Muscarinic Receptors and Mediation of
Hormonal Effects of Acetylcholine
Muscarinic Receptor Agonists and Antagonists:
Effects on Inflammation and Immunity
Norah G. Verbout and David B. Jacoby
Abstract In this chapter, we will review what is known about muscarinic regula-
tion of immune cells and the contribution of immune cell muscarinic receptors to
inflammatory disease and immunity. In particular, immune cell expression of
cholinergic machinery, muscarinic receptor subtypes and functional consequences
of agonist stimulation will be reviewed. Lastly, this chapter will discuss the
potential therapeutic effects of selective antagonists on immune cell function and
inflammatory disease in recent animal studies and human clinical trials.
Keywords Immune cells • Inflammatory disease • Non-neuronal cholinergic
It is increasingly apparent that cells of the immune system express muscarinic
receptors that directly regulate their function. Since immune cells play an important
role in defense against pathogens and disease pathophysiology, it seems likely that
muscarinic regulation of immune cells contributes to pathology. In particular,
muscarinic modulation of immune cell function may be a significant target under
inflammatory settings. Indeed, several muscarinic receptor agonists and antagonists
are approved to treat several clinical conditions, including glaucoma, Sjogren’s
syndrome, chronic obstructive pulmonary disease (COPD), and asthma. In this
chapter, we will review what is known about muscarinic regulation of immune
N.G. Verbout (*)
School of Public Health, Harvard University, 665 Huntington Avenue, Boston, MA 02115, USA
Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239,
A.D. Fryer et al. (eds.), Muscarinic Receptors,
Handbook of Experimental Pharmacology 208,
DOI 10.1007/978-3-642-23274-9_17, # Springer-Verlag Berlin Heidelberg 2012
cells and the contribution of immune cell muscarinic receptors to inflammatory
disease and immunity.
1.1 Non-neuronal Cholinergic System in Immune Cells
The emergence of a non-neuronal cholinergic system has expanded the conven-
tional assumption that acetylcholine production is limited to the nervous system.
Acetylcholine is synthesized by nearly all mammalian cells and can play an integral
role in regulating the interactions of non-neuronal cells with their external environ-
ment. Indeed, many immune cells have been demonstrated to express the molecular
machinery required to synthesize, store, and release acetylcholine. Evidence for
acetylcholine synthesis, storage, release, and breakdown in immune cells has been
demonstrated using multiple methods, including immunoreactivity for choline-
acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and
choline transferase (CHT1) in isolated immune cells, reviewed in (Kawashima
and Fujii 2004).
While the essential components of the cholinergic system are present in many
immune cells (Table 1), the method by which immune cells produce and release
acetylcholine differs from the nervous system. Neuronal cells store acetylcholine
in discrete neurosecretory vesicles and release acetylcholine via exocytosis.
In contrast, non-neuronal cells appear to actively transport acetylcholine directly
upon synthesis (Wessler et al. 1999). The current understanding of how non-
neuronal cells release acetylcholine is based upon human placenta as a model
Table 1 Expression of cholinergic machinery in immune cells
Immune cell ACh ChAT AChE ChT 1 VAChT References
Lymphocyte +++ + (Cl)+ Kawashima et al. (1993), Neumann et al.
(2007), and Tayebati et al. (2002)
Hecker et al. (2006) and Neumann et al.
Wessler and Kirkpatrick (2001)
Hagforsen et al. (2000) and Neumann
et al. (2007)
Kawashima et al. (2007)
Monocyte+ + (A) NRNRNR
NR+ (<) +
NR+ (A) +(A) NRNR
NR++ (Cl) NRNR Nechushtan et al. (1996) and Wessler
et al. (2003)
Durcan et al. (2006) and Hagforsen et al.
