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Airway Inflammation and Remodeling in Asthma 1
1
Airway Inflammation and Remodeling in Asthma
Current Concepts
Stephen T. Holgate
1. Introduction
Asthma is a heterogeneous disorder of the airways with intermittent airflow
obstruction frequently accompanied by increased responsiveness of the bron-
chi to a wide variety of exogenous and endogenous stimuli. A number of dif-
ferent types of asthma have been described, based largely on the clinical
manifestations of, or factors precipitating, the airflow obstruction. As the dis-
ease becomes more severe and chronic, it is suggested that the airflow obstruc-
tion may progressively lose some of its reversibility and, in this respect,
resemble some aspects of chronic obstructive pulmonary disease (COPD).
However, in a small proportion of patients with life-threatening asthma, the
airways are highly labile on account of greatly enhanced bronchial
hyperresponsiveness. At the other end of the disease severity spectrum there
are many patients with mild intermittent asthma whose disease only manifests
when exposed to particular sensitizing allergens to which they are sensitive
(e.g. outdoor allergens during the pollen season).
Over the last 50 yr, the prevalence of asthma and allied allergic disorders
have progressively increased on a worldwide scale, in both developed and
developing countries. In addition to an increase in asthma-related symptoms,
there is evidence of increased medication usage and a rise in hospital admis-
sions for asthma. The reasons for these rising trends may be multiple. Sugges-
tions include:
From: Methods in Molecular Medicine, vol. 56:
Human Airway Inflammation: Sampling Techniques and Analytical Protocols
Edited by: D. F. Rogers and L. E. Donnelly © Humana Press Inc., Totowa, NJ
1
2 Holgate
1. Increased allergen exposure (especially within the domestic setting).
2. Reduced exposure to childhood infections.
3. Changes to the diet (e.g., reduced antioxidant and δ 13 polyunsaturated fatty acids).
4. Alterations to the lung or gastrointestinal flora possibly linked to increased anti-
biotic prescribing in infancy.
Whatever the underlying causes for these rising trends, asthma has now
become a public health issue. Therefore, it is of the utmost importance that
a clear understanding is obtained of underlying cell and molecular mecha-
nisms in order that appropriate biomarkers are identified that can be used to
detect the disease earlier and follow its outcome. In childhood and early
adulthood, asthma occurs in association with atopy, which is characterized by
elevated circulating allergen-specific IgE and positive skin prick test responses
to allergen extracts. However, asthma in adults tends to lose its close associa-
tion with atopy, particularly in those patients with late onset and chronic dis-
ease. Recent estimates suggest that approx 50% of asthma can be linked to
immunological mechanisms associated with atopy. Compelling epidemiologi-
cal evidence indicates that atopy alone (i.e., the genetic susceptibility to gener-
ate allergen-specific IgE) is insufficient for the development of asthma and,
what is also required is a susceptibility of the airways to express and respond to
localized inflammatory responses. With the recent description of a subtype of
asthma manifesting as intermittent cough (cough-variant asthma) in which
there is airway inflammation in the absence of bronchial hyperresponsiveness,
it would seem that inflammation alone is also insufficient to produce the vari-
able airflow obstruction and accompanying symptoms of chronic asthma. It
would appear that some alteration to the airway structure (airway wall remod-
eling) upon which the inflammatory response is acting is also required.
2. The Epidemiology of Asthma and the Role of IgE
Although atopy is the single strongest risk factor for the development of
asthma increasing the risk up to 20-fold, only about one-fifth of atopic sub-
jects progress to develop chronic asthma requiring regular therapy. Both in
childhood and in adults, epidemiological associations have been shown
between asthma and IgE, whether assessed as total serum IgE or as allergen-
specific IgE. In many parts of the world it is exposure to indoor allergens
that appears to drive the expression of atopy linked to asthma and specifi-
cally exposure to dust mite, cat, and fungal antigens. Domestic exposure
in early life to the major dust mite allergen der P levels of >2 µg/g of
1
house dust has been shown to significantly increase the risk of initial aller-
gen sensitization and the development of asthma. Exposure to levels in excess
of 10 µg/g of dust increases the risk of acute exacerbations of preexisting dis-
ease. Exposure to Alternaria allergens has been linked to acute life-threaten-
Airway Inflammation and Remodeling in Asthma 3
ing attacks of asthma, whereas other allergens including animal dander, insect
dust, grass pollen, and molds have also been linked to asthma exacerbations.
