2018 年 41 巻 10 号 p. 1611-1614
Lymphocytic cholinergic system has important roles in T cell functions, including immune responses and proliferation and differentiation of immune cells. T lymphocytes exclusively produces acetylcholine (ACh) via choline acetyltransferase (ChAT), activating their muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) in an autocrine and paracrine manners. Hippocampal cholinergic neurostimulating peptide (HCNP) is an undecapeptide cleaved from N-terminal of phosphatidylethanolamine-binding protein 1 (PEBP1). HCNP enhances ACh synthesis through upreglation of ChAT expression in septo-hippocampal cholinergic neurons and participates in neuronal development and differentiation. Although PEBP1 and HCNP appears to be distributed ubiquitously in tissues and cells including spleen, its functions in immune cells have not been understood. In the present study, we observed that PEBP1 is also expressed in human and murine T cells. Long-term exposure to HCNP suppressed ChAT expression in MOLT3 human leukemic T cells, resulting in decreased release of ACh. HCNP also decreased the expression of extracellular signal-regulated kinase (ERK). Thus, HCNP appears to suppress lymphocytic cholinergic signaling, which might act as an immune modulator.
Lymphocytic cholinergic system plays a key role in regulating cytokine release from immune cells, as well as their proliferation and differentiation.1,2) Lymphocytic acetylcholine (ACh) is primarily synthesized in T cells through the action of choline acetyltransferase (ChAT).1,2) Within T cells, ChAT expression and activity are stimulated by such factor as phytohaemagglutinin, a T-cell receptor activator that binds to the T cell receptor (TCR)/CD3 complex, leading to the up-regulation of ChAT levels and its activation.3) These findings support the notion that immunological activation of T cells is related to ACh synthesis. Upon release from T cells, the ACh activates muscarinic and nicotinic ACh receptors (mAChR and nAChRs, respectively) on the T cells themselves, and on other immune cells including B lymphocytes, dendritic cells and macrophage.1,2) In fact, our recent study shows that autocrine ACh released from T lymphocytes evoked spontaneous increases in intracellular Ca2+ via nAChRs, which participates in interleukin (IL)-2 production and cell proliferation.3)
Hippocampal cholinergic neurostimulating peptide (HCNP) is an undecapeptide cleaved from the N-terminal domain of its 21 kDa phosphatidylethanolamine-binding protein 1 (PEBP1).4,5) HCNP was originally purified from young rat hippocampus. In the rat medial septal nucleus, HCNP is released from cholinergic neurons upon N-methyl-D-aspartate (NMDA) receptor stimulation, after which it stimulates ChAT expression, thereby increasing ACh synthesis.5) HCNP-mediated ACh synthesis may influence cholinergic development in the rat medial septal nucleus. Moreover, the expression levels of HCNP and its precursor protein were decreased in the brains of patients with Alzheimer’s disease.6) These findings suggest HCNP is a physiologically and pathologically important secretory peptide that coordinates neurological activity through ACh synthesis.
The distribution of PEBP1 is not limited to the central nervous system; indeed, PEBP1 is distributed in a variety of tissues and cells. PEBP1 levels are highest in testis and brain, followed by liver, kidney and spleen. It is also present in biological fluids, including rat haploid testicular germ cell secretion and testicular interstitial fluid.7) In bovine serum, the PEBP1 concentration is estimated at approximately 35 nM.8) Moreover, HCNP and PEBP1 are present within the chromaffin secretory granules in adrenal cells and are released into the circulation along with the catecholamines, which activates M2 mAChRs in the heart, exerting a negative inotropic effect under basal conditions and counteracting adrenergic-induced positive inotropism.8,9)
Given that PEBP1 appears to be expressed in spleen, in the present study we examined the expression and localization of PEBP1 and functions of HCNP in murine splenocytes and cells from the MOLT3 human leukemic T cell line. Long-term exposure to HCNP suppressed ChAT expression in MOLT3, resulting in decreased release of ACh. As lymphocytic cholinergic system contributes to the regulation of immune functions, our findings suggest HCNP acts as an immune modulator by inhibiting lymphocytic cholinergic system in T cells.
