Non-neuronal Cholinergic Muscarinic Acetylcholine Receptors in the Regulation of Immune Function

Abstract

Immune cells such as T and B cells, monocytes and macrophages all express most of the cholinergic components of the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase (AChE). Because of its efficient cleavage by AChE, ACh synthesized and released from immune cells acts only locally in an autocrine and/or paracrine fashion at mAChRs and nAChRs on themselves and other immune cells located in close proximity, leading to modification of immune function. Immune cells generally express all five mAChR subtypes (M₁–M₅) and neuron type nAChR subunits α2–α7, α9, α10, β2–β4. The expression pattern and levels of mAChR subtypes and nAChR subunits vary depending on the tissue involved and its immunological status. Immunological activation of T cells via T-cell receptor-mediated pathways and cell adhesion molecules upregulates ChAT expression, which facilitates the synthesis and release of ACh. At present, α7 nAChRs expressed in macrophages are receiving much attention because they play a central role in anti-inflammatory cholinergic pathways. However, it now appears that through modification of cytokine synthesis, G_q/11-coupled mAChRs play a prominent role in regulation of T cell proliferation and differentiation and B cell immunoglobulin class switching. It is anticipated that greater understanding of G_q/11-coupled mAChRs on immune cells will provide an opportunity to develop new and effective treatments for immunological disorders.

1. INTRODUCTION

Acetylcholine (ACh) is a well-characterized neurotransmitter in the central and peripheral nervous systems. Notably, however, ACh is also detectable in eukaryotes lacking nervous systems, such as sponges, yeast, fungi and plants, as well as in prokaryotes such as eubacteria and Archaea.^1–5) These findings suggest that cholinergic systems have been expressed in life-forms on earth as a local signaling molecule in non-neuronal cells and tissues for at least 2.5 billion years and that ACh has been utilized as a neurotransmitter since animals with nervous systems emerged about half a billion years ago.^1–5)

In mammalian species, non-neuronal cholinergic systems have been detected, for example, in reproductive organs (ovary, placenta, and amnion), immune cells, epidermal keratinocytes, airway and alimentary epithelial cells and vascular endothelial cells, as well as in cancers of the lung, female breast and gastrointestinal tract.⁴⁾ The first International Symposium on Non-Neuronal ACh (ISNNACh) was held in San Francisco, California in 2002, and four subsequent ISNNACh symposia have contributed greatly to the recognition and understanding of the biological significance of non-neuronal ACh and its function under both physiological and pathological conditions. Here we will briefly review findings reported at the ISNNACh^4,6–9) and in other relevant articles to summarize the roles played by muscarinic acetylcholine receptors (mAChRs) expressed on immune cells in the regulation of immune function.

2. CHOLINERGIC COMPONENTS EXPRESSED IN IMMUNE CELLS

The cholinergic system components found in the nervous system, which include 1) ACh, 2) the ACh-synthesizing enzyme choline acetyltransferase (ChAT), 3) muscarinic and nicotinic AChRs (mAChRs and nAChRs, respectively) and 4) acetylcholinesterase (AChE), are also detected in immune cells, including T and B cells, macrophages and dendritic cells (DCs) (see reviews by Fujii et al.)^10,11) (Fig. 1).

Fig. 1. Immune Cells Express Cholinergic Components

Immune cells express the ACh-synthesizing enzyme ChAT, which catalyzes the synthesis of ACh from choline (taken up into cells by CHT1) and acetyl-CoA. ChAT expression is upregulated by immunological stimulation of immune cells, especially Th cells, which promotes ACh synthesis and release. When ACh is released from immune cells, mAChRs and nAChRs are activated in an autocrine/paracrine manner. α7 nAChRs do not function as ion channels in immune cells but are associated with G proteins and the tyrosine kinases JAK2 and Lck, which may mediate anti-inflammatory effects. M₁, M₃ and M₅ mAChRs are coupled to G_q/11 proteins, which activate IP₃-mediated Ca²⁺ increase and PKC. M₂ and M₄ mAChRs are coupled to G_i/o proteins, which suppress PKA activation by inhibiting AC activity. mAChRs generally act to promote immune cell proliferation and differentiation and the secretion of pro-inflammatory cytokines from immune cells.

