSchlegel S. Airway remodeling and repair. Am J Respir Crit Care Med 1999, 160:1035-1042.

Campa JS, Harrison NK and Laurent GJ. Regulation of Matrix Production in the Airways. In: Jolles G, Karlsson JA and Taylor J (eds). T-Lymphocyte and Inflammatory Cell Research in Asthma: 1993, London, Academic Press Ltd., p. 221-239.

Carroll NG, Cooke C and James AL. Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 1997, 155:689-695.


Chu HW, Halliday JL, Martin RJ, Leung DY, Szefler SJ and Wenzel SE. Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 1998, 158:1936-1944.

Davidson JM. Biochemistry and turnover of lung interstitium. Eur Respir J 1990, 3:1048-1068.

Hoshino M, Nakamura Y and Sim JJ. Expression of growth factors and remodelling of the airway wall in bronchial asthma. Thorax 1998, 53:21-27.



Keene DR, Engvall E and Glanville RW. Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network. J Cell Biol 1988, 107:1995-2006.

Kuhn C3, Boldt J, King TE, Jr., Crouch E, Vartio T and McDonald JA. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989, 140:1693-1703.






Martin GR, Timple R, Müller PK and Kühn K. The genetically distinct collagens. IBS 1985, 285-287.

McAnulty RJ and Laurent GJ. Collagen and its regulation in pulmonary fibrosis. In: Phan SH and Thrall RS (eds). Pulmonary fibrosis: 1995, New York, NY, Marcel Dekker, Inc., p. 135-171.

McFawn PK and Mitchell HW. Bronchial compliance and wall structure during development of the immature human and pig lung. Eur Respir J 1997, 10:27-34.


Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, O' Byrne P, Dolovich J, Jordana M, Tamura G, Tanno Y and Shirato K. Eosinophils as a potential source of platelet-derived growth factor B-chain (PDGF-B) in nasal polyposis and bronchial asthma. Am J Respir Cell Mol Biol 1995, 13:639-647.

Orsida BE, Ward C, Li X, Bish R, Wilson JW, Thien F and Walters EH. Effect of a long-acting beta2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 2001, 164:117-121.

Palmans E, Kips JC and Pauwels RA. Prolonged allergen exposure induces structural airway changes in sensitized rats. Am J Respir Crit Care Med 2000, 161:627-635.

Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J 1994, 8:823-831.


Seow CY, Wang L and Pare PD. Airway narro wing and internal structural constraints. J Appl Physiol 2000, 88:527-533.

Vignola AM, Chanez P, Chiappara G, Merendino A, Pace E, Rizzo A, la Rocca AM, Bellia V, Bonsignore G and Bousquet J. Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis. Am J Respir Crit Care Med 1997, 156:591-599.

Welch MP, Odland GF and Clark RA. Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction. J Cell Biol 1990, 110:133-145.



Airway Structure after Repeated Bronchoconstriction 116

CHAPTER III

THE EFFECT OF REPEATED SMOOTH MUSCLE CONTRACTIONS

ON AIRWAY STRUCTURE OF BROWN NORWAY RATS

Els Palmans, Romain A. Pauwels and Johan C. Kips


1. Abstract

Remodeling of the extracellular matrix (ECM) in the airway wall is one of the

characteristics of asthma. Although it is suggested that alterations of the ECM proteins result

from the chronic inflammation present in the asthmatic airway wall, mechanical stress

associated with bronchoconstriction might also stimulate and amplify deposition of ECM

proteins in airway wall. The aim of the present study was therefore to examine whether the

structure of the airway wall of rats is altered after repeated induction of bronchoconstriction.

Brown Norway rats were injected daily with carbachol during two weeks. The carbachol

administrations resulted in constricted airways as shown by the increase of lung resistance.

Control animals received phosphate-buffered saline. After the treatment period, no

inflammation was present in the airways of carbachol-treated rats. Also, the composition of

the ECM proteins in the mucosa and adventitia or the airway wall dimensions did not change

after exaggerated smooth muscle constriction. Airway function was similar in carbachol-

treated and control animals. We concluded that bronchoconstriction in se did not alter the

airway structure in rats.

2. Introduction

The airways of chronic asthmatic patients display structural changes including a

thickened layer beneath the basement membrane that comprises predominantly collagen type

I, III and VI together with fibronectin and tenascin (Laitinen et al, 1997; Roche et al, 1989)

and increased deposition of collagen type III, V and VI in the bronchial submucosa (Lee et

al, 1999; Wilson and Li, 1997). Other characteristics of this so-called airway remodeling in

asthma are mucus cell hyperplasia, smooth muscle hypertrophy or hyperplasia and

neovascularization (Ebina et al, 1993; Jeffery et al, 1989; Li and Wilson, 1997). Airway

remodeling may contribute to airway hyperresponsiveness, the functional hallmark of

asthmatics (James et al, 1989).

It is generally thought that the increased amount of extracellular matrix proteins is

produced by myofibroblast in response to inflammation in the airway wall (Brewster et al,

1990; Zhang et al, 1999). However, an alternative hypothesis for the pathogenesis of the

altered deposition of ECM proteins and other structural airway changes has been proposed. It


is believed that the remodeling of the ECM and possibly also of other airway structures is not

only controlled by cellular responses but also by mechanical stress in the airway wall

(Chiquet, 1999). During bronchoconstriction the airway wall is subjected to abnormal

physical forces that might deform the shape of structural cells (Wirtz and Dobbs, 2000). In

vitro studies that mimic facets of the fibrotic response in asthmatic airways have shown that

shape deformations can stimulate cells to modify their phenotype and alter their production of

ECM proteins (Ressler et al, 2000; Swartz et al, 2001; Xu et al, 1999).

In a rat model that we developed to investigate the pathogenesis and functional

consequences of remodeling in asthmatic airways, allergen sensitization and exposure during

3 months resulted in increased deposition of collagen and fibronectin in the walls of large

airways (Palmans et al, 2000). We suggested that the altered production of ECM proteins was

caused by the ongoing inflammation present in the airways of the rats (Palmans et al, 2000).

Since mechanical stress that is imposed on the structural cells of the airway wall during

bronchoconstriction might itself induce the deposition of ECM proteins we evaluated the

structure of the airway wall in BN rats after repeated smooth muscle contractions induced by

treatment with carbachol.

3. Methods

3.1. Animals and study protocol

In specific pathogen-free, male BN rats (Harlan CPB, Zeist, the Netherlands) (n=10)

bronchoconstriction was induced once daily during 14 days by intraperitoneal (i.p.) injection

of carbachol (carbamylcholinehydrochloride; Merck, Darmstadt, Germany) (1 mg/kg body

weight) (CARBACHOL). This dose was shown to increase baseline lung resistance (RL) with

380%. Control animals (n=9) were injected i.p. with phosphate-buffered saline (PBS)

(CONTROL).

3.2. Outcome measures

On day 15, 24 hours after receiving carbachol or PBS, the rats were anaesthetized [60

mg/kg pentobarbital (Sanofi, Libourne, France), i.p.]. Airway responsiveness to increasing

concentrations (0.1 – 10 mg/ml) of aerosolized carbachol was assessed as previously

described (Palmans et al, 2000). Dose-response curves for the change in lung resistance (RL),


expressed as the change from baseline, were constructed. The concentration of carbachol

causing a 100% increase in baseline RL (PC100RL) was calculated via log-linear interpolation of

the dose-response curve.

Immediately after measuring airway responsiveness, bronchoalveolar lavage was

performed (Palmans et al, 2000).

3.3. Histology

Histological analysis was performed on 4% paraformaldehyde fixed lung tissue.

Histological and inflammatory changes were evaluated on sections stained with hematoxylin-

eosin (H&E) and Congo Red. Morphometric measurements were performed using a Zeiss

Axiophot microscope (Zeiss, Oberkochen, Germany) on 23 (CARBACHOL) to 24

(CONTROL) large airways. Total bronchial wall area (WAt), inner wall area (WAi) and outer

wall area (WAo) were normalized to the length of basement membrane (Pbm) (Palmans et al,

2000).

Collagen deposition in the airway wall was assessed on lung sections stained with Sirius-

Red. Quantitative measurements were performed using a computerized image analysis system

(LEICA Q500MC; Leica Cambridge Ltd., Cambridge, UK) on 20 (CONTROL) to 21

(CARBACHOL) large airways. The area of the total, inner and outer airway wall stained for

collagen (WCt, WCi and WCo respectively) is expressed per µm basement membrane

(Palmans et al, 2000).

3.4. Data analysis

Reported values are expressed as mean ± standard error of the mean (SEM). The

cumulative dose-response curves were compared through analysis of variance (ANOVA).

PC100RL values and cellular composition of BAL fluid of the CARBACHOL and

CONTROL group were compared using a Mann-Whitney U-test.

For the analysis of the morphometry and collagen deposition, the data of the different

rats in each group were pooled together. The mean values of WAt, WAi, WAo, WCt, WCi and

WCo were analyzed between the experimental groups by an unpaired t-test. P-values less than

0.05 were regarded as significant.

4. Results


Repeated i.p. injections with carbachol did not alter airway responsiveness to aerosolized

carbachol of the BN rats. The dose-response curve of these animals was not significantly

different with those of the control animals (Figure 1). Also, no change was observed in

PC100RL for carbachol (2.16 ± 0.45 mg/ml carbachol versus 1.90 ± 0.24 mg/ml carbachol in

PBS treated animals).

Figure 1. Bronchial responsiveness to aerosolized carbachol of BN rats after

repeated i.p. injections of carbachol (CARBACHOL, closed squares) or PBS

(CONTROL, open squares).

In the airways of the BN rats no inflammatory abnormalities were observed after repeated

carbachol injections. The total amount of cells in BAL fluid was similar in carbachol- and

PBS-treated animals. The distribution of macrophages, eosinophils, lymphocytes and

neutrophils in BAL fluid was not altered after carbachol-induced bronchoconstriction (Table

1). No peribronchial inflammatory infiltrate was observed in both experimental groups (data

not shown).

TABLE 1

TOTAL NUMBER AND DISTRIBUTION OF WHITE BLOOD CELLS IN BRONCHO-ALVEOLAR LAVAGE FLUID AFTER REPEATED BRONCHOCONSTRICTION

Total Cells Monocytes Eosinophils Neutrophils Lymphocytes

Treatment (x 103) (%) (%) (%) (%)

CONTROL 370.8±77.1 94.70±0.68 0.91±0.22 1.58±0.32 2.80±0.62

CARBACHOL 303.1±32.4 96.56±0.62 0.33±0.13 0.96±0.15 2.14±0.58

Histological evaluation of the lung tissue of carbachol and control rats did not reveal

structural changes within the airway wall. The epithelial layer had a normal appearance with no

signs of desquamation. The size of the different compartments in the airway wall was not

0.01 0.1 1 10 100

0

100

200

300

400


% in

crea

se in

RL


affected by carbachol treatment. Total airway wall area of the carbachol-treated group did not

change compared to control animals. Also inner and outer airway wall areas were similar in

rats treated with carbachol and control animals (figure 2A). Deposition of collagen in the

airway wall was not altered in rats injected with carbachol. Similar amounts of collagen were

present in inner and outer airway wall of carbachol- and PBS-injected BN rats (figure 2B).

A. AIRWAY WALL

total inner outer0

25

50

75

100

WA

/Pb

m (µ

m²/µ

m)

B. COLLAGEN DEPOSITION

total inner outer0

5

10

15

20

WC

/Pbm

(µm

²/µm

)

Figure 2. Effect of repeated carbachol injections (solid bars) or PBS injections (open bars) on (A) the total, inner and outer airway wall area and (B) on the deposition of collagen in the total, inner or outer

airway wall of large-sized airways.

5. Discussion

The main purpose of this study was to evaluate if repeated airway smooth muscle

contractions in se could induce the development of structural changes in the airway wall.

Exaggerated bronchoconstriction was repeatedly induced in BN rats via i.p. injections of

carbachol. Neither inflammatory nor structural alterations were observed in the airway wall of

these animals compared to control rats. Airway responsiveness to nebulized carbachol did

not change.

The administration of carbachol clearly increased RL as a result of the airway constriction

in the rats. Previously it was shown that the mucosa as well as the adventitia deformed during

bronchoconstriction induced by carbachol (James et al, 1988; Sasaki et al, 1996). The

present study in the BN rats thus gave us a tool to evaluate in vivo structural airway changes

that might have originated from repeated bronchoconstriction.


Airway smooth muscle shortening changes the architecture of the airway wall and results

in increased physical forces on the different wall compartments (Mitchell, 1997). In the lung,

physical forces can influence its structure, function and metabolism (Riley et al, 1990).

