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Acetylcholine beyond bronchoconstriction: a regulator of inflammation and remodelingKistemaker, Loes
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CHAPTER 1
GENERAL INTRODUCTION
Chapter 1
8
Preface
The objective of this thesis is to establish the role of acetylcholine and individual
muscarinic receptor subtypes in inflammation and remodeling of the airways.
Inflammation and remodeling are two important pathophysiological processes in asthma
and chronic obstructive pulmonary disease (COPD), affecting the decline in lung function
and severity of the disease. In the airways, acetylcholine acts as a parasympathetic
neurotransmitter, but also as an autocrine and/or paracrine hormone. Muscarinic
receptors are target receptors for acetylcholine and muscarinic receptor antagonists are
used as a therapy for COPD, and to a lesser extent also for asthma. Consequently, a
potential role for acetylcholine in inflammation and remodeling in COPD and asthma could
have important therapeutic implications.
Acetylcholine, a neurotransmitter and a hormone
Acetylcholine – a neurotransmitter
Acetylcholine is the primary parasympathetic neurotransmitter of the airways.
Acetylcholine was detected by Otto Loewi and Sir Henry Dale in the early 1920s, for which
they received the Nobel prize in 1936. As a neurotransmitter, acetylcholine is synthesized
in nerve endings from the substrates choline and acetyl‐CoA by the enzyme choline acetyl
transferase (ChAT) (1). The uptake of choline from the extracellular space is the rate‐
limiting step in this process (2). Synthesized acetylcholine is translocated into synaptic
vesicles via the vesicular acetylcholine transporter (VAChT), and released by exocytosis.
Exocytosis is triggered by a depolarizing stimulus and modulated by several regulatory
mechanisms in the neuroeffector junction (1). This results in the release of acetylcholine
from airway parasympathetic nerve endings in the extracellular space, where it interacts
with postsynaptic target receptors, but also with presynaptic receptors on cholinergic
nerve terminals themselves. Target receptors of acetylcholine include muscarinic
receptors and nicotinic receptors, which will be introduced in more detail below.
Acetylcholine in the synaptic cleft is rapidly degraded into acetate and choline by the
highly expressed enzyme acetylcholine esterase (AChE). Alternatively, acetylcholine can be
degraded by butyrylcholinesterase (BChE) (1). Choline can be taken up again by the nerve
for acetylcholine synthesis. The acetylcholine synthesis pathway in neurons is summarized
in figure 1A, together with the non‐neuronal synthesis pathway, which will be elaborated
on later.
General introduction
9
Figure 1. Schematic representation of acetylcholine synthesis, release, action and breakdown in neuronal cells at
a cholinergic nerve terminal (A) and in non‐neuronal cells, such as epithelial cells (B). In neurons, there is an
efficient metabolism of acetylcholine (ACh), involving the high‐affinity choline transporter‐1 (CHT1), choline
acetyltransferase (ChAT), the vesicular ACh transporter (VAChT) and ACh release via exocytosis. Although some
non‐neuronal cells also express these components, they are not common to all cell types and probably
alternative, less efficient, mechanisms predominate. Perhaps as a consequence, acetylcholine content is lower. In
non‐neuronal cells, choline is taken up not only via CHT1, but also via choline transporter‐like proteins (CTL),
including CTL1, CTL2 and CTL4. Carnitine acetyltransferase (CarAT) has been identified as an alternative
synthesizing enzyme of ACh. Organic cation transporters (OCT), including OCT1 and OCT2, have been shown to
transport ACh in and out of cells (3). MR: muscarinic receptor, NR: nicotinic receptor, AChE: acetylcholinesterase,
BChE: butyrylcholinesterase.
Airway nerves
Airway nerves regulate many aspects of airway function. One of the most prominent
effects is the regulation of airway smooth muscle tone, the neural component of which is
almost solely controlled by the parasympathetic nervous system (4, 5). Although airways
also express sympathetic and non‐adrenergic, non‐cholinergic (NANC) neural pathways,
focus of this thesis is on the parasympathetic neural pathway, which is the dominant
neural pathway in the airways and uses acetylcholine as a neurotransmitter. Airway vagal
nerve fibers can be divided into three major components: the primary afferent nerve
fibers, the integrating centers in the brain and the parasympathetic efferent nerve fibers
(see figure 2) (6). Afferent nerve fibers in the airways include stretch‐sensitive myelinated
nerve fibers or A‐fibers, and unmyelinated C‐fibers (7). A‐fibers conduct action potentials
Chapter 1
10
at a relatively high velocity and consist of rapidly adapting receptors (RARs) in the mucosal
layer, which respond to the dynamic phase of inspiration, and slowly adapting receptors
(SARs) in the smooth muscle layer, which respond to maintained inflation (7‐9). However,
most of the airway afferent nerves are unmyelinated C‐fibers, which are present
throughout the airways and conduct action potentials at a much slower velocity compared
to A‐fibers (6, 10). C‐fibers respond to noxious stimuli such as heat, cold or mechanical
forces, but also to mediators released upon tissue damage and inflammation (7).
