Asthma: Mechanisms and Protocols

332 Pages·2000·2.087 MB·English

by  Fan Chung K. (ed.), Adcock I. (ed.)

Asthma: Cell and Molecular Biology 1
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Asthma
Application of Cell and Molecular Biology Techniques
to Unravel Causes and Pathophysiological Mechanisms
Fan Chung and Ian Adcock
1. Introduction
The condition termed “asthma” has been difficult to define satisfacto-
rily. Much of this problem arises from poor understanding of its causes,
natural history, and pathophysiology, and also from a lack of a specific
marker(s) of the disease. To the clinician, the diagnosis of asthma is not
difficult in most cases, particularly if patients present early with symptoms
of intermittent wheeze and chest tightness, and if their symptoms respond
to particular treatments, such as β-adrenergic agonists. Early definitions of
asthma included the presence of airway obstruction that could spontane-
ously reverse with treatment, and also the increased narrowing of the airways
to non-specific bronchoconstrictor stimuli, i.e., bronchial hyperresponsive-
ness (BHR). The essential elements of this definition were useful in sepa-
rating asthma from other conditions, such as chronic bronchitis, chronic
obstructive pulmonary disease, and emphysema, which could sometimes
be diagnostically confused with asthma. More recently, the definition of
asthma has been enhanced by the recognition that the airway submucosa of
patients with asthma are chronically inflamed with a typical inflammatory
infiltrate, and that inflammatory processes are important causes of the chief
characteristics of asthma: airway obstruction and BHR. In addition, the loss
of reversibility of airway obstruction as a long-term effect of the chronic
inflammatory process is recognized:
From: Methods in Molecular Medicine, vol. 44: Asthma: Mechanisms and Protocols
Edited by: K. F. Chung and I. Adcock © Humana Press Inc., Totowa, NJ
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2 Chung and Adcock
Asthma is a common and chronic inflammatory condition of the airways whose cause
is not completely understood. As a result of inflammation the airways are hyper-
responsive and they narrow easily in response to a wide range of stimuli. This may result
in coughing, wheezing, chest tightness, and shortness of breath and these symptoms are
often worse at night. Narrowing of the airways is usually reversible, but in some patients
with chronic asthma the inflammation may lead to irreversible airflow obstruction. Char-
acteristic pathological features include the presence in the airway of inflammatory cells,
plasma exudation, oedema, smooth muscle hypertrophy, mucus plugging, and shedding
of the epithelium (1).
This working definition of asthma has helped to concentrate research work
on the characteristics of this inflammatory response, the potential causes, and
the mechanisms underlying this response. To address these issues, a number of
molecular and cell biological techniques have been applied. For the researcher
new to the field of asthma, it is important to first describe some of the epide-
miological and clinical aspects of the disease, prior to a description of the cel-
lular and molecular aspects.
2. Epidemiology of Asthma
Asthma is one of the most common chronic diseases worldwide. Prevalence
studies have centered on asking for a history of intermittent wheeze, and, on the
basis of this, the prevalence of asthma in childhood has been reported to be up to
40% in some areas of the United Kingdom, Australia, New Zealand, and Ireland;
in other less affluent countries, such as Indonesia, China, India, and Ethiopia,
this may be as low as 3% (2). In adults, prevalence rates are more difficult to
assess, particularly with the potential confusion of asthma with chronic bronchi-
tis, but up to 25% of adults questioned, aged 20–44 yr, reported wheeze in the
preceding 6 mo; in the United Kingdom, only 5.7% reported an attack of asthma
in the previous 12 mo (3). In several Western countries, the prevalence of asthma
among children has increased (4). Factors underlying this increase are unclear.
The likelihood of diagnosed asthma is increased by the presence of atopy, as
measured by positive skin-prick tests or elevated serum immunoglobulin E
(IgE) levels, by home exposure to passive cigarette smoke, by lower respira-
tory tract infections, and by the presence of reduced lung function. The in-
creased prevalence of asthma may be caused by changes in indoor or outdoor
environment, and may involve aeroallergens, particularly house dust mites. It
is possible that the increased prevalence of allergy and asthma may be caused
by the synergistic action of air pollution or tobacco smoking with allergic sen-
sitization(5). Passive smoking in infancy may predispose to allergic sensitiza-
tion to common aeroallergens (6). Urbanization has also been correlated with
increases in prevalence of asthma in some countries (7). Data from Ethiopia
indicate that westernization is associated with the appearance and increase in
asthma and that this may occur within a relatively short period of time (10 yr)
Asthma: Cell and Molecular Biology 3
(8). One possibility is that changes in the pattern of childhood infections
through westernization may influence the development of atopy through
changes in specific T-cell responses favoring the production of cytokines from
T-helper type-2 lymphocytes (Th2), such as interleukin 2 (IL-4) and IL-5, with
a reduction in Th1 cytokines, such as IFN interferon-γ. For example, children
with measles infection are less likely to be atopic than those receiving measles
immunization (9), and there is an inverse relationship between tuberculin
responses and atopy (10). Dietary factors have also been implicated (11).
