Abstract
The aim of this study was to investigate the effects of mechanisms of methionine enkephalin (MENK) on lymphocytes in human peripheral blood. We detected CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), dendritic cells (DCs), natural killer cells (NK), NKT cells and γδT cells before and after treatment with 10−12M MENK, in cell culture by FCM and RT-PCR. Our findings show that MENK stimulating expansion of lymphocyte subpopulationns by inhibiting CD4+CD25+ regulatory T cells (Treg), which is unique discovery of our study. We may use MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy.
Keywords: Interferon-γ, Interleukin-2, human peripheral blood, lymphocyte subpopulations, methionine enkephalin
Introduction
MENK, a penta-peptideis generated in adrenal gland and derived from proenkephalin. The endogenous neuropeptide, is suggested to be involved in the regulatory net between the immune and neuroendocrine systems, with modulation of various functions of cells related to both innate and adaptive immune systems in a naltrexone reversible manner.1 Since the pioneer discovery by Wybran, revealing that there were enkephalin receptors on human T cells and subsequent findings that the enkephalins indeed activated T cells and NK cells as well as LAK cells and TIL cells by Plotnikoff and many others,2 the endogenous opioid ligand interacting with more than one type of delta receptors have been detected to affect various immune cells, such as T cell,3 NK cells.4,7 macrophage8,9 and DCs10-13 at a moderate range of concentrations. MENK may work as a tonic activation agent that plays a role in immune modulation and cell proliferation. We recently demonstrated that MENK could exert positive modulation to the pathway of the DC-CD4+T cells in vitro and in vivo through increasing expression of delta receptors on the surface of the CD4+T cells and binding to delta receptors, which highlight a need to overcome the present bottleneck we are encountering in clinical application of IL-2 and IFN-γ for T cell mediated immune responses.14
Both IL-2 and IFN-γ are T cell activators via either autocrine or paracrine signaling, and play important roles in promoting T cell mediated immune responses in quite some of cancer patients. However, the applications of either IL-2 or IFN-γ in cancer therapy and other immune handicapped cases are very limited because of their side effects, limited efficacy or other unclear mechanisms.
Due to the great importance of restoring immune system in maintaining immune system homeostasis in the body and the role of balanced immunity in fighting cancers and other human-threatening diseases, like AIDS, especial role of the balanced immunity in cancer patient, whose immune system is damaged severely with chemotherapy and since the precise mechanisms of MENK’s effect on balanced immunity in lymphocyte subpopulations remain unclear.15 We endeavored to conduct following exploration to try to reveal mechanisms, by which MENK is regulating cells of immune system, so as to support clinical application of MENK as a new drug fighting cancers in the future.
Results
The expansion of the CD4+CD25+Foxp3+T cells were inhibited after treatment with 10−12 M MENK, and this was confirmed by FCM. The positive percentage for the Treg yielded 6.21 ± 0.75% in the MENK group, (p < 0.05) vs. 9.64 ± 0.86% in the RPMI 1640 group, (p < 0.01) vs. 10.38 ± 0.52% in the IL-2 group and (p < 0.05) vs. 9.57 ± 0.54% in the IFN-γ group. Neither IL-2 nor IFN-γ showed significant inhibition to Treg cells (Fig. 1A). Concurrently the expression of the CD4, CD25, Foxp3 molecules were reconfirmed at mRNA level by RT-PCR. These results indicated that MENK did downregulate the expression of these marker molecules on the Treg cells as shown in (Fig. 1B).
Figure 1. The percentage of Treg in the lymphocytes after treatment with 10−12M MENK for 48h by FCM (A) and reconfirmation of CD4, CD25, Foxp3 by RT-PCR at mRNA level (B). After treated with MENK, IL-2 and IFN-γ respectively in vitro, MENK inhibited Treg expansion in some direct ways as visualized by FCM profile. The number of Treg cells decreased substantially. Lane M: DNA marker; Lane 1: β-actin; Lane 2: RPMI-1640 group; Lane 3: β-actin; Lane 4: IFN-γ group; Lane 5: β-actin; Lane 6: IL-2 group; Lane 7: β-actin; Lane 8: MENK group. *p < 0.05 vs. in the RPMI1640 group; **p < 0.01 vs. in the RPMI1640 group; #p < 0.05 vs. in the MENK group; ##p < 0.05 vs. in the MENK group. Results represent the mean ± SD of six samples.