Epidemics of asthma, as occurred in Barcelona or following thunderstorms in
the United Kingdom, have also been linked to exposure to high concentrations
of allergens. These incidents have been attributed, respectively, to the unload-
ing of soy bean in Barcelona harbor and the release of pollen fragments into the
air following osmotic lysis of pollen grains at the peak of the pollen season.
3. The Pathological Features of Asthma
Asthma is classically an inflammatory disorder of the airways with infiltra-
tion of the submucosa and adventitia of both the large and small airways with
activated mast cells, tissue macrophages, eosinophils, and, in certain cases,
neutrophils. In acute severe asthma that may be provoked by respiratory virus
infections, the airways become infiltrated with neutrophils in addition to eosi-
nophils, an observation that has also been described in patients dying suddenly
of their disease. A second important characteristic of asthma is damage to the
ciliated stratified epithelium with a deposition of interstitial collagens (types I,
III, and V) as well as laminin and tenascin C beneath the true epithelial base-
ment membrane, which has been linked to the proliferation of subepithelial
myofibroblasts. The epithelium in asthma also undergoes metaplasia with the
acquisition of a repair phenotype and an increase in the number of goblet cells,
especially in chronic disease. A third pathological characteristic of chronic
asthma is hyperplasia of the formed elements of the airway, including
microvessels, afferent neurons, and smooth muscle as well as deposition of
matrix proteins and proteoglycans (versican, fibromodulin, biglycan, and
decorin) in the submucosa and outside the airway smooth muscle. Taken
together, the inflammatory response is superimposed on a remodeled airway,
and it is this that gives rise to clinical heterogeneity so characteristic of asthma.
Although once considered as purely a disease of airway smooth muscle,
asthma is now known to be a chronic inflammatory disorder of the airways,
orchestrated by CD4+ lymphocytes. There is evidence to support the view
that the asthmatic airway inflammation is driven by the persistence of chroni-
cally activated T cells of a memory phenotype (CD45RO+) with a propor-
tion of these being directed to allergenic, occupational, or viral antigens.
This is supported by a large number of studies using BAL and bronchial
mucosal biopsies from subjects with asthma. These studies revealed increased
transcription and product release of a discrete set of cytokines encoded on the
long arm of chromosome 5q , which includes interleukin (IL)-3, IL-4, IL-5,
31-33
IL-9, IL-13, and granulocyte macrophage colony stimulating factor (GM-CSF).
In more severe asthma and some cases of occupational asthma, CD8+ as well
as CD4+ T cells are the source of these cytokines.
4 Holgate
Although many of the cytokines associated with the Th-2 phenotype have
pleiotropic functions, there are specific aspects of each one that are worth high-
lighting when linking their function to airway pathology in asthma. In most
types the predominant inflammatory cell infiltrating the airway wall is the eosi-
nophil that is derived from CD34+ precursor cells, which are present in the bone
marrow, recruited from the circulation, and located in the airway wall itself.