In addition to prevalence, the severity of asthma appears to have increased,
as shown by the increase in hospital admissions for asthma and in the use of
anti-asthma drugs, such as β-agonists and inhaled steroids (12–14). Mortality,
however, is generally low, accounting for approx 5/100,000 population in 1990
in England and Wales. Although the mortality rates have been generally stable,
there have been substantial but transient increases in some countries, such as
New Zealand, in the late 1970s (15). Several reasons underlie continuing
asthma mortality rates, including the overall increase in severity, thus aug-
menting the pool of patients at risk of death; failure to use appropriate medica-
tion, because of health care professionals not evaluating the severity of disease
properly; poor access to medical care; and iatrogenic causes (16–19).
3. Natural History
There are relatively few cohort studies that have examined the natural history of
asthma. Between 30 and 70% of children with asthma become markedly improved
or become symptom-free by early adulthood, but significant disease will persist in
about 30% (20,21). Some may experience asymptomatic periods, before develop-
ing wheeze again as adults (22). Among predictors of persistent wheezing from
childhood to adulthood are low lung function in childhood and persistent BHR
(23). The more severe the asthma in childhood, the more severe is the asthma in
adulthood (20,24). Asthma can also start later in life, usually associated with a
nonatopic background. Often, these asthmatics are smokers, and therefore their
condition may be confused with chronic bronchitis or emphysema.
Asthmatics experience a more rapid decline in the lung function measure-
ment of forced expiratory volume in the first second (FEV ) than nonasthmat-
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ics, and smoking asthmatics have the greatest decline in FEV (25,26), which
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may reflect an irreversible process that occurs in asthma, and, although asthma
is predominantly a disease of reversible airway obstruction, it may become
irreversible(27).
4. Presentation of Asthma
Presentation of asthma can vary from patient to patient. Asthma may be
intermittent, with mild to severe episodes that may necessitate treatment. These
4 Chung and Adcock
Table 1
Different Types of Asthma
Atopic or nonatopic
Early onset (childhood) or late onset (adult)
Nocturnal
Exercise-induced
Aspirin-induced
Occupational
Seasonal
Cough variant
Acute severe
Chronic severe
Asthma deaths
Fixed irreversible
Brittle
Corticosteroid-resistant
Corticosteroid-dependent
episodes may be provoked by an upper respiratory viral infection or by an
exposure to an allergen to which the asthmatic is sensitive. Some cases of
asthma may be entirely seasonal, such as pollen-induced asthma in the summer
months. In children, exercise frequently provokes bronchoconstriction. Occu-
pational asthma, induced by specific chemicals or proteins encountered at the
workplace following sensitization, may also present in relation to exposures at
work. Severe episodes of asthma may occur very rapidly sometimes over a
period of a few minutes (brittle asthma), and may be life-threatening. Asthma
may also present with persistent chronic symptoms, often characterized by
worse symptoms at night or on waking in the morning. Some asthmatics
develop exacerbations of their asthma when taking aspirin and other nonsteroi-
dal anti-inflammatory drugs. These patients often develop asthma later in life,
and have concomitant rhinosinusitis and nasal polyps.
4.1. Different Types of Asthma
Given the varied presentation and course of the disease, it is not surprising
that asthma has been clinically classified in various ways, such as on the basis
of provoking factors, severity, pattern of asthma attacks, and even on response
to available treatments (Table 1). However, there is no real classification on
the basis of molecular mechanisms, because there is currently poor understand-
ing of these mechanisms. One central question is whether there are different
types of asthma, or whether there is only one central mechanism with varying
severity and interaction with other exogenous factors to create a varied pre-
Asthma: Cell and Molecular Biology 5
sentation and course. For example, in terms of the cellular inflammation in the
airway submucosa found in atopic and nonatopic asthma, there does not appear
to be any striking difference (28). However, the aspirin-sensitive asthmatic
appears to have an increased activity of the leukotriene (LT) C4 synthase, com-
pared to the nonaspirin-sensitive asthmatic (29). Classification according to
severity is probably most useful, since this can be used to determine not only
the amount of treatment a particular patient may need, but may also be used to
relate to the degree of inflammatory abnormalities in the airways. For example,
using a clinical score of severity, there is a significant positive correlation
between the number of eosinophils in bronchial biopsies or bronchoalveolar
lavage (BAL) fluid and the clinical severity of asthma (30). However, the mea-
surement of severity is not clearly established. A useful characterization of
severity is to use a combination of measurements of symptoms and lung func-
tion, and the number of acute attacks of asthma experienced.