2.1. ACh

ACh expression in immune cells reflects an essential and direct requirement for the cholinergic system of those cells. However, because of the difficulty of measuring the low concentrations of ACh with conventional HPLC-electrochemical detector (ECD) procedures, few have provided data on the ACh content of immune cells (see reviews by Kawashima and Fujii).^2,12,13) Moreover, it has been suggested that detection of ACh within immune cells does not prove that ACh was synthesized in those cells, as it could have been taken up from the extracellular space. However, that possibility is highly unlikely, given the extreme enzymatic and physicochemical lability of ACh in body fluids (see a review by Kawashima et al.).¹⁴⁾

2.2. ChAT

Immune cell expression of ChAT has been identified through 1) detection of ACh synthesizing activity, 2) detection of ChAT gene expression with RT-PCR or real-time PCR (quantitative (q)PCR), 3) detection of ChAT protein with Western blot analysis,¹⁵⁾ and 4) detection of fluorescent ChAT-reporter proteins in genetically engineered ChAT^BAC- enhanced green fluorescent protein (eGFP)¹⁶⁾ or ChAT-Cre-tdTomato mice.¹⁷⁾

1) Determination of ACh Synthesizing Activity

Detection of ACh synthesizing activity using the Fonnum procedure in rabbit buffy coat cells and the human blood mononuclear cell fraction revealed that immune cells have the ability to synthesize ACh.^18–20) Likewise, ACh synthesizing activity was found in rat T and B cells and in mouse and human immune cell lines.²¹⁾ Because both ChAT and the mitochondrial enzyme carnitine acetyltransferase (CarAT) are able to catalyze ACh synthesis from choline and acetyl-CoA,²²⁾ ACh synthesizing activity in the homogenates of immune cells as well as peripheral tissues determined with the Fonnum procedure (1975) represents the sum of these two enzyme activities.^19,20) Using bromo-ACh and bromo-acetylcarnitine, specific inhibitors of ChAT and CarAT, respectively, Fujii et al. detected far greater ACh synthesis by CarAT than by ChAT in various human leukemic cell lines.²³⁾ However, phytohemagglutinin (PHA)-induced activation of MOLT-3 human leukemic T cells via T-cell receptor (TCR)-mediated pathways enhanced only ChAT activity.²³⁾ This suggests ChAT expression in T cells is regulated by immune activity, and thus, CarAT is not involved in regulation of immune function.

2) Detection of ChAT mRNA Expression with RT-PCR or qPCR

Using RT-PCR, Fujii et al. for the first time detected expression of ChAT mRNA in the MOLT-3 T cell line,¹⁵⁾ confirming ACh synthesis by ChAT in immune cells. Later, ChAT mRNA expression was similarly detected in rat T and B cells,²⁴⁾ human lymphocytes,^25,26) and mouse DCs.²⁷⁾

3) Detection of ChAT Protein with Western Blotting

Using Western blot analysis, expression of ChAT protein was first detected by Fujii et al. in the MOLT-3 T cell line. Along with the data on ChAT mRNA expression in MOLT-3 cells,¹⁵⁾ these findings confirm the synthesis of ACh by ChAT in immune cells. Western blotting has also revealed the expression of ChAT protein in mouse CD4⁺ T cells.²⁸⁾

4) Detection of Fluorescent ChAT-Reporter Proteins

Tallini et al. detected fluorescent ChAT-reporter proteins in a subset of lymphocytes within the gastrointestinal and airway mucosa.¹⁶⁾ Moreover, Rosas-Ballina et al. observed ChAT-eGFP expression in 4.4% of splenic CD4⁺ T cells from ChAT^BAC-eGFP mice,²⁹⁾ while Gautron et al. found small populations of ChAT-expressing splenic T and B cells in ChAT-Cre-tdTomato mice.¹⁷⁾ These findings confirmed the discovery of the ChAT expression in T cells.^10,11)

