Macrophage polarization induced by neuropeptide methionine enkephalin (MENK) promotes tumoricidal responses

Abstract

The aim of this study is to investigate macrophages polarization induced by methionine enkephalin (MENK) that promotes tumoricidal responses in vivo and in vitro. Both phenotypic and functional activities of macrophages were assessed by the quantitative analysis of key surface molecules on macrophages with flow cytometry, immunofluorescent staining, and the production of cytokines with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Our results showed that MENK could down-regulate the expression of CD206 and the production of arginase-1 (the markers of alternatively activated (M2) macrophage) in tumor-associated macrophages in vivo, meanwhile it could significantly up-regulate the expression of CD64, MHC-II, and the production of induced nitric oxide synthase (the markers of classically activated (M1) macrophages). Furthermore, the studies on bone marrow-derived macrophages treated with MENK (10⁻¹² M) in vitro had demonstrated that MENK could markedly increase tumoricidal activity. MENK could also enhance the release of reactive oxidant species and the production of interleukin-12p40, tumor necrosis factor-α, while decrease the production of interleukin-10. In conclusion, MENK could effectively induce M2 macrophages polarizing to M1 macrophages, sequentially to modulate the Th1 responses of the host immune system. Our results suggest that MENK might have great potential as a new therapeutic agent for cancer.

Introduction

Opioid peptides are divided into three classes of active peptides, that is, enkephalins, dynorphins, and endorphins. The opioid peptides play their roles by binding to membrane-bound receptors, and several types of opioid receptors have been described. Especially, there was good evidence for DOR and KOR on immune cells in several species [1]. These receptors have all been found on the surface of different immune cells, like T cell, NK cell, macrophage, and dendritic cell. Recently, methionine enkephalin (MENK), an opioid growth factor (OGF), has been identified as a regulator related to proliferation, migration, and differentiation in a wide variety of tissues and cells [2, 3]. MENK, also known as the endogenous neuropeptide, is suggested to be involved in the regulatory loop between immune and neuroendocrine systems, modulating various functions of cells related to both the innate and adaptive immune systems as a messenger participating in the immunoregulation [4]. At moderate range of concentrations, MENK plays a role in immune modulation and cell proliferation as a tonic activating agent [5]. The studies in patients or mice bearing tumors treated with MENK have demonstrated reduction in tumor incidence, retardation in tumor progression, and prolonged survival [6–8].

Macrophages with diverse biological functions, including antigen presentation, cytotoxicity, debris removing and tissue remodeling, regulation of inflammation, induction of immunity, endocytosis [9], play an indispensable role in immunological defense of the organism against pathogens through participating in both innate and adaptive immunity, as well as antitumor activities. As described previously, macrophages can be functionally polarized into classically activated (M1) macrophages or alternatively activated (M2) macrophages [10, 11]. Classically activated macrophages can produce proinflammatory molecules, such as TNF-α, IL-1β, IL-6, and perform the functions to efficiently eliminate pathogens and tumor cells. In contrast, alternatively activated macrophages mainly produce anti-inflammatory molecules, such as IL-10 and IL-1 receptor antagonist (IL-1Ra), and counteract inflammatory response, promote angiogenesis as well as remodel impaired tissue. M1 and M2 macrophages are likely representatives of two extremes along a continuum of macrophage biological phenotypes. However, there is no report on the effect on macrophage polarization by MENK and related mechanisms via which MENK exerts antitumor activity. Due to the ever-increasing importance of MENK in immunoregulation and antitumor, we conducted the following studies to investigate whether MENK can induce an antitumor activity by reversing macrophages polarization from M2 to M1 and try to give an answer.

Materials and methods

Cell line

Murine Sarcoma 180 (S180) cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), where the cell line had been tested and authenticated by multilocus DNA fingerprinting and multiplex PCR DNA profiling analysis. Then the cells were immediately grown in the recommended media supplemented (DMEM, in the presence of 10 % fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin) in a humidified atmosphere of 5 % CO₂ and 95 % air at 37 °C.