The data shown in (Fig. 2A) indicated the proportion of CD8+CD28+ cells after treatment with 10−12M MENK for 48 h. RT-PCR analysis showed a significant increase of each molecule at mRNA level in the cells cultured in the presence of 10−12M MENK. Moreover, the mRNA level was much higher as compared with the levels in either IL-2 group or IFN-γ group as reflected in (Fig. 2B).
Figure 2. The percentage of CD8+CD28+ cells in the lymphocytes after treatment with 10-12 M MENK for 48h (A) by FCM and reconfirmation of CD8, CD28 molecules by RT-PCR (B). Lane M: DNA marker; Lane 1: β-actin; Lane 2: RPMI-1640 group; Lane 3: β-actin; Lane 4: IFN-γ group; Lane 5: β-actin; Lane 6: IL-2 group; Lane 7: β-actin; Lane 8: MENK group. *p < 0.05 vs. in the RPMI1640 group; **p < 0.01 vs. in the RPMI1640 group; #p < 0.05 vs. in the MENK group; ##p < 0.05 vs. in the MENK group. Results represent the mean ± SD of six samples.
The positive percentage for the NK cells yielded 7.47 ± 0.35% in the MENK group (p < 0.01) vs.4.98 ± 0.59% in the RPMI 1640 group, (p > 0.05) vs. 6.84 ± 0.54% in the IL-2 group and with p < 0.05 vs.5.94 ± 0.59% in the IFN-γ group, as shown in (Fig. 3A). Also the positive percentage for the NKT cells yielded 7.24 ± 0.65% in the MENK group, (p < 0.05) vs.5.74 ± 0.19% in the RPMI 1640 group, (p > 0.05) vs. 6.59 ± 0.51% in the IL-2 group, (p > 0.05) vs.6.45 ± 0.28% in the IFN-γ group (Fig. 3B).The result shown in Figure 3C reflected the mRNA levels of CD3, CD16, CD56, essentially consistent to the data by FCM.
Figure 3. The percentage of NK (A), NKT (B) cells in the lymphocytes after treatment with 10-12 M MENK for 48h by FCM and reconfirmation of CD3, CD16 and CD56 molecules by RT-PCR (C). The NK cells positive percentage generated in the MENK group was higher than these in the IFN-γ group (p < 0.05), however there was no significant difference with those in the IL-2 group (p > 0.05). Evidently either IL-2 or IFN-γ didn’t stimulate marked expansion of NKT cells. Lane M: DNA marker; Lane 1: β-actin; Lane 2: MENK group; Lane 3: β-actin; Lane 4: IL-2 group; Lane 5: β-actin; Lane 6: IFN-γ group; Lane 7: β-actin; Lane 8: RPMI-1640 group. *p < 0.05 vs. in the RPMI1640 group; **p < 0.01 vs. in the RPMI1640 group; #p < 0.05 vs. in the MENK group and ##p < 0.05 vs. in the MENK group. Results represent the mean ± SD of six samples.
The positive percentage for the γδT cells yielded 9.08 ± 0.43% in the MENK group, (p < 0.01) vs.6.21 ± 0.7% in the RPMI 1640 group, (p < 0.05) vs.7.43 ± 0.4% in the IL-2 group and (p < 0.05) vs.7.08 ± 0.38% in the IFN-γ group, as shown in (Fig. 4A). As shown in (Fig. 4B), RT-PCR analysis showed a significant increase of the γδT molecule at mRNA level in cells cultured in the presence of 10-12 M MENK.