Activation of these C‐fibers results in bronchoconstriction, mucus secretion and cough
(11). This can be a local reflex in the airways at the level of the parasympathetic ganglia,
which involves the release of peptide neurotransmitters, including substance P and other
tachykinins, directly from the nerve terminals (12). Next to this local reflex, a central reflex
arch via the brain stem exists (9). Parasympathetic efferent nerve fibers consist of
preganglionic neurons, which innervate the parasympathetic ganglia in or near the airway
wall of larger airways, and postganglionic neurons (6). Postganglionic neurons innervate
airway smooth muscle, mucus glands and the microvasculature throughout the airway
tree (figure 2). In addition, postganglionic parasympathetic neurons, which do not use
acetylcholine as a transmitter, exist. Instead, these NANC pathways use nitric oxide and/or
neuropeptides such as vasoactive intestinal peptide as a transmitter, and are in fact
bronchodilatory (6). However, the parasympathetic cholinergic nervous system is the
major neural regulator of airway smooth muscle tone (4, 9, 13).
Figure 2. The vagal nervous system in the airways. Mechanical forces, inhaled irritants and endogenous
inflammatory mediators activate afferent nerve fibres, which send signals to the central nervous system (CNS).
This results in the release of acetylcholine (ACh) in the airways. Parasympathetic, post‐ganglionic neurons are
located within the airway wall and innervate airway smooth muscle and submucosal glands to induce contraction
and mucus secretion, respectively. Blue: afferent nerve fibers; red: efferent nerve fibers. RAR: rapidly adapting
receptors; SAR: slowly adapting receptors.
General introduction
11
Acetylcholine ‐ a hormone
During the last decades, it has become clear that acetylcholine production in the airways is
not only restricted to the nerves. Bacteria, algae, protozoa, tubellariae and primitive
plants, all lacking a nervous system, express acetylcholine. This suggests an early and
widely distributed role of acetylcholine (14). Indeed, cells of higher organisms, including
cells of the human airway, have been shown to produce acetylcholine (14, 15). Thus,
acetylcholine can act as a local signaling molecule in an autocrine or paracrine fashion,
which is referred to as non‐neuronal acetylcholine. RNA expression of ChAT has been
detected in almost all cell types of the airways, including epithelial cells, airway smooth
muscle cells and inflammatory cells (16). Additionally, carnitine acetyltransferase (CarAT)
has been detected as an alternative, albeit less efficient, route for acetylcholine synthesis
in airway cells (2). In particular epithelial cells have been shown to express relatively high
levels of acetylcholine (17, 18), and the epithelial non‐cholinergic system has been
characterized in detail (2). Evidence for the actual release of non‐neuronal acetylcholine
from other airway cell types is still limited (15). Part of the biosynthesis pathway of non‐
neuronal acetylcholine overlaps with that used by the neuronal system; however, clear
differences exist, the former being less efficient (2). For example, neurons use the highly
efficient CHT1 transporter for choline uptake, whereas non‐neuronal cells mainly use
choline transporter‐like proteins (CTLs) and organic cation transporters (OCTs) for choline
uptake (2). In addition, in neurons, acetylcholine is stored and subsequently released from
vesicles, whereas in non‐neuronal cells acetylcholine is mainly released directly via OCTs
(2, 3) (figure 1B). As a consequence, acetylcholine levels seem to be lower in non‐neuronal
cells compared to neuronal cells. The functional relevance of non‐neuronal acetylcholine
in the airways still has to be established and it is unclear whether non‐neuronal
acetylcholine contributes to the classical cholinergic driven effects as described above (3).
However, the concept that acetylcholine is not only a neurotransmitter but also a
hormone has dramatically broadened the view on the role of acetylcholine in the airways,
and holds therapeutic promises for the future, which will be further elaborated on in
chapter 2 (19‐21).
Targets of acetylcholine
Acetylcholine can act via two different classes of receptors, namely muscarinic and
nicotinic receptors. Muscarinic receptors are G‐protein coupled receptors, characterized
by a seven transmembrane domain (1). Five different subtypes have been identified (M1‐
M5), of which M1‐M3 receptors are expressed abundantly in the airways (1, 16, 21). Almost
all cell types in the airways express muscarinic receptors, as discussed in detail below.
Activation of muscarinic receptors in the airways leads to bronchoconstriction and mucus
secretion (21). Nicotinic receptors are ligand gated ion channels, which are comprised of
Chapter 1
12
one to five different types of subunits (α, β, γ, δ and ε) (1). Multiple different nicotinic
receptor isotypes exist and at least ten different α‐subunits and four different β‐subunits
have been identified. In the airways, almost all cell types express nicotinic receptors (1,
15). Nicotinic receptors are involved in neurotransmission in the airways, and activation of
nicotinic receptors causes an influx of positively charged ions, leading to membrane
depolarization (1, 22). The nicotinic α7 receptor is highly expressed on multiple cell types
in the airways and plays an immunomodulatory role (15). Moreover, the nicotinic α7
receptor is thought to play a regulatory role in lung cancer (23). Current therapy for
obstructive airway diseases, however, is selectively directed to muscarinic receptors.
Therefore, the focus of this thesis is on muscarinic receptors, and nicotinic receptors will
not be discussed in more detail here.
Muscarinic receptors
Expression of muscarinic receptors
Expression of muscarinic M1, M2 and M3 receptors has been detected in the airways.