Synthetic HCNP was purchased from Bachem (H-8555).
Cell CultureC57BL/6J mice were obtained from Japan SLC, Inc. (Japan). The protocols used in this study were approved by the Ethical Committees of Doshisha Women’s College of Liberal Arts (No. Y15012, Y16002). Murine splenocytes were obtained from male C57BL/6J mice (3 months old) as described previously.3) MOLT3 human leukemic T cell and murine splenocytes were then cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37°C under a humidified atmosphere with 5% CO2. In addition, when culturing murine splenocytes, 2-mercaptoethanol (100 µM) was added to the medium.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western BlottingMurine splenocytes and MOLT3 cells were lysed in 20 mM Tris–HCl (pH 7.4) with 2% SDS. The lysates were subjected to 4–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, U.S.A.), after which the separated proteins were transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked using Blocking One (Nacalai Tesque, Japan) for 1 h at room temperature and incubated first with primary antibodies at 4°C overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The blots were developed using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific), after which the chemiluminescence was detected using an ECL system (FUJIFILM Las-3000, FUJIFILM, Japan).
ImmunocytochemistryMurine splenocytes and MOLT3 cells plated on poly-D-lysine-coated glass-bottom dishes were fixed with 4% paraformaldehyde for 20 min at 4°C, permeabilized, and blocked and then incubated first with rabbit polyclonal anti-PEBP1 antibody (Abgent, U.S.A.) overnight at 4°C and then with Alexa-488-conjugated goat anti-rabbit immunoglobulin G (IgG) (Thermo Fisher Scientific), phycoerythrin (PE)-conjugated anti-CD4 antibody (RM4-5, Thermo Fisher Scientific), and allophycocyanin (APC)-conjugated anti-CD8 antibody (53-6.7, Thermo Fisher Scientific) for 1 h at 4°C. Nuclei were stained using 300 nM 4′-6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Cells were imaged using a confocal microscope (Zeiss LSM 800 Meta, Carl Zeiss, Germany) equipped with an oil-immersion objective (40×, NA=1.3). Fluorescence images were processed using ImageJ 1.37a (National Institutes of Health, U.S.A.).
Real-Time PCRTotal RNAs were extracted using Sepasol RNA II Super (Nacalai Tesque), and cDNAs were prepared by reverse transcription using a Prime Script RT reagent Kit (TaKaRa Bio., Japan) in a S1000 Thermal Cycler (Bio-Rad, U.S.A.). Real-time PCR analysis was conducted using CYBR premix EX taq, FAM-labeled probes and predesigned primers (TaKaRa Bio.) with a Thermal Cycler Dice Real Time System (TaKaRa Bio.). The primer pairs were as follows: for human ChAT (HA221706), 5′-AGC CCT GCC GTG ATC TTT G-3′ and 5′-GCA CAG TCA GTG GGA ATG GAG T-3′; for mouse ChAT (MA028203), 5′-TGG ATG AAA CAT ACC TGA TGA GCA A-3′ and 5′-CGT GAA AGC TGG AGA TGC AGA A-3′; for PEBP1 (HA243336), 5′-CCA GCA GGA AGG ATC CCA AAT-3′ and 5′-CTG CTC GTA AAC CAG CCA GAC A-3′; for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (HA067812), 5′-GCA CCG TCA AGG CTG AGA AC-3′ and 5′-TGG TGA AGA CGC CAG TGG A-3′; for mouse GAPDH (MA050371), 5′-TGT GTC CGT CGT GGA TCT GA-3′ and 5′-TTG CTG TTG AAG TCG CAG GAG-3′
Radioimmunoassay for AChMOLT3 cells (1×105 cells) were incubated with HCNP (300 pg/mL) for 6 d in the presence of 1 µM diisopropylfluorophosphate, an ACh esterase inhibitor. The culture supernatant (1 mL) was mixed with 500 µL of 1.2 N perchloric acid, after which the solution was kept on ice for 15 min and then centrifuged at 15000×g for 30 min at 4°C. The ACh content of the extracts was determined with a RIA using [3H]ACh (specific activity: 2.81 TBq/mmol, GE Healthcare, U.S.A.) and antiserum against ACh raised in a rabbit immunized with choline hemiglutarate-bovine serum albumin conjugates.10)
Statistical AnalysisData are presented as means±standard error of the mean (S.E.M.) All representative experiments were repeated three times. Statistical analysis was performed using SigmaPlot (Systat Software Inc., U.S.A.). Differences between two groups were evaluated using Student’s t-test, and between three or more groups using one- and two-way ANOVA with post hoc Dunnett’s or Tukey’s test, respectively. Values of p<0.05 were considered significant.
