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Repetitive manual acupuncture increases markers of innate immunity in mice subjected to restraint stress
  1. Mayumi Watanabe1,
  2. Eisuke Kainuma2,
  3. Chikako Tomiyama3
  1. 1Department of Medical Informatics, Niigata University Medical & Dental Hospital, Niigata, Japan
  2. 2The House of Kainuma Acupuncture & Moxibustion, Oriental Medical Center Group.(Kainuma Shinkyu-in), Murakami, Niigata, Japan
  3. 3Graduate School of Health Sciences, Niigata University, Niigata, Japan
  1. Correspondence to Dr Mayumi Watanabe, Department of Medical Informatics, Niigata University Medical & Dental Hospital, Niigata 951-8520, Japan; watanabem62{at}gmail.com

Abstract

Objective To study the effects of repetitive manual acupuncture treatment on acute stress in mice and to explore its impact on the immune system.

Methods Thirty-six mice were randomly allocated to one of four groups: control, acupuncture, stress and acupuncture+stress (n=9 each). Mice in the two acupuncture groups were given daily acupuncture treatment superficially (to skin depth) at CV6, CV12 and bilateral ST25, LR14, GB20, GB21, BL10, BL11, BL13, BL14, BL19, BL23 and BL25 for 7 days. On the eighth day mice in the stress and acupuncture+stress groups were exposed to acute stress for 2 h by confinement in a 50 mL centrifuge tube. Body temperature, blood glucose, the number and subpopulation ratios of leucocytes in the liver, spleen and thymus, natural killer (NK) cell percentage cytotoxicity and serum corticosterone and interferon gamma IFNγ were quantified.

Results Mice exposed to stress (irrespective of acupuncture treatment) exhibited hypothermia and hyperglycaemia. However, the increase in glucose level was mitigated by repetitive acupuncture treatment (p<0.05). Percentage cytotoxicity and the level of corticosterone were significantly increased after stress but were unaffected by acupuncture. IFNγ levels did not differ between the groups. Hepatic innate immunity in the liver appeared to be stimulated by repetitive acupuncture treatment as proportions of extrathymic T cells, NK cells and NKT cells in the liver were greatest in the acupuncture+stress group and significantly increased relative to the control group.

Conclusions Repetitive manual acupuncture mitigated stress-induced hyperglycaemia and enhanced markers of innate immunity in the liver within the range of normal homoeostasis. As long as acupuncture stimuli were appropriately applied, they did not appear to be stressful to the mice.

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Introduction

Acupuncture has a long history and is widely practised worldwide. In recent years the potential effects of acupuncture on the immune system have attracted greater attention. Previous studies have indicated that acupuncture may prevent colds, increase local blood flow,1 activate natural killer (NK) cells and modify immunity via putative somato-autonomic effects.2–4 It is generally accepted that immune function can be negatively influenced by stress. Historically, acupuncture has been used for the treatment of stress. However, there are insufficient studies on its mechanism of action, especially for stress prevention.

The immune system comprises both innate and acquired responses. Leucocytes are broadly divided into three categories: monocytes, granulocytes and lymphocytes, which are differentially involved in these two principal types of immunity. Extrathymic T cells, NK cells and NKT cells belong to the innate immune system, whereas T cells and B cells mediate acquired immunity. NK and NKT cells both produce interferon gamma (IFNγ) and are cytotoxic.

Acute restraint in mice is an established animal model of stress and has previously been shown to suppress acquired immunity but augment innate immunity5–8 and to increase the production of corticosterone.8 ,9 When subjected to stress, both mice and humans show hypothermia and hyperglycaemia, which are considered markers of stress.7–11 The aim of this study was to determine the effects of repetitive manual acupuncture on various markers of innate immunity in mice exposed to restraint stress.

Methods

Experimental animals

The experimental protocol was approved by the animal ethics committee of Niigata University. C57BL/6 mice were purchased from Clea Japan (Tokyo, Japan). All mice were male, aged 9–12 weeks and weighed 21.5±1.6 g (range 19.5–23.5). Mice were housed in a room at a constant temperature (26°C) and humidity (50–70%) with a 12 h light/dark cycle (light on from 8:00 to 20:00) and fed under specific pathogen-free conditions in the animal facility of Niigata University (Niigata, Japan).