Although ChAT activity is expressed in a variety of immune cells, including T cells, B cells, DCs, and macrophages, its expression and activity are particularly high in helper T (Th) cells.^21–23) In addition, various immune stimuli such as antigen presentation and cytokines increase both the expression and activity of ChAT in Th cells,²³⁾ which enhances ACh release in both the short and long terms. Thus, immune cell function is affected by both cytokines and ACh released from activated Th cells, which acts on AChRs expressed in other immune cells.^10,11,30)

2.3. mAChRs and nAChRs

Both mAChRs and nAChRs are expressed in lymphocytes, monocytes and macrophages (see reviews, Kawashima and Fujii; Kawashima et al.; Fujii et al.).^{2,10–13,27)} Because the expression patterns and levels of each mAChR subtype and nAChR subunit vary depending on the specific tissue and its immunological status,^31,32) to fully understand their functions, it is important that they are investigated under a variety physiological and/or pathophysiological conditions. Furthermore, it is noteworthy that interindividual variations in the ACh content of blood, plasma and mononuclear leukocytes (MNLs) in general populations of humans and animals are typically far larger than in genetically homogeneous animals bred in aseptic laboratories.^15,33,34) This suggests that the interindividual variations in the expression patterns and levels of mAChR subtypes and nAChR subunits are also far larger than those observed in laboratory animals. Therefore, when analyzing clinical data, it is essential that adequate numbers of samples are studied when assessing the significance of changes in the expression patterns or levels of mAChR subtypes and nAChR subunits.

1) mAChRs

mAChRs consist of a five-member family of seven transmembrane G protein-coupled receptors (M₁–M₅)^35,36) (Fig. 1). M₁, M₃ and M₅ mAChRs are G_q/11-coupled receptors. Upon their activation, phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate to produce diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). IP₃ binds to its receptors on the endoplasmic reticulum membrane to induce Ca²⁺ release from intracellular stores. DAG binds to and activates protein kinase C (PKC), which then catalyzes the phosphorylation of various proteins. On the other hand, M₂ and M₄ mAChRs are G_i/o-coupled receptors that inhibit cAMP production by suppressing adenylate cyclase activation, leading to inactivation of protein kinase A (PKA). Based on the physiology and distribution of the five mAChR subtypes, it is apparent that mAChRs are able to selectively trigger different signal transduction pathways in a cell- and tissue-specific manner.

All five mAChR subtypes are expressed in immune cells from humans, mice and rats.^10,11,27,31) The M₄ and M₅ subtypes appear to be constitutively expressed in healthy adult MNLs, whereas the expression patterns of the M₁–M₃ subtypes vary among individuals¹⁴⁾ (Table 1), and the same expression patterns are seen in human T cell lines.³¹⁾ Although the expression patterns of the five mAChR subtypes differ during immunological activation, it has been shown using genetically engineered mice that some are involved in regulating immune responses (also see reviews by Fujii et al.).^{10,11,37–39)} But because mAChRs are ubiquitously expressed throughout the body, and it has proved difficult to develop specific agonists and antagonists for the different mAChR subtypes, there has been less research interest in the role of mAChRs in the regulation of immune function than in the role of nAChRs. However, the regulation of antibody synthesis by M₁/M₅ mAChRs suggests that M₁/M₅ mAChR agonists could be utilized to facilitate antibody production and/or lymphocyte proliferation in ex vivo culture settings.³⁹⁾