Although muscarinic receptor antibodies have been proven not to be very useful in the
detection of subtype specific expression of muscarinic receptors because of lack of
specificity (24, 25), other techniques, including binding studies and gene expression
analyses, have elucidated the expression pattern of muscarinic receptors throughout the
airways. From these studies, it is clear that M1 receptors appear to be expressed
particularly in peripheral lung tissue, M2 receptors are expressed throughout the airways,
whereas expression of M3 receptors is highest in the larger airways (1, 26). Almost all cells
of the airways express muscarinic receptors, with high expression on airway smooth
muscle cells, epithelial cells and submucosal glands. Moreover, in parasympathetic
ganglia, M1 receptors are expressed, as well as autoinhibitory prejunctional M2 receptors
(21, 22). Expression of muscarinic receptors per cell type, and their major functional
effects, are summarized in table 1.
Signal transduction of muscarinic receptors
Muscarinic receptors couple to heterotrimeric G proteins. G proteins are composed of an
α‐, β‐ and γ‐subunit, and are classified according to the α‐subunit. Four different families
have been identified: Gαs, Gαi/o, Gαq/11 and Gα12/13 (30, 31). M2 receptors couple primarily
to Gαi and M1 and M3 receptors couple primarily to Gαq (32). Receptor activation
promotes the exchange of guanosine diphosphate (GDP), bound to the α‐subunit, with
guanosine triphosphate (GTP). This will lead to dissociation of the α‐subunit from the βγ‐
heterodimer and subsequent activation of second messengers (30). Activation of Gαi via
M2 receptors results in inhibition of adenylyl cyclase (AC), which in turn inhibits the second
General introduction
13
messenger cyclic AMP (cAMP), and Gβγ mediated inhibition of potassium channels (33,
34). Activation of Gαq via M1 and M3 receptors results in activation of phospholipase C
(PLC). PLC converts phosphatidylinositol‐4,5‐bisphosphate (PIP2) into inositol 1,4,5‐
trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of calcium from
internal endoplasmic reticulum stores, whereas DAG activates protein kinase C (PKC). The
initial calcium release is followed by a more sustained calcium release mediated via
ryanodine receptors on the endoplasmic reticulum, and via an increase in the open
probability of calcium channels in the cell membrane, mediated via PKC (35).
Functional roles in the airways
Neurotransmission
M1 receptors expressed in parasympathetic ganglia contribute to neurotransmission, by
facilitating depolarization via inhibition of potassium currents. The magnitude of
muscarinic depolarization may not be sufficient to initiate an action potential by itself, but
rather enhances or facilitates nicotinic‐induced depolarization (1). It should be noted
therefore that there is still debate about the functional relevance of M1‐mediated
neurotransmission (1). Evidence for the auto‐inhibitory role of prejunctional M2 receptors
is more convincing, since M2 selective antagonists enhance electrical field stimulation‐
induced acetylcholine release from guinea pig and human trachea (36, 37). The
autoinhibitory M2 receptor is dysfunctional in asthma, which contributes to the enhanced
cholinergic tone in this disease (16). No such mechanism has been observed in COPD (38).
Table 1. Expression of muscarinic receptors on airway cells and their major effects (16, 27‐29).
Cell Muscarinic receptor
expression
Functional effect
Neuron M1, M2 Neurotransmission
Airway smooth muscle cell M2, M3 Bronchoconstriction via M3
Epithelial cell M1, M2, M3 Mucus secretion via M3
Submucosal gland M1, M3 Mucus secretion via M3
Fibroblast M1, M2, M3 Proliferation, ECM production
Mast cell M1, M3 Inhibition of histamine release
Macrophage M1, M2, M3 Cytokine production
Lymphocyte M1, M2, M3 Cytokine production
Neutrophil M1, M2, M3 Cytokine production
Eosinophil M1, M2, M3 ?
Chapter 1
14
Airway smooth muscle contraction
Muscarinic receptors on airway smooth muscle mediate airway smooth muscle
contraction, and therefore play an important role in bronchoconstriction associated with
COPD and asthma. Although M2 receptors represent the majority of muscarinic receptors
expressed on airway smooth muscle, airway smooth muscle contraction is primarily
mediated by M3 receptors (39). This is clear from affinity profiles of selective muscarinic
antagonists in different species, including humans (16). Moreover, the introduction of
specific muscarinic receptor subtype deficient animals confirmed that this is an M3‐
mediated effect, as M3 receptor deficient mice (M3R‐/‐), and not M2R
‐/‐ mice, lack
methacholine and vagally‐induced bronchoconstriction in vivo (40).
Mucus secretion
Muscarinic receptors regulate mucus secretion in the airways. Mucus secretion is
enhanced in COPD and asthma, which contributes to airflow obstruction of the airways
(41). Mucus is secreted by airway submucosal glands, which are in connection to the
airway lumen, and by goblet cells, which are embedded in the airway epithelium.
Submucosal glands are innervated by parasympathetic nerves (see also figure 2), and
mucus secretion from glands is under cholinergic control, mediated via M3 receptors.
Thus, electrical field stimulation increases mucus secretion from airway preparations,
which can be inhibited by the M3 antagonist 4‐DAMP (1,1‐Dimethyl‐4‐diphenylacetoxy‐pi‐
peridinium iodide) (42, 43). Acetylcholine can also induce mucus secretion from goblet
cells, again mediated primarily via M3 receptors (21, 41). There is still debate whether
acetylcholine‐induced mucus secretion from goblet cells is mediated by neuronal or non‐
neuronal acetylcholine, or by a combination of both. M1 receptors are also expressed on
epithelial cells and submucosal glands, and mediate electrolyte and water secretion in
cooperation with M3 receptors (21, 42).