Tumor model and MENK administration

The male C57BL/6 mice (aged 8–10 week-old, 18–21 g) were obtained from Slac Laboratory Animals Co. Ltd (Shanghai, China). All experiments with animals were conducted in pathogen-free condition per the guidelines of NIH Guide for the Care and Use of Laboratory Animals. S180 cells (1.0 × 10⁶/ml) were injected subcutaneously into C57BL/6 mice (0.2 ml/mouse) and after establishment of tumor model for 24 h, mice were divided into two groups (8 mice/group). The mice in the MENK group were injected (i. p) with MENK (20 mg/kg, Penta Biotech Inc. USA, ≥97 purity) once a day for consecutive 21 days [12]. The mice in the vehicle control group received normal saline only. The mice were observed daily, and when tumors became palpable, the shortest/longest diameter of tumors was measured with a vernier caliper every 3 days. Finally, tumor sizes (mm³) were calculated via the following formula: (the shortest diameter)² × (the longest diameter) × 0.5 [13]. The mean survival time and long-term survival rate were assessed at the end of the experiment.

Tumor morphology

Tumor tissues from each mouse were fixed in 4 % paraformaldehyde at 4 °C for histological assessment. After fixation, the tissues were dehydrated with a graded series of ethanol and embedded in paraffin. Thin sections (4 mm) prepared from the paraffin embedded tissues were mounted on glass slides, deparaffinized with xylene, and then stained with H&E for light microscopic examination. For morphological studies, three randomly selected sections were photographed using a 40× objective lens (Olympus BX-51) as described previously [14].

Analysis of macrophages and Th lymphocyte subsets by FCM

The tumor cells and splenocytes were prepared as described previously [14, 15]. The digested tumors and the processed splenocytes were filtered through a 200-μm nylon cell strainer (BD Bioscience) in PBS containing 2 % FCS to obtain single-cell suspensions. Isolated splenocytes and tumor cells (1 × 10⁶ cells) were labeled with multiple fluorescently monoclonal antibodies (Biolegend, CA, USA) for surface markers to delineate macrophages of M1 and M2 phenotypes, as described previously [16–18]. The following antibodies were utilized: anti-mannose receptor (CD206)-FITC, anti-F4/80-PE, anti-CD64-APC, anti-MHC-II-PerCP Cy5.5.

For FCM analysis of Th lymphocyte subsets, splenocytes were incubated for 5 h at 37 °C with phorbol ester (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and GolgiStop (Monensin 2 μmol/L, BD Pharmingen). The surfaces were stained with the corresponding fluorescence-labeled anti-CD3-APC/anti-CD4-PerCP for 15 min on ice. For intracellular staining, the Cytofix/Cytoperm buffer set (BD Pharmingen) was used, and the cells were fixed and made permeable for 30 min at 21 °C, subsequently were stained with 0.25 μg of anti-IFN-γ-FITC/anti-IL-4-PE for 30 min at 21 °C [19].

All cells were analyzed by using FACS Calibur (BD Biosciences). As for analysis of macrophage phenotypes, the results were expressed as fluorescent intensity of different molecular expression on the membrane obtained with the gated population staining positive for F4/80 (macrophage marker). As for analysis of Th lymphocyte subsets, forward and side light scatter properties were used to gate of morphological splenocytes. Four-color flow cytometry is used to identify the percentages of intracellular cytokines (IFN-γ or IL-4) in CD3⁺CD4⁺ lymphocytes. The percentage of positive cell stained for each protein was determined by comparing testing samples with the isotype control. Data were analyzed by FCS Express Version 3 software.

Analysis of tumor cells/splenocytes apoptosis by FCM

Tumor cells and splenocytes were treated as described above. The 1 × 10⁶ cells were stained per manufacturer’s instructions (Annexin V-FITC/PI kit, BD Biosciences). Briefly, the cells were suspended in 1× binding buffer, and then followed by incubation with Annexin V-FITC and propidium iodide in dark for 20 min. The cells were analyzed by FCM, and finally, the cell sorting and data processing were carried out on 10,000 cells as mentioned above.

Immunofluorescent staining for the expression of surface molecules on tumor-associated macrophages (TAMs)

The tumors were collected and treated with Bouin’s fixative. Cryostat sections (8 μm) were incubated with anti-CD206-FITC (1:100) and anti-F4/80-PE (1:100). Fluorescent images were obtained using a laser scanning confocal microscope (LEICA TCS SP5). The density of specific immunoreactive cells of tumors was determined using Image-Pro Plus image analysis software 6.0 as reported previously [20].

mRNA analysis for TAMs by RT-PCR

After mononuclear cells from tumors were incubated for 1 h, adherent TAMs were collected. Total RNA was extracted by using Trizol (Invitrogen, Carlsbad, CA) and quantified by OD at 260 nm using a Gene Quant Pro spectrophotometer (Beijing Technologies, China). The cDNA was synthesized by using RNA PCR Kit (AMV) Ver.3.0 (Takara, Japan), and reverse transcription was then performed per manufacturer’s instruction. The subsequent PCR amplification was performed in MyCycler Thermal Cycler (Bio-Rad Laboratories, USA). Initial denaturation was followed by 35 cycles of DNA amplification. The primers (synthesized by Takara, Japan) used for each gene validation are shown in Table 1. Ethidium bromide-stained PCR products were separated by 1.5 % (w/v) agarose gel electrophoresis and followed by UV visualization in a transilluminator. The mRNA levels of specific genes were analyzed using Bandscan software.