5. Chronic Inflammation of Asthma
It has been recognized for a long time that patients who die of asthma attacks
have grossly inflamed airways, with occlusion of the airway lumen by a tena-
cious plug made of plasma proteins exuded from airway vessels and mucus
glycoproteins(31). The airway wall is oedematous and infiltrated with inflam-
matory cells predominantly composed of eosinophils, lymphocytes and neu-
trophils. Over the past 15 yr, it has been possible to examine the airways of
asthmatic patients, using rigid bronchoscopy under general anesthesia, but
more usually using a fiberoptic bronchoscope, which can be undertaken with
sedation. Studies of the bronchial mucosa of patients with mild and even
asymptomatic asthma have established asthma as a chronic inflammatory dis-
ease of the airways, characterized by an airway submucosal infiltration of lym-
phocytes and eosinophils, epithelial shedding, subepithelial reticular fibrosis,
and edema (30,32–35). Immunostaining using the monoclonal antibody EG2,
which specifically stains the cleaved, secreted form of eosinophil cationic pro-
tein, has identified increased numbers of activated eosinophils, both within the
submucosal and the epithelial mucosal layers. A consistent increase in CD25+
(IL-2 receptor-bearing) cells, representing activated T-lymphocytes in the bron-
chial submucosa of extrinsic asthmatics, has been shown (35). An increase in
activated monocytes, probably recruited from the circulating blood compart-
ment, has also been reported in bronchial mucosal biopsies (36). An increase in
the number of mast cells has also been demonstrated (32). BAL of the lower
airways, with 0.9% saline solution, usually yields an excess of eosinophils,
mast cells, and T-lymphocytes, with evidence of activation of macrophages
(30,37). Alveolar macrophages from asthmatics express an excess of various
markers on their surface as determined by flow cytometric analysis, including
6 Chung and Adcock
CD16, CD18, CD32, CD44, histocompatibility leukocyte antigen (HLA) Class 1,
HLA-DR, and HLA-DQ (38).
Recent studies in patients with more severe disease indicate that there is an
eosinophilic inflammation that involves not only the mucosa of the proximal
airways, but also the more distal airways, together with the alveolar inflamma-
tion (39). In addition, there appears to be a predominance of neutrophils in
more severe asthmatic patients needing high doses of oral corticosteroids (40).
This has also been confirmed in the examination of expectorates obtained from
such patients, following induction with inhaled hypertonic saline (41).
5.1. Airway Wall Remodeling
Together with the cellular abnormalities, there are changes indicative of an
ongoing repair process (42). There is an increase in the number of myofibro-
blasts in the subepithelial areas (43), together with an increase in the thickness
of the lamina reticularis, which is composed of collagen, types III and V, and
fibronectin(44). There has been some dispute as to the presence of shedding of
the airway epithelium. It is likely that the epithelium is more fragile and likely
to shed with the slightest trauma in asthma (45). The proportion of the bron-
chial wall area occupied by mucous glands is increased in the lungs of fatal
cases of asthma (46–48); an increase in the number of goblet cells in the airway
epithelium of mild asthmatics has been reported (34).
In the lungs of patients with fatal asthma, the area of airway smooth muscle
(ASM) is substantially increased in both large and small airways (47–51).
Detailed morphometric analysis indicates the presence of two distinct patterns
of smooth muscle thickening: in those cases in which the process is confined to
the central airways, and those in which the changes involve the whole bron-
chial tree (51). In the first pattern, the increase in ASM occurs from hyperpla-
sia; in the latter pattern, there is predominant hyperplasia (52). An excess of
blood vessels in the airways of patients with asthma is also reported (53).