Experimental design

Thirty-six mice were randomly allocated to one of four groups (n=9 each): control, acupuncture, stress and acupuncture+stress. Experiments involved three mice per group and were repeated three times. Mice in the two acupuncture groups received acupuncture treatments once a day for 1 week, whereas mice in the control and stress groups were left untreated. Rectal temperature and blood glucose levels were determined on day 1 and on day 8 using Thermac Sensor (Shibaura Denki Co, Tokyo) and Precision Xtra (Abbott Japan Co, Ltd, Chiba, Japan) instruments, respectively.

Acupuncture treatment

Disposable acupuncture needles of 13 mm length and 0.24 mm diameter (Carbo deluxe Yangyi Trading Co., Ltd., Japan) were inserted to skin depth at the following 13 acupuncture points based on “Kurono's standard points for the whole body regulation”: CV6 (Qihai), CV12 (Zhongwan) and bilateral ST25 (Tianshu), LR14 (Qimen), GB20 (Fengchi), GB21 (Jianjing), BL10 (Tianzhu), BL11 (Dazhu), BL13 (Feishu), BL14 (Jueyinshu), BL19 (Danshu), BL23 (Shenshu) and BL25 (Dachangshu). WHO standard acupuncture point locations were used and all acupuncture treatment was provided by nationally licensed experienced Japanese acupuncturists.

Acute stress

We used a conventional method to administer restraint stress to mice.12–14 On the eighth day of the study, mice were exposed to acute stress for 2 h (9:00–11:00) by confinement in a 50 mL centrifuge tube with a sufficient number of holes to facilitate ventilation and to prevent an acute rise in body temperature. The animals were not squeezed or compressed, and therefore this stress was considered to be psychological rather than physical.12–15 Mice in the (non-stressed) acupuncture and control groups were handled in the same way as the other two groups but were not subjected to any restraint.

Cell preparation

Mice were anaesthetised with ether and killed by exsanguination from the subclavian artery and vein. Thereafter, various organs were removed for harvest of leucocytes. Hepatic lymphocytes were prepared as previously described.16 Briefly, the liver was pressed through a 200 gauge stainless steel mesh and suspended in Eagle's minimum essential medium supplemented with 5 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; Nissui Pharmaceutical, Tokyo, Japan) and 2% fetal calf serum. After one wash, the pellet was resuspended in 35% Percoll solution containing 100 U/mL heparin and centrifuged at 424 g for 15 min. The pellet was resuspended in red blood cell lysis solution (155 mM NH4Cl; 10 mM KHCO3; 1 mM EDTA; 170 mM Tris, pH 7.3) and then washed twice with medium. Splenocytes and thymocytes were obtained by forcing the spleen and thymus through a stainless steel mesh. Splenocytes were treated with 0.2% NaCl solution to remove the red blood cells.

Flow cytometric analysis

In parallel with measurements of the number and ratio of leucocytes, the phenotype of lymphocytes was identified by two-colour immunofluorescence tests.5 Cells from the liver and spleen were categorised into the following groups based on immunostaining: extrathymic T cells (CD3int IL2Rβ+), NK cells (CD3 IL2Rβ+ or CD3 NK1.1+), NKT cells (CD3int NK1.1+), T cells (B220 CD3+), B cells (B220+ CD3), monocytes (Mac-1+) and granulocytes (Gr-1+). Additional staining for CD4/CD8 (T cell subtypes) was carried out in cells derived from the thymus. Reagents used included anti-CD3ε (145–2C11), anti-IL2Rβ (TMβ-1), anti-NK1.1 (PK136), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-B220 (RA3-6B2), anti-macrophage (M1/70) and anti-granulocyte (RB6–8C5) monoclonal antibodies (mAbs) from BD Pharmingen (BD Biosciences, San Diego, California, USA). All mAbs were used in fluorescein isothiocyanate (FITC)- and phycoerythrin-conjugated-forms. To prevent non-specific binding of mAbs, anti-CD16/CD32 (2.4 G2) mAb was added before staining with labelled mAbs. The suspended lymphocytes (5×105−2×106/tube) were stained with mAbs and then analysed with a FACScan (Becton–Dickinson, Franklin Lakes, New Jersey, USA). Dead cells were excluded by forward scatter, side scatter and propidium iodide gating.