Asthma is characterized by exaggerated narrowing in response to a bronchoconstrictor agent

defined as bronchial hyperresponsiveness (James et al, 1989). The mechanical stress on the

tissue and cells of the asthmatic airway is therefore elevated compared to normal subjects

(Wiggs et al, 1997). Although it is known that in response to mechanical stress cells can

increase their production of ECM proteins, we did not observe higher amounts of collagen in

repeatedly constricted airway walls. Most information on the influence of mechanical stress

on cellular ECM turnover however is derived from in vitro models (Kolpakov et al, 1995;

Swartz et al, 2001; Xu et al, 1999). These experiments describe the increased production of

several ECM proteins in cell cultures containing airway epithelial cells and fibroblasts (Xu et al,

1999) or pulmonary vascular smooth muscle cells (Kolpakov et al, 1995) in response to

physical forces such as elongation or stretch. Airway epithelial cells subjected to

transmembrane pressure released growth factors associated with remodeling in asthma

(Ressler et al, 2000) and fibroblast co-cultured with these epithelial cells increased their

production of fibronectin and collagen types III and V (Swartz et al, 2001). The mechanical

stress sensed by cells in vitro and in vivo might however be different. How mechanical forces

can be sensed by cells and converted into biochemical signals is not known, but it has been

suggested that stress signals can be transmitted from ECM to cells via an ECM-integrin-

cytoskeleton complex (Ingber, 1997). Organ structures are complex with a variety of cell

types and a variety of forces exposed to these cells. The complexity of the interactions

cannot be fully mimicked in cell cultures and this might explain the difference between our in

vivo study and results obtained via in vitro models.

Our experiments show that mechanical stress alone in the absence of inflammation is

insufficient to induce structural airway changes. This contrasts with in vitro data in which

remodeling responses are induced in mechanically stressed cells without the presence of other

inflammatory cells (Swartz et al, 2001). Previously we showed that BN rats could develop

alterations in epithelium and ECM proteins of the airway wall in response to sensitization and

repeated allergen exposure. The structural alterations appeared after 2 weeks of exposure and

were associated with peribronchial inflammation and airway hyperresponsiveness (Palmans et

al, 2000). Moreover, the remodeling in the airway wall was prevented by anti-inflammatory

treatment (Vanacker et al, 2001). Since allergen-induced structural changes were induced after


2 weeks, the period of challenge with carbachol was judged to be sufficient to evaluate the

effect of repeated bronchoconstriction on ECM protein production.

In conclusion, we showed that in BN rats repeated bronchoconstriction over 2 weeks

did not alter the structure of the airway wall.

6. References

Brewster CE, Howarth PH, Djukanovic R, Wilson JW, Holgate ST and Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990, 3:507-511.

Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biology 1999, 18:417-426.


Ingber DE. Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol 1997, 59:575-599.

James AL, Hogg JC, Dunn LA and Pare PD. The use of the internal perimeter to compare airway size and to calculate smooth muscle shortening. Am Rev Respir Dis 1988, 138:136-139.



Kolpakov V, Rekhter MD, Gordon D, Wang WH and Kulik TJ. Effect of mechanical forces on growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role of individual cell types. Circ Res 1995, 77:823-831.





Mitchell HW. Physiology of airway narrowing. Clin Exp Pharmacol Physiol 1997, 24:256-260.


Ressler B, Lee RT, Randell SH, Drazen JM and Kamm RD. Molecular responses of rat tracheal epithelial cells to transmembrane pressure. Am J Physiol Lung Cell Mol Physiol 2000, 278:L1264-L1272.

Riley DJ, Rannels DE, Low RB, Jensen L and Jacobs TP. NHLBI Workshop Summary. Effect of physical forces on lung structure, function, and metabolism. Am Rev Respir Dis 1990, 142:910-914.


Sasaki F, Saitoh Y, Verburgt L and Okazawa M. Airway wall dimensions during carbachol-induced bronchoconstriction in rabbits. J Appl Physiol 1996, 81:1578-1583.

Swartz MA, Tschumperlin DJ, Kamm RD and Drazen JM. Mechanical stress is communicated between different cell types to elicit matrix remodeling. Proc Natl Acad Sci U S A 2001, 98:6180-6185.

Vanacker NJ, Palmans E, Kips JC and Pauwels RA. Fluticasone inhibits but does not reverse allergen-induced structural airway changes. Am J Respir Crit Care Med 2001, 163:674-679.



Wirtz HR and Dobbs LG. The effects of mechanical forces on lung functions. Resp Physiol 2000, 119:1-17.

Xu J, Liu M and Post M. Differential regulation of extracellular matrix molecules by mechanical strain of fetal lung cells. Am J Physiol 1999, 276:L728-L735.


Airway Wall Changes in Inbred Mouse Strains 125

CHAPTER IV

ALLERGEN-INDUCED INFLAMMATORY AND STRUCTURAL

AIRWAY CHANGES IN TWO INBRED MOUSE STRAINS

Els Palmans, Nele J. Vanacker, Romain A. Pauwels and Johan C. Kips

Clinical and Experimental Allergy, Submitted


1. Abstract

The mechanisms responsible for the development of airway remodeling in asthma are not

well understood. In the present study we evaluated to what extent differences in immune and

inflammatory responses can influence the remodeling process in airways. We therefore

compared structural airway changes after prolonged allergen exposure in two genetically

distinct mouse strains. BALB/c and C57BL/6 mice were sensitized and exposed to

ovalbumin (OA) and phosphate-buffered saline (PBS) for 2 to 9 weeks. After 2 weeks of OA

exposure, the allergic response was more pronounced in BALB/c and the number of BALF

eosinophils was higher in C57BL/6 mice. The number of peribronchial eosinophils and

goblet cells were equally increased in both BALB/c and C57BL/6 mice. BALB/c, but not

C57BL/6 showed an increase in airway wall thickness and airway hyperresponsiveness

(AHR) to methacholine (p<0.005 versus PBS). Upon further allergen exposure, peribronchial

eosinophilia disappeared in both mice strains and AHR was abolished in BALB/c. The

number of goblet cells remained elevated and collagen deposition in the airway wall was

increased (p<0.005 versus PBS) in OA-exposed BALB/c and C57BL/6. Also airway wall

thickness remained increased in both mouse strains after longer OA exposure. In conclusion,

these data indicate that despite different inflammatory responses to prolonged allergen

exposure, BALB/c as well as C57BL/6 mice displayed comparable structural airway changes.

2. Introduction

Asthmatic airways display, in addition to signs of an acute inflammatory process, more

structural alterations coined airway remodeling (Bousquet et al, 2000). Remodeling is thought

to have important consequences on airway behavior in asthma (Pare and Bai, 1995). The

pathophysiology of the remodeling process is largely unknown but it is thought to result

from inappropriate attempts at tissue repair in response to inflammation (Kips and Pauwels,

1999; Vignola et al, 2000). To what extent remodeling develops uniformly in all asthmatic

patients also is unclear. The limited data currently available indicate a substantial degree of

variability between remodeled asthmatic airways (Amin et al, 2000; Carroll and James, 2001).

Moreover, remodeling of the airways is not necessarily clinically demonstrated in all asthmatic

patients (Bousquet et al, 2000). Several studies of familial aggregation have suggested a


genetic etiology for each of asthma, atopy, and bronchial hyperresponsiveness and it is likely

that several or many genes, as well as interactions with environmental factors, are involved

(Clarke et al, 2000; Sandford and Pare, 2000). However, the importance of genetic alterations

in the development of allergen-induced remodeling remains unknown.

In vivo mouse models might help to understand the pathogenesis of airway remodeling

and its functional implicatio ns in asthma. In view of the well characterized genetic differences

in immunological and smooth muscle responses to allergen exposure between inbred mouse

strains (Brewer et al, 1999; Duguet et al, 2000; Takeda et al, 2001; Zhang et al, 1997), murine

models can also address the impact of genetic differences on the remodeling process.

In the present study we compared to what extent differences in the immune and

inflammatory responses can influence the remodeling process in airways of mice. We

compared allergen induced airway inflammation and remodeling in two genetically distinct

mouse strains.

3. Methods

3.1. Immunization and exposure of animals

Male C57BL/6 and BALB/c mice aged 6 to 8 weeks (Harlan CPB; Zeist, the

Netherlands) were actively sensitized by the intraperitoneal injection of 10 µg ovalbumin (OA)

(Grade III; Sigma Chemical Co., Poole, Dorset, UK) absorbed to 1 mg Al(OH)3 0.9% NaCl

on day 0 and day 7. From day 14 on all animals were exposed three times per week for 2, 6

and 9 weeks to aerosolized OA (1% wt/vol) or phosphate-buffered saline (PBS) (Palmans et

al, 2000). Group sizes varied from 6 to 12.


Assessment of airway responsiveness

Twenty-four hours after the last aerosol exposure, airway responsiveness to

methacholine was assessed in awake animals using a whole body plethysmograph system

(Buxco; Buxco Electronics Inc., Troy, NY). Before performing readings, the system was

calibrated by rapid injection of 1 ml air. Pressure differences between the main chamber

containing awake mice and a reference chamber were recorded using the software BioSystem

XA (version 157; Buxco). This pressure signal is caused by flow changes in the main box


during the respiratory cycle of the animal. The value of ‘enhanced pause’ (Penh) is used to

monitor airway function (Hamelmann et al, 1997). Penh = [(Te-Tr)/Tr] x (PEP/PIP) where Te

is expiratory time (s), Tr is relaxation time or time of the pressure decay to 36% of total box

pressure during expiration, PEP is peak expiratory pressure (ml/s), and PIP is peak inspiratory

pressure (ml/s).

For each mouse, data of Penh were recorded at baseline, and after exposure to PBS or

increasing concentrations of methacholine (2 – 81 mg/ml). The solutions were nebulized

through an inlet of the main chamber during 1 min. Readings were taken during 5 min after

each nebulization. Between readings Penh value returned to baseline.

Bronchoalveolar lavage

After assessment of airway responsiveness the animals were anesthetized intraperitoneal

with 60 mg/kg pentobarbital (Sanofi, Libourne, France) and bronchoalveolar lavage (BAL)

was performed with 4 ml Hanks’ balanced salt solution for total and differential cell count.

OA-specific IgE

Blood was collected by cardiac puncture for the measurement of OA-specific IgE with

ELISA as previously described (Kips et al, 1995).

Histological analysis of lung tissu e

Following lavage, lungs were fixed with 4% paraformaldehyde and slices from all lobes

were embedded in paraffin for histological analysis. Sections of 2 µm were stained with Sirius

Red, Periodic Acid Shiff (PAS) and Congo Red.

Quantitative measurements in the airway wall

For each animal quantitative measurements were performed in all airways with a length of

basement membrane (Pbm) greater than 1000 µm and cut in reasonable cross sections (ratio

of minimal to maximal internal diameter greater than 0.5). Measurements were performed on

the digital representation of the airways via a computerized image analysis system (KS400;

Zeiss, Oberkochen, Germany).

Airway morphometry and collagen deposition were measured in Sirius Red-stained

sections. Morphometrical parameters (Bai et al, 1994) were marked manually on the digital

representation of the airway: Pbm, the area defined by the basement membrane (Abm) and the


area defined by the total adventitial perimeter (Ao). The total bronchial wall area (WAt) was

calculated (WAt = Ao - Abm) and was normalized to the squared length of basement

membrane. For the quantification of collagen deposition, the area in the airway wall covered

by the stain was determined by the software (KS400; Zeiss) and its value calculated (Palmans

et al, 2000). The area of collagen deposition (WCt) was normalized to Pbm. Goblet cells were

quantified in PAS-stained sections and expressed as the number of goblet cells per mm Pbm.

Peribronchial infiltration with eosinophils was evaluated in lung sections stained with Congo

Red and expressed as total number of eosinophils per mm² WAt.

3.3. Data analysis

Reported values are expressed as mean ± standard error of the mean (SEM). Data of

quantitative measurements in the airway wall of different mice in one experimental group were

pooled together. Mean values of WAt and WCt were compared between experimental groups

using one-way analysis of variance (ANOVA) with post-hoc (LSD and Scheffé) tests. For

bronchial responsiveness, cumulative dose-response curves were constructed for changes in

Penh after increasing doses of methacholine. Changes in Penh are expressed as percentage

increase of maximum Penh following methacholine challenge compared to the average Penh

after PBS-exposure (Analyst 1.29; Buxco). Dose-response curves of Penh were compared

using ANOVA. The concentration of methacholine causing a 250% increase in baseline Penh

(PC250Penh) was calculated by log-linear interpolation of the dose-response curve. Mean

values of cellular composition of BAL fluid, goblet cells, peribronchial eosinophils,

PC250Penh and OA-specific IgE of different groups were compared through the Kruskal-

Wallis test for multiple comparisons. When significant differences were observed, pairwise

comparisons were made, using the Mann-Whitney U-test with Bonferroni’s corrections. P-

values less than 0.05 were considered significant.