Vagal dysregulation
Activity of the neuronal system is altered in disease, including asthma and COPD, via
several mechanisms, leading to exaggerated acetylcholine release and airway
hyperresponsiveness (9, 13, 44). This can be via [1] changes in activity of afferent nerves,
[2] changes in synaptic transmission and [3] changes in neurotransmitter content in the
synaptic cleft. First, changes in activity of afferent nerves have been observed in response
to inflammatory mediators, present in the airways of patients with COPD or asthma. This
results in enhanced release of acetylcholine from vagal nerve endings and an increase in
cholinergic tone (6). This effect can be mediated via several inflammatory mediators,
including histamine, tachykinins, prostaglandins and thromboxane A2, which stimulate
General introduction
15
sensory nerve fibers directly (6, 45). Moreover, epithelial damage will expose afferent
sensory nerve endings in the subepithelial layer to the airway lumen (46). Second,
inflammation can also change synaptic transmission, by increasing synaptic efficacy and by
increasing acetylcholine release, via the release of mediators including tachykinins, which
interact with receptors on nerve terminals (6, 12). Moreover, autoinhibitory prejunctional
M2 receptors, which limit acetylcholine release under healthy conditions, are
dysfunctional in asthmatic airways. This has been shown in animal models of allergen
exposure, viral infection and ozone exposure (47), but also in patients with asthma (16,
48). M2 receptor dysfunction is probably mediated via several mechanisms, but convincing
evidence exists for the involvement of major basis protein. Major basic protein is secreted
by eosinophils that are recruited to airway nerves, and acts as an allosteric antagonist for
the M2 receptor (49). In support, treatment with an antibody against major basic protein
prevents M2 receptor dysfunction in guinea pigs (50). Third, acetylcholine content in the
synaptic cleft is altered. In part this is due to dysfunctional M2 receptors as described
above, but it has also been shown that activity of AChE is reduced in tracheal smooth
muscle homogenates obtained from ragweed pollen‐sensitized dogs compared to sham
controls (51). Together, this results in enhanced acetylcholine concentrations in the
synaptic cleft and prolonged effects on postjunctional target receptors.
There is little evidence for altered muscarinic receptor expression in asthma or COPD.
Using radioligand binding studies, no significant changes were observed in affinity or
density of muscarinic receptors in the central or peripheral airways of patients compared
to healthy controls (52, 53). Moreover, until now, there is no evidence for genetic
polymorphisms of muscarinic receptors, as at least M2 and M3 receptors seem to be highly
conserved, and no significant differences between healthy and asthmatic subjects were
detected (54).
Besides these acute effects on vagal nerves, there is also evidence for neural remodeling.
Growth and development of airway nerves continues well into adolescence, and exposure
to allergens might alter airway nerve structure (55). Pan et al. demonstrated that allergen
exposure in guinea pigs results in a shift from a NANC phenotype, which does not use
acetylcholine as a neurotransmitter, towards a cholinergic phenotype (56). In addition,
alterations in airway nerve structure might start very early in life, which may affect the
incidence of airway diseases later in life (57). A recent paper demonstrated that early‐life
exposure of mouse neonates to ovalbumin leads to a two‐fold increase in airway smooth
muscle innervation in adulthood (58). Furthermore, exposure of infant rhesus monkeys to
ozone and/or house dust mite results in changes in airway innervation in the epithelial
region (59). Thus, exposure to allergen or environmental stress, either early in life or in
Chapter 1
16
adulthood, may lead to dysregulated vagal innervation, and might thereby affect airways
diseases.
Although most of these studies focus on asthmatic airways, evidence suggests that
innervation is also altered in the airways of COPD patients (7). The inflammatory response
in the airways of patients with COPD might trigger acetylcholine release via above
described mechanisms. Moreover, altered breathing patterns and changes in gas tensions
in patients with COPD activate RAR‐fibers and SAR‐fibers (7), and the ability of SARs to
respond to lung inflation seems to be be altered in patients with COPD (13). Therefore,
dysregulation of vagal nerves, leading to an increased cholinergic tone, largely contributes
to both COPD and asthma pathophysiology. Strikingly, increased cholinergic tone is the
major reversible component of airflow limitation in COPD (60, 61).
Muscarinic receptors in the treatment of COPD and asthma
Therapeutic targets in COPD and asthma
As described above, muscarinic receptors play a key role in regulating bronchoconstriction
and mucus secretion, and cholinergic tone is increased in patients with COPD and asthma.
For these reasons, muscarinic receptors represent key therapeutic targets for COPD and
asthma. Muscarinic receptor antagonists, referred to as anticholinergics, have already
been used for centuries for the treatment of obstructive airway diseases. This started with
the use of naturally occurring medicinal plants containing anticholinergic alkaloids such as
atropine. Fumes of these medicinal plants were inhaled, and later on even smoked via
cigarettes, until the middle of the 20th century (62, 63). Since then, more safe and effective
synthetic anticholinergics have been introduced to the market.
Anticholinergics
Ipratropium was the first synthetic anticholinergic introduced to the market. Because of
the quaternary ammonium structure, ipratropium is much safer compared to the natural
occurring anticholinergic atropine. Atropine can easily pass the blood‐brain barrier leading
to numerous side effects, which is prevented by the quaternary structure of ipratropium.