Under the influence of IL-3, IL-5, and GM-CSF, eosinophils acquire a mature
phenotype with the capacity to secrete a range of preformed and newly gener-
ated mediators. Interleukin-4 and IL–13 are intimately involved in the isotype
switching of B cells from IgM to IgE and also in the upregulation of a specific
adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), involved in
the recruitment of basophils, eosinophils, and Th2-like T cells. Following
allergen exposure, it is the interaction of the inflammatory cell integrin VLA-4
with VCAM-1 that plays a key role in the recruitment of eosinophils, baso-
phils, and Th-2 cells from the circulation into the airways. An additional prop-
erty of IL-4 (which is not shared by IL-13) is the ability of this cytokine to
support the development of Th-2 T cells and enhance their survival. Two IL-4
and two IL-13 receptors have been described on a variety of cell types with
subtle differences in their ability to initiate intracellular activation mechanisms.
These involve the transcription factor signal transducer and activator of tran-
scription (STAT)-6 and insulin receptor substrate (IRS)-1/2 that are involved
in IL-4/IL-13 induced gene transcription and cell proliferation, respectively.
Bronchial epithelial cells and fibroblasts also express IL-4 and IL-13 receptors
and are highly responsive to these cytokines in vitro. In vivo overexpression of
IL-13 into the bronchial epithelium of transgenic mice not only leads to
increased IgE production but also goblet cell metaplasia, subepithelial fibrosis,
and bronchial smooth muscle hyperplasia, linked to the acquisition of bron-
chial hyperresponsiveness.
A Th-2 cytokine that is gaining importance in asthma is GM-CSF, which
serves as a growth factor for eosinophils, basophils, macrophages, and
dendritic cells and is also a key cytokine for rescuing eosinophils from pro-
grammed cell death (apoptosis).
4. An Important Role for Chemokines in Asthma
Apart from Th-2 cytokines, a second group of low-molecular-weight pro-
teins are required for the recruitment of inflammatory cells into the asthmatic
airway — the chemokines. At the transcriptional and protein levels, both C-C
and C-X-C chemokines are generated by asthmatic airways but, of particular
relevance, are the C-X-C chemokines regulated on activation normal T cell
expressed and secreted (RANTES), eotaxin, and monocyte chemoattractant
protein (MCP)-1, -3, and -4. These five chemokines interact with the CCR-3
Airway Inflammation and Remodeling in Asthma 5
receptor that is expressed in high numbers on eosinophils, basophils, and a
subset of Th-2 cells. Besides promoting local migration of leukocytes within
the airway wall, the same chemokines are also involved in the release of eosi-
nophils and their precursors from the bone marrow when appropriate signals
are received from the lung via the circulation.
Although both immune and inflammatory cells have the capacity to release
CCR-3 ligands, the most abundant sources of these chemoattractants are the
formed airway elements: the bronchial epithelium, microvascular endothelium,
and myofibroblast/fibroblast. There is also in vitro evidence to suggest that
proliferation of airway smooth muscle can generate chemokines as well as a
number of other cytokines, including those encoded in the IL-4 gene cluster.
5. Antigen Processing and Presentation:
The Role of Dendritic Cells
Activation of the mucosal immune response involving CD4+ T cells is a cen-
tral feature of chronic asthma. In order for mucosal T cells to either proliferate
and/or generate appropriate cytokines, they require activation usually via the
T-cell receptor, CD3. Uptake of antigen by dendritic cells, in allergic disease, is
enhanced by the ability of these cells to express both high and low affinity IgE Fc
receptors that increase the efficiency of allergen capture by 50–1000-fold. On
internalization, a peptide sequence is selected and presented in the groove of
major histocompatibility complex (MHC) class II to the T-cell receptor (signal
1). However, in order for T cells to respond to this antigen-specific stimulus,
engagement of a second signal is required involving both the costimulatory mol-
ecules B7.1 (CD80) or B7.2 (CD86) that engage the homodimeric molecule
CD28 on the T-cell (signal 2). In contrast to peripheral blood mononuclear cells
that preferentially utilize CD86 for Th-2 cytokine production, CD28 on T cells
in the bronchial mucosa of asthmatics engage either CD80 or CD86 in augment-
ing local Th-2 cytokine production. In the presence of IL-4, a monomeric adhe-
sion molecule CTLA-4 is induced on T cells that has an increased affinity for
both CD80 or CD86 and is able to “steal” the CD28 signal from the B7 ligands,
thereby rendering the T-cell anergic or initiate its programmed cell death.