4. Results

4.1. Airway inflammation

Bronchoalveolar lavage fluid (BALF)

Over time the total number of cells increased in both mouse strains (Table 1). Ovalbumin

exposure altered the total amount of white blood cells in BALF of C57BL/6 mice and the

composition of the BALF in both inbred mouse strains (Table 1). The total cellularity of

BALF recovered from OA-exposed BALB/c was not significantly different from control

animals. However, throughout the experimental period, the BALF of these OA-exposed

BALB/c mice contained increased numbers of eosinophils and lymphocytes (p<0.05 versus

PBS) (Table 1), but not of macrophages and neutrophils (data not shown). The number of

eosinophils and lymphocytes slightly decreased after OA exposure of 6 weeks and longer

(p<0.05 versus 2 weeks). When compared to BALB/c, OA exposure during 2 weeks

resulted in a greater influx of total white blood cells in BALF of C57BL/6 mice (p<0.05).

This increase was also reflected in increased numbers of eosinophils and lymphocytes in the

C57BL/6 mice (Table 1). The differences between BALB/c and C57BL/6 mice observed at 2

weeks were no longer present after a longer exposure period during which the numbers of

total cells, eosinophils and lymphocytes returned to baseline.

Airway eosinophilia

Both inbred mouse strains developed a peribronchial infiltrate that contained increased

numbers of eosinophils after repeated OA-exposure (Table 2). In contrast to the findings in

BALF, the number of peribronchial eosinophils was similar in BALB/c and C57BL/6 mice

(Table 2). After 2 weeks of allergen exposure the increase in number of eosinophils was

maximal (p<0.001 versus PBS). Prolonged exposure resulted in decreased amounts of

peribronchial eosinophils in both allergen exposed and control animals, albeit that the

difference between both groups remained significant until 9 weeks in BALB/c (p<0.05 versus

PBS) and 6 weeks in C57BL/6 (p<0.001 versus PBS).


Lym

phoc

ytes

(x10

³)

0.6±

0.3

68.7

±16.

2†

4.5±

0.5§

‡

22.0

±3.0

*

8.8±

4.1

9.3±

1.6‡

ll

E

osin

ophi

ls

(x10

³)

0.0±

0.0

1115

.9±2

50.3

†

0.0±

0.0

1.5±

0.4

* ‡

2.4±

1.6

1.9±

1.2

‡ ll

C57

BL/

6

Tot

al c

ells

(x10

³)

170.

65±1

2.66

1542

.86±

273.

74*

333.

93±8

4.02

424.

13±3

3.23

‡

364.

50±1

6.98

‡

288.

08±2

8.95

‡ ll

Lym

phoc

ytes

(x10

³)

2.4±

1.2

23.3

±4.7

† ¶

1.5±

0.3

8.1±

2.0

* ‡

¶

1.7±

0.9

7.1±

1.2*

‡

Eos

inop

hils

(x10

³)

0.3±

0.1

18.9

±7.5

† ¶

0.0±

0.0

1.4±

0.6

* ‡

0.0±

0.0

1.5±

0.5

* ‡

BA

LB/c

Tot

al c

ells

(x10

³)

235.

2±37

.2

239.

2±30

.6¶

248.

9±17

.5

256.

0±47

.0

476.

7±62

.9‡

ll

445.

6±55

.6‡

ll

PBS

OA

PB

S

OA

PB

S

OA

TA

BL

E 1

BA

LF

AN

AL

YSI

S O

F T

OT

AL

WH

ITE

BL

OO

D C

EL

LS,

EO

SIN

OP

HIL

S

AN

D L

YM

PH

OC

YT

ES

IN B

AL

B/C

AN

D C

57B

L/6

MIC

E

Tre

atm

ent

2 w

eeks

6

wee

ks

9 w

eeks

* p<

0.05

, † p

<0.0

05 O

A v

ersu

s PB

S

‡ p<

0.05

, § p

<0.0

05 6

and

9 w

eeks

ver

sus 2

wee

ks

ll p<

0.05

9 w

eeks

ver

sus 6

wee

ks

¶ p<

0.01

BA

LB/c

ver

sus C

57B

L/6


C57

BL

/6

(x10

³)

0.08

±0.0

8

56.5

9±4.

70†

3.79

±1.3

8‡

31.6

6±4.

94†

§

9.62

±3.9

5ll

42.9

0±7.

16†

Gob

let c

ells

(cel

ls/m

m b

asem

ent m

embr

ane)

BA

LB

/c

(x10

³)

0.05

±0.0

3

71.7

5±6.

21†

0.03

±0.0

3¶

38.2

7±5.

57†

ll

2.18

±1.4

5¶

35.3

2±6.

50†

ll

C57

BL

/6

(x10

³)

104.

89±1

7.05

661.

45±4

5.98

†

56.9

8±9.

09‡

110.

72±9

.32†

ll

69.0

2±10

.30

78.9

9±8.

22ll

Eos

inop

hils

(cel

ls/m

m²a

irw

ay w

all)

BA

LB

/c

(x10

³)

149.

01±1

7.56

506.

19±3

0.49

†

93.4

2±10

.93

150.

72±1

3.13

* ll

79.2

3±11

.35ll

111.

03±9

.00*

ll

PBS

OA

PBS

OA

PBS

OA

TA

BL

E 2

PE

RIB

RO

NC

HIA

L E

OSI

NO

PH

ILS

AN

D G

OB

LE

T C

EL

LS

IN E

PIT

HE

LIU

M I

N B

AL

B/C

AN

D C

57B

L/6

MIC

E

Tre

atm

ent

2 w

eeks

6 w

eeks

9 w

eeks

* p<

0.05

, † p

<0.0

01 O

A v

ersu

s PB

S

‡ p<

0.05

, § p

<0.0

05, l

l p<0

.001

6 a

nd 9

wee

ks v

ersu

s 2

wee

ks

¶ p<

0.05

BA

LB

/c v

ersu

s C

57B

L/6


4.2. Goblet cells

Ovalbumin exposure induced goblet cell hyperplasia in both inbred mouse strains

(p<0.001 versus PBS) (Table 2). In BALB/c mice a maximal increase in goblet cells was

observed after 2 weeks of OA-exposure (p<0.001 versus PBS). Moreover, the number of

goblet cells after more prolonged allergen exposure was lower compared to OA-exposed

BALB/c during 2 weeks (p<0.001). Also in OA-exposed C57BL/6 mice goblet cell

hyperplasia was significantly smaller after a period of 6 weeks (p<0.005 versus 2 weeks). At

no time point any difference was observed in the number of goblet cells between allergen-

exposed mice of both strains. In contrast to PBS-exposed BALB/c mice, the number of

goblet cells increased in control C57BL/6 when exposed for a longer period (p<0.05 versus

2 weeks PBS).

4.3. Morphometry and collagen deposition

Morphometry

After prolonged allergen exposure both mouse strains showed an increased in total wall

area of large-sized airways (figure 1A and 1B). In BALB/c mice the increase was already

observed after 2 weeks of OA exposure and was similar after the different allergen exposure

periods (figure 1A). The airway wall of C57BL/6 mice was thickened in animals exposed to

OA from 6 weeks onwards (figure 1B). Within PBS- and OA-exposed C57BL/6 groups no

difference was observed between 2 and 6 weeks but the airway wall of both control and OA-

exposed C57BL/6 mice increased upon further exposure (figure 1B). A difference between

control as well as between OA-exposed BALB/c and C57BL/6 mice was only observed at 9

weeks (p<0.01) (figure 1A and 1B).

Collagen

Ovalbumin exposure increased the total amount of collagen in the airway wall (figure 2A

and 2B). This change was observed after 6 weeks OA exposure in both mouse strains. Only

in BALB/c mice the increased amount of collagen was also present after further allergen

exposure (figure 2A). In contrast to BALB/c, in which the amount of collagen was similar in

the different OA-exposed groups or in the different control groups (figure 2A), C57BL/6


showed a change in collagen content over time in both PBS- and OA-exposed mice (figure

2B).

A. BALB/c

2 weeks 6 weeks 9 weeks0.00

0.01

0.02

0.03

0.04

p<0.005

p<0.005 p<0.001

exposure period

WA

t/Pbm

2 (µm

²/µm

²)B. C57BL/6

2 weeks 6 weeks 9 weeks0.00

0.01

0.02

0.03

0.04

°p<0.005

p<0.05

exposure period

WA

t/Pbm

(µm

²/µm

²)

Figure 1. Effect of sensitization and prolonged allergen exposure to OA (solid bars) or PBS (open bars) on airway wall area of (A) BALB/c or (B) C57BL/6 mice for 2, 6 or 9 weeks.

A. BALB/c

2 weeks 6 weeks 9 weeks0

5

10

15

20

p<0.001 p<0.05

exposure period

µm² c

olla

gen/

µm P

bm

B. C57BL/6

2 weeks 6 weeks 9 weeks0

5

10

15

20

p<0.001

p<0.005

p<0.05

exposure period

µm² c

olla

gen/

µmP

bm

Figure 2. Effect of sensitization and prolonged allergen exposure to OA (solid bars) or PBS (open bars) on amount of collagen in the airway wall of (A) BALB/c or (B) C57BL/6 mice for 2, 6 or 9 weeks.

4.4. Airway responsiveness

BALB/c mice developed a significant increase in airway responsiveness to inhaled

methacholine after 2 weeks OA exposure (ANOVA p<0.005 versus PBS) (figure 3A). After a

longer exposure period the airway responsiveness of OA-exposed BALB/c mice returned to

baseline responsiveness (figure 3B). The airway responses of C57BL/6 exposed to OA for

the different periods were not changed compared to control animals (figure 3C and 3D).

Overall, C57BL/6 were less responsive to methacholine than BALB/c mice. At 2 weeks

the concentration of methacholine that caused a 250% increase in Penh from baseline in


allergen-exposed C57BL/6 was 17.40±5.92 mg/ml methacholine versus 4.69±1.81 mg/ml

methacholine in BALB/c mice (p<0.05). Results for both the other exposure periods were

similar (data not shown).

A. BALB/c - 2 weeks

1 10 100

0

250

500

750

ANOVA: p<0.005

concentration metacholine (mg/ml)

% in

crea

se in

bas

elin

e

C. C57BL/6 - 2 weeks

1 10 100

0

500

1000

1500

2000

2500


% in

crea

se in

bas

elin

eB. BALB/c - 6 weeks

1 10 100

0

500

1000

1500


% in

crea

se in

bas

elin

e

D. C56BL/6 - 6 weeks

1 10 100

0

500

1000

1500


% in

crea

se in

bas

elin

e

Figure 3. Airway responsiveness to nebulized methacholine in sensitized BALB/c (A and B) and C57BL/6 (C and D) exposed to OA (closed squares) and PBS (open squares) during 2 weeks (A and C)

and 6 weeks (B and D) (ANOVA OA versus PBS at 2 weeks: p<0.005).


OA-specific IgE levels in serum of BALB/c and C57BL/6 mice were significantly

increased after repeated OA exposure (Table 3). After 2 weeks of allergen exposure the levels

of OA-specific IgE were similar in both mouse strains. Extending the exposure period

resulted in BALB/c but not in C57BL/6 mice, in a further increase of OA-specific IgE serum

levels in both OA-exposed and control animals (Table 3). When compared to BALB/c,

C57BL/6 mice had also lower levels of OA-specific IgE in PBS-exposed animals.

TABLE 3

OA-SPECIFIC IGE-SERUM LEVELS AFTER SENSITIZATION

AND OA-EXPOSURE OF BALB/C AND C57BL/6 MICE


OA-specific IgE (U/ml)

Treatment BALB/c C57BL/6

2 weeks PBS 7.60±1.02ll 2.22±0.55

OA 41.75±5.28† 33.50±7.19†

6 weeks PBS 29.11±2.54§ ll 6.38±1.80

OA 96.30±10.67† ‡ 49.18±9.00*

9 weeks PBS 23.00±1.90§ ll 3.43±1.14

OA 102.20±10.63† § ll 21.83±4.01*

* p<0.05, † p<0.005 OA versus PBS

‡ p<0.05, § p<0.005 6 and 9 weeks versus 2 weeks

ll p<0.01 BALB/c versus C57BL/6

5. Discussion

Repeated allergen exposure in sensitized BALB/c and C57BL/6 mice induced in

addition to IgE synthesis and eosinophil influx around the airways, more structural alterations

including goblet cell hyperplasia and increased collagen deposition. These changes resulted in

an increase of airway wall thickness and affected airway behavior to methacholine.

The present study confirms that the genetic background of mice has an impact on

immunological responses and inflammatory responses in BALF after allergen exposure. As

previously reported a similar protocol for OA sensitization and challenges resulted in BALB/c

in relative higher levels of IgE and in a lower amount of cells in the BALF compared to

C57BL/6 mice (Brewer et al, 1999; Herz et al, 1998; Takeda et al, 2001; Zhang et al, 1997).