Abbreviations: ACh acetylcholine; ChAT choline acetyltransferase; AChE acetylcholinesterase;
VAChT vesicular acetylcholine transporter; ChT1 high affinity choline transporter; NRnotreported
Symbols: + present; ? absent; +/? conflicting reports; Cl detected in cell lines, but not primary
cells; A detected in experimental animal; < detected in very low amounts
404 N.G. Verbout and D.B. Jacoby
system (Wessler et al. 2001), in which active transport of acetylcholine is
mediated by members of the organic cation transporter (OCT) family. Inhibitors
of organic cation transporters suppress acetylcholine release in human placenta
(Wessler et al. 2001), organic cation transporter-transfected oocytes (Lips et al.
2005) and airway epithelium (Kummer et al. 2006; Lips et al. 2005). Although
it is not yet confirmed that organic cation transporters control acetylcholine
release from immune cells, these transporters are expressed on nearly every
cell type, making them a probable candidate for immune cell acetylcholine
1.2 Acetylcholine in the Blood
Leukocytes are derived from a multipotent progenitor cell in the bone marrow.
Once produced, leukocytes migrate to various tissues throughout the body via
the systemic circulation and the lymphatic system, which consists of the thymus,
spleen, and lymphatic vessels. Although there is no evidence of cholinergic
innervation of the bone marrow or lymphatic system, it is likely that leukocytes
are exposed to acetylcholine in the blood. Indeed, human blood contains phy-
siologically relevant concentrations of acetylcholine (8.66 +/? 1.02 nM in whole
blood and 3.12 nM in plasma). This concentration is similar to values measured
in blood of chimpanzees, pigs, and rabbits (Fujii et al. 1995; Kawashima et al.
The source of acetylcholine in the circulation is not definitively known.
Kawashima et al. purport that lymphocytes are the main source of blood acetylcho-
line, since 60% of blood acetylcholine has been located in lymphocytes
(Kawashima et al. 1993). Acetylcholine degradation is regulated in part by acetyl-
cholinesterase (AChE), an enzyme that is present at sufficiently high concentrations
in the blood. The local concentration of acetylcholine at sites of muscarinic
receptors on immune cells in the blood is not known, but it is likely that acetylcho-
line hydrolysis in the plasma occurs at a much slower rate than what occurs at
neuromuscular junctions (Kawashima and Fujii 2000). Thus, physiologic levels of
acetylcholine are present in the blood, and may affect immune cells during migra-
tion to sites of inflammation.
Inflammation is one of the initial responses of the immune system to infection.
Symptoms of inflammation include redness and edema, caused by increased blood
flow into tissues. Injured or infected cells produce specific mediators that attract
immune cells to the site of inflammation. These pro-inflammatory mediators
prostaglandins that cause vasodilation of the local vasculature and leukotrienes
that attract leukocytes. Cytokines mediate communication between leukocytes and
chemokines promote leukocyte chemotaxis. In addition, growth factors and other
cytotoxic factors are present at sites of inflammation. These mediators act in concert
Muscarinic Receptor Agonists and Antagonists 405
to selectively recruit immune cells from the blood to the site of inflammation to
clear pathogens and promote healing of damaged tissue.
1.3 Early Evidence of Muscarinic Regulation of Immune Cells
Muscarinic modulation of the immune system is an evolving story. In 1976, Levy
et al. demonstrated that vagotomy and atropine protect against histamine shock and
lethal anaphylaxis in rats (Levy et al. 1976). Anaphylactic shock is an acute
physiological response to an allergen, characterized by systemic release of inflam-
matory mediators, leading to circulatory and respiratory collapse. Thus, this study
suggested that muscarinic blockade may modulate the immune response to
A subsequent study conducted in guinea pigs demonstrated that carbacholine
increased granulocytes and lymphocytes in venous splenic blood and decreased
spleen weight (Sandberg 1994). During an immune response, lymphoid tissues,
such as spleen are sites of immune cell proliferation. The splenic vein drains
blood from the spleen into the portal vein. Atropine alone had no effect on any
other parameters, but blocked the effects of carbacholine, indicating an effect on
muscarinic receptors. It is known that the spleen has adrenergic innervation,
though direct cholinergic innervation of the spleen is sparse or unreported.