Figure 4. The percentage of γδT cells in the lymphocytes after treatment with 10-12 M MENK for 48h (A) by FCM and reconfirmation by RT-PCR of the expression of γδT cells at mRNA level (B). A higher percentage of γδT cells as visualized by FCM profile indicated that the cell number increased substantially. Lane M: DNA marker; Lane 1: RPMI-1640 group; Lane 2: β-actin; Lane 3: IFN-γ group; Lane 4: β-actin; Lane 5: IL-2 group; Lane 6: β-actin; Lane 7: MENK group; Lane 8: β-actin. *p < 0.05 vs. in the RPMI1640 group; **p < 0.01 vs. in the RPMI1640 group; #p < 0.05 vs. in the MENK group and ##p < 0.05 vs. in the MENK group. Results represent the mean ± SD of six samples.
The positive percentage for the DCs, CD1a+ yielded 1.46 ± 0.09% in the MENK group, (p < 0.01) vs.0.77 ± 0.09% in the RPMI 1640 group, (p < 0.05) vs.0.65 ± 0.2% in the IL-2 group and (p < 0.05) vs.0.91 ± 0.09% in the IFN-γ group, as reflected in (Fig. 5A). The mRNA level of CD1a molecule was much higher compared with this in the IL-2 group and that in the IFN-γ group (Fig. 5B). The result indicated that MENK did enhance the expression of CD1a molecule.
Figure 5. The percentage of DCs after treatment with 10-12 M MENK for 48h by FCM (A) and reconfirmation by RT-PCR of the expression of CD1a molecule at mRNA level (B). Lane M: DNA marker; Lane 1: β-actin; Lane 2: MENK group; Lane 3: β-actin; Lane 4: IL-2 group; Lane 5: β-actin; Lane 6: IFN-γ group; Lane 7: β-actin; Lane 8: RPMI-1640 group. *p < 0.05 vs. in the RPMI1640 group; **p < 0.01 vs. in the RPMI1640 group; #p < 0.05 vs. in the MENK group and ##p < 0.05 vs. in the MENK group. Results represent the mean ± SD of six samples.
Discussion
As an important transmitter connecting the endocrine and immune systems, MENK plays an important role in both endocrine and immune regulation. Moreover, the influence of this molecule is shared between the neuroendocrine and immune systems.1 In fact, MENK’s function of upregulating immune system at suitable doses had been reported previously.16-18 The data of their findings proved that MENK could upregulate the functions of both innate and adaptive immune cells, including: (A) activation of neutrophils.19,20 NK cells,4-7,16,17 macrophages8,9,21 and T cells;3,22,23 (B) upregulation of the production of cytokines such as IL-2, IFN-γ, TNF-α17,24 and IL-6 and (C) enhance of release of hydrogen peroxide and nitric oxide by macrophage.9
Our previous study had already shown that MENK could markedly stimulate CD4+T cell expansion and induce maturation of DCs with higher level of IL-12 production, which would intensify DC-CD4+T cell pathway, resulting in increased Th1 responses.14 Also we found, by our another study, MENK restored immune cells of peripheral blood in 21 cancer patients whose immune systems had been severely damaged by chemotherapy.25
The present results obtained above, show that MENK’s stimulating effect on the expansion of lymphocyte subpopulations in human peripheral blood was present and functional. In fact, what we found from our data are the following findings: (1) MENK at used concentration, could markedly stimulate the expansion of CD4+T cells(Fig. 1), CD8+T cells(Fig. 2), NK, NKT cells (Fig. 3), γδT cells (Fig. 4) and DCs (Fig. 5), which will directly result in increased immunity to remove antigens out of body and keep the body with perfect anti-defense, homeostasis and surveillance, (2) MENK at used concentration could markedly inhibit the expansion of Treg, which reveals a new mechanism by which MENK exerts positive regulation to the cells of immune system and therefore this is unique discovery of this study with great potential in cancer therapy, (3) the marked expansion of γδT cells is with great importance in cancer therapy according to recently increasing number of data elucidating this point, and (4) MENK is more active than either IL-2 or IFN-γ in stimulating expansion of lymphocyte subpopulations by inhibiting Treg cells, which is a new mechanism to activate cellular immune system. MENK can act on human lymphocytes, promoting lymphocyte growth, improve and enhance human immunity. Also present data will help us with better understanding the mechanism, via which MENK is working in restoring human immune system, especially in concrete subpopulations of lymphocytes, such as DCs, NK, NKT cells and γδT cells.