Despite these differences between BALB/c and C57BL/6 mice, both inbred strains displayed

a similar degree of goblet cell hyperplasia and collagen deposition after sensitization and

allergen exposure. The development of these structural alterations in BALB/c and C57BL/6

mice shows differences in kinetics. Goblet cell hyperplasia and increased airway wall

thickness were observed after two weeks of allergen exposure. Collagen deposition could not

be observed prior to OA exposure of 6 weeks. These observations correspond to findings in

previous studies in BALB/c mice. Some describe a rapid onset of goblet cell hyperplasia in


response even to a single allergen challenge (Blyth et al, 1996; Haile et al, 1999). Others

reported, using different sensitization protocols, the development of goblet cell hyperplasia

and increased subepithelial collagen deposition. Again goblet cells hyperplasia preceded the

alterations in collagen deposition (Blyth et al, 1996; Temelkovski et al, 1998). Far less data are

available on airway remodeling in C57BL/6 and only few studies compared structural

changes between both strains after sensitization and a single allergen challenge (Foster et al,

1996; Trifilieff et al, 2000). Light microscopical analysis revealed that C57BL/6 exposed to

OA showed changes in the structural integrity of the airway wall, thickening of the basement

membrane region and smooth muscle band. However, these alterations were not quantified

(Foster et al, 1996). Trifilieff et al evaluated the proliferative index of epithelium and the

presence of cellular fibronectin in BALF as markers for airway remodeling in C57BL/6 and

BALB/c mice. They found that BALB/c revealed more pronounced structural alterations

compared to C57BL/6 after a single OA exposure (Trifilieff et al, 2000; Trifilieff et al, 2001).

A potential cause of remodeling is the release of growth factors and cytokines from

infiltrating eosinophils (Eickelberg et al, 1999; Minshall et al, 1997; Vignola et al, 1997). Of

note is that in the current comparative study the intensity of eosinophilic infiltration of the

airways was similar and that the number of peribronchial eosinophils decreased both in

BALB/c and C57BL/6 when collagen deposition was increased. The presence of the

eosinophils in the airways could have initiated the process of altered collagen synthesis

(Minshall et al, 1997). The waning of peribronchial eosinophilia despite further allergen

exposure seems similar to previous data. An acute-on-chronic allergic inflammation was

observed in the airways of BALB/c mice after prolonged allergen exposure and the infiltrating

cells in the lamina propria of intrapulmonary conducting airways consisted of lymphocytes,

macrophages and plasma cells but not of eosinophils (Foster et al, 2000; Sakai et al, 2001;

Temelkovski et al, 1998). A significantly increased but small amount of eosinophils was only

observed in the epithelium of BALB/c mice exposed to allergen for 6 weeks (Foster et al,

2000). A possible explanation for this observation is that chronically challenged BALB/c and

C57BL/6 can develop antigenic tolerance characterized by loss of airway eosinophilia, shifts

in the lymphocyte population in the airway and diminished IgE levels (McMenamin et al,

1994; Sakai et al, 2001; Tsitoura et al, 2000; Yiamouyiannis et al, 1999). The persistent IgE

production in OA-exposed mice would argue against tolerance development as being the

only mechanism explaining the reduction in airway eosinophilia.


Airways of BALB/c mice were more sensitive to nebulized methacholine as compared to

C57BL/6 mice. After a 2 week allergen exposure period, BALB/c but not C57BL/6

developed airway hyperresponsiveness despite the influx of peribronchial eosinophils in both

inbred strains. A significant genetic variability exists between inbred mouse strains in their

development of enhanced bronchial responses after airway antigen sensitization and exposure

(Brewer et al, 1999; Wills-Karp and Ewart, 1997; Zhang et al, 1997). The finding that OA-

exposed C57BL/6 had similar airway responses compared to control animals is in contrast to

previous data from our lab, using a similar sensitization and exposure protocol in C57BL/6

mice but using a different parameter of airway caliber (Kips et al, 1995). This again illustrates

one of the main weaknesses of murine models, namely the difficulty in reliably assessing

airway caliber and lack of an invariably accepted gold standard lung function index (Kips et

al, 2000).

The evolution of airway hyperresponsiveness over time in BALB/c is somewhat similar

to data obtained in BN rats (Palmans et al, 2000). In both models hyperresponsiveness wanes

despite the persistent presence of eosinophils in the BN rats and to a lesser extent in BALB/c

mice. The loss of hyperresponsiveness parallels the increased collagen deposition in the

airway wall. It is tempting to speculate that the remodeling process might protects against

airway hyperresponsiveness because increased collagen deposition could increase airway wall

stiffness and oppose against extended narrowing of the airway wall (Lambert et al, 1994;

Milanese et al, 2001; Seow et al, 2000). Although the increase in airway wall thickness could

affect airway responses in OA-exposed BALB/c mice, enhanced collagen deposition in the

airway wall might have compensated the increased wall thickness and contribute more to the

loss in airway hyperresponsiveness observed after prolonged allergen exposure.

In conclusion, the inbred mouse strains BALB/c and C57BL/6 showed different allergic,

BALF inflammatory and airway responses after a similar allergen sensitization and exposure.

Despite the different allergic response between BALB/c and C57BL/6 mice, both inbred mice

strains developed similar structural airway changes. The mechanisms that predispose the

inbred mouse strains to the development of the different allergic and functional responses did

not affect the occurrence and magnitude of the remodeling process in the airways of these

mice.


6. References




Bousquet J, Jeffery PK, Busse WW, Johnson M and Vignola AM. Asthma - From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000, 161:1720-1745.

Brewer JP, Kisselgof AB and Martin TR. Genetic variability in pulmonary physiological, cellular, and antibody responses to antigen in mice. Am J Respir Crit Care Med 1999, 160:1150-1156.

Carroll NG and James AL. The use of morphometry to assess airway wall remodeling in asthma. In: Howarth PH, Wilson JW, Bousquet J, Rak S and Pauwels RA (eds). Airway remodeling: 2001, New York, NY, Marcel Dekker, Inc., p. 27-44.

Clarke JR, Jenkins MA, Hopper JL, Carlin JB, Mayne C, Clayton DG, Dalton MF, Holst DP and Robertson CF. Evidence for genetic associations between asthma, atopy, and bronchial hyperresponsiveness: a study of 8- to 18-yr-old twins. Am J Respir Crit Care Med 2000, 162:2188-2193.

Duguet A, Biyah K, Minshall E, Gomes R, Wang CG, Taoudi-Benchekroun M, Bates JH and Eidelman DH. Bronchial responsiveness among inbred mouse strains. Role of airway smooth-muscle shortening velocity. Am J Respir Crit Care Med 2000, 161:839-848.

Eickelberg O, Kohler E, Reichenberger F, Bertschin S, Woodtli T, Erne P, Perruchoud AP and Roth M. Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3. Am J Physiol 1999, 276:L814-L824.

Foster PS, Hogan SP, Ramsay AJ, Matthaei KI and Young IG. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996, 183:195-201.

Foster PS, Ming Y, Matthei KI, Young IG, Temelkovski J and Kumar RK. Dissociation of inflammatory and epithelial responses in a murine model of chronic asthma. Lab Invest 2000, 80:655-662.

Haile S, Lefort J, Joseph D, Gounon P, Huerre M and Vargaftig BB. Mucous-cell metaplasia and inflammatory-cell recruitment are dissociated in allergic mice after antibody- and drug-dependent cell depletion in a murine model of asthma. Am J Respir Cell Mol Biol 1999, 20:891-902.



Herz U, Braun A, Ruckert R and Renz H. Various immunological phenotypes are associated with increased airway responsiveness. Clin Exp Allergy 1998, 28:625-634.

Kips JC, Brusselle GG, Joos GF, Peleman RA, Devos RR, Tavernier J and Pauwels RA. Importance of interleukin-4 and interleukin-12 in allergen- induced airway changes in mice. Int Arch Allergy Immunol 1995, 107:115-118.

Kips JC and Pauwels RA. Airway wall remodelling: does it occur and what does it mean? Clin Exp Allergy 1999, 29:1457-1466.

Kips JC, Tournoy KG and Pauwels RA. Gene knockout models of asthma. Am J Respir Crit Care Med 2000, 162:S66-S70.


McMenamin C, Pimm C, McKersey M and Holt PG. Regulation of IgE responses to inhaled antigen in mice by antigen-specific gamma delta T cells. Science 1994, 265:1869-1871.





Sakai K, Yokoyama A, Kohno N, Hamada H and Hiwada K. Prolonged antigen exposure ameliorates airway inflammation but not remodeling in a mouse model of bronchial asthma. Int Arch Allergy Immunol 2001, 126:126-134.

Sandford AJ and Pare PD. The genetics of asthma. The important questions. Am J Respir Crit Care Med 2000, 161:S202-S206.


Takeda K, Haczku A, Lee JJ, Irvin CG and Gelfand EW. Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung. Am J Physiol 2001, 281:L394-L402.



Trifilieff A, El Hashim A and Bertrand C. Time course of inflammatory and remodeling events in a murine model of asthma: effect of steroid treatment. Am J Physiol 2000, 279:L1120-L1128.

Trifilieff A, Fujitani Y, Coyle AJ, Kopf M and Bertrand C. IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation. Clin Exp Allergy 2001, 31:934-942.

Tsitoura DC, Blumenthal RL, Berry G, DeKruyff RH and Umetsu DT. Mechanisms preventing allergen-induced airways hyperreactivity: role of tolerance and immune deviation. J Allergy Clin Immunol 2000, 106:239-246.


Vignola AM, Kips J and Bousquet J. Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 2000, 105:1041-1053.

Wills-Karp M and Ewart SL. The genetics of allergen-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med 1997, 156:S89-S96.

Yiamouyiannis CA, Schramm CM, Puddington L, Stengel P, Baradaran-Hosseini E, Wolyniec WW, Whiteley HE and Thrall RS. Shifts in lung lymphocyte profiles correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model. Am J Pathol 1999, 154:1911-1921.

Zhang Y, Lamm WJ, Albert RK, Chi EY, Henderson WR, Jr. and Lewis DB. Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am J Respir Crit Care Med 1997, 155:661-669.

Structural Airway Changes in Young Rats 143

CHAPTER V

EFFECT OF AGE ON ALLERGEN-INDUCED STRUCTURAL

AIRWAY CHANGES IN BROWN NORWAY RATS

Els Palmans, Nele J. Vanacker, Romain A. Pauwels and Johan C. Kips

American Journal of Respiratory and Critical Care Medicine, 2002, 165:1280-1284


1. Abstract

It remains to be fully established whether allergen-induced airway inflammation and

remodeling are influenced by age. The aim of the present study was to compare allergen-

induced airway changes in young and adult rats. Brown Norway rats were sensitized at 4

weeks of age (young) or 13 weeks of age (adult) and exposed to aerosolized ovalbumin (OA)

or phosphate-buffered saline for 2 weeks. In both age groups OA exposure induced an

increase in OA-specific Immunoglobulin E and in the number of peribronchial eosinophils.

OA-challenged animals also developed an increase in total airway wall area, enhanced

fibronectin deposition and goblet cell hyperplasia. Both inflammatory and structural

alterations were more pronounced in the airways of young compared with adult OA-exposed

rats. The number of peribronchial eosinophils was increased in young animals (685.4±75.0

versus 389.9±37.8/mm² in adult rats; p<0.001). A higher degree of goblet cell hyperplasia

was observed in young rats (65.37±4.68 versus 34.74±3.68/mm basement membrane in adult

rats; p<0.001) and area of fibronectin deposition in the airway wall was higher in young

compared with adult animals (5.08±0.46 versus 3.62±0.29 µm²/µm basement membrane;

p<0.005). In conclusion, in young rats airways are more susceptible to allergen-induced

inflammatory and structural airway changes.

2. Introduction

Asthma is an inflammatory disorder of the airways, and several studies have confirmed a

similar type of inflammation in children and adults with asthma (Cai et al, 1998; Louis et al,

2000; Twaddell et al, 1996). However, it is unknown whether developing airways in children

are more prone to the development of allergic inflammation, nor has it been fully established

whether a similar environmental exposure will induce a similar severity of airway inflammation

in children and adults. Part of the inflammatory process in asthmatic airways consists of

structural airway changes, the so-called airway remodeling. This is thought to result from

attempts at repair in response to repetitive bouts of allergen-induced inflammation and tissue

injury (Vignola et al, 2000b). Again, it is unknown whether developing airways in children are

more susceptible to remodeling. This aspect would seem particularly relevant for the natural

history of asthma as remodeling is thought to have important functional consequences on


lung function, contributing to loss of airway distensibility and bronchial hyperresponsivess

(Boulet et al, 1997; Macklem, 1996). Both low baseline FEV1 and severe bronchial

hyperresponsiveness are known risk factors for the persistence of asthma into adulthood

(Grol et al, 1999; Palmer et al, 2001; Roorda et al, 1994).

Representative in vivo animal models might provide interesting information in this

respect. We and others have previously shown that structural airway changes can be induced

in Brown Norway (BN) rats after sensitization and chronic exposure to the allergen ovalbumin

(OA) (Palmans et al, 2000; Salmon et al, 1999; Sapienza et al, 1991). In the present study we

compared the allergen-induced airway changes in prepuberal and adult rats. The results

indicate that sensitized rats exposed to allergen before they reach puberty develop more

profound inflammatory and structural airway changes.

