After discovery of ACh in the peripheral blood and plasma of humans and animals, the presence of ACh in immune cells was first demonstrated in the human peripheral blood mononuclear leukocyte (MNL) fraction, which consists mainly of lymphocytes and a small monocyte fraction (18–23); also see a review (9). Later, the presence of ACh in immune cells was confirmed by detection of ACh in various human leukemic cell lines (24, 25) and rat lymphocytes, including T and B cells (26). These findings provided an explanation for the seemingly enigmatic observation of Dale and Dudley (6) that ACh was present in the spleen, though that organ is not cholinergically innervated.

In general, human leukemic T cell lines had higher ACh contents than B cell lines, prelymphoma cell lines, or a monocytic cell line (Table 1) (9, 25). Among rat lymphocytes, the ACh content in T cells was significantly higher than in B cells, and the ACh content in CD4⁺ T cells was significantly higher than in CD8⁺ T cells (26). The higher ACh content observed in rat T cells than B cells reflects the higher ChAT activity in T cells (27). Little information is available on the intracellular ACh contents in macrophages and DCs.

It is important to note that, with the molecular weight of 146 Da, ACh is small, water soluble, and both physicochemically and enzymatically fragile. Furthermore, the chemical nature of ACh is quite different from that of catecholamines and serotonin, which are able to be fixed to a tissue using paraformaldehyde. At present, no technique is available to fix ACh to the tissue. It is therefore currently impossible to detect ACh in tissues or cells using immunohistochemical or immunocytochemical techniques. Although ACh may bind to an antibody, because it is not anchored to its site, it will be washed away with the antibody. Nonetheless, Takahashi et al. (28) reported a successful attempt to localize ACh in the mouse gut sections using a tandem imaging mass spectrometry.

Kajiyama et al. (29) observed that a major portion of rabbit blood ACh is present in the buffy coat along with ACh-synthesizing activity. On the basis of that observation, Rinner and Schauenstein (27) confirmed the expression of ACh-synthesizing activity in rat T and B cells in the thymus, spleen, and blood, suggesting the presence of ChAT in lymphocytes.

ChAT is primarily responsible for ACh synthesis within the nervous systems of mammalian species (30). However, in peripheral tissues and non-neuronal cells, the mitochondrial enzyme carnitine acetyltransferase (CarAT, EC 2.3.1.7) also contributes to ACh synthesis along with ChAT (30, 31). ACh-synthesizing activity determined in peripheral samples using the so-called Fonnum method (32) with [³H]acetyl coenzyme A and choline reflects the total activities of ChAT and CarAT. It is therefore recommended that one determines ChAT and CarAT activities in the presence of respective specific inhibitors, bromoacetylcholine and bromoacetylcarnitine. ChAT activity is proportional to the ACh content in cells of the T cell lines expressing ChAT mRNA (Table 1) (10, 25). However, Daudi B cells, which do not express ChAT mRNA, contained little ACh despite of a high CarAT activity. This suggests that ChAT is responsible for ACh synthesis in immune cells (10).

Fujii et al. (33) provided definitive evidence for the synthesis of ACh by ChAT in T cells by demonstrating expression of ChAT mRNA and the enzyme protein in MOLT-3 human leukemic T cells, using reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis (Figure 1A). Later, constitutive ChAT mRNA expression was detected in other human leukemic T cell lines (25), human blood CD4⁺ T cells (Figure 1B) (34), rat T and B cells (24, 26), and rat MNLs isolated from the renal vasculature (35). These findings support the idea that ACh production catalyzed by ChAT is occurring in lymphocytes, including T and B cells.

Reverse transcription-polymerase chain reaction revealed ChAT mRNA expression in C57BL/6J mouse spleen-derived MNLs activated with concanavaline A (ConA) and bone marrow-derived DCs activated with lipopolysaccharide (LPS) (36). However, there was no detectable expression of ChAT mRNA in these cells under resting conditions, which suggests that immunological activation is required for ChAT transcription in these cells. While no ChAT mRNA was detected in peritoneal macrophages under either resting or LPS-activated conditions, even after an amplification protocol entailing 40 cycles (36), Koarai et al. (37) detected expression of ChAT mRNA in human lung and alveolar macrophages and monocytes using an RT-PCR protocol entailed 45 cycles, which suggests marginal ChAT mRNA expression in these cells. ChAT mRNA and protein were also detected in human mature and immature DCs using RT-PCR and immunocytochemistry (38). These findings confirm the expression of ChAT mRNA in T and B cells, DCs, and macrophages.