Measurement of serum corticosterone and IFNγ concentrations

The concentration of corticosterone was measured in the serum from each mouse using enzyme immunoassay (IDS Ltd, Boldon Tyne & Wear, UK), according to the manufacturer's instructions.17 The concentration of IFNγ (a marker of NK and NKT cell function) was measured in the serum from each mouse using a LEGEND MAX ELISA kit with precoated plates (BioLegend, Inc, San Diego, California, USA), according to the manufacturer's instructions.18

NK and NKT cell cytotoxicity assay

To evaluate the functional activities of NK and NK T cells, a cytotoxicity assay was performed with a lactate dehydrogenase cytotoxicity detection kit (Roche Diagnostics GmbH, Germany), according to the manufacturer's instructions. As the effector cells, liver mononuclear cells were conventionally purified, serially diluted with RPMI-1640 medium without phenol red (Sigma-Aldrich, St Louis, Missouri, USA) and mixed with target cells (NK-sensitive YAC-1) in 96-well, round-bottomed microculture plates. The plates were briefly centrifuged and incubated for 4 h at 37°C. At the end of the culture, 100 μL of the supernatant was retrieved and lactate dehydrogenase released from the target cells was measured colorimetrically.19

Statistical analysis

Differences between values were determined by Bartlett's test, Student's t test, Welch's t test, Mann–Whitney test, Wilcoxon signed-rank test or one-way analysis of variance followed by post hoc comparisons, as appropriate. The level of significance was set at p<0.05 and p<0.01, and all data are presented as mean±SEM.

Results

Body temperature, glucose and leucocyte numbers

There were no baseline differences between the four groups in body weight (data not shown) and no obvious differences in the response to restraint between the stress and acupuncture+stress groups. Before stress, no significant differences were found between any of the four groups in body temperature or glucose levels (figure 1). After stress, body temperature was not significantly affected. However, glucose levels were higher than the baseline values (on day 1) in the stress group and the acupuncture+stress groups (both p<0.001). This increase was lower in the acupuncture+stress group than in the (untreated) stress group (p=0.029).

Figure 1

Body temperature and glucose levels. Rectal body temperature (°C) and blood glucose (mg/dL) measurements on day 1 and day 8 in 18 healthy control mice receiving 1 week of repetitive manual acupuncture (acupuncture group, n=9) or no treatment (control group, n=9) and 18 mice subjected to acute restraint stress (day 8) with or without pretreatment with repetitive manual acupuncture (acupuncture+stress and stress groups, respectively, n=9 each). The left panel shows changes between day 1 and day 8 and the right panel, changes before and after stress. Data are mean±SEM. *p<0.05, **p<0.01.

Figure 2 shows the effects of stress and acupuncture treatment on lymphocyte yields from liver, spleen and thymus. No significant differences were found between the four groups in cell numbers from these three organs.

Figure 2

Numbers of leucocytes. Leucocyte cell yields from the liver, spleen and thymus in 18 healthy control mice receiving 1 week of repetitive manual acupuncture (acupuncture group (yellow), n=9) or no treatment (control group (white), n=9) and 18 mice subjected to acute restraint stress (day 8) with or without pretreatment with repetitive manual acupuncture (acupuncture+stress (blue) and stress (red) groups, respectively, n=9 each). Data are mean±SEM.