3. Methods

3.1. Immunization and exposure of animals

Specific pathogen-free, male BN rats (Harlan CPB, Zeist, the Netherlands) were actively

sensitized by intraperitoneal injection of OA (4 mg/kg body weight) (Grade III; Sigma, Poole,

Dorset, UK) together with Al(OH)3 (800 µg/kg body weight) in 0.5 ml 0.9% NaCl on Day 0

and Day 7. On Day 0 one group of rats (n=20) was 4 weeks of age (young rats) and another

group of rats (n=19) was 13 weeks of age (adult rats). From Day 14 to Day 28 animals were

exposed seven times to aerosolized OA or phosphate-buffered saline (PBS).


On Day 29, 24 hours after the last exposure to OA or PBS, rats were weighed prior to

instrumentation and anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital

(Sanofi, Libourne, France). Bronchoalveolar lavage (BAL) was performed for analysis of

total and differential cell count; blood was collected for the measurement of OA-specific

Immunoglobulin E (IgE).

Histological analysis of lung tissue

After lavage, lungs were fixed with 4% paraformaldehyde, slices from different lobes

were embedded in paraffin and cut in 2-µm-thick sections. Histological changes were

evaluated on sections stained with hematoxylin and eosin. In addition, Congo red was used


to determine the number of eosinophils in the bronchial wall and the number of goblet cells in

epithelium was assessed in sections stained with periodic acid-Schiff reagent. Fibronectin was

stained immunohistochemically. Details of the methodology used have been previously

described (Palmans et al, 2000).

Analysis of airway morphometry and fibronectin deposition

Hematoxylin- and eosin-stained tissue sections were examined by light microscopy and

morphometric measurements were performed in three sections per animal, using a Zeiss

Axiophot microscope (Zeiss, Oberkochen, Germany) as previously describ ed (Palmans et al,

2000). The perimeter of epithelial basement membrane (Pbm) and the total bronchial wall area

(WAt) were measured in all large airways (Pbm > 2 mm) cut in reasonable cross-sections (ratio

of minimal to maximal internal diameter greater than 0.5). Smooth muscle area (WAm) was

measured by specifically tracing around the smooth muscle bundles.

Fibronectin deposition was examined by light microscopy and quantitative

measurements were performed using a computerized image analysis system (LEICA

Q500MC; Leica Cambridge, Cambridge, UK). Fibronectin deposition was analyzed in three

lung sections per rat as previously described (Palmans et al, 2000). The amount of protein

deposited in the total airway wall (WFt) was measured for all large airways cut in reasonable

cross-sections.

3.3. Data analysis

Reported values are expressed as mean ± standard error of the mean. The weight of

young and adult rats was compared using a Mann-Whitney U-test. The cellular composition

of the bronchoalveolar lavage fluid (BALF) and concentrations of OA-specific IgE in the

serum of the different groups were compared via Kruskal-Wallis test for multiple

comparisons. When significant differences were observed, pairwise comparisons were made

by using a Mann-Whitney U-test with Bonferroni corrections.

For analysis of morphometry and fibronectin deposition, the data of the different rats in

each experimental group were pooled. Measurements were performed in a mean of 27 large

airways (range, 24 to 32) for morphometry and in a mean of 23 large airways (range, 17 to 31)

for quantification of the fibronectin. To ensure that a similar range of airway sizes was being

compared, the frequency distribution of Pbm values between different experimental groups

was compared by the Kolmogorov-Smirnov test. The mean values of WAt, WAm and WFt


were normalized to Pbm; the number of goblet cells per mm Pbm and number of eosinophils

per square mm WAt were compared between the experimental groups, using one-way analysis

of variance with post hoc (least significant difference and Scheffé) tests (p values less than

0.05 were considered significant).

4. Results

4.1. Weight

On Day 29, young rats reached a mean weight of 181.0±2.5 g, whereas adult rats

weighed 266.6±5.5 g (p<0.001).


Serum levels of OA-specific IgE were elevated in all sensitized rats exposed to OA

compared with control animals (Table 1). The IgE levels of OA-exposed animals were not

different when rats sensitized at 4 weeks were compared with the group of rats sensitized at 13

weeks. Increased levels of OA-specific IgE were observed in adult control animals compared

with young control animals (Table 1).

4.3. Inflammatory cell infiltrate in BALF

More cells were recovered in the BALF from all sensitized and OA-exposed animals

compared with those exposed to PBS (Table 2). In young animals the amount of cells after

OA exposure was twice the amount of BALF cells in control animals. In adult rats a 3-fold

increase was observed in OA-exposed animals compared with control animals. Moreover, the

total cellularity of OA-exposed rats immunized at 13 weeks was significantly increased

compared with OA-exposed rats sensitized at the age of 4 weeks (Table 2). The amount of

macrophages, eosinophils, and neutrophils changed in both OA-exposed animal groups. In

OA-exposed young animals a 3-fold increase in the number of eosinophils was observed

compared with PBS-exposed rats ([500.0±121.5]x103 cells versus [182.4±74.0]x103 cells;

p<0.05); in OA-exposed adult animals we observed a 10-fold increase of eosinophils

([538.3±85.4]x103 cells versus [58.1±26.9]x103 cells in PBS-exposed rats; p<0.005). The

total amount of BALF eosinophils was similar in both age groups ([500.0±121.5]x103 for

young rats versus [538.3±85.4]x103 for adult rats). Total cellularity of BALF was not


different between both PBS-exposed control groups. However, the percentage of eosinophils

was higher in control rats sensitized at 4 weeks compared with those sensitized at 13 weeks

(Table 2).

TABLE 1

OVALBUMIN-SPECIFIC IMMUNOGLOBULIN E LEVELS IN SERUM AFTER IMMUNIZATION AT DIFFERENT AGES AND REPEATED OA EXPOSURE

Age at immunization OA-specific IgE

(weeks) Treatment (µg/ml)

4 PBS 1.083±0.194

OA 17.486±2.578*

13 PBS 2.675±0.402†

OA 10.612±0.869* * p < 0.001, OA versus PBS † p < 0.001, PBS at age 13 weeks versus PBS at age 4 weeks

4.4. Lung histology

The difference in BALF composition was not reflected by histological analysis.

Inflammatory infiltrates were observed in the peribronchial area of sensitized and OA-exposed

animals. In addition to mononuclear cells, these infiltrates contained increased numbers of

eosinophils. The number of eosinophils was higher around the bronchi of young rats. The

number of peribronchial eosinophils was similar in both control groups (Table 3).

There was no epithelial desquamation observed, but OA exposure resulted in goblet cell

hyperplasia. The increase in goblet cells was more pronounced in OA-exposed animals

immunized at 4 weeks. The number of goblet cells in young and adult control rats was not

different (Table 3).

TABLE 2

TOTAL NUMBER OF WHITE BLOOD CELLS AND CELLULAR DISTRIBUTION IN BRONCHOALVEOLAR LAVAGE FLUID

Total cells Monocytes Eosinophils Neutrophils Lymphocytes

Age* Treatment (x 106) (%) (%) (%) (%)


4 PBS 1.12±0.18 79.8±4.4 14.2±4.2 1.5±0.3 4.6±0.8

weeks OA 2.36±0.41† 43.8±4.5 § 20.4±3.4† 29.3±4.4 ‡ 6.6±0.9

13 PBS 3.54±0.95 94.7±0.7 ¶ 1.5±0.4 ** 0.4±0.2 ll 3.4±0.4

weeks OA 12.18±2.0 †

¶ 57.5±5.3 § 4.5±0.5‡ ** 33.7±5.3 § 4.3±0.5

* Age = age at immunization † p < 0.05, ‡ p < 0.005 and § p < 0.001 OA versus PBS ll p < 0.05, and ¶ p < 0.005 and ** p < 0.001 age 13 weeks versus same treatment age 4 weeks

TABLE 3 NUMBER OF GOBLET CELLS IN THE EPITHELIUM AND NUMBER OF AIRWAY WALL

EOSINOPHILS AFTER IMMUNIZATION AT DIFFERENT AGES AND REPEATED OVALBUMIN EXPOSURE

Age at Immunization Peribronchial eosinophils Goblet cells

(weeks) Treatment (cells/mm2 airway wall area) (cells/mm basement membrane)

4 PBS 34.30±5.45 287.2±35.7

OA 65.37±4.68† 685.4±75.0 †

13 PBS 24.83±3.92 295.5±55.9

OA 34.74±3.68* § 389.9±37.8 * ‡ * p<0.05 and † p < 0.001 OA versus PBS ‡ p<0.005 and § p<0.001 OA age 13 weeks versus OA age 4 weeks

4.5. Fibronectin deposition

Fibronectin deposition in large airways was significantly increased after 2 weeks of

allergen exposure (5.08±0.46 versus 1.50±0.26 µm²/µm Pbm in young rats and 3.62±0.29

versus 2.27±0.37 µm²/µm Pbm in adult animals; p<0.001 and p<0.01 respectively) (Figure

1A). The fibronectin deposition after allergen exposure in rats immunized at 4 weeks was

higher compared with OA-exposed adult animals (p<0.005) (Figure 1A). The amount of

fibronectin in control animals was similar in both age groups (Figure 1A).


4.6. Morphometry

The total airway wall area was increased in OA-exposed adult animals compared with the

control rats (94.04±11.53 versus 73.33±4.27 µm²/µm Pbm; p<0.05) (Figure 1B). In young

animals, the difference in total wall area between control and OA-exposed rats showed a

strong trend toward significance (92.89±5.93 versus 70.26±3.89 µm²/µm Pbm; p=0.056)

(Figure 1B). No difference in airway wall thickness was observed between allergen-exposed

rats of both age groups. There was no difference between age groups in airway wall thickness

in control animals (Figure 1B).

B. total airway wall

4 weeks 13 weeks0

50

100

150

*

age at immunization

WA

t/P

bm (µ

m²/

µm)

A. fibronectin

4 weeks 13 weeks0.0

2.5

5.0

7.5

+ §

++

age at immunization

µm²

fibro

nect

in/µ

mba

sem

ent m

embr

ane

Figure 1. Effect of age at immunization on fibronectin deposition in the airway wall (A) and airway wall

area (B) in large airways after OA exposure (closed bars) versus PBS exposure (open bars) (* p<0.05 versus PBS, + p<0.01 versus PBS, ++ p<0.001 versus PBS and § p<0.005 versus OA immunization at 4

weeks). WAt = total bronchial wall area; Pbm = length of epithelial basement membrane.

No difference in airway smooth muscle layer thickness was observed between control

and OA-exposed adult rats (2.78±0.28 and 2.00±0.26 µm2/µm Pbm respectively). Although

the thickness of the smooth muscle was increased in young animals compared with the adult

animals (p<0.001) no difference was observed between PBS- and OA-exposed young rats

(5.83±0.65 versus 4.14±0.55 µm2/µm Pbm).

5. Discussion


The main purpose of this study was to evaluate the effect of age on the development of

structural airway changes in BN rats after allergen sensitization and exposure. Synthesis of

allergen-specific IgE was similar in both OA-exposed groups. However, in young animals

sensitization and repeated exposure to aerosolized allergen resulted in the development of

more pronounced acute inflammation reflected by increased peribronchial eosinophils as well

as more structural alterations, including goblet cell hyperplasia and fibronectin deposition.

Several elements could contribute to the different responses observed in young and adult

BN rats. A first potentially confounding factor is a technical one. In view of the weight

difference that we observed between young and adult rats, it is likely that the lung size in both

groups was also different. We therefore cannot exclude the possibility that airways of slightly

different generations are compared between both groups. However, we have previously

shown that the extent of alterations in small, medium and large airways of sensitized BN rats

after OA exposure was similar (Palmans et al, 2000). This indicates that allergen-induced

airway changes are uniformly distributed throughout the bronchial tree and that comparison

of airways in both age groups can provide valid data. In addition, an increased lung size

could also result in a larger lung surface being sampled by the BAL procedure. Yet, as was

also reported by Yagi et al(Yagi et al, 1997)), the total cell number in BALF was not different

between young and adult rats. To us, these results indicate that lung size differences do not

significantly influence the results obtained.

Another element that needs to be considered is a possible difference in the immune

response to sensitization in both age groups. It is known that experimental animals fail to

develop helper T cell type 2-induced allergic responses at an older age (Yagi et al, 1997)(Ide

et al, 1999; Smith et al, 2001). Throughout the age range chosen in the present study

however, the immune response to inhaled allergen is helper T cell type 2 driven (Nelson et al,

1994; Nelson and Holt, 1995). The observation that young and adult rats develop similar IgE

serum levels is also an indirect argument that differences in the susceptibility of the airway

mucosa rather than the immune response explain the observed results.