The recent development of ChAT^BAC-eGFP transgenic mice (39) and ChAT-Cre-tdTomato mice (40) provide the opportunity to detect ChAT-expressing cells using fluorescent reporter proteins. Tallini et al. (39) observed eGFP expression in a subset of lymphocytes in Peyer’s patches, leading to the detection of ChAT-GFP in splenic CD4⁺ T cells (15) and CD8⁺ T cells and B cells (41). In addition, Gautron et al. (40) observed expression of the reporter protein in T cells within Peyer’s patches in mice expressing tdTomato fluorescent protein in ChAT-expressing cells. These results confirm the earlier findings that peripheral blood CD4⁺ and CD8⁺ T cells and B cells express ChAT mRNA and contain ACh (26, 36).

ChAT expression in splenic DCs was confirmed using ChAT^BAC-eGFP mice (41). That finding along with the ChAT gene expression in DCs described above (36) suggests DCs are able to synthesize ACh using ChAT. So far, however, no additional data on ACh synthesis and release in DCs has been reported, and the physiological significance of ACh in DCs is yet to be determined.

ChAT expression was also observed in splenic macrophages using ChAT^BAC-eGFP transgenic mice (41). These findings are in line with those from Koarai et al. (37), who reported the expression of ChAT mRNA in human lung and alveolar macrophages and monocytes. However, Gautron et al. (40) did not find reporter expression in macrophages from gut-associated lymphoid tissue or the spleen of ChAT-Cre-tdTomato mice. As mentioned, we did not detect ChAT mRNA expression in either resting or activated mouse peritoneal macrophages (36). These findings suggest ChAT expression in macrophages may vary depending upon strain, species, tissue, cell processing procedure, and/or immunological status, or that the levels of ChAT expression are marginal in macrophages. Information on the physiological significance of ACh synthesis in macrophages is not available at present.

The diversity of mAChR functions prompted investigation of their molecular basis using cloning techniques and led to the identification of five distinct mAChR subtypes (M₁–M₅) (70–72). These five mAChRs have been divided into two groups based on their functional coupling. The M₁, M₃, and M₅ subtypes are coupled to pertussis toxin-insensitive G_q/11 proteins, which mediate activation of PLC activity. Upon activation of these mAChR subtypes, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate, leading to the formation of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. These products then act as second messengers by, respectively, mobilizing Ca²⁺ from intracellular stores and activating PKC (73). The M₂ and M₄ subtypes are coupled to pertussis toxin-sensitive G_i/o protein, which mediates inhibition of adenylate cyclase, and thus a decrease in cAMP formation, upon activation (74, 75).

Nicotinic AChRs are located on the plasma membranes of skeletal muscle cells, neurons, and non-neuronal cells. Activation of nAChRs elicits membrane depolarization and excitation due to a rapid increase in membrane permeability to Na⁺, K⁺, and Ca²⁺. The molecular cloning of nAChR subunits (78) prompted a series of proteomic and genomic investigations of the various nAChR subunit proteins. At present, 10 α (α1–10), 4 β (β1–4), γ, δ, and ε nAChR subunits have been detected through molecular cloning. The α subunits can be subdivided according to their sensitivity to α-BTX: α1, α7, and α9 are α-BTX-sensitive, while α2–α6 are α-BTX-insensitive. nAChRs exist as pentamers composed of 1–5 different subunits. At least two copies of the α subunit are always present among these subunits, and multiple ACh binding sites are formed at the interface of each α subunit and a neighboring subunit.