It may be further noted that both IL-2 and IFN-γ do upregulate the expansion of CD8+T cells, DCs, NK, NKT cells and γδT cells. However, the impacts on these cells are limited, especially on γδT cells and they show no great inhibition to the Treg cells, which might be key obstacle for them to play more role and less efficacy observed in a large number of cases clinically.
As we know, the increased DCs not only augment the increase of CD4+T cells, but also increase the stability of the pathway between DCs and CD4+T cells, which, in turn, will result in more secretion of cytokines like IL-2 and IFN-γ by the CD4+T cells, which will trigger a chain of immune responses. Furthermore, it should also be noted that DCs have emerged as themost potent and professional antigen presenting cells that possess the ability to stimulate naive T cells and initiate T cell responses, acting as messengers between the innate and adaptive immunities. They can thus potentially be used in therapeutic vaccines in cancer immunotherapy and for other threatening diseases.26-30
In contrast, Treg cells are known for their suppressive capacity on various immune reactions and are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.31-33 The identification of Treg cells represents a milestone in the field of immunology and provides an explanation for T-cell mediated immunosuppression. Although Treg cells were originally identified for their ability to prevent organ-specific autoimmune disease in mice, emerging evidence suggests that Treg cells play a pivotal role in tumor immunity and contribute to tumor growth and progression, thereby having an important impact on the outcome of cancer patients.34-38 Finally, despite unique discovery for MENK in inhibiting Treg cells there are still more mechanisms for us to investigate further to gain thorough understanding on MENK, such as concrete impact on signals to inhibit Treg cells (studying in process).
The immune system is a very complicated and diverse entity, controlled by endocrine system via signaling molecules that act as activating agents, and thus, formulating a coordinated interaction between the immune and endocrine systems. In addition, there are internal interactions among the immune cells through a variety of cytokines that regulate or even police each other as part of a dynamic immune system.
MENK works as an immune modulator in restoring human immune system and we may use MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy. Also we may consider MENK as a chemotherapy additive, which will sustain immune system of a cancer patient during the process of chemotherapy to get maximized treatment with minimized side effects at same time. In this way the life of a cancer patient could be prolonged markedly or even saved.
This study can therefore contribute to the understanding in depth, of MENK’s positive modulating effects on the immune system. Our results also provide a meaningful mechanism of action for MENK and highlight the clinical significance of MENK in cancer immunotherapy, especially for terminal cancer patients and patients whose immune systems are severely damaged by chemotherapy.39-41 According to our previous study in cancer therapy with MENK, the application of MENK should include: (1) the application of MENK, before chemotherapy or with chemotherapy would sustain the immune system, show great synergistic with drugs, and minimize side effects caused by drugs during process of chemotherapy, (2) the application of MENK, post chemotherapy would restore immune system much fast, and minimize side effects caused by drugs during process of chemotherapy. Likewise, we may consider MENK as a possible adjuvant to be used in vaccine preparations against life-threatening diseases like AIDS.
Materials and Methods
Reagents
MENK was provided by Penta Biotech. Inc. US (≥ 97% purity). In our previous pre-experiments we tested a range of concentrations of MENK from 10−1 to 10−15 M on the proliferation of human lymphocyte in vitro and proved the optimal concentration of 10−12 M. So based on our previous studies, the optimal concentration of 10−12 M of MENK was used in this study.14 For MENK solution the powdered MENK was dissolved in RPMI1640 to make a 10−1 M solution and would be diluted to designated concentration for use. Recombinant human IFN-γ (Catalog number: 20070809), and recombinant human IL-2 (Catalog number: 20070806) were purchased from PEPROTECH Inc. The mAbs used in this study: FITC anti-human CD8a, PE anti-human CD28, PerCP anti-human CD3, FITC anti-human CD16, PE anti-human CD56, FITC anti-human TCRγ/δ, FITC anti-human CD1a and Human Treg FlowTM Kit were all purchased from Biolegend. Ficoll-Paque solution was a product of sigma-Wisconsin. RT-PCR Kits were purchased from Takra. RT-PCR primer was prepared by Nanking Kinsit Gene Tech. Co. China. Trizol was a product of Invitrogen. Other chemicals frequently used in our laboratory were all products from sigma-Aldrich or BD PharMingen.