Table 1. Gene Expression of mAChR Subtypes and nAChR Subunits in Human Immune Cells

mAChR subtypes
Gender	Cell type	Sample	M1	M2	M3	M4	M5
Female	MNLs	1	+	+	+	+	+
		2	−	+	−	+	+
		3	+	+	+	+	+
		4	+	−	+	+	+
Male	MNLs	1	+	+	−	+	+
		2	+	−	+	+	+
		3	−	+	+	+	+
nAChR subunits
Gender	Cell type	Sample	α3	α5	α7	α9	α10
Female	T cells	1	+	+	+	+	+
		2	+	+	−	+	−
		3	+	+	−	−	−
		4	+	+	+	+	−
		5	+	+	−	+	−
		6	−	−	−	−	−
		7	+	−	−	−	−
		8	−	+	+	+	+
	B cells	1	+	+	+	−	+
		2	+	+	+	+	+
		3	+	+	+	+	+
		4	+	+	+	+	+
		5	+	−	−	+	−
		6	+	+	+	+	+
		7	−	+	+	+	+
		8	+	+	+	+	+

+, positive expression; −, negative expression; F, female; M, male; MNLs, mononuclear leukocytes. Arranged from data presented in Ref. 14.

2) nAChRs

nAChRs are composed of hetro- or homopentameric assemblies of various subunits (α1–α10, β1-β4, γ, δ, and ε), which form ion channels permeable to Na⁺, K⁺ and Ca²⁺.^40,41) The nAChRs expressed on immune cells are mainly composed of the neuron-type α2, α5, α6, α7, α9, α10, β2 and β4 subunits.¹⁰⁾ Similar to the mAChR subtype, the expression patterns of nAChR subunits vary among individuals (Table 1). The homopentameric α7 nAChR is one of the most abundant nAChRs in the nervous system and is also expressed in many non-neuronal cells (see reviews by Grando et al.; Fujii et al.).^9,10) Prominent features of α7 nAChRs expressed in nerve cells are 1) high Ca²⁺ permeability; 2) high sensitivity to α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), two specific α7 nAChR antagonists; and 3) rapid desensitization.⁴²⁾ In particular, α7 nAChRs involved in cholinergic anti-inflammatory pathways that downregulate 1) the synthesis of proinflammatory cytokines such as tumor necrosis factor (TNF)-α in macrophages; 2) the lethality of lipopolysaccharide (LPS)-induced sepsis; and 3) antigen presentation on DCs.^43–48) Moreover, the enhancement of serum antigen-specific immunoglobulin G₁ (IgG₁) and proinflammatory cytokine production in α7 subunit gene-deficient mice immunized with ovalbumin (OVA) suggest a role for α7 nAChR in the regulation of immune function.⁴⁹⁾

The nAChR subunit contains an intracellular loop between M3 and M4 that is involved in subunit clustering and localization to the plasma membrane, but also binds proteins involved in intracellular signaling.^50–52) The M3-M4 loop is less homologous among nAChR subunits, which binds different intracellular proteins. The M3-M4 loop in α7 nAChRs interacts with G_αq, which can directly activate G_αq and downstream signaling via PLC- and IP₃-induced Ca²⁺ release in PC12 cells.^50–52) Therefore, nAChRs have long been recognized to be ligand-gated ion channels, but α7 nAChRs may also act as metabotropic receptors. Consistent with that idea, 1) nicotine-evoked activation of α7 nAChRs on T cells induces no electric currents ascribable to the entry of extracellular Ca^{2+53, 54)}; 2) α7 nAChRs form a functional TCR/CD3 complex with leukocyte-specific tyrosine kinase (Lck), activation of which leads to Ca²⁺ release from intracellular Ca²⁺ stores in T cells⁵⁵⁾; 3) α7 nAChRs are associated with phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), which exert anti-inflammatory effects in macrophages⁵⁶⁾; and 4) α-BTX and MLA, specific inhibitors of ionotropic α7 nAChRs, do not reverse the suppression of proinflammatory cytokine synthesis induced by GTS-21, an α7 nAChR agonist, in macrophages.⁵⁷⁾ Taken together, these findings suggest that α7 nAChRs on immune cells function as metabotropic receptors in a cholinergic anti-inflammatory pathway that is independent of ion channel signaling.^53,54,57)

2.4. AChE

AChE rapidly hydrolyzes ACh into choline and acetate, terminating the action of ACh at its receptors within a few milliseconds. Expression of AChE mRNA has been detected in murine lymphocytes, macrophages and DCs²⁷⁾ and in human leukemic T and B cell lines.²⁾ The ability of PHA to enhance AChE activity in human peripheral blood lymphocytes⁵⁸⁾ suggests T cell activation via TCR/CD3-mediated pathways regulates expression of such cholinergic elements as ChAT and AChE.