Alterations in the resident cells of the airways therefore constitute airway
wall remodeling, and this altered structure may result in altered lung function,
in a number of ways. With an increased thickening of the airways resulting
from an increase in the amount of ASM, the degree of smooth muscle shorten-
ing required to occlude the airways would be expected to be lower (54). An
increase in the adventitial area could also lead to uncoupling of the distending
forces of parenchymal recoil from the forces that narrow the airways (55). Thus,
these factors may contribute to the airway hyperresponsiveness of asthma. How
the other remodeling features of the airways relate to airflow obstruction is not
clear. Finally, structural cells must now be considered as potential important
sources of cytokines. For example, ASM cells are capable of releasing several
chemokines, including regulated on actuation normal T-cell expressed and
Asthma: Cell and Molecular Biology 7
secreted (RANTES), IL-8, eotaxin, and macrophage chemoattractant protein-1
(MCP-1) and -3, and granulocyte-macrophage colony-stimulating factor
(GM-CSF) together with prostoglandin E (PGE2) (56–58) which indicates
2
that the ASM may participate in the inflammatory response.
5.2. Overexpression of Cytokines
Increased gene expression of IL-3, IL-4, IL-5, and GM-CSF, presumably in
T-lymphocytes, has been observed in mucosal biopsies (59). Elevated num-
bers of mRNA cells for IL-3, IL-4, IL-5, and GM-CSF in BAL fluid of symp-
tomatic asthmatic patients were found, compared to asymptomatic subjects
(60). However, there were no differences in the expression of IL-2 and IFN-γ,
indicating that there was a predominance of cytokines derived from Th2, such
as IL-3, IL-4, and IL-5, rather than from Th1-lymphocytes, such as IFN-γ and
IL-2. An increase in the number of cells in bronchial biopsies of asthmatics
expressing the IL-5 receptor has been reported, mostly on eosinophils (61).
IL-5 is an important cytokine, involved as an eosinophil-differentiating fac-
tor, particularly on late-committed eosinophil precursors (62,63), and can pro-
long the survival of eosinophils (64). IL-4 is important in the class switch of
B-cells to the synthesis of IgE and promotes the development of Th2-like CD4+
T-cells(65,66). Factors identified as consisting of IL-5 and GM-CSF activities
in BAL fluid from patients with asthma can prolong eosinophil survival;
GM-CSF appears to be the most important contributor (67), and is predomi-
nantly expressed in airway epithelium and macrophages (68,69). IL-5 and
GM-CSF can prime eosinophils, e.g., to increase the release of granule-associated
proteins, such as eosinophil-derived neurotoxin and eosinophil cationic pro-
tein (ECP) from stimulated eosinophils (70,71). GM-CSF can also enhance the
production of leukotrienes from eosinophils (72).
Increased mRNA expression of the chemoattractant cytokine, RANTES, and
eotaxin has been reported, particularly in the airway epithelium (73,74). These
chemokines are important in causing the chemotaxis of inflammatory cells,
such as T-cells, monocytes, and eosinophils, into the airway submucosa, with
eotaxin being very selective for eosinophils. Cooperation between IL-5 and
chemokines, such as eotaxin, has been described in terms of eosinophil mobi-
lization from the bone marrow and to the airways (75,76). Such cooperation
may occur in terms of the development of BHR (77). The airway epithelium of
patients with asthma also expresses another chemokine, MCP-1, compared to
airway epithelium from nonasthmatic subjects. Thus, release of chemokines,
such as RANTES and eotaxin, and other cytokines, such as IL-5 and GM-CSF,
may lead to the recruitment of eosinophils to the airways, with prolonged sur-
vival, which are activated to release LTs and eosinophilic proteins. Eosino-
philic proteins may in turn damage airway epithelium and contribute to BHR.
8 Chung and Adcock
Alveolar macrophages obtained by BAL from patients with mild asthma
release more proinflammatory cytokines, such as GM-CSF, IL-8, MIP-1α,
tumor necrosis factor-α (TNF-α), IL-1, and IFN-γ(78–80). Lymphocytes and
alveolar macrophages from BAL of asthmatic patients demonstrate an aug-
mented expression of TNF-α, IL-6, and GM-CSF following allergen challenge
(81,82). Increased amounts of IL-1, IL-6, and GM-CSF have been measured in
bronchoalveolar fluid of patients with symptomatic asthma, and the source of
these cytokines appears to be epithelial cells (ECs) and macrophages (83).
Normally, airway macrophages are poor at antigen presentation, and suppress
T-cell proliferative responses, possibly via the release of cytokines, such as
receptor antagonist (IL-1[ra]), but in asthma there is evidence for reduced sup-
pression after exposure to allergen (84,85). The expression of IL-1ra in the
airway epithelium is reduced in asthma (86). Both GM-CSF and IFN-γ increase
the ability of macrophages to present allergen and express HLA-DR (87).