HCNP is generated from PEBP1 by the action of chymotrypsin-like enzyme intracellularly or after being secreted extracellularly.5,8) To assess expression of PEBP1 in T cells and splenocytes, we performed Western blotting using anti-PEBP1 antibodies. PEBP1 appears to be expressed in both MOLT3 human leukemic T cells and murine splenocytes (Fig. 1A). PEBP1 was expressed in several types of cells, including CD4+ and CD8+ T cells, in murine spleen (Fig. 1B) and was broadly distributed in the cytoplasm of MOLT3 cells (Fig. 1C). HCNP was previously described as a cytoplasmic protein that also associates with the plasma and endoplasmic reticulum membranes,8) which is consistent with our observed cytoplasmic localization of PEBP1 in MOLT3 cells and splenocytes.
A. Western blotting showing PEBP1 expression in MOLT3 cells and splenocytes. GAPDH served as a loading control. B. PEBP1 was expressed in CD4+ and CD8+ T cells in murine spleen. Splenocytes were immunostained with antibodies against PEBP1 (green), CD4 (red) and CD8 (white). Nuclei were stained using DAPI (blue). Scale bar: 20 µm. C. Intracellular localization of PEBP1 in MOLT3 cells. Nuclei were stained with DAPI. Scale bar: 20 µm.
In hippocampal cholinergic neurons, HCNP produced through cleavage of the N-terminal domain of PEBP1 fosters development of the cholinergic phenotype by increasing ChAT expression.4,5) To assess the effect of HCNP in T cells, MOLT3 cells were exposed to HCNP (1–300 pg/mL) for 1, 3 or 6 d. Exposure to HCNP for 6 d, but not 1 and 3 d, suppressed levels of ChAT mRNA in MOLT3 cells at concentrations ranging from 3 to 300 pg/mL (approximately 2.56 to 256 pM), which is consistent with the concentrations that increased ChAT expression in cultured hippocampal neurons4) (Fig. 2A). This suppressive effect of HCNP was also observed in splenocytes (Fig. 2B). Consistent with its ability to suppress ChAT mRNA expression, exposure to 300 pg/mL HCNP for 6 d reduced ChAT proteins by 40% and the ACh content as compared to control in MOLT3 cells (Figs. 2C, D). Long-term exposure to HCNP did not affect cell viability in MOLT3, indicating that the suppressive effect of HCNP on ChAT expression results from its cytotoxic effect (data not shown).
A and B. ChAT mRNA levels following long-term exposure to HCNP in MOLT3 and splenocytes, respectively. Cells were exposed to the indicated concentration (1–300 pg/mL) of HCNP for indicated times. ChAT mRNA levels were assessed using real-time PCR. Shown are means±S.E.M. (n=3). * p<0.05 vs. Control. C. Western blots showing ChAT protein levels in MOLT3 cells exposed to 300 pg/mL HCNP for 6 d. GAPDH was used as a loading control. The graph shows the relative expression of ChAT protein after 6 d with or without 300 pg/mL HCNP. ChAT was quantified using densitometry and normalized to GAPDH. Shown are means±S.E.M. (n=3). ** p<0.01 vs. Control. D. ACh content in MOLT3 cells exposed to 300 pg/mL HCNP for 6 d. ACh contents were measured using a RIA. Shown are means±S.E.M. (n=3). * p<0.05 vs. Control.