3. ROLES OF mAChRs IN THE REGULATION OF IMMUNE FUNCTION

3.1. G_q/11-Coupled mAChRs Mediate Th1 and Th2 Cell Proliferation and Differentiation

1) Plasticity of mAChR Expression in Activated T Cells

PHA enhances ChAT and M₅ mAChR gene expression in MOLT-3 human leukemic T cells, while stimulation with phorbol 12-myristate 13-acetate (PMA) plus ionomycin upregulates gene expression of both M₃ and M₅ mAChRs.^25,59) In addition, stimulation via lymphocyte function-associated antigen-1 (CD11a) enhances the gene expression of ChAT and M₅ mAChRs in both the CCRF-CEM and MOLT-3 human leukemic T cell lines.⁶⁰⁾ These findings indicate that the pattern and levels of mAChR subtype expression in T cells change in response to immunological stimulation. Qian et al. observed that activation of murine splenic CD4⁺ cells via TCR-mediated pathways enhances gene expression of M₁ and M₅ mAChRs.³²⁾ Moreover, in vitro differentiation of CD4⁺ T cells into Th1, Th2 and Th17 cells reduces expression of M₃ mAChRs, while Th17 cells also exhibit increased expression of M₁ and M₅ mAChRs. These observations reveal the plasticity of mAChR expression in Th cells during differentiation.

2) Proliferation

mAChR agonists, including ACh, oxotremorine-M (Oxo-M), bethanechol and carbachol, all induce rapid and sustained increases in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) via IP₃-mediatred Ca²⁺ release from Ca²⁺ stores in the CCRF-CEM and Jurkat human T cell lines and in the Daudi human B cell line.^61–64) In CCRF-CEM cells, which express M₃ and M₅ mAChRs, but not the M₁ subtype, Oxo-M induces an initial transient increase in [Ca²⁺]_i followed by repetitive [Ca²⁺]_i oscillations.⁶⁴⁾ Removal of extracellular Ca²⁺ or pharmacological blockade of Ca²⁺ release-activated Ca²⁺ channels (CRACs) abolishes the [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i transient elicited by Oxo-M. This suggests IP₃-mediated Ca²⁺ release induces extracellular Ca²⁺ influx through CRACs, which generates repetitive [Ca²⁺]_i oscillations.⁶⁴⁾ mAChR-induced increases in [Ca²⁺]_i enhances the gene expression of c-fos and interleukin (IL)-2. Oxo-M enhances IL-2 production in PHA-activated human peripheral blood lymphocytes and in PMA/A23187 (Ca²⁺ ionophore)-activated Jurkat cells.^65,66) The Oxo-M-induced effects are abolished by an mAChR antagonist, 4-DAMP, suggesting the involvement of G_q/11-coupled mAChRs in IL-2 production. Chronic administration of Oxo-M promotes antibody production and T cell proliferation in rats, which is inhibited by atropine.⁵⁵⁾ In addition, preincubating T cells with physostigmine, an AChE inhibitor, enhances PHA-induced IL-2 production in a mAChR-dependent manner.⁶⁷⁾ These findings suggest G_q/11-coupled mAChR activation promotes T cell proliferation through IL-2 production.