However, there is some evidence indicating that lymphoid tissues are innervated
by parasympathetic fibers in rodents (Bulloch and Pomerantz 1984; Kendall and
al-Shawaf 1991). Indeed, in vitro, acetylcholine at 10?9to 10?4mol/l signifi-
cantly increased spleen cell proliferation induced by concanavalin A (Con A), a
lymphocyte mitogen (Qiu et al. 1995, 1996). Altogether, these studies suggested
that muscarinic stimulation has an immunomodulatory effect on cells of the
2 Expression of Muscarinic Receptors on Immune Cells
Table 2 gives an overview of the expression of muscarinic receptors on immune
cells in several different species. Recently, the specificity of antibodies raised
against subtype specific muscarinic receptors has been questioned (Jositsch et al.
2009). However, it should be noted that in the majority of the references given in
Table 1, more than one method had been utilized to demonstrate expression of
muscarinic receptors. The different methods used are indicated in the legend.
406 N.G. Verbout and D.B. Jacoby
2.1.1 Role in the Immune System
Lymphocytes are derived from a hematopoietic precursor in the thymus.
Lymphocytes can be broadly characterized into three major types: T cells, B
cells, and natural killer (NK) cells. NK cells are a part of innate immune system
and play a major role in host defense against tumors and viruses. T cells and B cells
are the major immune cells of the adaptive response. T cells participate in cell-
mediated immunity and B cells are associated with antibody production.
B and T lymphocytes coordinate the immune response to “non-self” antigens,
during a process known as antigen presentation. B cells respond to pathogens by
producing large quantities of antibodies that can neutralize foreign objects such as
bacteria and viruses. In response to pathogens, T helper cells secrete cytokines that
Table 2 Expression of muscarinic receptor subtypes in immune cells
Immune cellM1 M2M3M4 M5 SpeciesSource Detection References
Lymphocytes + (V) + (V)
W, I, B
Tayebati et al. (2002),
Ricci et al. (2002),
Tayebati et al.
et al. (2007), and
Costa et al. (1994)
Kawashima et al.
(2007), Costa et al.
(1994), and Pahl
et al. (2006)
Profita et al. (2005),
Gwilt et al. 2007,
et al. (2007)
Profita et al. (2005) and
Bany et al. (1999)
Neutrophils++ + (S)
Liu et al. (2010), Ma
et al. (2007),
et al. (2007)
Masini et al. (1983) and
Nemmar et al.
Profita et al. (2005),
Durcan et al.
(2006) and Profita
et al. (2005)
Abbreviations: B binding experiments; F functional experiments with agonists and antagonists;
I immunoreactivity; M detection of subtype specific mRNA; W Western blot
Symbols: +present; ?absent; V varied by individual subject; < present in very low amounts;
S present in smoker or COPD
Muscarinic Receptor Agonists and Antagonists 407
coordinate the immune response, and cytotoxic T cells release toxic proteins that
induce the death of pathogen-infected cells. Of the immune cells, lymphocytes are
the best characterized with regard to regulation by muscarinic receptors.
2.1.2 Cholinergic Components
Lymphocytes express most cholinergic components found in the nervous system,
including muscarinic, nicotinic, acetylcholine, choline acetyltransferase, vesicular
acetylcholine transporter, choline transferase 1, and acetylcholinesterase (Fujii
et al. 1999; Kawashima and Fujii 2004). Expression of muscarinic receptors has
been demonstrated in lymphocytes isolated from blood, lymph nodes, spleen and
thymus of mouse, rat, and human (Kawashima and Fujii 2000).
2.1.3 Muscarinic Receptors
The presence of muscarinic receptors has been detected via multiple methods in
lymphocytes obtained from experimental animals and humans. Radioligand bind-
ing studies demonstrate muscarinic binding in mouse (Atweh et al. 1984; Genaro
et al. 1993; Gordon et al. 1978; Kawashima et al. 2007) and rat lymphocytes (Costa
et al. 1994; Krzystyniak et al. 1982; Maslinski et al. 1980; Tominaga et al. 1992). In
human lymphocytes, muscarinic receptors have been detected by multiple methods,
including radioligand binding (Adem et al. 1986; Bidart et al. 1983; Ferrero et al.