nAChRs are classified into muscle and neuron types based on their expression sites. Muscle type nAChRs are expressed mainly in skeletal muscle and contain four different subunits within the pentameric complex: (α1)₂/β1/δ/γ in embryonic and denervated muscle, and (α1)₂/β1/δ/ε in innervated adult muscle. By contrast, neuron type nAChRs expressed in the nervous system and non-neuronal cells, including immune cells, are composed of only α (α2–α7, α9, and α10) and β (β2–β4) subunits. The α8 subunit is found only in the visual areas of the avian brain (79). nAChRs consisting of α3β2 and α3β4 are abundantly expressed in peripheral ganglia, while α4β2 subunits are expressed in the brain (80). Within the mammalian brain, about 80% of nAChRs are composed of α4β2 subunits, and 10–15% are composed of α7 (81). The α7 nAChR gene (CHRNA7) is expressed widely in the central and peripheral nervous system, and in non-neuronal cells, and the homomeric α7 nAChR exhibits uniquely high Ca²⁺ permeability upon activation (82, 83). The α9 and α10 subunits were first identified in the mechanosensory hair cells of the rat auditory system (84, 85). The α9 subunit was initially postulated to form a homomeric nAChR. However, subsequent studies identified expression of heteromeric α9α10 nAChR subtypes within the hair cells. Unlike other nAChRs, which mediate excitatory neurotransmission, α9α10 nAChR activation elicits hair cell hyperpolarization evoked by Ca²⁺ entry through the receptor, leading to activation of a small-conductance SK2 Ca²⁺-dependent potassium channel (85).

SLURP-1 and -2 mRNAs are detected in nearly every organ in the C57BL/6J mouse (123). Gene expression for SLURP-1 and -2 was also detected in MNLs, DCs, and macrophages, but neither the T cell activator Con A nor the DC and macrophage activator LPS modified the levels of their expression (36, 123). This suggests that SLURP-1 and -2 are constitutively expressed in these cells, and their expression is independent of immunological stimulation.

Intense SLURP-1 immunoreactivity (SLURP-1⁺) was detected in DC-like cells residing mainly in the interfollicular zone surrounding the germinal center of human tonsils, and in a few cells scattered within the germinal center (Figure 3) (126). Some SLURP-1⁺ cells in the tonsil interfollicular zone also showed immunopositivity for CD205 (Figure 3A), a marker of mature DCs that mediates efficient antigen presentation (127), and these SLURP-1⁺ CD205⁺ DCs were surrounded by CD4⁺ T cells. These observations support the notion that ACh synthesized and released from T cells and activated macrophages during antigen presentation acts on α7 nAChRs expressed in immune cells, and that SLURP-1 potentiates the ACh activity at α7 nAChRs in both T cells and DCs (16, 126).

Upregulation of M₅ mAChR mRNA expression in human T and B cells during immunological stimulation strongly suggests that mAChRs, including M₅, are involved in regulating immune function (33, 47). Thus far, however, there have been few investigations of the roles played by mAChRs in regulation of immune function. The involvement of M₁ mAChRs in the differentiation of CD8⁺ T cells into cytolytic T cells was first postulated by Zimring et al. (129), but it was later found that neither M₁ nor M₅ mAChRs are required for expansion of antigen-specific CD8⁺ T cells, in response to viral infection in C57BL/6 mice (130). On the basis of these observations, the authors concluded that the extent to which one can draw a generalized conclusion that M₁ and M₅ mAChRs are not involved in antiviral immunity mediated by CD8⁺ T cells, depends upon issues of antigen strength, genetic background, and receptor redundancy (130).

Fujii et al. (56) investigated the roles of M₁ and M₅ mAChRs in the regulation of immune function by immunizing combined M₁ and M₅ mAChR gene-knockout (M₁/M₅-KO) and WT mice with ovalbumin (OVA). One week after the immunization, serum concentrations of anti-OVA-specific IgG₁ in the M₁/M₅-KO mice were significantly lower than in WT mice, though the serum concentrations of anti-OVA-specific IgM did not differ between the two genotypes. Spleen cells from M₁/M₅-KO mice activated with OVA secreted significantly lower amounts of TNF-α, IFN-γ, and IL-6 than those from WT mice. These observations suggest that M₁ and/or M₅ mAChRs contribute to the regulation of pro-inflammatory cytokine production related to adaptive immunity.