3) Th Cell Differentiation

Upon antigenic challenge eliciting a Th1 or Th2 response, M₃ mAChRs play crucial roles to induce optimal polarization and cytokine production in CD4⁺ T cells.⁶⁸⁾ M₃ mAChR-deficient mice infected with helminth Nippostrongylus brasiliensis, which is commonly used to elicit a predominant Th2 immune reaction, exhibited an attenuated immune response compared with that in wild-type (WT) control mice. J104129, a selective M₃ mAChR antagonist, attenuated helminth-induced production of the Th2 cytokine IL-4 in activated splenic immune cells from WT mice, confirming the role of M₃ mAChRs in the regulation of CD4⁺ T cell differentiation into Th2 cells. Furthermore, M₃ mAChR-deficient mice infected with Salmonella enterica, which primarily elicit a Th1 response, exhibited an impaired ability to resolve their infection. In addition, ex vivo stimulation of lymphocytes from Salmonella typhimurium-infected WT mice with Oxo-M enhanced secretion of the Th1 cytokine interferon (IFN)-γ, and the effects were abolished by J104129. Taken together, these findings suggest M₃ mAChRs are involved in regulating the magnitude and efficacy of the optimal adaptive response to infection.

3.2. G_q/11-Coupled mAChRs Mediate B Cell Differentiation and Immunoglobulin Class Switching

B cells mature in the bone marrow and migrate to lymphoid organs, where their differentiation into plasma cells is induced by antigen presentation and various cytokines. Naive B cells express membrane-bound IgM and IgD as B cell receptors, but once differentiated into plasma cells, they change their antibody production from IgM to IgA, IgG or IgE, depending on the functional requirements (immunoglobulin class switching). B cells, including the Daudi human leukemic B cell line, express all five mAChR subtypes.^10,11) Pansorbin (i.e., heat-killed, formalin-fixed Staphylococcus aureus coated with protein A) triggers B cell activation by binding to toll-like receptor 2 (TLR2).⁶⁹⁾ Pansorbin induces increases in the levels of M₁–M₄ mAChR mRNAs, but not M₅ mAChR mRNA. Oxo-M promotes the differentiation of Pansorbin-activated Daudi cells into plasma cells and the release of IgG through immunoglobulin class switching.⁷⁰⁾ These effects are blocked by scopolamine, a non-selective mAChR antagonist, and 4-DAMP, a G_q/11-coupled mAChR antagonist, which suggests the involvement of G_q/11-coupled mAChRs in B cell maturation and antibody class switching (Fig. 2).

Fig. 2. mAChR Activation Enhances TLR2-Mediated Immunoglobulin Class Switching in B Cells

TLR2 activation increases mAChR expression, IL-6 release and immunoglobulin class switching from IgM (BCR) to IgG in B cells. Activation of mAChRs using Oxo-M, a mAChR agonist, enhances IL-6 production and immunoglobulin class switching from IgM to IgG.

IL-6 was first identified as a cytokine that induces B cells to differentiate into antibody-producing plasma cells.⁷¹⁾ Subsequent studies have shown that IL-6 is produced by T cells, B cells, macrophages and fibroblasts has a variety of biological activities and plays a central role in inflammatory responses.⁷²⁾ TLR2 activation enhances the production of IL-6 in B cells by activating extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NFκB) signaling.^73,74) In Daudi cells, Oxo-M enhances Pansorbin-induced IL-6 production. As mentioned above, G_q/11-coupled mAChRs activate PKC and, in turn, mitogen-activating protein kinase kinase (MEK)/ERK. This suggests activation of G_q/11-coupled mAChRs causes redundant activation of MEK/ERK signaling via TLR2, which enhances IL-6 production and then B cell maturation into plasma cells. These findings are in line with studies showing that following immunization with OVA, serum levels of IgG₁ and IL-6, but not IgM, are lower in M₁/M₅ mAChR double-deficient mice than WT mice³⁹⁾ and that M₁ mAChR-deficient mice harbor significantly fewer IgG-producing B cells.⁷⁵⁾ Thus, either or both M₁ and M₅ mAChRs modulate B cell differentiation and antibody class switching from IgM to IgG but are not involved in B cell proliferation or the initial generation of the antibody response.