Dendritic cells are characterized by their cell surface expression of CD1,
high density of MHC class II, and costimulatory molecules. They form a net-
work within the bronchial epithelium and submucosa. On chronic exposure to
antigen, the number of dendritic cells increases, thereby enhancing mucosal
responsiveness to sensitizing antigens. These cells are seen to be obligatory for
the development of primary allergen-specific airway responses, although, once
sensitization has occurred, there are other cells in the airways including B cells
and possibly epithelial cells, that may also provide an antigen-processing
and -presenting function.
6 Holgate
There are strong genetic determinants for the development of Th-2 polariza-
tion within the lower airways. Not only do antigen-presenting cells provide T
cells with antigen and costimulatory signals, but also soluble signals that polar-
ize their subsequent differentiation (signal 3). The Th-2 biased responses
observed in atopic diseases appear to be associated with a decreased IL-12/pros-
taglandin (PG)-E ratio and, as a consequence, downregulation of Th-2 cytokine
2
production. The presence of interferon-γ (IFN-γ) enhances the ability of imma-
ture dendritic cells to produce IL-12, which in turn creates the environment for
preferential Th-1 T cell maturation. In contrast, PGE primes for a reduced IL-12,
2
producing ability and consequently, biasing T-cell development in favor of a Th-
2 phenotype. Thus, it is suggested that antigens provoke either a Th-1 or Th-2
response by inducing the production of a pattern of inflammatory dendritic cell
mediators with the capacity to direct at the local site of exposure. This concept
may be of particular relevance to the early life origins of allergic disease because,
in children destined to develop atopic disorders, their cord blood mononuclear
cells have an impaired capacity to respond to IL-12 and, as a consequence, to
generate IFN-γ efficiently. Because IFN-γ provides such a strong negative signal
to Th-2 development, its impaired production may predispose the infant to per-
sistence into postnatal life of Th-2 responsiveness that in normal infants shuts
down efficiently prior to birth. It has been shown that the impaired production of
T-cell IFN-γ production in atopic children persists into late childhood providing
further evidence that reduction in this inhibitory pathway rather than enhance-
ment of the excitatory is responsible for the persistence of the Th-2 phenotype
linked to the allergic phenotype.
6. The Role of IgE
The identification of the passive sensitizing agent “reagin” as immunoglobu-
lin E has provided the immunological basis for type I hypersensitivity disorders.
Immunoglobulin-E directed to specific allergens is generated by B cells and
plasma cells through an interaction with antigen-specific T cells in the presence
of IL-4 or its homolog (IL-13) together with engagement of the costimulatory
molecules CD40 on B cells and CD40 ligand on T cells. Immunoglobulin E
binds to both high-affinity Fc receptors (α ,β ,γ : FcεR1) expressed on mast cells,
1 1 2
basophils, dendritic cells, and eosinophils and also to monomeric low-affinity
receptors (FcεR2 or CD23) expressed on a wide variety of cell types including
epithelial cells, B cells, monocytes, T cells, neutrophils, and eosinophils. Although
the role of IgE in the manifestation of many allergic diseases such as anaphylaxis
and rhinoconjunctivitis is without dispute, there is considerable debate over the
role of this signaling molecule and its receptors in asthma. The availability of a
fully humanized blocking monoclonal antibody against IgE (E25) has provided
considerable insight into the role of IgE-mediated mechanisms in allergic asthma.