Flow cytometric analysis

Figure 3 shows representative flow cytometry outputs from all four groups, illustrating the effect of repetitive acupuncture with or without subsequent acute stress on the distribution of lymphocyte subsets. The most prominent change appeared to be in the proportion of extrathymic T cells (CD3int IL2Rβ+) and NKT cells (CD3int NK1.1+) in the liver tissue of the stress and acupuncture+stress groups (indicated by arrows, figure 3A, B). The ratio of these lymphocyte subsets in the liver of the stress and acupuncture+stress groups appeared to be higher than those of the control and acupuncture groups. Furthermore, the ratio of these lymphocyte subsets in the acupuncture+stress group appeared to be higher than that of the stress group (50.3% vs 36.7% and 42.3% vs 34.0%, respectively). The proportion of NK cells (CD3 IL2Rβ+ or CD3 NK1.1+) in the liver appeared to be increased by stress and slightly increased further by repetitive acupuncture (indicated by arrowheads, figure 3A, B). Hepatic T cells (B220 CD3+ cells) showed a step-like increase across groups (37.6% <44.7% <59.9% <66.7%) while hepatic B cells (B220+ CD3 cells) decreased (53.1% >46.0% >26.9% >18.8%) (figure 3C). No significant differences were found between the four groups in the ratio of monocytes (Mac-1+) and granulocytes (Gr-1+) in the liver (figure 3D) or in any subset ratios in the spleen. There was no apparent reduction in the proportion of double-positive cells (CD4+ CD8+) in the thymus (a sign of immunosuppression) in any group relative to controls (figure 3E).

Figure 3

Flow cytometric analyses. Representative distributions of two-colour stained lymphocytes in the liver, spleen and thymus in 18 healthy control mice receiving 1 week of repetitive manual acupuncture (acupuncture group, n=9) or no treatment (control group, n=9) and 18 mice subjected to acute restraint stress (day 8) with or without pretreatment with repetitive manual acupuncture (acupuncture+stress and stress groups, respectively, n=9 each). Lymphocytes were isolated and immunofluorescence tests were conducted to identify subsets. Hepatic and splenic lymphocytes were stained for CD3 and (A) IL2Rβ (or (B) NK1.1 or (C) B220), (D and E) Mac-1 and Gr-1 and thymocytes were stained for CD4 and CD8. Numbers represent the percentages of fluorescence-positive cells in corresponding areas.

Leucocyte subsets in the liver and cytotoxicity

Figure 4 shows the absolute cell numbers of each lymphocyte subpopulation, calculated by multiplying the percentage of each subset (figure 3) by total cell number (figure 2). The acupuncture+stress group had significantly greater populations of extrathymic T cells, NK and NKT cells in the liver than the control group (p≤0.001–0.035). Moreover, both stressed groups (stress and acupuncture+stress groups) demonstrated greater percentage cytotoxicity than both non-stressed groups (control and acupuncture groups) (figure 5A, p<0.001).

Figure 4

Leucocyte subsets in the liver. Absolute number of hepatic lymphocyte subsets (natural killer (NK) cells, extrathymic T cells (CD3int IL2Rβ+) and NKT cells) in 18 healthy control mice receiving 1 week of repetitive manual acupuncture (acupuncture group (yellow), n=9) or no treatment (control group (white), n=9) and 18 mice subjected to acute restraint stress (day 8) with or without pre-treatment with repetitive manual acupuncture (acupuncture+stress (blue) and stress (red) groups, respectively, n=9 each). Data are mean±SEM. *p<0.05, **p<0.01.

Figure 5

Cytotoxicity, corticosterone and interferon gamma (IFNγ). Percentage (A) natural killer (NK) cell cytotoxicity, (B) serum corticosterone and (C) IFNγ concentrations in 18 healthy control mice receiving 1 week of repetitive manual acupuncture (acupuncture group (yellow), n=9) or no treatment (control group (white), n=9) and 18 mice subjected to acute restraint stress (day 8) with or without pretreatment with repetitive manual acupuncture (acupuncture+stress (blue) and stress (red) groups, respectively, n=9 each). Data are mean±SEM. *p<0.05, **p<0.01.

Serum corticosterone and IFNγ concentration

Figure 5B shows the effects of stress and acupuncture on serum corticosterone levels. Corticosterone was higher in the stress and acupuncture+stress groups compared with the control and acupuncture groups (both p<0.001). By contrast, there were no significant differences between the four groups in the serum level of IFNγ (figure 5C).

Discussion

This study examined the effects of restraint stress on mice. Stress resulted in increased glucose and corticosterone levels and increased cytotoxicity. Acupuncture mitigated stress-induced hyperglycaemia and increased the proportions of extrathymic T cells, NK cells and NKT cells as determined by flow cytometric analysis.