The safety profile of ipratropium is further enhanced by applying the drug via inhalation,
thereby limiting systemic exposure. Besides dry mouth, ipratropium, but also other
anticholinergics, have limited side effects (64). Thereafter, oxitropium was introduced.
Both drugs have relatively short durations of action (4‐8 hours), and there was a need for
long‐acting anticholinergics. Tiotropium is the first long‐acting anticholinergic introduced
to the market in the early 2000s. Tiotropium has been shown to be a potent muscarinic
General introduction
17
Table 2. Binding affinity and half‐life time at the M1, M2 and M3 receptor for the different anticholinergics against
human M1, M2 and M3 receptors.
Binding affinitiy (‐log M) t1/2 (h)
M1R M2R M3R M1R M2R M3R
Ipratropium 9.40 9.53 9.58 0.1 0.03 0.22
Tiotropium 10.80 10.69 11.02 10.5 2.6 27
Aclidinium 10.78 10.68 10.74 6.4 1.8 10.7
Glycopyrronium 10.09 9.67 10.04 2.0 0.37 6.1
Binding affinity was determined in heterologous competition experiments against [3H]NMS. Data represent pKi
values of at least three independent experiments performed in triplicate and the standard error was 0.1 or less.
Dissociation half‐life was determined by the dissociation constants, by analyzing competition kinetics curves in
the presence of [3H]NMS and different concentrations of antagonist. At least three independent experiments
were performed in triplicate. Data from Casarosa et al (66).
receptor antagonist; the affinity of tiotropium for muscarinic receptors is around 10‐fold
higher compared to ipratropium. However, onset of action is slower compared to
ipratropium (65). Although the steady‐state affinity of tiotropium for M1, M2 and M3
receptors is similar, dissociation from the M3 receptor is much slower compared to the
other receptor subtypes, in particular compared to M2 receptors (table 2) (66, 67). Used as
a bronchodilator, this is a desired property, since inhibition of M3 receptors inhibits airway
smooth muscle contraction, whereas antagonizing M2 receptors would enhance
acetylcholine release and thereby enhance airway smooth muscle contraction. Moreover,
this long duration of action at the M3 receptor allows for once daily dosing. Slow
dissociation of tiotropium from the M3 receptor is attributed to interactions at the binding
site, which prevents rapid dissociation via a snap‐lock mechanism (68). Recently, inhaled
glycopyrronium and aclidinium were also introduced to the market. As becomes clear
from table 2, like tiotropium, glycopyrronium and aclidinium are kinetically selective for
the M3 receptor, and similarly, there is no difference in affinity of both drugs for individual
muscarinic receptors. However, the dissociation half‐life from the M3 receptor of these
compounds is smaller compared to tiotropium (66). Glycopyrronium is given once daily,
whereas aclidinium is given twice daily. Several new anticholinergics have recently
reached the market, as well as combinations of long‐acting anticholinergics with long‐
acting β2‐agonists and/or corticosteroids (69).
Use of anticholinergics in COPD
In COPD, anticholinergics represent a first‐line of treatment according to the Global
Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (70). Long‐acting
anticholinergics and long‐acting β2‐agonists are the most frequently prescribed
bronchodilators for COPD treatment. Anticholinergics have a particular value in the
Chapter 1
18
treatment of COPD because they block the increased cholinergic tone, which is the major
reversible component of the disease (60, 61). The first randomized controlled clinical trial
into the efficacy and safety of tiotropium in patients with COPD was published in 2000
(71). In this study, tiotropium was compared to ipratropium. Both tiotropium and
ipratropium induced an increase in lung function over a 13 week period, and the long‐
acting tiotropium was more effective compared to the short‐acting ipratropium. The
safety profile of both drugs was similar (71). Since then, a number of studies have been
undertaken, of which the UPLIFT (Understanding Potential Long‐term Impacts on Function
with Tiotropium) trial was the most extensive study. In this study, a total of 5993 patients
were randomly assigned to placebo or tiotropium, and followed for 4 years. Tiotropium
significantly improved lung function and quality of life, and reduced the number of
exacerbations compared to placebo. Tiotropium did not alter the rate of decline in forced
expiratory volume in 1 second (FEV1) (72). More recently, new anticholinergics were
introduced, including glycopyrronium and aclidinium. Glycopyrronium (NVA237) was
shown to improve lung function, quality of life and dyspnoea, and to reduce the risk of
exacerbations compared to placebo in patients with moderate to severe COPD (73), with
efficacy comparable to tiotropium (74). Similarly, aclidinium was shown to improve lung
function, quality of life and dyspnoea compared to placebo in moderate to severe COPD
patients (75, 76), which was comparable to the effects of tiotropium over a 15 day period
(77). Fixed‐dose combinations of several long‐acting anticholinergics and long‐acting β2‐
agonists are currently under development or have recently reached the market, and large
trials are evaluating the efficacy and safety of fixed‐dose combinations (35, 78). First
evidence suggests that the fixed‐dose combinations of tiotropium and olodaterol (79),
glycopyrronium and indacaterol (80, 81), aclidinium and formoterol (82, 83), and
umeclidinium and vilanterol (84, 85) are more effective than monotherapy.