Airway Inflammation and Remodeling in Asthma 7
This humanized monoclonal antibody was directed to that part of IgE that, under
normal circumstances, binds with high affinity to the α chain of the tetrameric
FcεR1 receptor. Thus, following a single injection of Mab E-25, there occurs a
>90% decrease in circulating levels of IgE with a composite increase in IgE/E25
complexes. These small complexes are rapidly cleared by the reticuloendothe-
lial system and, so far, have not been associated with any adverse immune com-
plex deposition effects. Over periods of 9–12 wk the regular administration of
Mab E25 produced profound attenuation of both the early and late phase bron-
choconstrictor responses following allergen provocation of the airways of
patients with atopic asthma, as well as causing a reduction in allergen-induced
acquired bronchial hyperresponsiveness (BHR). A clinical trial involving over
300 patients in whom E25 was administered over a period of 12 wk has demon-
strated improvement in all parameters of asthma, including the requirement of
inhaled and oral corticosteroids. Not all patients responded equally to this treat-
ment, although there appear to be no particular features that identify “respond-
ers” from “nonresponders.”
Although IgE has been classically associated with asthma of the atopic
extrinsic type, in patients with intrinsic nonallergic asthma Th-2 development
in the bronchial mucosa is also accompanied by an increase in the number of
cells bearing the FcεR1 receptor. This indicates that local IgE production may
contribute to this form of the disease. The putative role of local airway IgE
synthesis is further supported by the increased expression of ε germ line tran-
scripts (Iε) and mRNA for ε heavy chain of IgE in bronchial biopsies from both
atopic and nonatopic asthmatics. However, in the latter case the initiating anti-
gen or antigens have yet to be identified. It has been suggested that the inflam-
matory response in nonatopic asthma is preferentially promoted by the
activation of IgE receptors on monocytes/macrophages rather than mast cells
and involves putative “autoimmune” processes.
7. The Role of the Mast Cell in Asthma
Immunoglobulin-E-dependent activation of mast cells provides the basis for
the early asthmatic response. Crosslinkage of FcεR1 on the surface of sensi-
tized mast cells by allergen results in the noncytotoxic secretion of both pre-
formed and newly generated mediators. In addition to preformed histamine
and heparin, the mast cell secretory granule also contains a range of enzymes,
including exoglycosidases, endoglycosidases, and serum proteases. The
mucosal type mast cell (MC ) that predominates in the asthmatic airway con-
T
tains the unique four-chained neutral protease tryptase that is stabilized by heparin.
Tryptase has the capacity to cleave a number of soluble substrates, but in
asthma its main function may well be to activate protease-activated receptors
(specifically PAR-2), through cleavage of a small peptide from the tethered
8 Holgate
ligand. This enables the receptor to stimulate cell proliferation and cytokine
production. PAR-2 receptors are found on bronchial epithelial, endothelial,
neural ganglia, smooth muscle, and fibroblast cells and their function may be
to initiate and maintain airway wall remodeling. Activation of PAR-2 recep-
tors in the epithelium and endothelium leads to the release of chemokines such
as RANTES and IL-8, which may provide one mechanism for chemokine
release when sensitized airways are exposed to allergen.
In addition to releasing granule-associated preformed mediators, activated
mast cells have the capacity to generate an array of newly generated products
including the cysteinyl leukotriene LTC and PGD . The combination of LTC ,
4 2 4
LTD , LTE (together comprising slow reacting substance of anaphylaxis;
4 4
SRS-A), histamine, and PGD accounts for the majority of the early asthmatic
2
response following allergen challenge. Thus, inhibitors of receptor activation
or mediator synthesis, either by themselves or, more effectively, in combina-
tion, or mast cell stabilizers, such as sodium cromoglycate and nedocromil
sodium, attenuate the early asthmatic response. The same mediators are also
implicated in the pathogenesis of exercise-induced asthma. The most popular
prevailing hypothesis to explain exercise-induced asthma is that increased ven-
tilation impacts on a damaged epithelium resulting in water loss from the air-
way lining fluid that is inadequately replaced and, as a consequence, causes
activation of primed mast cells by a hyperosmolar environment.