Importantly, the acupuncture stimuli used in this study did not appear to be stressful to the mice, given that they did not show any signs of stress (such as hypothermia or hyperglycaemia) relative to the two untreated groups. Indeed, compared with the control group, the acupuncture group showed evidence of increased innate immunity, reflected by the altered lymphocyte phenotype. Furthermore, there were no decreases in cell numbers in the thymus, which is a sign of immunosuppression. Reassuringly, this suggests that, as long as acupuncture stimuli are applied appropriately, the treatment is not a severe stress to the body. These results are consistent with previous reports.20

In this study, mice that were exposed to stress did not show prominent hypothermia compared with the unstressed groups; however, they did show hyperglycaemia, which was partially ameliorated by pretreatment with acupuncture. In addition, the acupuncture+stress group had evidence of greater innate immunity, reflected by significant differences in the proportions of hepatic leucocyte subsets (extrathymic T cells, NK cells and NKT cells). There was no measurable effect of acupuncture on percentage cytotoxicity, reflecting NK cell function using NK-sensitive YAC-1,19 which increased after exposure to stress (stress and acupuncture+stress groups). Furthermore, cells for acquired immunity (other types of T cells and B cells) did not change significantly, suggesting that the effects of acupuncture stimulation in this study were confined to the innate immune system.

Stress induces the enhancement of leucocyte trafficking and immune activation.21 This enhancement is mediated by the innate immune response, which triggers a wide range of non-specific defences. In contrast, acquired immunity is effective only for specific foreign antigens. During a fight-or-flight response, immediate action of the white muscles is needed. These muscles are supported by increases in blood glucose, mediated by glucocorticoids such as corticosterone or cortisol.22 Moreover, the adrenocortical axis (adrenaline and glucocorticoid) affects NKT cells.23 In this study, corticosterone and glucose levels in stressed mice were higher than in non-stressed mice, in keeping with this observation. The acupuncture-treated group had lower levels of glucose than the untreated group after stress exposure. The lack of any change in circulating corticosterone may reflect a more subtle effect, which we were underpowered to detect, or relative corticosterone resistance. Possibly, mice in the acupuncture+stress group might have grown accustomed to the acupuncture stimuli after 1 week and consequently, developed a higher stress threshold. However, further study is needed to support or refute this hypothesis.

It is recognised in biomedicine that stress can influence the liver,8 ,9 ,23 and we have previously reported that exposure to hypoxic stress (ie, organ reperfusion after acute ischaemia) results in liver damage that is associated with qualitative overactivation of NKT cells (excessive IFNγ production and high cytotoxicity) and granulocytes (superoxide production).24 Therefore, we hypothesised that repetitive acupuncture stimuli would protect the liver from damage caused by restraint stress. In our study, however, no increase in IFNγ was seen secondary to restraint stress and acupuncture increased the numbers of innate immunity cells in the liver in the absence of visible tissue damage. Further studies of models of hypoxic stress are required to assess whether acupuncture stimulation is effective in preventing the associated tissue damage.

Many researchers and clinicians consider that acupuncture stimuli are effective only within the range of normal homoeostasis and can thereby move a deviated value towards a normal value (eg, leucocyte pattern).2 Accordingly, it is a potential limitation of this study that healthy young mice were used as their clinical test values remained inside the homeostatic range and we may therefore have been underpowered to detect small differences. Consequently, further research, including larger-scale studies, is required to further characterise the mechanisms of action behind acupuncture treatment of stress. Our study was also limited by the lack of any behavioural tests to assess the severity of stress and whether it can be ameliorated by acupuncture. Ultimately, studies in human subjects will be required to assess the potential role of repetitive acupuncture stimuli in the medical treatment of stress.

In conclusion, repetitive manual acupuncture increases hepatic markers of innate immunity in mice subjected to restraint stress. When applied appropriately, is does not appear to be stressful to mice.

Acknowledgments

We thank Dr Yasuzo Kurono (Oriental Medical Center (Toyo Igakukennkyu-sho), Nagoya, Japan) for medical advice.

References

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Footnotes

  • MW and EK contributed equally.

  • Contributors MW and EK equally conceived the study, collected data, conducted the statistical analysis and prepared the draft. CT contributed to the literature review. All authors critically edited drafts of this manuscript and approved the final manuscript.

  • Competing interests None declared.

  • Ethics approval Approved by the animal ethics committee of Niigata University (reference number 63) and all procedures were carried out in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals”.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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