The pathogenesis of remodeling remains to be established, but it is thought to result from

attempts at tissue repair after bouts of repetitive injury caused by allergen inhalation (Vignola et

al, 2000b). A characteristic feature of allergen-induced acute airway inflammation is the

occurrence of airway eosinophilia. The present study indicates that for a same intensity of

allergen exposure, the airways of young animals are infiltrated with larger numbers of

eosinophils than those of adult rats. A previous study, using a different experimental protocol,


reported similar findings (Yagi et al, 1997). Comparative studies in human asthma are hardly

available in this respect. The data available indicate that cellular composition of the airway

inflammation in children resembles that observed in adult asthmatics (Cai et al, 1998; Louis et

al, 2000; Twaddell et al, 1996). Similarly, analysis of induced sputum in asthmatic children

showed, as in adults, an increase in the percentage of eosinophils (Cai et al, 1998; Twaddell et

al, 1996). From the limited studies currently available, sputum eosinophilia in untreated

asthmatic children also falls within a similar wide range compared with adults (Cai et al, 1998;

Louis et al, 2000). To what extent this fully reflects tissue eosinophilia is, however, unknown

(Grootendorst et al, 1997). Of note is that in the present study the difference in eosinophils in

the airway wall between both age groups was not reflected in BALF counts.

Airways in young rats develop a more pronounced remodeling as indicated by the

number of goblet cells and fibronectin deposition. Biopsy studies have confirmed the

presence of remodeling in airways of children (Cokugras et al, 2001; Cutz et al, 1978). A

preliminary report even indicates that remodeling might be present prior to the development of

asthma symptoms (Pohunek et al, 1997). This somewhat challenges the concept that

remodeling results from inflammation, but raises the idea that eosinophilic airway inflammation

and remodeling might be two independent processes (Pohunek et al, 1997). The present

study confirms that remodeling is induced by allergen exposure, but obviously does not

prove a causal role of the eosinophils in this process.

The age-dependent difference between animals in terms of infiltrating inflammatory cells

and structural changes is concordant with observations relating to other repair processes.

During healing of cutaneous punch biopsies in humans the inflammatory response was

delayed in adult compared with young subjects (Ashcroft et al, 1998). In vivo models of

wound healing also showed that repair processes are associated with increased amounts of

inflammatory cells in young compared with older animals (Ashcroft et al, 1997; Marcus et al,

2000). In young rabbits excision of tissue in the ear resulted in a higher amount of proliferative

cells in the healing dermis compared with old animals (Marcus et al, 2000). In mice, the

inflammatory response to cutaneous incision wounds was less pronounced in older animals,

as was the deposition of the extracellular matrix (ECM) components fibronectin and collagen

type I and III (Ashcroft et al, 1997).

That young animals develop more pronounced remodeling in response to allergen

exposure could have profound implications. To date, the functional consequences of

remodeling have been related mainly to altered airway behavior (Laprise et al, 1999). Although


it is generally assumed that remodeling contributes to bronchial hyperresponsiveness, it has

been shown that depending on the exact extent and location, changes in ECM can also

protect against bronchial hyperresponsiveness (Milanese et al, 2001; Palmans et al, 2000;

Seow et al, 2000). We have previously reported that the degree of fibronectin deposition after

exposing adult rats to OA for 12 weeks is three times higher than in animals exposed for 2

weeks. This coincided with a change from airway hyperresponsiveness at 2 weeks to

hyporesponsiveness after 12 weeks of exposure (Palmans et al, 2000). In the present study,

we did not measure airway responsiveness. However, the difference between the intensity in

fibronectin deposition is only 1.5. We would therefore speculate that this relatively limited

difference is insufficient to significantly influence lung function.

ECM proteins in the airway wall not only determine the structural integrity and

mechanical properties of the airway wall, they can also interact with inflammatory cells

(Roman, 1996). The components of the ECM can affect migration, proliferation,

differentiation, and activation state of inflammatory cells and enhance their survival (Anwar et

al, 1993; Lavens et al, 1996; Meerschaert et al, 1999b; Neeley et al, 1994; Zhang and Phan,

1999). Modulation of the ECM by remodeling its structure can therefore affect the behavior

of cells residing within it, and the changes of ECM composition could have an important

influence on the persistence of the allergic inflammation, anchoring it firmly within the airway

wall. The matrix adhesion molecule fibronectin promotes recruitment and attachment of cells

(Armstrong and Armstrong, 2000). As in the current model, asthmatic airways have increased

levels of fibronectin (Meerschaert et al, 1999a; Roche et al, 1989). Fib ronectin is known to be

released by proinflammatory cytokines and is thought to play an important role in epithelial

repair (Vignola et al, 2000a). In addition, however, the presence of fibronectin in the ECM

facilitates the adhesion of inflammatory cells and prolongs eosinophil survival (Meerschaert et

al, 1999b). The more pronounced remodeling in response to allergen exposure at a young

age might therefore increase the vulnerability of the lower airways to the development of a

persistent allergic inflammation. This also fits with the observation that sensitization at young

age increases the likelihood of developing asthma in adulthood compared with those who

become sensitized later (Peat et al, 1990; Roorda et al, 1994; Sherrill et al, 1999). As

remodeling promotes the ongoing inflammatory process, the current observations further

strengthen the importance of avoiding stimuli that can induce remodeling in young children;

this includes not only allergens, but also stimuli such as cigarette smoke (Rennard, 1999).


In vivo animal models obviously have limitations, one of which is to what extent the

observations can be extrapolated to human asthma. However, the observations that allergen

avoidance measures are far more effective at young age than in adults would seem to support

the idea that the increased susceptibility to allergen induced airway changes observed in the

present model might also apply in humans.

6. References

Anwar AR, Moqbel R, Walsh GM, Kay AB and Wardlaw AJ. Adhesion to fibronectin prolongs eosinophil survival. J Exp Med 1993, 177:839-843.

Armstrong PB and Armstrong MT. Intercellular invasion and the organizational stability of tissues: a role for fibronectin. Biochim Biophys Acta 2000, 1470:O9-O20.

Ashcroft GS, Horan MA and Ferguson MW. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol 1997, 108:430-437.

Ashcroft GS, Horan MA and Ferguson MW. Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing. Lab Invest 1998, 78:47-58.


Cai Y, Carty K, Henry RL and Gibson PG. Persistence of sputum eosinophilia in children with controlled asthma when compared with healthy children. Eur Respir J 1998, 11:848-853.

Cokugras H, Akcakaya N, Seckin, Camcioglu Y, Sarimurat N and Aksoy F. Ultrastructural examination of bronchial biopsy specimens from children with moderate asthma. Thorax 2001, 56:25-29.

Cutz E, Levison H and Cooper DM. Ultrastructure of airways in children with asthma. Histopathology 1978, 2:407-421.

Grol MH, Gerritsen J, Vonk JM, Schouten JP, Koeter GH, Rijcken B and Postma DS. Risk factors for growth and decline of lung function in asthmatic individuals up to age 42 years. A 30-year follow-up study. Am J Respir Crit Care Med 1999, 160:1830-1837.

Grootendorst DC, Sont JK, Willems LN, Kluin-Nelemans JC, Van Krieken JH, Veselic-Charvat M and Sterk PJ. Comparison of inflammatory cell counts in asthma: induced sputum vs bronchoalveolar lavage and bronchial biopsies. Clin Exp Allergy 1997, 27:769-779.


Ide K, Hayakawa H, Yagi T, Sato A, Koide Y, Yoshida A, Uchijima M, Suda T, Chida K and Nakamura H. Decreased expression of Th2 type cytokine mRNA contributes to the lack of allergic bronchial inflammation in aged rats. J Immunol 1999, 163:396-402.

Laprise C, Lavioette M, Boutet M and Boulet LP. Asymptomatic airway hyperresponsiveness: relationships with airway inflammation and remodelling. Eur Respir J 1999, 14:63-73.

Lavens SE, Goldring K, Thomas LH and Warner JA. Effects of integrin clustering on human lung mast cells and basophils. Am J Respir Cell Mol Biol 1996, 14:95-103.

Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M and Djukanovic R. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med 2000, 161:9-16.


Marcus JR, Tyrone JW, Bonomo S, Xia Y and Mustoe TA. Cellular mechanisms for diminished scarring with aging. Plast Reconstr Surg 2000, 105:1591-1599.

Meerschaert J, Kelly EA, Mosher DF, Busse WW and Jarjour NN. Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid. Am J Respir Crit Care Med 1999a, 159:619-625.

Meerschaert J, Vrtis RF, Shikama Y, Sedgwick JB, Busse WW and Mosher DF. Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro. J Immunol 1999b, 163:6217-6227.


Neeley SP, Hamann KJ, Dowling TL, McAllister KT, White SR and Leff AR. Augmentation of stimulated eosinophil degranulation by VLA-4 (CD49d)- mediated adhesion to fibronectin. Am J Respir Cell Mol Biol 1994, 11:206-213.

Nelson DJ and Holt PG. Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals. J Immunol 1995, 155:3517-3524.

Nelson DJ, McMenamin C, McWilliam AS, Brenan M and Holt PG. Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)-negative precursors: differential regulation of Ia expression at different levels of the respiratory tract. J Exp Med 1994, 179:203-212.


Palmer LJ, Rye PJ, Gibson NA, Burton PR, Landau LI and Lesouef PN. Airway responsiveness in early infancy predicts asthma, lung function, and respiratory symptoms by school age. Am J Respir Crit Care Med 2001, 163:37-42.


Peat JK, Salome CM and Woolcock AJ. Longitudinal changes in atopy during a 4-year period: relation to bronchial hyperresponsiveness and respiratory symptoms in a population sample of Australian schoolchildren. J Allergy Clin Immunol 1990, 85:65-74.

Pohunek P, Roche WR, Turzikova J, Kudrmann J and Warner JO. Eosinophilic inflammation in the bronchial mucosa of children with bronchial astma [Abstract]. Eur Respir J 1997, 10:160s.

Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999, 160:S12-S16.


Roman J. Extracellular matrix and lung inflammation. Immunol Res 1996, 15:163-178.

Roorda RJ, Gerritsen J, van Aalderen WM, Schouten JP, Veltman JC, Weiss ST and Knol K. Follow-up of asthma from childhood to adulthood: influence of potential childhood risk factors on the outcome of pulmonary function and bronchial responsiveness in adulthood. J Allergy Clin Immunol 1994, 93:575-584.

Salmon M, Walsh DA, Koto H, Barnes PJ and Chung KF. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling. Eur Respir J 1999, 14:633-641.



Sherrill D, Stein R, Kurzius-Spencer M and Martinez F. On early sensitization to allergens and development of respiratory symptoms. Clin Exp Allergy 1999, 29:905-911.

Smith P, Dunne DW and Fallon PG. Defective in vivo induction of functional type 2 cytokine responses in aged mice. Eur J Immunol 2001, 31:1495-1502.

Twaddell SH, Gibson PG, Carty K, Woolley KL and Henry RL. Assessment of airway inflammation in children with acute asthma using induced sputum. Eur Respir J 1996, 9:2104-2108.

Vignola AM, Bonsignore G, Siena L, Melis M, Chiappara G, Gagliardo R, Bousquet J, Bonsignore G and Merendino AM. ICAM-1 and alpha3beta1 expression by bronchial epithelial cells and their in vitro modulation by inflammatory and anti-inflammatory mediators. Allergy 2000a, 55:931-939.

Vignola AM, Kips J and Bousquet J. Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 2000b, 105:1041-1053.

Yagi T, Sato A, Hayakawa H and Ide K. Failure of aged rats to accumulate eosinophils in allergic inflammation of the airway. J Allergy Clin Immunol 1997, 99:38-47.


Zhang HY and Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am J Respir Cell Mol Biol 1999, 21:658-665.

Is there an Animal Model? 159

CHAPTER VI

IS THERE AN ANIMAL MODEL OF AIRWAY REMODELING?

Johan C. Kips, Els Palmans, Nele J. Vanacker and Romain A. Pauwels

In: PH Howarth, JW Wilon, J Bousquet, S Rak and RA Pauwels (eds.) Airway

Remodeling. Lung Biology in Health and Disease: 2001, Volume 155, 261 – 270


1. Introduction

Airway remodeling encompasses a number of changes to the airway structure, found in

asthma. This includes goblet cell hyperplasia, pseudo-thickening of the basement membrane

due to subepithelial fibrosis, neovascularization, changes in composition of the extracellular

matrix, and smooth muscle hyperplasia and/or hypertrophy (Chetta et al, 1997; Ebina et al,

1993; Jeffery et al, 1989; Laitinen et al, 1997; Li and Wilson, 1997; Redington and Howarth,

1997; Roche et al, 1989). What exactly causes these structural changes to occur is unclear.

The amount of mediators present in asthmatic airways that can activate and induce or enhance

proliferation of fibroblasts and smooth muscle cells is seemingly endless (Hirst, 1996;

Minshall et al, 1997; Redington et al, 1997; Vignola et al, 1997), ranging from mediators such

as leukotrienes, or proinflammatory cytokines including TNFa and IL-1ß, to a variety of

growth factors. These can be divided in nonfibrinogenic mediators, such as GM-CSF, and

fibrogenic ones, including epidermal growth factor (EGF), insulin -like growth factor (IGF),

and transforming growth factor ß (TGF-ß). The exact functional importance of each of these

mediators in the pathogenesis of remodeling is unknown. This is a first area to which data

derived from in vivo models can contribute valuable information.