Use of anticholinergics in asthma
In asthma, the use of anticholinergics is limited to the treatment of exacerbations, and
anticholinergics are not approved for chronic treatment (86). Short‐acting anticholinergics
have a significant but small effect on peak expiratory flow (PEF) compared to placebo in
patients with stable asthma (87). The effects of short‐acting anticholinergics are smaller
than the effects of short‐acting β2‐agonists in patients with asthma (64, 87). According to
the Global Initiative for Asthma (GINA) guidelines, ipratropium can be used in stable state
as an alternative bronchodilator to short‐acting β2‐agonists, but is usually less effective. In
case of an exacerbation, the addition of a short‐acting anticholinergic to the short‐acting
β2‐agonists is recommended (86). More recently, clinical trials into the effects of the long‐
acting anticholinergic tiotropium on lung function in patients with asthma have started. In
one of the first studies, Peters et al. demonstrated that the addition of tiotropium to
General introduction
19
glucocorticoid therapy in patients with mild to moderate asthma was more effective than
doubling of the glucocorticoid dose by means of morning peak expiratory flow, the
proportion of asthma‐control days, FEV1 and daily symptom scores, and very similar to the
effects of adding salmeterol (88). Moreover, it was shown in a phase 2 study in patients
with severe uncontrolled asthma that addition of tiotropium to standard therapy with
inhaled corticosteroids and long‐acting β2‐agonists improved lung function over an 8 week
period (89). These findings were confirmed by two replicate phase 3 randomized
controlled trials involving 912 severe asthma patients over a period of 48 weeks.
Tiotropium induced bronchodilation on top of the use of inhaled corticosteroids and long‐
acting β2‐agonists. Furthermore, tiotropium reduced exacerbations and episodes of
worsening of asthma (90). Two phase 2 studies in patients with moderate persistent
asthma demonstrated that tiotropium can also induce bronchodilation in this patient
group, on top of the use of corticosteroids (91, 92). These results have been confirmed in
two large replicate phase 3 trials in 2100 patients with uncontrolled asthma on medium
dose corticosteroids, of which the results have only been presented as abstracts (93).
Tiotropium induced sustained bronchodilation after 24 weeks, accompanied by
improvements in asthma control, all very similar to salmeterol (93). Together these data
indicate that asthma patients might benefit from addition of tiotropium to standard
therapy.
Regulation of inflammation and remodeling by muscarinic receptors
COPD and asthma are associated with airway inflammation and remodeling. Increasing
evidence suggests a role for acetylcholine in inflammation and remodeling in both
obstructive airway diseases (16, 21). This suggests that the role of acetylcholine in patients
with COPD or asthma might be much broader than previously appreciated.
Inflammation and remodeling in COPD
COPD is associated with persistent inflammation and remodeling of the airways.
Inflammation is present throughout the airways (94), and the degree of inflammation
correlates to the severity of the disease (95). Upon inhalation of cigarette smoke, which is
the main risk factor for COPD, epithelial cells are activated and macrophages are
attracted, resulting in the release of chemotactic factors. Macrophages are thought to
orchestrate the inflammatory response in COPD (96). Amongst others, macrophages
release CC‐chemokine ligand 2 (CCL2), also known as monocyte chemotactic protein
(MCP1), to attract more monocytes to the lung, and CXC‐chemokine ligand (CXCL)‐1 and
CXCL‐8, to attract neutrophils (94, 96). Moreover, both macrophages and epithelial cells
release CXCL9, CXCL10 and CXCL11, which attract T‐cells to the lung (94, 97). In COPD, T‐
Chapter 1
20
cells are mainly T‐helper type 1 (TH1) and type 1 cytotoxic T (TC1) CD8+ cells (94). The
inflammatory response is thought to contribute to the remodeling of the airways, by the
release of TGF‐β and proteases such as matrix metalloproteinase 9 (MMP9) (94, 98).
Fibrosis, by means of peribronchiolar deposition of extracellular matrix proteins, mainly
occurs around the small airways, and is an important factor contributing to the irreversible
airway narrowing observed in COPD (99). Moreover, alveolar walls in the lung parenchyma
are destructed due to the chronic inflammation, which is called emphysema. Emphysema
results in enlargement of parenchymal airspaces and loss of elastic recoil, and is a major
contributor to morbidity and mortality in COPD (100, 101). Remodeling of the epithelium
also occurs, leading to squamous and mucous metaplasia (100). Together with mucus
gland hypertrophy, this leads to mucus hypersecretion in patients with COPD (102, 103).
Remodeling of the airways starts later in life in patients with COPD, but is progressive and
irreversible, and is the major contributor to the decline in lung function (100).