3.3. M₁ mAChRs in CD8⁺ Cells Mediate Development of Cytotoxic T Cells

Naive CD8⁺ T cells are activated by alloantigen and differentiate into cytotoxic T cells (CTLs), which recognize and destroy cells that are foreign to the host (e.g., transplanted cells, virus-infected cells and cancer cells). Naive CD8⁺ T cells isolated from spleen express M₁, M₃, M₄ and M₅ mAChRs, and in vitro activation of CD8⁺ T cells via TCR/CD3-mediated pathways enhances expression of M₁ and M₄ mAChRs and suppresses expression of M₃ mAChR.³²⁾ Zimring et al. reported that CD8⁺ T cells from M₁ mAChR-deficient mice developed virtually no detectable lytic activity when subjected to weak or moderate TCR stimulation, while those from WT mice develop a strong lytic response.³⁷⁾ However, strong TCR stimulation was able to overcome this defect in M₁ mAChR-deficient mice, resulting in normally functioning CTLs. In their follow-up report, Vezys et al. observed that neither M₁ nor M₅ mAChRs are required for expansion of antigen-specific CD8⁺ T cells associated with lymphocytic choriomeningitis virus or vesicular stomatitis virus infections, which potently activate the immune system.³⁸⁾ This suggests that M₁ mAChRs are involved in CTL development only when CD8⁺ cells receive a weak immune stimulus.

3.4. M₃ mAChR in DCs Enhance Inflammatory Responses through Antigen Presentation, Th2 Cell Differentiation, and Inflammatory Cytokine Production

DCs are specialized cells for antigen processing and presentation and are present in mucosal, skin and lymphoid tissues. Acting as messengers between the innate and adaptive immune systems, DCs play a major role in the initiation of immune responses.⁷⁶⁾ Activation of mAChRs on immature DCs promotes T cell priming by increasing cell surface expression of human lymphocyte antigen (HLA)-DR and CD86 as well as production of TNF-α and IL-8. However, activation of mAChRs in LPS-activated DCs suppresses their expression and cytokine production in LPS-activated DCs.⁷⁷⁾ This suggests the effect of mAChR activation of DCs may differ depending on their stage of maturation.

Nasal polyposis is characterized by chronic immune inflammation of the nasal mucosa, usually arising in patients with chronic rhinosinusitis.⁷⁸⁾ In over 90% of these patients, the condition is caused by Staphylococcal aureus infection and the presence of enterotoxin B (SEB) within the nasal mucosa. Expression of M₃ mAChRs is markedly upregulated in DCs within the nasal mucosa of these patients and in isolated DCs exposed to SEB.⁷⁹⁾ Activation of mAChRs on the DCs with methacholine, a non-specific mAChR agonist, upregulates expression of the cytokine OX40 ligand (OX40L also known as CD252). Both OX40 (CD134) and OX40L (CD252) are members of the TNF receptor (TNFR)/TNF superfamily and are expressed in activated CD4⁺ and CD8⁺ T cells as well as other lymphoid cells. OX40L acting on CD4⁺ T cells promotes the release of TNF-α and IL-4. TNF-α not only causes IgE production and mast cell degranulation, it also induces apoptosis and is involved in local inflammatory responses. IL-4 promotes immunoglobulin class switching in B cells and polarizes naive Th cells toward Th2. These findings suggest that M₃ mAChRs in DCs are involved in regulating local inflammation within the nasal mucosa of patients with nasal polyposis.

mAChR activation in mouse peripheral blood DCs increases production of cytokines, such as OX40L, as well as the Th2 chemokines MDC (macrophage-derived chemokine/CCL22) and TARC (thymus and activation-regulated chemokine/CCL17), which promotes the synthesis of IL-4, IL-5 and IL-13 in T cells, thereby polarizing Th2 differentiation.⁸⁰⁾ In lung DCs, ACh upregulates expression of M₃ mAChRs and MHC II IAd as well as production of TNF-α and monocyte chemoattractant protein-1 (MCP-1), which are strongly associated with inflammatory infiltration and tissue damage.⁸¹⁾ These effects of ACh are prevented by tiotropium, a selective M₃ mAChR antagonist, suggesting M₃ mAChRs on DCs are involved in enhancement of inflammatory responses that results from antigen presentation, Th2 cell differentiation, and inflammatory cytokine production.