Fujii et al. (133) observed a significantly higher serum anti-OVA-specific IgG₁ levels in α7-KO than WT C57BL/6J mice 2 weeks after immunization with OVA. Moreover, antigen-stimulated spleen cells from α7-KO mice produced significantly greater amounts of TNF-α, IL-6, and IFN-γ responsible for antibody class switch induction to IgG₁ than those from WT mice. These observations suggest the involvement of α7 nAChRs in regulating pro-inflammatory cytokine production, leading to modification of antibody production. In line with these findings, a recent study showed that nicotine attenuates production of TNF-α, IL-1β, and IL-12 in murine bone marrow-derived monocytes via α7 and α9 nAChR-mediated pathways (134). In addition, activation of B cells with anti-CD40 antibody elicited a greater proliferative response in α7-KO mice than in the WT, and the suppression of α7 nAChRs with methyllycaconitine (MLA) evoked a greater proliferative response in B cells stimulated either with anti-CD40 antibody or antibody against B cell receptor (135–138), suggesting a role for α7 nAChRs in downregulation of B cell proliferation. Taken together, the above findings suggest the possibility that a higher serum antigen-specific IgG₁ concentration observed in α7-KO mice (133) can be ascribed to the upregulation of pro-inflammatory cytokine production and the increased number of B cells in α7-KO mice (135–138).

MOLT-3 human leukemic T cells and cultured spleen cells from C57BL/6J mice constitutively produce amounts of ACh sufficient to elicit autocrine changes in [Ca²⁺]_i and upregulation of IL-2 mRNA and protein expressions (139). Mecamylamine, a nAChR inhibitor, suppressed the [Ca²⁺]_i transients, IL-2 release, and cell proliferation. These findings indicate that T cells utilize ACh as a tool to interact with one another and that autocrine ACh-activated nAChRs are involved in regulating immune cell functions such as cytokine synthesis and cell proliferation.

Methyllycaconitine, a specific α7 nAChR antagonist, did not suppress the above described autocrine ACh-induced changes in [Ca²⁺]_i, suggesting the involvement of nAChRs other than α7 nAChRs in the [Ca²⁺]_i transients in freshly isolated spleen cells and resting MOLT-3 cells leading to upregulation of IL-2 production (139). In fact, Qian et al. (77) detected the expression of α7 nAChR mRNA in CD4⁺ and CD8⁺ T cells only after the activation via TCR/CD3 cross-linking, but not in freshly isolated CD4⁺ and CD8⁺ T cells. Taken together, these findings suggest the possibility that the pattern of nAChR subtype expression is variable depending on the immunological status. Furthermore, nicotine modified IFN-γ and IL-17 production in T cells activated with TCR/CD3 cross-linking (77, 140). These findings suggest that nAChRs including α7 nAChR contribute to immunomodulation through modification of T cell differentiation by altering cytokine production.

T cell activation with anti-CD3/anti-CD28 mAbs of peripheral blood MNLs isolated from MdM patients with SLURP-1 mutation showed a defect in their proliferative response (125). Moreover, addition of WT recombinant SLURP-1 (rSLURP-1) to cultures of T cells from MdM patients restored the normal T cell activation response, showing that SLURP-1 plays a key role during normal activation of T cells induced by immunological stimulation enhancing the actions of ACh via α7 nAChRs (125).

Recombinant SLURP-1 increases ChAT gene expression and the ACh content in MOLT-3 human leukemic T cells and human peripheral blood MNLs, and these effects are abolished by the α7 nAChR antagonist MLA. This suggests that, working as a positive allosteric ligand, SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action on α7 nAChRs, thereby facilitating functional development of T cells (126). rSLURP-1 induces a slight but significant attenuation of cell growth in peripheral blood MNLs and MOLT-3 cells and that is abolished by MLA. These findings support the notion that SLURP-1 acts as a key modulator of T cell activity.

Chernyavsky et al. (122) found that rSLURP-1 decreases production of TNF-α in CEM human leukemic T cells, downregulates IL-1β and IL-6 secretion in U937 macrophages, and moderately upregulates IL-10 production in these immune cells. rSLURP-2 downregulates TNF-α and IFN-γ-receptors in CEM cells and reduces IL-6 production in U937 macrophages. These results demonstrate that SLURP-1 and -2 exert anti-inflammatory effects on T cells and macrophages.