A second element with regard to remodeling that needs to be further evaluated is the

impact of these structural changes on airway function. It has been hypothesized, largely based

on mathematical models, that the remodeling process underlies bronchial

hyperresponsiveness (Pare and Bai, 1995). The two main determinants of bronchial

hyperresponsiveness are hypersensitivity and hyperreactivity of the airways (Woolcock et al,

1985). Hypersensitivity is reflected in a leftward shift of the dose-response curve toward a

bronchoconstrictor agonist. This can be attributed, in adaptation of Poiseuille’s law, to

thickening of the airway wall, especially the area inner to the smooth muscle layer (Lambert et

al, 1993). Increased stiffness of the subepithelial layer could additionally contribute to this

phenomenon, by influencing mucosal folding upon smooth muscle contraction (Wiggs et al,

1997). Hyperreactivity of the airways is reflec ted by an increased slope of the dose-response

curve of methacholine and exaggerated maximal airway narrowing (Macklem, 1996). This has

been ascribed to an increase in airway smooth muscle mass and/or changes in the adventitial

matrix, leading to a loss of elastic recoil on contracting airway smooth muscle (Lambert et al,

1993; Macklem, 1996). These assumptions are based on superb mathematical models, but

require further functional confirmation, an issue were in vivo animal models again can prove


very useful. This in turn implies that the animal model displays not only morphological

characteristics of remodeling, but also a degree of airway hyperresponsiveness, comparable

to human asthma, and that from a methodological point of view, airway responsiveness is

measured in such a way that both sensitivity and reactivity of the airways can be assessed.

When combining both these morphological and physiological requirements, it is quite clear

that the ideal animal model of airway remodeling does not yet exist. An obvious disadvantage

is that animal models displaying naturally occurring asthma-like syndromes are nearly

inexistent. Horses are known to develop airway obstruction after exposure to mold but the

airway histology is different from asthma, as it resembles more bronchiolitis (Derksen et al,

1985).

The only species that seems to spontaneously develop an airway disorder that bears

similarities to asthma, both functionally and histologically, are cats. Sensitization followed by

repeated exposure to Ascaris has been shown to increase airway responsiveness in

conjunction with histological changes that mimic those observed in naturally occurring feline

asthma. This includes goblet cell hyperplasia, eosinophil infiltration in the airway mucosa, and

an increase in the thickness of the airway smooth muscle layer. The overall airway wall

thickness was not increased (Padrid et al, 1995). However, the cat has not gained widespread

acceptance as an animal model of asthma, and the vast majority of animal models of allergic

airway inflammation have been experimentally induced in small rodents, mice in particular.

This choice of animal species is in part dictated by economical and ecological

considerations, but mainly attributable to the huge range of immunologic tools available in

mice. The development of monoclonal antibodies and, more recently, gene deletion

technology makes it possible to effectively neutralize a given mediator or cell, thus fully

establishing its functional importance in the disease process (Kips and Pauwels, 1997).

However, the degree of hyperresponsiveness obtained in most of the currently available

models by no means compares to the generally far larger differences in airway responsiveness

that exist between asthmatic and nonasthmatic individuals. This could relate at least in part to

the pattern of airway inflammation induced in these models. In the murine models of allergic

airway inflammation developed so far, only acute inflammatory changes have been induced,

without any structural changes that might affect responsiveness to a larger degree. The

distribution of the inflammation is also different from human asthma, concentrating not only

in or around the airways, but frequently also including lung parenchymal, mainly perivascular,

changes. Furthermore, in the vast majority of these currently available murine models,


sensitivity as opposed to reactivity of the airways has not been specifically evaluated. This is

predominantly due to methodological problems. No ‘gold standard’ lung function test has

been invariably accepted for small animal models, mice in particular. The indices used include

lung resistance, based on the principles described by Amdur and Mead, overflow of

insufflation pressure as described by Konzett and Rösler, sometimes expressed as the APTI

(airway pressure over time index) or the penh value (Amdur and Mead, 1958; Hamelmann et

al, 1997; Konzett and Rössler, 1940; Levitt and Mitzner, 1988). These various indices reflect

to a variable and ill-defined degree the changes in airway sensitivity and reactivity.

2. Rat models of airway remodeling

Although as already explained, the ideal animal model of airway remodeling does not yet

exist, the issue of structural airway changes has been addressed in a number of models. Most

of these studies have been conducted in rats. In a series of studies conducted in Martin’s

laboratory, Brown Norway (BN) rats were sensitized to ovalbumin on day 0, by injecting 1

mg of ovalbumin + 200 mg Al(OH)3 subcutaneous, and 1 ml containing 6x109 Bordetella

pertussis organisms intraperitoneally. The animals were then exposed to a number of

inhalation challenges with ovalbumin, ranging from three to six, usually every 5 days, starting

2 weeks after sensitization (Bellofiore and Martin, 1988; Du et al, 1996; Sapienza et al, 1991;

Wang et al, 1993). This protocol led to an increase in airway responsiveness in those animals

that developed an early response to ovalbumin inhalation, as judged by the dose of inhaled

methacholine required to double baseline resistance (PD200RL) in animals ventilated via a

tracheal cannula (Bellofiore and Martin, 1988). In one of these studies (Sapienza et al, 1991),

the maximal increase in RL that could be achieved was measured, revealing no difference

between ovalbumin-exposed and control animals. Noteworthy is that the increase in airway

responsiveness in some of these animals persisted for up to 17 days after the sixth challenge.

The altered airway behavior in these rats was not accompanied by a clear eosinophil influx

into the airways, nor increased airway wall thickness. The basement membrane thickness was

not specifically measured. What was reported was an increase in the thickness of the smooth

muscle layer when expressed as a percentage of the squared basement membrane length.

Further analysis, using BrdU pretreatment to identify replicating cells, suggested this was due

to smooth muscle hyperplasia (Panettieri, Jr. et al, 1998). In these studies a weak correlation


was found between the quantity of airway smooth muscle in the large airways and the

PD200RL. No correlation was found with the maximal increase in RL obtained in individual

animals. Both the increase in airway smooth muscle mass and the increase in airway

responsiveness could be blocked by pretreating animals with a CysLT1-receptor antagonist

(Wang et al, 1993).

In our hands, repeated allergen exposure of BN rats also induced components of airway

remodeling, albeit somewhat different in nature. We sensitize animals by the intraperitoneal

injection of 1 mg ovalbumin + 200 µg Al(OH)3. A booster is given on day 7. The animals are

then exposed to aerosolized ovalbumin 3 times per week for 2 weeks from day 14 onward

(Palmans et al, 2000). The protocol induces quite patchy inflammatory infiltrates, which are

located predominantly around the airways and contain mononuclear cells in addition to

eosinophils. Airway epithelium shows an increase in the relative proportion of goblet cells. In

addition, the epithelium contains an increased number of BrdU+ cells, indicating increased

proliferative activity. The basement membrane does not appear thickened. Collagen

deposition in the airway mucosa is slightly, but not significantly increased. The smooth

muscle layer is not thickened and, in contrast to the findings from Martin’s group, does not

show signs of hyperplasia, as judged by BrdU staining. However, morphometric analysis

shows an overall increase in the thickness of the airway wall area, in both small and large

airways. This coincides with an increase in airway responsiveness to aerosolized carbachol, as

illustrated by a leftward shift of the dose-response curve. We could not reach a plateau. Of

particular interest is that the increase in airway wall thickness and airway responsiveness

persisted for at least 14 days after the last of seven ovalbumin exposures.

In addition, the airway changes induced in this model prove to be relatively insensitive to

the effect of inhaled steroids. Pretreatment with inhaled fluticasone (FP) (10 mg), prior to each

allergen inhalation, reduces the ovalbumin-induced peribronchial eosinophilic infiltration, the

increase in BrdU+ cells in airway epithelium, and goblet cell hyperplasia. However, the increase

in airway wall thickness and airway responsiveness are not fully prevented (Vanacker et al,

2001). Furthermore, once the increase in airway wall thickness and hyperresponsiveness have

been induced, they remain unaffected to treatment with FP (10 mg) during the ensuing 14

days. These observations arguably add to the value of the model, as this lack of

responsiveness to steroids is quite similar to the limited and variable effect of steroids on

airway responsiveness and subepithelial fibrosis observed in human asthma. This also seems

to indicate that the thickening of the airway wall area and increase in airway responsiveness are


not caused by acute inflammatory changes and cannot be readily reversed by steroids. What

the exact pathophysiological mechanism is that cause both phenomena and whether they are

causally linked remain to be further established.

However, that the increased airway wall area can indeed be the cause of this altered

airway behavior is further suggested by experiments during which the sensitized BN rats were

exposed to ovalbumin over more prolonged time periods. When animals are repeatedly

exposed to ovalbumin over 12 weeks, as opposed to 2 weeks, the airway morphology

further changes. Collagen deposition increases further in the mucosa, whereas in the

adventitial area large amounts of fibronectin were present. The smooth muscle layer remains

unaffected, showing no signs of hyperplasia or hypertrophy. Strikingly, the overall thickness

of the airway wall is reduced in comparison to animals exposed to allergen over a 2-week

period, and is not different from control animals. Correlating with these morphological

observations, airway responsiveness also wanes, and even turns into a slight

hyporesponsiveness of the airways, in comparison to control animals (Palmans et al, 2000).

These observations suggest not only that the elements in the remodeling process that cause an

increase in airway wall thickness are indeed the cause of airway hyperresponsiveness, but also

that when these elements are replaced by a scar-like tissue containing large amounts of

collagen or fibronectin, this could possibly, through increased stiffness, appear to affect the

airway smooth muscle, thus protecting against instead of enhancing increased airway

responsiveness.

3. Other animal models of airway remodeling

3.1. Antigen-induced structural airway changes in other animal species

The majority of the currently developed murine models have focused on acute

inflammatory changes. However, in a few models, the effect of more prolonged allergen

exposure has been evaluated. Blyth and co-workers reported that repeated intratracheal

instillation of ovalbumin induced not only eosinophil infiltration in the airways, but also goblet

cell hyperplasia in the airway epithelium. In contrast to the eosinophil infiltration, this was not

influenced by treatment with dexamethasone. In this model, airway reactivity was not

measured (Blyth et al, 1996). Temelkovski and co-workers recently reported that repeated

exposure of BALB/c mice to low-dose antigen over 6-8 weeks induced goblet cell


hyperplasia as an early characteristic of airway remodeling, followed by epithelial thickening

and subepithelial fibrosis. These changes are paralleled by increased sensitivity to

methacholine. Of interest is that altered airway behavior was also observed in nonimmunized

allergen-exposed animals, despite the absence of clear inflammatory airway changes

(Temelkovski et al, 1998).

3.2. Non-antigen-induced models of remodeling in genetically manipulated mice

Overexpression of cytokines in murine airway epithelium, using transgene technology

has been reported to induce structural airway changes. Interleukin-11 overexpression causes

subepithelial fibrosis, due to increased expression of collagen I and III (Tang et al, 1996).

This was accompanied by an increase in airway responsiveness, as indicated by a decreased

PC100RL to inhaled methacholine. Others have reported some degree of subepithelial fibrosis

in addition to eosinophil accumulation in IL-5 transgenic animals (Lee et al, 1997). In this

model, penh as marker of airway caliber was used to demonstrate increased airway

responsiveness. Others have reported that adenovirus-mediated gene transfer of TGF-ß or

GMC-SF induces widespread pulmonary fibrosis in rats (Sime et al, 1997; Xing et al, 1997).

These models obviously are of interest, illustrating the potential effect of high

concentration of a given cytokine in inducing structural airway changes. However, they do

not address the role of physiological concentrations of endogenously released cytokines in

the pathogenesis of the remodeling process.

3.3. Models of remodeling, using nonantigenic inhaled substances

A number of models have exposed animals over prolonged periods to non-specific

irritants including SO2 or endotoxin (Stolk et al, 1992), thus inducing structural airway

changes that resemble more similarities to the changes observed in chronic bronchitis than in

asthma. In dogs, prolonged exposure to SO2 increases the thic kness of epithelium and the

smooth muscle layer as well as the size of the mucous glands. However these changes are not

accompanied by altered airway behavior (Scanlon et al, 1987). In contrasts, in rats, increased

airway responsiveness following chronic SO2 exposure has been observed, together with

histological changes that are quite similar to those reported in dogs (Shore et al, 1995). In

addition to epithelial changes, the thickness of the airway smooth muscle layer is also

increased provided C fibers are destroyed by neonatal capsaicin treatment (Long et al, 1997).


To date the role of matrix -degrading enzymes, including collagenase and elastase, has

been predominantly evaluated in animal models of emphysema, either by administering the

enzymes exogenously or by evaluating the effect of cigarette smoke in genetically manipulated

knockout mice (Otto Verberne et al, 1992). In developing S-D rats, chronic hyperoxia

exposure resulted in an increased epithelial and airway smooth muscle layer thickness and

increased airway wall thickening that correlated with airway (D'Armiento et al, 1992;

Hautamaki et al, 1997; Massaro and Massaro, 1997). In most of these models, histological

changes in the airways either were absent or were not specifically addressed. Likewise, altered

airway reactivity was related to changes in the histology of the lung parenchyma (Bellofiore et

al, 1989). These models are therefore less suited for investigating the interrelationship between

airway remodeling and bronchial responsiveness as observed in asthma.

