Yasuhiro Moriwaki

Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function.

Lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and stimulation of mAChRs and nAChRs produces various biochemical and functional changes. Although it has been postulated that parasympathetic cholinergic nerves directly innervate immune cells, no evidence has supported this hypothesis. We measured ACh in the blood of various animal species and determined its localization in T cells using a sensitive and specific radioimmunoassay. Furthermore, we showed that T cells express choline acetyltransferase (ChAT), an ACh synthesizing enzyme. Immunological T cell activation enhances ACh synthesis through the up-regulation of ChAT expression, suggesting lymphocytic cholinergic activity is related to immunological activity. Most immune cells such as T cells, B cells, and monocytes express all five subtypes of mAChRs (M(1)-M(5)), and various subunits of the nAChR, such as α3, α5, α7, α9, and α10. Studies on serum antibody production in M(1) and M(5) combined mAChR gene knockout (KO) mice immunized with ovalbumin (OVA) revealed that M(1)/M(5) mAChRs up-regulate TNF-α, IFN-γ and IL-6 production in spleen cells, leading to an elevation of serum anti-OVA specific IgG(1). In contrast, studies of nAChR α7 subunit gene KO mice immunized with OVA show that α7 nAChRs down-regulate these proinflammatory cytokines, thereby leading to a reduction of anti-OVA specific IgG(1). Taken together, these findings demonstrate that both mAChRs and nAChRs modulate production of cytokines, such as TNF-α, resulting in a modification of antibody production. These findings support the notion that a non-neuronal cholinergic system is involved in the regulation of immune cell function.

The Loss of PGAM5 Suppresses the Mitochondrial Degeneration Caused by Inactivation of PINK1 in Drosophila

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinsons disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.

Enhanced serum antigen-specific IgG1 and proinflammatory cytokine production in nicotinic acetylcholine receptor α7 subunit gene knockout mice

Human and murine immune cells such as mononuclear leukocytes consisting of mainly T and B cells, bone marrow derived dendritic cells (DCs) and macrophages all express various nicotinic acetylcholine (ACh) receptor (nAChR) subunits. Activated T cells and DCs have the ability to synthesize ACh by choline acetyltransferase, suggesting the role of non-neuronal cholinergic system expressed in immune cells in the regulation of immune cell function. Stimulation of human leukemic T and B cell lines with nicotine causes a transient Ca(2+)-signaling that is antagonized by alpha-bungarotoxin, suggesting the involvement of alpha7 subunit. Furthermore, alpha7 nAChRs have been shown to negatively regulate synthesis and release of tumor necrosis factor (TNF)-alpha in macrophages. These findings suggest that immune cell function is regulated by its own non-neuronal cholinergic system, at least in part, via alpha7 nAChR-mediated pathways. In the present study, we tested the role of alpha7 nAChRs in the regulation of immune function by measuring total serum and antigen-specific IgG(1) and IgM, and production of TNF-alpha, gamma interferon (IFN-gamma) and interleukin (IL)-6 in activated spleen cells of nAChR alpha7 subunit gene knockout (alpha7 KO) and wild-type C57BL/6J mice immunized with ovalbumin (OVA). We found that serum levels of total and anti-OVA-specific IgG(1) were significantly elevated in alpha7 KO mice, though there were no significant differences in serum levels of total and anti-OVA-specific IgM between the two genotypes. Production of TNF-alpha, IFN-gamma and IL-6 in spleen cells was significantly facilitated in alpha7 KO mice. Expression of AChE mRNA was not different between the two genotypes. These results suggest that alpha7 nAChRs are involved in the regulation of cytokine production, through which modulates TNF-alpha, IFN-gamma and IL-6 productions, leading to modification of antibody production, but are not involved in expression of cholinergic components in immune cells.

Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M1/M5 mAChR signaling upregulates IgG1 and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP‐1, also appears to be involved in modulating normal T cell function.

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand.

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the alpha7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

Diminished antigen-specific IgG1 and interleukin-6 production and acetylcholinesterase expression in combined M1 and M5 muscarinic acetylcholine receptor knockout mice

Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca(2+)-signaling and up-regulation of c-fos expression; moreover, M1 mAChRs play a crucial role in the differentiation of CD8(+) T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M1 and M5 mAChR knockout (M1/M5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG1 were significantly lower in M1/M5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M1/M5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M1/M5 KO mice. These results suggest that M1 and/or M5 mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.