We next investigated the HCNP-involved signaling pathway modulating ChAT expression. HCNP activates M2 mAChRs in the heart, exerting a negative inotropic effect under basal conditions and counteracting adrenergic-induced positive inotropism.8,9) We tested whether scopolamine, a mAChR antagonist, suppressed HCNP-induced ChAT gene suppression. However, scopolamine did not affect the HCNP effect (Fig. 3A). ChAT gene expression is regulated by mitogen-activated protein kinase (MAPK) cascade.11) In fact, U0126, a MEK inhibitor, decreased ChAT mRNA levels in MOLT3 cells (Fig. 3B). Western blotting showed that exposure to HCNP for 6 d decreased ERK expression, leading to decreases in the level of phosphorylated (activated) ERK (Fig. 3C). This suggests HCNP acts by suppressing ERK transcription, which in turn leads to decrease in T cell expression of ChAT.
A. ChAT mRNA levels in MOLT3 cells exposed to 300 pg/mL HCNP in the absence or the presence of 10 µM scopolamine for 6 d. ChAT mRNA levels were assessed using real-time PCR. Shown are means±S.E.M. (n=3). *** p<0.001 vs. Control. B. ChAT mRNA levels in MOLT3 cells exposed to 300 pg/mL HCNP or 10 µM U0126 for 6 d. ChAT mRNA levels were assessed using real-time PCR. Shown are means±S.E.M. (n=3). *** p<0.001 vs. Control. C. Left: Western blots showing ChAT and ERK levels in MOLT3 cells exposed to 300 pg/mL HCNP for 6 d. GAPDH was used as a loading control. Right: Graph showing relative levels of ChAT in MOLT3 cells with or without exposure to 300 pg/mL HCNP for 6 d. ChAT protein was quantified using densitometry and normalized to GAPDH. Shown are means±S.E.M. (n=3). * p<0.05, *** p<0.001 vs. Control.
In summary, PEBP1, a HCNP precursor protein, is expressed in several immune cell types present in the murine spleen. Although it is unknown whether HCNP is released from its precursor protein expressed in these cells, long-term exposure to HCNP has a negative effect on ACh synthesis by suppressing ChAT expression in T lymphocytes. In hippocampal neurons, HCNP increases ChAT expression, resulting in the increased ACh production.5) This discrepancy is probably due to multiple ChAT mRNA transcripts (R-, N1-, N2-, and M-types) initiated by three distinct promoter regions and produced by alternative splicing of 5′-noncoding exons.12) M-type is the most abundant transcript, followed by R-type and N-types, in the brain and spinal cord, while only N2-type is expressed in MOLT-3.13) The different promoter for ChAT transcription might account for the different response to HCNP between T lymphocytes and neurons.
Long-term, but not short-term, exposure to HCNP is required for the down-regulation of ChAT expression in T cells. We suppose that HCNP might be able to regulate cell metabolism rather than receptor activation and cell signals, because it suppressed ERK expression, but not its phosphorylation level. Further study will be required to identify the targets of HCNP to modulate ChAT expression.
Our previous study using mAChR and nAChR-deficient mice revealed that lymphocytic cholinergic system influences diverse immune responses.1) Given the fact that HCNP suppresses ChAT expression and then ACh contents in T lymphocytes, HCNP acts as an immune modulator by inhibiting cholinergic signaling in T cells.
This study was supported by Grants-in-Aid for Scientific Research (15K07979, 18K06903) from Japan Society for the Promotion of Science (JPSP), and by Individual Research Grants from the Doshisha Women’s College of Liberal Arts (TF).
The authors declare no conflict of interest.