1991; Rabey et al. 1986; Zalcman et al. 1981), reverse-transcription-polymerase
chain reaction (RT-PCR) and immunocytochemistry with monoclonal antibodies
(see Table 2 for subtypes).
All five muscarinic subtypes have been detected in human lymphocytes; how-
ever, receptor subtype expression appears to vary by individual. For example,
Tayebati et al. detected expression of M3, M4, and M5 muscarinic receptor
subtypes in all subjects tested, whereas expression of M1 and M2 varied by
individual (Tayebati et al. 2002). In healthy individuals, relative expression of
each subtype exhibited a pattern, with M3 being the most abundantly expressed,
followed in order by M5, M4, and M2, as determined by immunocytochemistry
(Tayebati et al. 2002) and radioligand binding (Ricci et al. 2002; Tayebati et al.
1999) techniques. It may be that receptor subtype differs under pathophysiological
conditions, since asthmatic patients have greater expression of M2 and M5 subtypes
in mononuclear lymphocytes (Ricci et al. 2002).
2.1.4Functional Changes in Lymphocytes Induced by Muscarinic Agonists
Most available data of the effects of muscarinic stimulation on lymphocytes have
been derived from in vitro studies (see review (Kawashima and Fujii 2004)).
Stimulation of muscarinic receptors on lymphocytes by acetylcholine or other
408 N.G. Verbout and D.B. Jacoby
agonists initiates intracellular signaling via increased inositol-1, 4,5-triphosphate
(IP3) content, inhibition of cAMP production, and increased intracellular calcium
[Ca2+]ivia muscarinic receptor coupling to either phospholipase C (PLC) (M1, M3,
M5) or adenylyl cyclase (M2, M4).
Muscarinic receptor subtype expression has been well characterized in human
lymphocyte cell lines (Kawashima and Fujii 2004), and these have been used as a
model system to examine subtype specific responses to muscarinic stimulation in
lymphocytes. In human B and T cell lines, both acetylcholine and oxotremorine-M
(Oxo-M), a non-selective muscarinic agonist, induced intracellular calcium release
and increased c-fos gene expression, a transcription factor upregulated in activated
lymphocytes (Fujii and Kawashima 2000a, b). These effects of muscarinic stimu-
lation were inhibited by 4-DAMP (M1, M3, M4, M5), YM905 (M1, M3) and
atropine (Fujii and Kawashima 2000a, b, c). Conversely, neither pirenzipine (M1)
nor AF-DX 16 (M2, M4) had any effect. Taken together, these experiments suggest
that muscarinic agonists increase intracellular signaling and increase gene expres-
sion in lymphocytes via M3 and/or M5 muscarinic receptors.
2.1.5 Cytokine Production and Proliferation
During an immune response, lymphoid tissues, such as the thymus or spleen are
sites of lymphocyte proliferation. Activated cytotoxic T cells undergo proliferation
induced by IL-2, a cytokine that induces growth and differentiation. This IL-
2 mediated activation increases the number of antigen-specific lymphocytes,
thereby enhancing the immune response.
In vitro, acetylcholine enhances mitogen (ConA)-induced T-cell proliferation in
rat spleen cell cultures, an effect blocked by atropine (Qiu et al. 1995). Similarly,
IL-2 production is enhanced by Oxo-M following stimulation with phytohaemag-
glutinin (PHA), a T cell mitogen (Fujino et al. 1997). Pretreating T cells with the
acetylcholine or Oxo-M in the presence of an acetylcholinesterase inhibitor
enhances mitogen-induced IL-2 production (Nomura et al. 2003; Okuma and
Nomura 2001), suggesting that acetylcholine produced by lymphocytes acts in a
paracrine/autocrine fashion. It is probable that IL-2 mediated signal transduction in
lymphocytes is also regulated by muscarinic receptors since treatment with Oxo-M
also increased gene expression of the IL-2 receptor. Similarly, Fujino et al. (1997)
reported stimulatory effects of Oxo-M on IL-2 production and proliferation in T
cells (Fujino et al. 1997). Altogether, these studies suggest that IL-2 acts as an
autocrine factor via muscarinic receptors during immunological interactions. It is
known that many immune tasks performed by T cells depend on IL-2 production,
which is a key cytokine for regulating immunity.