Expression of SLURP‐1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells

Mammalian secreted lymphocyte antigen‐6/urokinase‐type plasminogen activator receptor‐related peptide‐1 (SLURP‐1) is a positive allosteric ligand for α7 nicotinic acetylcholine (ACh) receptors (α7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP‐1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP‐1 is postulated to be involved in regulating tumor necrosis factor‐α (TNF‐α) release from keratinocytes and macrophages via α7 nAChR‐mediated pathways. In the present study, we assessed SLURP‐1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP‐1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP‐1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5‐kDa SLURP‐1 protein in whole‐lung tissue and trachea. In addition, high‐affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP‐1 and ACh. We also observed direct contact between F4/80‐positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1‐positive nerve elements. Collectively, these results suggest that SLURP‐1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF‐α release from macrophages in bronchial tissue.

PINK1, a gene product of PARK6, accumulates in α-synucleinopathy brains

α-Synucleinopathy is an entity of neurodegenerative diseases such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), that involves accumulation of α-synuclein in the brain. PINK1 (PTEN induced kinase 1) is a novel gene recently identified as causative in autosomal recessive early onset parkinsonism (PARK6). In the present study, we examined the localisation of PINK1 in the brains of patients with α-synucleinopathy and found PINK1 in glial cytoplasmic inclusions (GCIs) in MSA, as well as in Lewy bodies (LBs) in PD and DLB. These findings imply that PINK1 may be involved in the formation of LBs and GCIs, suggesting that PINK1 is one of the major pathological proteins in α-synucleinopathy. The cDNA of PINK1, corresponding to 112–520 amino acids of the protein, was subcloned in a vector pET30(a) with a His tag. Anti-PINK1 antibody was generated against recombinant His tagged PINK1 by immunising a rabbit. The obtained antibody was affinity purified. A postmortem brain sample from a normal patient was homogenised, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a membrane. After blocking in Tris buffered saline with 5% dry milk, the membrane was incubated with anti-PINK1 antibody (1:1000). The membrane was then incubated with a secondary antibody (1:2500; Amersham, Buckinghamshire, UK), and visualised with an enhanced chemiluminescent substrate (Pierce, Rockford, Illinois, USA). Immunohistochemical analysis was carried out with paraffin embedded midbrain sections from patients with sporadic PD, …

T cells down-regulate macrophage TNF production by IRAK1-mediated IL-10 expression and control innate hyperinflammation

Significance Appropriate development of inflammation is essential to protect hosts from microbial infections, but inflammation can occasionally overshoot and cause collateral damage in hosts. Such hyperinflammation happens during endotoxemia and sepsis, and is attributed to excessive production of proinflammatory cytokines by host cells, such as macrophages. In this study, we demonstrate an unconventional mechanism by which T cells regulate cytokine production of macrophages. It is our general understanding that fast-acting immune cells (such as macrophages) “instruct” how T cells should behave through the step termed antigen presentation. However, what we show here is that T cells instruct macrophages to down-regulate a key proinflammatory cytokine, TNF, within hours after the initiation of endotoxemia. Endotoxemia is caused by excessive inflammation, but the immune system has various mechanisms to avoid collateral organ damage in endotoxemia. A handful of reports have shown that innate immune responses are suppressed by the adaptive immune system. However, the molecular mechanism by which adaptive immune cells suppress innate inflammatory responses is not clear. Here, we report that T cells are shown to interact with macrophages at the early stage of enodotoxemia and to prolong survival of mice through controlling TNF and IL-10 levels by macrophage CD40 stimulation. The cross-talk between CD40 and toll-like receptor (TLR4) signaling first mediates IL-1 receptor-associated kinase 1 (IRAK1) nuclear translocation and its binding to the IL-10 gene promoter in macrophages, without interfering with the NFκB pathway. IL-10 is then detected by macrophages in an autocrine fashion to destabilize Tnfa mRNA. To induce IRAK1-mediated IL-10 expression, signals from both CD40 and TLR4 are essential. CD40 signaling induces IRAK1 sumoylation in the presence of TNF receptor-associated factor 2 (TRAF2) and intracellular isoform of osteopontin (iOPN) whereas TLR4 signaling provides IFN regulatory factor 5 (IRF5) as a chaperone for sumoylated IRAK1 nuclear translocation. Interaction of T cells with macrophages was observed in the spleen in vivo after endotoxemia induction with LPS injection. Our study demonstrates a mechanistic basis for the immunosuppressive role of macrophage CD40 in LPS endotoxemia.

Non-neuronal cholinergic system in regulation of immune function with a focus on α7 nAChRs

In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animals body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4(+) T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including α7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M(1)/M(5) mAChR signaling up-regulates IgG(1) and pro-inflammatory cytokine production, while α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric α7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205(+) DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating α7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.