Because the efferent vagus nerve that should carry the efferent signals terminates in the celiac ganglion, Rosas-Ballina et al. (15) postulated that the efferent signals carried by the efferent vagus nerve are transmitted to the postganglionic sympathetic splenic nerve innervating the spleen in the celiac ganglion. This would activate the splenic nerve to release NE within the spleen, which would promote Ach release from a subset of CD4⁺ T cells that relay the neural signal to other immune cells—e.g., through activation of α7 nAChRs on macrophages, resulting in inhibition of the synthesis and release of pro-inflammatory cytokines, including TNF-α. Consistent with those ideas, they found that electrical vagus stimulation elevated ACh concentrations in the spleen, and attenuated serum TNF-α levels during endotoxemia. Their observations further confirmed that T cells mediate the inflammatory reflex. They found that vagus nerve stimulation did not diminish serum TNF-α concentrations during endotoxemia in nude mice lacking functional T cells, but that the transfusion of a subset of ChAT⁺ T cells from normal mice to the nude mice restored the ability of vagus nerve stimulation to suppress serum TNF-α concentrations. They, therefore, proposed that efferent vagus nerve signals are transferred to ChAT⁺ T cells via NE released from the splenic nerve in the spleen, leading to elevation of ACh synthesis through activation of β-adrenoceptors on the ChAT⁺ T cells. The elevated ACh in turn acted on α7 nAChRs to suppress synthesis and release of TNF-α in macrophages (15).

Based on the observation that electrical stimulation of the vagus nerve did not elicit action potentials in the splenic nerve (158), Martelli et al. (159) proposed that efferent vagal stimulation and/or antidromic vagal afferent activation elicits lymphocyte mobilization to the spleen from a deposit of lymphocytes (vagal target). Then within the spleen, ACh-synthesizing CD4⁺ T cells among the mobilized lymphocytes release ACh to activate α7 nAChRs on the sympathetic nerve terminals leading to NE release. NE released from sympathetic nerve terminals acts on β₂-adrenoceptors on macrophages to suppress TNF-α synthesis and release [see reviews in Ref. (13, 14)]. However, considering the extreme enzymatic fragility of ACh, it is essential to confirm the formation of synapse-like structures between ACh-synthesizing CD4⁺ T cells and sympathetic nerve terminals in the spleen, and between vagus nerve terminals and the vagus target. It is also necessary to demonstrate α7 nAChR expression in the sympathetic nerve terminals in the spleen and their functional release of NE.

SLURP-1 acts as a positive allosteric ligand for α7 nAChRs (117, 118) and increases the potency and efficacy of ACh at α7 nAChRs more than 2.5 times (117). SLURP-1 co-localizes with subsets of sensory neurons containing substance P (SP) and calcitonin gene-related peptide (CGRP) (160), which have been detected among afferent vagus nerve fibers (161, 162). In the white pulp of the spleen, SP⁺ nerve fibers have been identified within the marginal zone and the outer regions of the periarteriolar lymphoid sheaths, which are filled with T cells (163). In mice, CGRP⁺ nerve fibers have been detected in the spleen and lymph nodes (164, 165). Furthermore, the significant increase in splenic CGRP levels seen during the initial swelling phase of antigen-induced arthritis in mice suggests inflammation-induced release of CGRP from the sensory neurons (166). These findings provide evidence for splenic innervation by sensory neurons containing both SP and CGRP, and suggest the possibility of co-localization of SLURP-1 in the SP and CGRP nerve fibers innervating the spleen.

A subset of CD205⁺ DCs located within the marginal zone of human tonsils contain immunoreactive SLURP-1 and are surrounded by CD4⁺ T cells and CD68⁺ macrophages (126) (Figure 3C). Detection of SLURP-1 in human plasma, urine, sweat, saliva, and tears is suggestive of the protein’s stability and mobility (167). Moreover, SLURP-1 potentiates ACh synthesis in T cells (126). It is therefore reasonable to suggest that by potentiating the action of ACh at α7 nAChRs, SLURP-1 released from CD205⁺ DCs during antigen presentation and from SP/CGRP-containing neurons may participate in the inflammatory reflex leading to suppression of TNF-α synthesis in splenic macrophages (Figure 5). Because CD4⁺ T cells can directly interact with splenic macrophages through antigen presentation or cell-to-cell adhesion via LFA-1 (CD11a), ACh released from CD4⁺ T cells should act effectively on α7 nAChRs on macrophages in the presence of SLURP-1, leading to the suppression of TNF-α synthesis and release.