Inflammation and remodeling in asthma
Like COPD, asthma is characterized by airway inflammation and remodeling. However,
there are marked differences between the pattern of inflammation and remodeling in
asthma compared to COPD. In asthma, inflammation and remodeling is mainly present in
the larger conducting airways. Depending on the severity of disease, small airways can
also be affected, however, the lung parenchyma is generally not affected in most patients
with asthma (94). The nature of the inflammation and the cell types involved in the
inflammatory response in asthma are different compared to COPD. Upon allergen
challenge, which is the main trigger for an inflammatory response in allergic asthma, mast
cells are activated. Mast cells release bronchoconstricting agents, including histamine and
lipid mediators. Moreover, CD4+ T‐helper type 2 (TH2) cytokines IL‐4, IL‐5 and IL‐13 are
released by mast cells (94, 97). In addition to mast cells, TH2 cells also release these
cytokines, and TH2 cells play a central role in orchestrating the inflammatory response in
asthma (94). IL‐4 release stimulates B cells to synthesize IgE, the driver of allergic
inflammation that binds to high‐affinity Fc receptors for IgE (FcεRI) on mast cells. The
release of IL‐5 attracts eosinophils, whereas the release of IL‐13 contributes to an
enhancement of airway hyperresponsiveness and an increase in goblet cell number (104,
105). There is also an increase in the size of submucosal glands (106), and together this
leads to excessive mucus production. Enhanced mucus secretion significantly contributes
to airflow limitation in asthma by obstructing the airways (41, 107). Furthermore, there is
thickening of the basement‐membrane, as a result of collagen deposition under the
epithelium, and thickening of the airway smooth muscle layer, as a result of airway
smooth muscle hyperplasia and hypertrophy (100). The former is observed in all patients
with asthma, whereas the latter is mainly observed in patients with severe asthma (108).
General introduction
21
Airway smooth muscle thickness is related to severity of the disease, but not to its
duration, since airway smooth muscle thickening is already present in children with
asthma (109, 110). Pulmonary vascular remodeling and increased angiogenesis are also
observed, which is mediated by various angiogenic proteins, including several growth
factors, like vascular endothelial growth factor (VEGF) (100, 111). Structural changes in the
airways of patients with asthma are considered an important component of airflow
limitation and may accelerate the decline in lung function (112, 113).
Muscarinic receptor regulation of inflammation and remodeling
Increasing evidence suggests a role for acetylcholine in regulating airway inflammation
and remodeling. From in vitro studies it is known that muscarinic receptor stimulation can
enhance the release of cytokines from inflammatory cells and structural cells of the
airways, either alone or in concerted action with stimuli including cigarette smoke and
growth factors. Moreover, acetylcholine has been shown to enhance remodeling
parameters in vitro. Thus, muscarinic receptor stimulation of airway smooth muscle cells
and fibroblast enhances proliferation and the production of extracellular matrix proteins
(16, 21). This is further supported by evidence from in vivo studies, demonstrating that
airway inflammation and remodeling can be inhibited by anticholinergic intervention. It
has been shown that cigarette smoke‐induced inflammation in mice, and LPS‐induced
inflammation and remodeling in guinea pigs can be inhibited by tiotropium, indicating the
potential relevance of anticholinergic treatment for patients with COPD (114, 115). In
addition, allergen‐induced inflammation and remodeling can be inhibited by tiotropium in
both mice and guinea pigs, indicating the potential relevance of anticholinergic treatment
for patients with asthma (116‐118). The role of acetylcholine in inflammation and
remodeling is extensively reviewed in chapter 2, and will therefore not be elaborated on
in more detail here.
Clinical implications
As briefly described above and reviewed in chapter 2, there is convincing evidence from in
vitro and in vivo animal studies that acetylcholine contributes to airway inflammation and
remodeling. This might be relevant for patients with COPD and asthma, especially since
acetylcholine release is enhanced in these disease states. However, this hypothesis still
needs to be proven by clinical studies investigating the effect of acetylcholine or
anticholinergic therapy on airway inflammation and remodeling. In the UPLIFT study,
COPD patients treated with tiotropium for 4 years showed improvements in lung function,
quality of life and exacerbation frequency (72). The latter might suggest an anti‐
inflammatory effect of tiotropium, as the inflammatory response is enhanced during
exacerbations, with increased expression of pro‐inflammatory cytokines, including IL‐8
Chapter 1
22
(119, 120). However, Powrie et al. were not able to demonstrate a reduction in sputum IL‐
6 or IL‐8 levels after tiotropium use for one year, even though the exacerbation frequency
was reduced (121). In this study, tiotropium reduced the amount of sputum, which might
result in a concentration of cytokines in the sputum of patients treated with tiotropium, as
proposed by the authors to explain this discrepancy. Therefore, further studies are needed
to elucidate the mechanism by which tiotropium reduces the number of exacerbations in
patients with COPD and whether a direct anti‐inflammatory effect of tiotropium is
involved. Structural changes in the airways of COPD patients may accelerate the decline of
lung function. Initially, it was thought that tiotropium might affect this process, since a
retrospective study suggested that the use of tiotropium was associated with a reduction
in decline of lung function after 1 year (122). However, this was not replicated in the large,
prospective UPLIFT trial, in which no significant effect on the rate of decline in lung
function was observed in the overall study population (72). Pre‐specified post‐hoc
subgroup analysis revealed that tiotropium did inhibit the accelerated decline in lung
function in young patients and in patients with moderate disease (123, 124). Future
studies are needed to understand the effects of tiotropium on lung function decline in
patients with COPD in these subgroups.
Recently, clinical studies into the effects of tiotropium in patients with asthma have
started. Addition of tiotropium to standard therapy for severe asthma patients with long‐
acting β‐agonists and corticosteroids increased the time to the first exacerbation and
reduced the risk of a severe exacerbation (90). Moreover, it has been shown that repeated
inhalation of the muscarinic receptor agonist methacholine induces airway remodeling in
mild asthma patients (125). This suggests that acetylcholine might also affect airway
inflammation and remodeling in patients with asthma, but clearly more studies are
needed to further elucidate the effects of acetylcholine or anticholinergic therapy on
airway inflammation and remodeling in patients with asthma.