It is interesting to note that different muscarinic agonists affect lymphocyte
function in diverse ways. For instance, arecoline, a partial muscarinic agonist
used clinically in Alzheimer’s disease, has immunosuppressive, rather than stimu-
latory effects on lymphocytes. Chronic administration of arecoline reduced spleen
size compared to untreated control mice (Wen et al. 2006). Other lymphoid organs
Muscarinic Receptor Agonists and Antagonists409
including the thymus and mesenteric lymph nodes have also been found to have
modest weight reductions after chronic arecoline treatment (Selvan et al. 1989). In
vitro, Wen et al. found that chronic arecoline treatment decreased splenocyte
proliferation induced by Con A or lipopolysaccharide (LPS), and reduced IL-
2 secretion, effects that were reversed by pretreatment with atropine. Yet another
study found that pilocarpine, an M2 agonist, had no effect on IL-2 production;
however, it did decrease the number of IL-2 receptor expressing cells (Prync et al.
The underlying reason for opposing effects of different muscarinic agonists on
IL-2 expression and signaling is not known, but it may reflect differences in
receptor subtypes on discrete lymphocyte subpopulations, since it is known that
an array of subtype combinations are expressed among lymphocytes within indi-
vidual subjects (Tayebati et al. 2002). Alternatively, it may be that lymphocyte
populations differ in their phenotype, for example, the muscarinic receptor targeted
by the arecoline could be expressed on a suppressor lymphocyte, and the musca-
rinic receptor targeted by Oxo-M found on an activating type.
Upon stimulation, naı ¨ve CD8+ lymphocytes differentiate into cytolytic T cells,
which are the main effector mechanism by which the immune system clears
pathogen-infected cells. It is known that differentiation of naı ¨ve CD8+ lymphocytes
into cytolytic T cells requires activation. Since acetylcholine regulates T cell
proliferation via muscarinic receptors, it is conceivable that muscarinic signaling
may modulate generation of cytolytic T cells. In support of this, Zimring et al.
reported that CD8+ T cells from M1 receptor knockout mice were defective in their
ability to differentiate into cytolytic T lymphocytes in vitro (Zimring et al. 2005).
However, a subsequent study published by the same authors found evidence to the
contrary. In vivo, there was no identifiable defect in virus-induced CD8+ T cell
expansion in M1 mice knockout mice (Vezys et al. 2007). Some potential
explanations for these discrepancies include differences in experimental stimulus
conditions (standard proliferation assay versus whole animal viral infection) and
differences in antigenicity, mouse strain, or compensation by redundant pathways.
Despite these potential reasons for contrasting results, it appears that the data
supporting muscarinic regulation of T cell differentiation are somewhat limited.
2.1.7 Immunoglobulin Class Switching
There is evidence that muscarinic receptors on lymphocytes may modulate anti-
body class switching. Fujii et al. examined this hypothesis in dual M1/M5 knockout
mice exposed to ovalbumin (OVA) protein (Fujii et al. 2007). They found that
serum levels of total and OVA specific IgG were significantly lower in M1/M5
knockout mice compared to wildtype mice. In addition, IL-6 secretion was reduced
410 N.G. Verbout and D.B. Jacoby
in activated spleen cells from M1/M5 KO mice, suggesting that M1 and or M5
receptors contribute to IL-6 production, leading to modulation of antibody class
switching from the IgM type to the IgG1. There were no differences in serum level
of total and anti-OVA specific IgM between the KO and WT, thus M1 and M5 do
not appear to contribute to the initial generation of antibodies.