4. References

Amdur MO and Mead J. Mechanics of respiration in anaesthetized guinea pigs. Am J Physiol 1958, 192:364-368.


Bellofiore S, Eidelman DH, Macklem PT and Martin JG. Effects of elastase-induced emphysema on airway responsiveness to metacholine in rats. J Appl Physiol 1989, 66:606-612.



D'Armiento J, Dalal SS, Okada Y, Berg RA and Chada K. Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell 1992, 71:955-961.

Derksen FJ, Robinson NE, Armstrong PJ, Stick JA and Slocombe RF. Airway reactivity in ponies with recurrent airway obstruction (heaves). J Appl Physiol 1985, 58:598-604.

Du T, Sapienza S, Wang CG, Renzi PM, Pantano R, Rossi P and Martin J. Effect of nedocromil sodium on allergen-induced airway responses and changes in the quantity of airway smooth muscle in rats. J Allergy Clin Immunol 1996, 98:400-407.




Hautamaki RD, Kobayashi DK, Senior RM and Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997, 277:2002-2004.



Kips JC and Pauwels RA. Animal models of asthma. Clinical Asthma Reviews 1997, 1:45-53.

Konzett H and Rössler R. Versuchsanordung zur Untersuchungen an der Bronchialmuskulatur. Arch Exp Pathol Pharmkol 1940, 71-74.



Lee JJ, McGarry MP, Farmer SC, Denzler KL, Larson KA, Carrigan P, Brenneise IE, Horton MA, Haczku A, Gelfand EW, Leikauf GD and Lee NA. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997, 185:2143-2156.

Levitt RC and Mitzner W. Expression of airway hyperreactivityto acetocholine as a simple autosomal recessive trait in mice. FASEB J 1988, 2605-2608.


Long NC, Martin JG, Pantano R and Shore SA. Airway hyperresponsiveness in a rat model of chronic bronchitis: role of C fibers. Am J Respir Crit Care Med 1997, 155:1222-1229.


Massaro GD and Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nature Med 1997, 3:675-677.



Otto Verberne CJ, Ten Have Opbroek AA, Franken C, Hermans J and Dijkman JH. Protective effect of pulmonary surfactant on elastase-induced emphysema in mice. Eur Respir J 1992, 5:1223-1230.

Padrid P, Snook S, Funicane T, Shiue P, Cozzi P, Solway J and Leff AR. Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats. Am J Respir Crit Care Med 1995, 151:184-193.

Palmans E, Kips JC and Pauwels RA. The effect of chronic allergen exposure on airway structure and responsiveness in rats [Abstract]. Am J Respir Crit Care Med 1998, 157:A822.




Redington AE and Howarth PH. Airway wall remodelling in asthma. Thorax 1997, 52:310-312.

Redington AE, Madden J, Frew AJ, Djukanovic R, Roche WR, Holgate ST and Howarth PH. Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid. Am J Respir Crit Care Med 1997, 156:642-647.



Scanlon PD, Seltzer J, Ingram RH, Jr., Reid L and Drazen JM. Chronic exposure to sulfur dioxide. Physiologic and histologic evaluation of dogs exposed to 50 or 15 ppm. Am Rev Respir Dis 1987, 135:831-839.

Shore S, Kobzik L, Long NC, Skornik W, Van Staden CJ, Boulet L, Rodger IW and Pon DJ. Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis. Am J Respir Crit Care Med 1995, 151:1931-1938.

Sime PJ, Xing Z, Graham FL, Csaky KG and Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997, 100:768-776.

Stolk J, Rudolphus A, Davies P, Osinga D, Dijkman JH, Agarwal L, Keenan KP, Fletcher D and Kramps JA. Induction of emphysema and bronchial mucus cell hyperplasia by intratracheal instillation of lipopolysaccharide in the hamster. J Pathol 1992, 167:349-356.


Tang W, Geba GP, Zheng T, Ray P, Homer RJ, Kuhn C, Flavell R and Elias JA. Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest 1996, 98:2845-2853.


Vanacker NJ, Palmans E, Kips JC and Pauwels RA. Fluticasone inhibits but does not reverse allergen-induced structural airway changes. Am J Respir Crit Care Med 2001, 163:674-679.


Wang CG, Du T, Xu LJ and Martin JG. Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in the rat. Am Rev Respir Dis 1993, 148:413-417.


Woolcock AJ, Salome CM and Yan K. The shape of the dose-response curve to histamine in asthmatic and normal subjects. Am J Respir Crit Care Med 1985, 130:71-75.

Xing Z, Tremblay GM, Sime PJ and Gauldie J. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation. Am J Pathol 1997, 150:59-66.

General Discussion 171

PART III

GENERAL DISCUSSION


1. Summary

Airway morphology in asthma not only displays characteristics of an acute inflammatory

process, but also more structural changes. The functional consequences of this so-called

airway wall remodeling in asthma remain however largely unknown. Hence, the necessities to

further identify the characteristics and pathogenesis of airway remodeling and to link

morphological alterations to physiological data. The application of representative in vivo

animal models can help to better understand the processes of remodeling occurring in the

asthmatic airway wall.

In chapter 1 ovalbumin sensitized Brown Norway rats were exposed over increasing

time periods to aerosolized ovalbumin. The animals developed an increase of ovalbumin-

specific IgE and a persistent eosinophilic airway inflammation. After 2 weeks exposure,

limited structural changes were observed. Allergen exposure during this period resulted in

goblet cell hyperplasia and a limited increase in fibronectin deposition was noted. Total airway

wall thickness was increased. These alterations coincided with airway hyperresponsiveness.

Repeated exposure over a period of up to 12 weeks induced far more pronounced structural

changes, including increased mitogenic activity in the epithelium and an enhanced deposition

of extracellular matrix components. Fibronectin deposition was far more intense than after 2

weeks. Collagen deposition increased from a 4 week exposure period onwards, resulting after

12 weeks exposure in an even more pronounced deposition both in the inner and outer

airway wall. At the same time, the increase in airway wall thickness observed at 2 weeks

subsided and the airway hyperresponsiveness waned, turning into hyporesponsiveness.

Based on these observations, we speculate that in rats the airway wall stiffness increases

through deposition of increased amounts of collagen and fibronectin both within and around

the smooth-muscle layer. This altered deposition of extracellular matrix proteins together with

the reduction of the airway wall thickness might constitute an attempt to protect against airway

hyperresponsiveness. These observations therefore suggest that depending on the extent and

distribution of airway wall fibrosis, airway remodeling might oppose against, rather than

enhance airway hyperresponsiveness.

The changes in different collagen types in the airway wall of sensitized and OA-exposed

Brown Norway rats were characterized in chapter 2. After 12 weeks of allergen exposure, the


airways contained increased amounts of collagen type I, V and VI. An increase of the three

collagen types was observed in the outer airway wall. Collagen type I was also deposited in

the inner airway wall. Since type VI collagen forms fibrillar connections between other

extracellular matrix components, the presence of more type VI collagens in the airway wall

could increase the adhesion between interstitial collagen fibrils and other matrix structures,

thus increasing the rigidity of the airway wall. In addition, Type I collagen forms a relatively

rigid linear fibrillar network and changes in its deposition could further increase the stiffness of

the airway wall. These changes could contribute to the loss of airway hyperresponsiveness

observed in the rat model after 12 weeks of ovalbumin exposure. A positive correlation was

found between the amounts of collagen type I and VI and the number of eosinophils

infiltrating the airway wall. This suggests a role for eosinophils in the process of airway

remodeling and in the deposition of collagen in the extracellular matrix. Which eosinophil

derived mediators possibly contribute to the process remain to be further evaluated. In the rat

model blood vessels were identified by immunostaining for collagen type IV that is the major

collagen subtype present in the vascular basement membrane (chapter 2). Using this

approach, we could not identify an increase in the total amount of vessels per square mm.

However, in rats exposed to allergen for 12 weeks an increase in the total area of the mucosa

occupied by blood vessels was found. This increase in vascular perfusion is in line with the

findings of increased vascularity in the airway wall of asthmatics.

Repeated smooth muscle contractions during bronchoconstriction in asthmatics might

contribute to the development of structural alterations in the airway wall, as this can result in an

altered deposition of extracellular matrix proteins. To address this hypothesis in our rat model,

we evaluated the effect of repeated smooth muscle contractions on airway structure in Brown

Norway rats in chapter 3. Bronchoconstriction was repeatedly induced in the rats via

intraperitoneal injections of carbachol. Neither inflammatory nor structural alterations were

observed in the airway wall of these animals compared to control rats. Airway responsiveness

to nebulized carbachol did not change. This experiment therefore indicates that repeated

airway smooth muscle contractions in the absence of airway inflammation do not induce

structural airway changes.

In chapter 4, using a similar approach of allergen sensitization and repeated ovalbumin

exposure a model of structural airway changes was developed in two inbred mouse strains.


After ovalbumin exposure for a period of up to 9 weeks both BALB/c and C57BL/6 mouse

strains displayed structural changes in the airway wall including goblet cell hyperplasia and

collagen deposition. Inbred mouse strains have well characterized genetic dissimilarities in

immunologic, inflammatory and smooth muscle responses. This was confirmed in the present

study. BALB/c had relative higher levels of OA-specific IgE and lower amount of cells in the

bronchoalveolar lavage fluid compared to C57BL/6. Also at baseline, airways of BALB/c

mice were more sensitive to nebulized methacholine as compared to C57BL/6 mice and

BALB/c but not C57BL/6 mice developed airway hyperresponsiveness after a 2 week

ovalbumin exposure period. These murine models are therefore of particular interest to

address the impact of genetic differences on the remodeling process in the airway wall.

However, despite the differences in the inflammatory response and in lung function alterations

between BALB/c and C57BL/6 mice, both inbred mouse strains displayed a similar degree of

goblet-cell hyperplasia and collagen deposition.

Chapter 5 evaluated the effect of age on allergen induced airway inflammation and

remodeling. The rat model was used to compare allergen induced inflammatory and structural

airway changes in prepubertal and adult animals. The study indicated that for a same intensity

of allergen exposure, synthesis of allergen-specific IgE was similar in both young and adult

OA-exposed groups. In contrast, the airways of young animals were infiltrated with a larger

number of eosinophils than those of adult rats. The airways of young rats also displayed

more pronounced remodeling as indicated by the degree of goblet cell hyperplasia and

fibronectin deposition. As the presence of fibronectin in the extracellular matrix facilitates the

adhesion of inflammatory cells and prolongs eosinophil survival, the more pronounced

remodeling in response to allergen exposure at a young age might increase the vulnerability of

the lower airways to the development of a persistent allergic inflammation and promote its

persistence. The current observations further strengthen the importance of avoiding stimuli in

young atopic children.

Chapter 6 discusses the shortcomings but also the potentials of in vivo animal models

for the study of human asthma. An overview is also given of the existing models of airway

wall remodeling. The observation in our rat model is compared to data obtained by other

groups. Finally, a brief description is given of experiments that have evaluated the effect of

steroids on the allergen induced structural changes in our model. These data indicate that


pretreatment with inhaled steroids partly prevents but does not reverse the allergen induced

airway remodeling.

In conclusion, we propose that the specific aims of the study were met, in that:

1. By repeatedly exposing sensitised rats or mice to aerosolised allergen, we were able

to induce, in addition to acute inflammatory changes, structural alterations that bear

similarities to airway remodeling observed in asthma. These structural changes

cannot merely be attributed to repeated airway contraction, but seem to result from

the presence of a chronic allergic inflammation.

2. The structural changes relate to altered airway behaviour, the overall effect being

dependent on the exact extent and location of the remodeling process.

3. For a same degree of allergen-induced acute inflammatory changes, the airways of

young animals are more prone to the development of structural changes than those

of adult rats.

2. Future perspectives

We believe that this model represents a valid instrument for the further investigation of the

mechanisms involved in the pathogenesis of remodeling, as well as for the evaluation of the

effect of therapeutic measures on the prevention or regression of remodeling and the

associated effects on lung function.

To us, future experiments should include:

1. Evaluation of the allergen induced expression of growth factors that are

potentially responsible for the remodeling process. In a first approach, this will be

based on an immunohistological assessment. Depending on the results obtained,

the functional role of a given growth factor can then be assessed in specific

knockout mice.

2. Further evaluation of the functional consequences of remodelling, e.g. by relating

morphological changes to indices of airway wall stiffness.

3. Evaluation of the effect of currently available asthma maintenance treatment on

the development, persistence and/or progression of remodeling. As a first


priority, the effect of inhaled steroids, alone or in combination with long-acting

inhaled beta-agonists would need to be assessed.

Animal models of airway remodeling in asthma - Els Palmans…

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