Bronchoconstriction provoked by isocapnic ventilation, cold dry air, and
hypertonic sodium chloride (or mannitol) aerosols is produced by a similar
mast cell-dependent mechanism. Similarly, in asthma, adenosine generated by
mast cells (and also by other cells) interacts with adenosine A receptors on
2B
primed mast cells leading to autacoid release. H -antihistamines and mast cell
1
suppressor drugs (e.g., sodium cromoglycate and nedocromil sodium) can also
inhibit this early response.
A characteristic feature of asthma associated with known environmental sen-
sitizing agents is the occurrence of delayed bronchoconstriction 4–24 h fol-
lowing inhalation exposure, which is accompanied by a progressive increase in
bronchial hyperresponsiveness that may last up to 3 wk following challenge.
This late asthmatic response is, in part, mast cell dependent because it is also
inhibited by sodium cromoglycate and nedocromil sodium and also by
nonanaphylactogenic antihuman IgE. The most likely explanation for the onset
of a late asthmatic response is the release of cytokines from activated mast
cells, specifically TNFα, IL-4, IL-5, GM-CSF, and chemokines active at the
CCR3 receptor. Human mast cells store small quantities of cytokines within
their secretory granules that can be released rapidly. In addition, activation of
their IgE receptors in the presence of stem cell factor (a mast cell growth fac-
tor) leads to increased cytokine transcription with subsequent product release
Airway Inflammation and Remodeling in Asthma 9
that may persist for up to 48–72 h. Mast cell-derived histamine and leukotrienes
express the preformed adhesion molecule P selectin on endothelial cells result-
ing in leukocyte “rolling.” TNFα upregulates the expression of the adhesion
molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and
VCAM-1, the latter also requiring either IL-4 or IL-13 for stabilization of
expression. By interacting with their complementary ligands on neutrophils,
eosinophils, basophils, T cells, and monocytes, these adhesion molecules
enable leukocytes to be recruited selectively into the airway wall.
There is strong evidence that cytokine release from T cells also contributes
to the latter period of the late phase bronchoconstrictor response and the
accompanying increase in BHR. Antigen specific T cells have the capacity to
be selectively recruited into exposed airways, possibly involving novel
chemokine and epithelial homing receptors.
8. The Eosinophil
The presence of eosinophils in the walls and lumen of the conducting air-
ways is a characteristic feature of chronic asthma and has placed this cell at the
center of the mediator cascade in all types of asthma irrespective of etiology.
The extent of airway eosinophilia is closely linked to epithelial damage and
disease severity, as reflected by eosinophil counts and the presence of granule
proteins in lavage fluid, mucosal tissue, and induced sputum. In chronic asthma
increased eosinophil survival by locally produced GM-CSF, IL-3, and IL-5 is
at least as important in maintaining airway eosinophilia as is the recruitment of
new cells from the circulation. More recently, the identification of CD3+ leu-
kocyte precursors in the airway wall bearing receptors for the same
eosinophilopoietic cytokines suggests that part of the tissue eosinophilia may
be locally generated.
The eosinophil granules contain a number of arginine rich proteins (major
basic protein [MBP]; eosinophil cationic protein [ECP]; eosinophil, derived
neurotoxin [EDN]; and eosinophil peroxidase [EPO]). At high concentrations
these proteins are cytotoxic to the bronchial epithelium whereas when present
in smaller amounts they activate epithelial stress signaling pathways and have
the capacity to interfere with muscarinic neurotransmission. It has been sug-
gested that MBP and possibly ECP are responsible for the epithelial disruption
in asthma. However, there is also evidence that detachment of columnar cells
from basal cells occurs through weakening of cell adhesion complexes, possi-
bly mediated by an altered epithelial phenotype, proteolytic attack, or increased
apoptosis. Eosinophils and neutrophils are rich sources of the metalloproteinase
MMP-9 whose inhibition by the endogenous tissue inhibitor TIMP-1 has been
shown to be impaired in chronic asthma. MMP-9 is also induced in epithelial
cells when they are engaged in a repair response.
