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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
2002
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
Review of the Literature 5
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
Review of the Literature 6
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
Review of the Literature 7
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).
Review of the Literature 9
B
D
A
C
Fig
ure
1. S
chem
atic
rep
rese
ntat
ion
of a
cro
ss-s
ectio
n of
a n
orm
al (
A)
and
rem
odel
ed (
B)
airw
ay w
all.
Det
aile
d ill
ustr
atio
n of
the
dif
fere
nt
com
part
men
ts in
the
norm
al (C
) and
rem
odel
ed (D
) air
way
wal
l.
Review of the Literature 11
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).
Review of the Literature 12
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).
Review of the Literature 13
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
Review of the Literature 14
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.
Review of the Literature 15
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).
Review of the Literature 16
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.
Review of the Literature 17
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
Review of the Literature 18
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
Review of the Literature 19
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
Review of the Literature 20
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.
Review of the Literature 21
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
Review of the Literature 22
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,
Review of the Literature 23
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
Review of the Literature 24
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).
Review of the Literature 25
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
Review of the Literature 26
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
Review of the Literature 27
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.
Review of the Literature 28
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
Review of the Literature 29
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
Review of the Literature 30
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
Review of the Literature 31
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.
Review of the Literature 32
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.
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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.
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.
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 and Hogg JC. The mechanisms of airway narrowing in asthma. Am Rev Respir Dis 1989, 139:242-246.
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
Prolonged Allergen Exposure in Sensitized Rats 73
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
Prolonged Allergen Exposure in Sensitized Rats 74
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
Prolonged Allergen Exposure in Sensitized Rats 75
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).
Prolonged Allergen Exposure in Sensitized Rats 76
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
Prolonged Allergen Exposure in Sensitized Rats 77
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
Prolonged Allergen Exposure in Sensitized Rats 78
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
Prolonged Allergen Exposure in Sensitized Rats 79
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
Prolonged Allergen Exposure in Sensitized Rats 80
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)
Prolonged Allergen Exposure in Sensitized Rats 81
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
concentration carbachol (mg/ml)
% i
ncr
ease
in
RL
C. 12 weeks
0.01 0.1 1 10
0
50
100
150
200
concentration carbachol (mg/ml)
% i
ncr
ease
in
RL
Prolonged Allergen Exposure in Sensitized Rats 82
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.
Prolonged Allergen Exposure in Sensitized Rats 83
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
Prolonged Allergen Exposure in Sensitized Rats 85
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).
Prolonged Allergen Exposure in Sensitized Rats 87
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.
Prolonged Allergen Exposure in Sensitized Rats 88
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
)
Prolonged Allergen Exposure in Sensitized Rats 89
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).
Prolonged Allergen Exposure in Sensitized Rats 91
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
)
Prolonged Allergen Exposure in Sensitized Rats 92
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
Prolonged Allergen Exposure in Sensitized Rats 93
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
Prolonged Allergen Exposure in Sensitized Rats 94
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.
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Prolonged Allergen Exposure in Sensitized Rats 95
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Prolonged Allergen Exposure in Sensitized Rats 97
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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
Collagen Deposition in Rat Airway Wall 101
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
Collagen Deposition in Rat Airway Wall 102
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).
Collagen Deposition in Rat Airway Wall 103
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.
Collagen Deposition in Rat Airway Wall 104
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.
Collagen Deposition in Rat Airway Wall 105
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).
Collagen Deposition in Rat Airway Wall 107
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).
Collagen Deposition in Rat Airway Wall 109
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
Collagen Deposition in Rat Airway Wall 110
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
Collagen Deposition in Rat Airway Wall 111
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.
Collagen Deposition in Rat Airway Wall 112
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
Collagen Deposition in Rat Airway Wall 113
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.
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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.
Roche WR, Beasley R, Williams JH and Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989, 1:520-524.
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.
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.
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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
Airway Structure after Repeated Bronchoconstriction 117
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
Airway Structure after Repeated Bronchoconstriction 118
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),
Airway Structure after Repeated Bronchoconstriction 119
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
Airway Structure after Repeated Bronchoconstriction 120
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
concentration carbachol (mg/ml)
% in
crea
se in
RL
Airway Structure after Repeated Bronchoconstriction 121
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 Structure after Repeated Bronchoconstriction 122
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
Airway Structure after Repeated Bronchoconstriction 123
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.
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.
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.
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.
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.
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.
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.
Li X and Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997, 156:229-233.
Airway Structure after Repeated Bronchoconstriction 124
Mitchell HW. Physiology of airway narrowing. Clin Exp Pharmacol Physiol 1997, 24:256-260.
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.
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.
Roche WR, Beasley R, Williams JH and Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989, 1:520-524.
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.
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.
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.
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.
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
Airway Wall Changes in Inbred Mouse Strains 127
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
Airway Wall Changes in Inbred Mouse Strains 128
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.
3.2. Outcome measures
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
Airway Wall Changes in Inbred Mouse Strains 129
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
Airway Wall Changes in Inbred Mouse Strains 130
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.
Airway Wall Changes in Inbred Mouse Strains 131
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).
Airway Wall Changes in Inbred Mouse Strains 132
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
Airway Wall Changes in Inbred Mouse Strains 133
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
Airway Wall Changes in Inbred Mouse Strains 134
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
Airway Wall Changes in Inbred Mouse Strains 135
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
Airway Wall Changes in Inbred Mouse Strains 136
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
concentration metacholine (mg/ml)
% in
crea
se in
bas
elin
eB. BALB/c - 6 weeks
1 10 100
0
500
1000
1500
concentration metacholine (mg/ml)
% in
crea
se in
bas
elin
e
D. C56BL/6 - 6 weeks
1 10 100
0
500
1000
1500
concentration metacholine (mg/ml)
% 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).
4.5. OA-specific IgE
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
Airway Wall Changes in Inbred Mouse Strains 137
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
Airway Wall Changes in Inbred Mouse Strains 138
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.
Airway Wall Changes in Inbred Mouse Strains 139
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.
Airway Wall Changes in Inbred Mouse Strains 140
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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.
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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.
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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.
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Vignola AM, Kips J and Bousquet J. Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 2000, 105:1041-1053.
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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
Structural Airway Changes in Young Rats 145
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
Structural Airway Changes in Young Rats 146
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).
3.2. Outcome measures
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
Structural Airway Changes in Young Rats 147
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
Structural Airway Changes in Young Rats 148
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).
4.2. OA-specific IgE
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
Structural Airway Changes in Young Rats 149
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) (%) (%) (%) (%)
Structural Airway Changes in Young Rats 150
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).
Structural Airway Changes in Young Rats 151
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
Structural Airway Changes in Young Rats 152
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,
Structural Airway Changes in Young Rats 153
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
Structural Airway Changes in Young Rats 154
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).
Structural Airway Changes in Young Rats 155
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.
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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
Is there an Animal Model? 161
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
Is there an Animal Model? 162
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,
Is there an Animal Model? 163
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
Is there an Animal Model? 164
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
Is there an Animal Model? 165
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
Is there an Animal Model? 166
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).
Is there an Animal Model? 167
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.
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General Discussion 171
PART III
GENERAL DISCUSSION
General Discussion 173
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
General Discussion 174
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.
General Discussion 175
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
General Discussion 176
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
General Discussion 177
priority, the effect of inhaled steroids, alone or in combination with long-acting
inhaled beta-agonists would need to be assessed.
Recommended