3.5. mAChRs on Macrophages Mediate Proliferation and Release of Inflammatory Mediators

Macrophages are key components of the innate immune system. They are released from bone marrow as monocytes, circulate in the bloodstream, and then migrate into tissues where they eventually differentiate into resident macrophages. Monocytes, monocyte-derived macrophages, and lung and alveolar macrophages express high affinity choline transporter 1 (CHT1), ChAT and vesicular ACh transporters.⁸²⁾ Expression of mAChR subtypes in macrophages varies with the cells’ differentiation stage and the tissue in which they are localized.⁸²⁾ Lung and alveolar macrophages express M₁ and M₃ mAChR mRNAs. Carbachol, a nonspecific AChR receptor agonist, stimulates the release of the proinflammatory mediator leukotriene B4 (LTB4) from lung macrophages, and this response is attenuated by 4-DAMP, an M₃ mAChR antagonist. This suggests the involvement of M₃ mAChRs in lung macrophage-mediated inflammatory responses.⁸²⁾

In lung diseases such as chronic obstructive pulmonary disease (COPD), the numbers of macrophages infiltrating the lungs are increased more than 10-fold. Moreover, they are highly activated, which leads to increased release of inflammatory mediators.⁸³⁾ Tiotropium, a long-acting mAChR antagonist, has been shown to be effective in COPD patients and to inhibit the release of inflammatory mediators from macrophages, suggesting the inflammatory response is at least in part ACh-mediated.⁸⁴⁾ Consistent with that idea, macrophages and other cells (e.g. neutrophils) in the sputum of COPD patients exhibit elevated release of LTB4 following ACh stimulation. This effect is inhibited by oxitropium, a mAChR antagonist, and is not seen in samples from non-patients without inflammation.⁸⁵⁾ This suggests that mAChRs expressed on macrophages contribute to the pathogenesis of COPD by facilitating release of inflammatory mediators, such as LTB4. In addition, atropine and 4-DAMP ameliorate LPS-induced acute lung injury, at least in part by inhibiting LPS-induced neutrophil infiltration as well as increases in pulmonary vascular permeability and secretion of IL-6 and TNF-α from alveolar macrophages.⁸⁶⁾ Thus, blockade of M₃ mAChRs exerts its anti-inflammatory effects by suppressing the inflammatory responses of macrophages.

mAChRs are also involved in macrophage proliferation. Carbachol, for example, promotes the proliferation of peritoneal macrophages through activation of arginase and prostaglandin E₂ (PGE₂) production via M₁–M₃ mAChR activation.⁸⁷⁾ Carbachol also activates M₂ mAChRs on macrophages, which, in turn, activates the G_βγ/phosphatidylinositol-3 kinase (PI3K)/PKC pathway, leading to moderate PGE₂ release.⁸⁷⁾

4. CONCLUSION

Although immune function is generally thought to be regulated mainly by cytokines and glucocorticoids, it appears that a non-neuronal cholinergic system in immune cells also plays a crucial role in the regulation of immune cell function via modification of the synthesis and release of relevant cytokines. Along with α7 nAChRs expressed on macrophages, which play a central role in the cholinergic anti-inflammatory pathway, recent findings indicate that G_q/11-coupled M₁, M₃ and, possibly, M₅ mAChRs expressed in T cells, B cells, DCs and macrophages are involved in the upregulation of immune cell proliferation and differentiation, antibody class switching, and modification of cytokine synthesis, leading to immune regulation. These findings provide an opportunity through the use of mAChR agonists in ex vivo immune cell culture settings to explore novel methods for harvesting large numbers of appropriate Th cells for adoptive immunity and production of large quantities of antibodies.

Acknowledgments

This study was supported by funding from Smoking Research Foundation (KK, MM, TF) and JSPS KAKENHI Grant No. JP21K06539 (MM).

Conflict of Interest

The authors declare no conflict of interest.

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