2.2.1 Role in the Immune System
Monocytes are produced by the bone marrow, travel via the bloodstream to
populate various tissues where they differentiate into macrophages or dendritic
cells. Monocytes and their macrophage and dendritic cell progeny serve three main
functions in the immune system. These are phagocytosis, antigen presentation, and
2.2.2 Cholinergic Components
Human mononuclear cells (monocytes and lymphocytes) isolated from peripheral
blood produce acetylcholine (Neumann et al. 2007). In rat monocytes, expression of
ChAT has been detected (Hecker et al. 2006); however, there are no reports of
ChAT expression in human primary monocytes or in a monocytic cell line (Fujii
et al. 1999).
2.2.3 Muscarinic Receptors
Expression of muscarinic receptors on monocytes appears to be mixed. Early
studies suggested that human peripheral monocytes do not express muscarinic
receptors. Using radiolabeling techniques, Eva et al. found that human peripheral
monocytes do not bind NMS (Eva et al. 1989) and neither was mRNA for any of the
five subtypes detected by RT-PCR (Hellstrom-Lindahl and Nordberg 1996). These
reports contrast with a study conducted by Pahl et al., which found that human
monocytes express mRNA for M3, M4 and M1 muscarinic receptors and possibly
M2 (Pahl et al. 2006). M5 mRNA was not detected. Inflammatory status may also
affect receptor subtype expression in monocytes, since treatment with LPS, a
component of bacterial cell walls, modulates gene expression of muscarinic recep-
tor subtypes (Pahl et al. 2006). Despite the conflicting reports, functional data
suggest that monocytes probably do express muscarinic receptors.
Muscarinic Receptor Agonists and Antagonists411
2.2.4 Functional Changes in Monocytes Induced by Muscarinic Agonists
Acetylcholine stimulates ERK1/2 signaling and leukotriene (LTB4) production in
blood monocytes, an effect blocked by oxitropium bromide (Profita et al. 2005).
2.3.1 Role in the Immune System
Macrophages are derived from a monocytic precursor produced in the bone mar-
row. In the blood, monocytes are recruited to the tissues, where they differentiate
into tissue-specific resident macrophages. Macrophages play an important role in
the innate response to pathogens by phagocytosing cellular debris and pathogens
and releasing factors that stimulate lymphocytes and other immune cells.
2.3.2 Cholinergic Components
In the lung, human alveolar macrophages express ChAT (Wessler and Kirkpatrick
2001) and likely produce acetylcholine (Wessler et al. 1999). This may not be the
case in other species, since peritoneal macrophages from C57BL/6J mice do not
appear to express mRNA for ChAT (Kawashima et al. 2007).
2.3.3 Muscarinic Receptors
Lung macrophages (Gwilt et al. 2007) and macrophages isolated from human
sputum express M2 and M3 receptors (Profita et al. 2005). Similarly, bovine
alveolar macrophages express M3 receptors (Sato et al. 1998). In mice, mRNAs
encoding all five muscarinic receptor subtypes are expressed in peritoneal
macrophages from C57BL/6J mice (Kawashima et al. 2007).
2.3.4Functional Changes in Macrophages Induced by Muscarinic Agonists
In bovine alveolar and human alveolar and sputum macrophages, acetylcholine
stimulates release of lipoxygenase-derived inflammatory mediators, in particular
leukotriene B4 (LTB4) acting via M3 muscarinic receptors (Buhling et al. 2007;
Profita et al. 2005; Sato et al. 1998). LTB4 is a potent inflammatory mediator that
increases leukocyte adhesion, activation, and neutrophil recruitment. Thus acetyl-
choline acting via muscarinic receptors may increase inflammation by recruiting
inflammatory cells via release of macrophage-derived chemotactic mediators.
412 N.G. Verbout and D.B. Jacoby
2.4.1 Role in the Immune System
Neutrophils are granulocytic leukocytes that are produced in the bone marrow.
They are the most abundant leukocyte found in the blood. Neutrophils are part of
the acute inflammatory response and are robustly recruited from the vasculature to
sites of injury, inflammation, or infection via chemoattractant mediators.