Scope of the thesis
The above mentioned findings suggest that acetylcholine is not only a neurotransmitter
involved in bronchoconstriction and mucus secretion in the airways, but might also be a
mediator of airway inflammation and remodeling, which possibly involves non‐neuronal
acetylcholine. This will have implications for therapy of obstructive airways diseases like
COPD and asthma, in which muscarinic antagonists are frequently used. This thesis will
further elucidate the role of acetylcholine in inflammation and remodeling in COPD and
asthma. Specifically, the aim of this thesis is to investigate the role of individual muscarinic
General introduction
23
receptor subtypes, as well as the contribution of neuronal versus non‐neuronal
acetylcholine, to airway inflammation and remodeling in COPD and asthma.
Chapter 2 provides a comprehensive review on the regulation of airway inflammation and
remodeling in COPD and asthma by muscarinic receptors. In addition, therapeutic
implications of muscarinic receptor regulation of inflammation and remodeling are
discussed.
The following chapters deal with the role of acetylcholine in inflammation in COPD. As
stated above, there is evidence for a pro‐inflammatory role of acetylcholine in
inflammation. However, it is not known which individual muscarinic receptor subtypes are
involved in this response. There is a focus for anticholinergics on M3 receptor selectivity,
because of involvement of this receptor subtype in bronchoconstriction, however, there is
no evidence that the pro‐inflammatory effects of acetylcholine are also mediated via this
receptor subtype. Therefore, in chapter 3, we investigated the contribution of individual
muscarinic receptor subtypes to cigarette smoke‐induced inflammation. Because of
limited selectivity of available muscarinic receptor antagonists, muscarinic receptor
subtype specific knock‐out animals were used. In this way, the contribution of individual
muscarinic receptors can be investigated in detail. Knock‐out animals were exposed to
cigarette smoke for four days and the inflammatory response in the airways was analyzed.
The contribution of the M3 receptor to cigarette smoke‐induced inflammation was
investigated in more detail in chapter 4. As discussed above, practically all cells in the
airways express muscarinic receptors. It is not known which cell types contribute to the
pro‐inflammatory effect of the M3 receptor as described in chapter 3. To distinguish
between effects of structural cells and inflammatory cells, bone marrow chimeric animals
were generated and exposed to cigarette smoke, using a similar protocol as described in
chapter 3. In this way, the contribution of structural cells versus inflammatory cells to the
pro‐inflammatory effect of the M3 receptor can be defined.
In chapter 5, we further elucidated the role of acetylcholine in inflammation in patients
with COPD. In this chapter, we investigated the effects of targeted lung denervation (TLD)
on inflammation in patients with COPD. TLD is a novel potential therapy for patients with
COPD, in which parasympathetic airway nerves are ablated by locally applying radio‐
frequency energy in the main bronchi using bronchoscopy, to inhibit acetylcholine‐
induced bronchoconstriction. We hypothesized that TLD would also inhibit airway
inflammation. Specifically, we studied whether TLD affects inflammatory cell number and
pro‐inflammatory cytokine expression in bronchial wash fluid and gene expression of pro‐
inflammatory cytokines in bronchial brush specimen.
Chapter 1
24
The second half of the thesis is focused on the role of acetylcholine in inflammation and
remodeling in asthma. As is described above for COPD, it is not known which muscarinic
receptor subtypes mediate inflammation and remodeling in asthma. Therefore, in chapter
6, we investigated the effects of individual muscarinic receptor subtypes on allergen‐
induced inflammation and remodeling. To this aim, muscarinic receptor subtype specific
knock‐out animals were exposed to ovalbumin, and lungs were collected for analysis of
inflammation and remodeling.
Increasing evidence suggests that airway remodeling might not only be a consequence of
inflammation, but might also or alternatively be a consequence of bronchoconstriction,
which triggers TGF‐β release via biomechanical activation. In chapter 7, we aimed to
investigate the effect of TGF‐β and bronchoconstriction on remodeling, and the
involvement of the M3 receptor in this response, by using murine precision cut lung slices
(PCLS). TGF‐β was used to induce remodeling in PCLS from wild‐type animals. In addition,
PCLS were stimulated with methacholine, to investigate the effect of bronchoconstriction
on remodeling. Moreover, PCLS from M3R‐/‐ mice, in which bronchoconstriction is
abolished, were exposed to TGF‐β and methacholine, to investigate the involvement of
the M3 receptor in this response. After two days, expression of remodeling parameters, as
well as the functional impact on bronchoconstriction, was analyzed.
In chapter 8, we investigated the effects of endogenous non‐neuronal acetylcholine on
epithelial cell differentiation. There is evidence from in vivo studies which suggests an
indirect role for acetylcholine in epithelial cell differentiation and goblet cell metaplasia.
Here, we aimed to investigate direct effects of endogenous non‐neuronal acetylcholine on
epithelial cell differentiation and possible mechanisms involved. Human airway epithelial
cells were isolated from healthy donors and cultured at an air‐liquid interface. During
differentiation at the air‐liquid interface, cells were exposed to tiotropium, to investigate
the effects of acetylcholine on epithelial cell differentiation following air exposure, and to
IL‐13 and tiotropium, to investigate the effects of acetylcholine on IL‐13‐induced goblet
cell metaplasia.
Finally, in chapter 9, the results of this thesis are summarized and discussed. Perspectives
for future studies are provided.
General introduction
25
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