Subject
All reported data are derived from experiments using PBMC of the 6 healthy individuals.
Lymphocytes isolation and culture in vitro
Peripheral blood mononuclear cells PBMCs were isolated from heparinized human peripheral blood of six healthy volunteers aged from 18 y to 35 y (the mean age was 25.8 ± 5.6 y), who all signed informed consent, by Ficoll density gradient centrifugation. Freshly isolated PBMCs were washed twice with phosphate-buffered saline (PBS) and the cell vitality was tested by trypan blue dye exclusion assay. The cell concentration was adjusted to 1 × 106/ ml and the cells were grown in RPMI1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1.2% sodium bicarbonate. All media contained antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml kanamycin). Unless otherwise indicated, all cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Analysis by flow cytometry (FCM)
Lymphocyte subpopulations were measured by their immunophenotypes. We recorded the major lymphocyte subpopulations with CD8+CD28+ T cells and Treg (CD4+CD25+Foxp3+), NK cells (CD16+CD56+), NKT cells (CD3+CD56+), γδT cells (γδTCR+), DCs (CD1a+). The lymphocytes grown as previously described, were then performed to the following groups; MENK 10-12M, IL-2 (200 U/ml), IFN-γ (400 U/ml) and RPMI1640 (as control). Diversification of the lymphocyte subpopulations after 48 h were checked with FACS Calibur (Becton Dickinson) as well as confirmed by the RT-PCR analysis at mRNA level for each molecule marker. Typically 10,000 events were acquired in the gating region. Each subpopulation was expressed as percentage of lymphocytes and the data were then analyzed using WinMDI 2.9 software (Joseph Trotter, BD Biosciences).
Confirmation of each molecule by RT-PCR at mRNA level
The analysis of each molecule in a final volume of 50μl sample was performed by Takra two-step RT-PCR Kit per manufacturer’s instructions. The RT-PCR reaction was performed as shown in Table 1.
Table 1. RT-PCR reaction of each molecule at mRNA level.
Meanwhile, the total cellular RNA was extracted from the lymphocytes with TRIZOL, precipitated and resuspended in 50 μl diethyllpyrocarbonate treated-dH2O, and the concentration was determined by optic density lecture at 260 nm using a GeneQuantPro spectrophotometer. The integrity of the RNA was tested by applying 1μl in 2% agarose gel containing ethidium bromide, and subsequently was visualized under UV transilluminator.
Statistical analysis
Statistical analysis was performed using the statistical program SPSS (Statistical Package for Social Sciences, Version 16.0) for Windows. All variables are presented as mean ± sd. Differences were evaluated by ANOVA for multiple groups and by the Student test for two groups using the Prism (Graph Pad Software). Tukey test used for post hoc analysis indicated significance when p < 0.05 by ANOVA. The results will be discussed later.
Acknowledgments
This work was supported financially by China Liaoning provincial foundation for international collaboration, No.2006305007 (to Fengping Shan).
Glossary
Abbreviations:
- MENK
methionine enkephalin
- FCM
flow cytometry
- IL-2
Interleukin-2
- IFN-γ
Interferon-γ
- DC
dendritic cell
- NK
natural killer
- Treg
CD4+CD25+ regulatory T cells
- PBML
peripheral blood mononuclear cell
- RT-PCR
reverse transcriptase polymerase chainreaction
Disclosure of Potential Conflicts of Interest
The authors have no financial conflicts of interest with any party.
Footnotes
Previously published online: www.landesbioscience.com/journals/vaccines/article/20759
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