2.4.2 Cholinergic Components
Choline acetyltransferase has been detected in human skin neutrophils, peripheral
blood neutrophils (Hagforsen et al. 2000), and peripheral granulocytes (Wessler
et al. 1999). Human peripheral blood granulocytes have also been found to contain
low levels of acetylcholine (Neumann et al. 2007).
2.4.3 Muscarinic Receptors
M3, M4, and M5, but not M1 or M2 muscarinic receptors have been detected on
neutrophils by immunocytochemistry (Profita et al. 2005) and RT-PCR (Bany et al.
2.4.4Functional Changes in Macrophages Induced by Muscarinic Agonists
There is little evidence to indicate that neutrophil function is regulated by musca-
rinic receptors. According to Profita et al., acetylcholine increased chemotactic
activity and LTB4 production in sputum neutrophils, though it is not clear whether
this is a muscarinic or nicotinic effect, since specific antagonists were not tested
(Profita et al. 2005).
2.5 Dendritic Cells
2.5.1 Role in the Immune System
Dendritic cells are professional antigen-presenting cells produced in the bone
marrow. Immature dendritic cells migrate via the bloodstream to the tissues. As a
part of their antigen-sensing role, dendritic cells are located in tissues that interface
with the external environment, for example the skin, the nose, lungs, stomach, and
Muscarinic Receptor Agonists and Antagonists413
intestines. Once activated, dendritic cells migrate to the lymph node where they
interact with lymphocytes and initiate the adaptive immune response.
2.5.2 Cholinergic Components
In mice, unstimulated bone-marrow derived dendritic cells do not appear to express
ChAT; however, stimulation with the bacterial wall component, LPS induces ChAT
expression, suggesting that acetylcholine synthesis is an outcome of dendritic cell
activation (Kawashima et al. 2007). AChE mRNA is expressed in mouse bone
marrow-derived dendritic cells (Kawashima et al. 2007), and dendritic cells may
also contain acetylcholine (Wessler et al. 1999).
2.5.3 Muscarinic Receptors
In mice, mRNAs encoding all five muscarinic receptor subtypes are expressed in
bone marrow-derived dendritic cells from C57BL/6J mice (Kawashima et al. 2007),
and M2 muscarinic receptors have been detected in dendritic cells in mouse gut (Ma
et al. 2007). In humans, dendritic cells from nasal mucosa express M3 muscarinic
receptors; however, very little expression was detected by flow cytometry in
peripheral blood dendritic cells (Liu et al. 2010), underscoring the observation
that the local environment may modulate subtype expression.
2.5.4 Functional Changes in Dendritic Cells Induced by Muscarinic Agonists
Methacholine induces dendritic cells to produce OX40L, a ligand expressed on
activated dendritic cells that contributes to immune cell interactions (Liu et al.
2010). It is likely that this effect is mediated by muscarinic receptors, since
methacholine has little effect on nicotinic receptors.
2.6 Mast Cells
2.6.1 Mast Cells in the Immune System
Mast cells are resident tissue cells derived from a hematopoetic precursor produced
in the bone marrow. Immature mast cells migrate to the tissues via the blood stream
and mature in the tissues. Mast cells play a key role in the inflammatory process by
producing large quantities of protein mediators. Activated mast cells rapidly release
protein granules and various inflammatory mediators into the interstitium. Mast cell
degranulation is triggered by tissue injury, cross-linking of Immunoglobulin E
(IgE) receptors, or by activated complement proteins. Mast cells are a major source
414 N.G. Verbout and D.B. Jacoby
of histamine, which dilates post capillary venules, activates the endothelium and
increases blood vessel permeability. This leads to local edema, warmth, redness,
and recruitment of other inflammatory cells to the site of release.
2.6.2 Cholinergic Components
At present, there is no direct evidence that mast cells produce acetylcholine;
however, ChAT immunoreactivity has been detected in human mast cells in the
skin (Wessler et al. 2003) and mRNA for AChE has been detected in a murine mast
cell line (Nechushtan et al. 1996), suggesting that mast cells may participate in
2.6.3 Muscarinic Receptors
Muscarinic receptors have been identified on rodent mast cells (Masini et al. 1983),
with M1 as the best characterized subtype. Data from studies on airway disease
suggest that mast cells from humans and rabbits also express M1 muscarinic
receptors (see below).