IL-1 is important in activating T-lymphocytes, and is an important co-stimu-
lator of the expansion of Th2 cells after antigen presentation (88). Airway mac-
rophages may be an important source of first-wave cytokines, such as IL-1,
TNF-α, and IL-6, which may be released on exposure to inhaled allergens via
the low-affinity IgE receptors (FcεRII). These cytokines may then act on ECs
to release a second wave of cytokines, including GM-CSF, IL-8, and RANTES,
which then amplifies the inflammatory response and leads to an influx of sec-
ondary cells, such as eosinophils, which themselves may release multiple cyto-
kines. Mast cells can also express IL-4, IL-5, IL-6, and TNF-α in asthma (89).
Cytokines may exert an important regulatory effect on the expression of
adhesion molecules, both on endothelial cells of the bronchial circulation and
on airway ECs. IL-4 increases the expression of vascular cell adhesion mol-
ecule-1 (VCAM-1) on endothelial cells and ECs, which may be important for
the regulation of eosinophil and lymphocyte trafficking (90). On the other hand,
IL-1 and TNF-α increase the expression of intercellular adhesion molecule-1
(ICAM-1) in both vascular endothelium and airway epithelium (91). Following
allergen challenge, there is increased expression of ICAM-1 and E-selectin, with
no increase in VCAM-1 in asthmatic biopsies (92). In asthmatics, E-selectin,
ICAM-1, and VCAM-1 can be detected in atopic, but not in nonatopic asth-
matics (93–95). ICAM-1 expression is generally increased in the airway epi-
thelium of patients with asthma (96,97). The importance of the integrin, very
late antigen-4 (VLA4), has been demonstrated in several animal models of
airway eosinophilia (77,98).
5.3. Transcription Factors in Asthma
Increased gene expression in asthma raises the possibility that there is
increased activation of transcription factors that bind to regulatory sequences,
Asthma: Cell and Molecular Biology 9
usually on the 5'-upstream promoter region of target genes, to increase or
decrease transcription. Transcription factors are involved in the regulation of
expression of cytokine genes, and play an important role in the long-term regu-
lation of cell function, growth, and differentiation. c-fos, a nuclear proto-
oncogene and constituent of the transcriptional activator protein, AP-1, has
been shown to be overexpressed in the airway epithelium of patients with
asthma(99). Overexpression of c-fos in circulating blood mononuclear cells of
patients with steroid-resistant asthma has been described (100).
Nuclear factor κB (NF-κB) is another family of transcription factors impor-
tant in the induction of a wide array of genes, including chemokines, cytok-
ines, enzymes, receptors, and stress proteins. It consists of dimeric complexes
composed of various members, but the p50/p65 heterodimer is usually the most
abundant of the transactivating complexes. NF-κB DNA-binding activity in
cells, such as macrophages from induced sputum, and in biopsies of mild asth-
matic patients, is increased, and the expression of this transcription factor was
increased in the airway epithelium of patients with mild asthma (101). The
epithelium in asthma has been the site of enhanced expression of several pro-
teins, including cytokines such as GM-CSF, RANTES, and MCP-1, enzymes
such as inducolde macrophages-type nitric oxide synthase (iNOS) and cyto-
chrome oxidase-2, and adhesion molecules such as ICAM-1 (68,73,93,102–104),
and the transcriptional control of these genes is partly dependent on NF-κB
activation. A crucial role for NF-κB has been demonstrated in the p50(–/–)
knockout mice which were defective in their capacity to mount an allergic eosi-
nophil response because of lack of production of the Th2 cytokine, IL-5, and
the chemokine, eotaxin (105). Other transcription factors of interest include
GATA3, which is also expressed in the Th2, but not Th1, cells, and is crucial
for activation of IL-5 promoter gene by different stimuli. Ectopic expression of
GATA-3 is sufficient to drive IL-5, but not IL-4, gene expression (106).
5.4. Inflammatory Mediators in Asthma
Many different mediators have been implicated in asthma and possess a
variety of effects on the airways that could account for the pathophysiological
features of asthma (Figs. 1 and 2). Mediators, such as histamine, PGs, and LTs,
contract ASM, increase microvascular leakage, cause airway mucus secretion,
and attract inflammatory cells (107). The role of individual mediators in asthma
is not clear. Recently, much attention has been given to the cysteinyl-LTs
LTC4, LTD4, and LTE4, which are potent constrictors of human airways and
can induce BHR (108,109). In addition, other effects of cysteinyl-LTs have
been described, including chemotactic effects on eosinophils, and a permissive
effect on ASM proliferation (110,111). Potent LTD4 antagonists protect against
exercise- and allergen-induced bronchoconstriction, indicating the contribu-