2.6.4 Functional Changes in Mast Cells Induced by Muscarinic Agonists
Muscarinic regulation of mast cell degranulation differs by species. For example, in
rabbits and rats, muscarinic agonists stimulate mast cell degranulation (Masini et al.
1985; Nemmar et al. 1999). Similarly, carbachol induces degranulation in a rat
basophil leukemic cell line transfected with M1 receptors (Jones et al. 1991). There
is also some indication that allergy may affect rodent mast cell responses to
acetylcholine, since mast cells in allergen-sensitized rats are more sensitive to
acetylcholine-induced histamine release compared to non-sensitized rats (Masini
et al. 1985).
While muscarinic agonists promote histamine release in rats and rabbits, in
human airways, muscarinic receptors are inhibitory. Acetylcholine inhibits iono-
phore induced histamine release in human mucosal mast cells through an M1-
mediated pathway (Reinheimer et al. 2000). Allergen-induced histamine release is
similarly inhibited in human airways (Reinheimer et al. 1997). Altogether, these
studies suggest that this inhibitory pathway may be important in pathological
conditions, such as asthma or COPD. Wessler et al. have examined histamine
release in mucosal mast cells in tracheas. In healthy controls, oxotremorine reduced
ionophore induced histamine release, but had little effect in COPD patients,
suggesting that muscarinic inhibition of mast cells in COPD patients is
dysregulated (Wessler et al. 2007). It is not known whether the interaction between
acetylcholine and mast cells, a key effector cell in asthma, contribute to the
pathophysiology of asthma. One possibility is that inhibitory muscarinic receptors
Muscarinic Receptor Agonists and Antagonists415
on mast cells normally limit allergen-induced histamine release, but in asthma, this Download full-text
pathway is dysfunctional. If this were the case, blockade of M1 muscarinic
receptors on mast cells may worsen allergic asthma. It may be important to
emphasize that studies using human mast cells have only been performed in
whole tissue (Reinheimer et al. 1997, 2000; Wessler et al. 2007), therefore one
cannot rule out the contribution of other cell types, for instance structural cells and
nerves within the trachea.
2.7.1 Role in the Immune System
Eosinophils are granulocytic leukocytes that play a role in immune defense and are
implicated in pathogenesis of allergic disorders including asthma, rhinitis, and
atopic dermatitis (Hogan 2007). In the bone marrow, eosinophils develop and
mature in response to specific cytokines, IL-3, IL-5, and GM-CSF. Following
maturation, eosinophils circulate in the blood and migrate to inflammatory sites
within tissues in response to chemoattractant mediators, including CCL11 and
CCL2, and CCL5. Activated eosinophils release granular proteins and inflamma-
tory mediators at sites of infection or inflammation.
2.7.2 Cholinergic Components
There is conflicting evidence of cholinergic components or muscarinic receptors in
eosinophils. Very small amounts of ChAT have been reported to be present in
peripheral blood eosinophils (Hagforsen et al. 2000) via Western blot. This
contrasts with Durcan et al., who did not detect mRNA for ChAT or VAChT in
human peripheral blood eosinophils via RT-PCR (Durcan et al. 2006). There are no
reports of eosinophils producing acetylcholine.
2.7.3 Muscarinic Receptors
Profita et al. (2005) report that human sputum eosinophils are negative for M1, M2
and M3 muscarinic receptors (Profita et al. 2005). However, it may be that musca-
rinic receptors on eosinophils are inducible, since the same study detected positive
M1 immunostaining in sputum eosinophils from patients with COPD. In human
peripheral blood eosinophils, mRNA and protein expression of M3, M4, and M5, but
not M1, M2muscarinic receptors has been detected (Verbout 2008). In addition,
guinea pig peritoneal and blood eosinophils express M3and M4, but not M1, M2, or
M5muscarinic receptors (Verbout 2008).
416N.G. Verbout and D.B. Jacoby