Table 1.

The primers of genes

Gene	Sequence	GC (%)	Tm (°C)	Products length (bp)
β-Actin	5′-TGCTGTCCCTGTATGCCTCT-3′	55.00	56.0	462
	5′-GGTCTTTACGGATGTCAACG-3′	50.00
TNF-α	5′-GGCGGTGCCTATGTCTC-3′	64.71	52.0	362
	5-GCAGCCTTGTCCCTTGA-3′	58.82
iNOS	5′-GAGCGAGTTGTGGATTGTC-3′	52.63	54.0	208
	5′-TCTGCCTATCCGTCTCGTC-3′	57.89
Arg-1	5′-TGGGAAGACAGCAGAGGA-3′	55.56	52.0	132
	5′-CACAGTCACTTAGGTGGTTTA-3′	42.86

Induction of BMDMs in vitro

BMDMs were prepared as described previously [21, 22]. The bone marrow cells were incubated for 4 h, and non-adherent bone marrow cells were collected, subsequently cultured in RPMI 1640, containing rGM-CSF (100 U/ml, Pepro Tech Inc., Rocky Hill, NJ, USA).

Determination of optimal MENK concentration

The BMDMs were incubated with MENK ranging from 10⁻¹⁵ to 10⁻⁸ M for 24 and 48 h, respectively, as previously described [23, 24]. BMDMs were cultured in the RPMI1640 medium alone as a control. After incubation, BMDMs were washed with PBS, and subsequently, 10 μl of 5 mg/ml MTT in FBS-free medium was added to each well. After incubation for 4 h, the supernatants were removed, and cells were resolved with 150 μl/well DMSO. Finally, the optical density was measured using bichromatic microplate reader (Bio-rad, USA) at 570 nm.

Polarization of BMDMs in vitro

The polarization was obtained by culturing the BMDMs in RPMI 1640 supplemented with 5 % FCS and LPS plus IFN-γ (for M1 polarization) or IL-4 (for M2 polarization). The cells (2 × 10⁵/ml) were incubated for 48 h with either Escherichia coli 055:B5 LPS (5 ng/ml, Sigma-Aldrich, USA), plus IFN-γ (100U/ml, Pepro Tech Inc., Rocky Hill, NJ, USA) [25] or IL-4 (10 ng/ml, Pepro Tech Inc., Rocky Hill, NJ, USA) [26], with optimal 10⁻¹² M MENK. The supernatants were collected for assay of inflammatory cytokine using ELISA. Fresh single-cell suspensions were immediately utilized for FCM and monolayers for immunofluorescent staining. The aliquot cells were frozen at −80 °C for mRNA analysis by RT-PCR. All the experiments were conducted in accord with the protocols as described in vivo.

Measurement of intracellular reactive oxidant species (ROS) in BMDMs

The determination of ROS was done based on the oxidative conversion of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) by peroxide. Briefly, the treated BMDMs were incubated with DCFH-DA for 20 min, and the fluorescence of 2,7-dichlorofluorescein (DCF) was detected using FCM with excitation at 488 nm and emission at 530 nm, and all measurements were repeated in triplicate. Finally, the data were processed with CellQuest program (BD Biosciences). The fluorescence was meanwhile monitored using a fluorescence microscope (Olympus BX51) with FITC filter, and the average intensity values were measured (200×) in three randomly chosen fields of each replicated experiment.

The assay of inflammatory cytokines with ELISA

The productions of inflammatory cytokines, IL-12, TNF-α, and IL-10, in the supernatants of BMDMs were measured using ELISA kits (eBioscience, San Diego, CA).

The cytotoxicity mediated by BMDMs

The assay for macrophage cytotoxicity was performed as described previously [27]. Briefly, the treated BMDMs were co-incubated with B16 mouse melanoma cells (1 × 10⁴/wells) at ratio of effector:target 10:1 for 24 h, stained with crystal violet containing 10 % formaldehyde for 15 min. Finally, OD₅₄₀ was determined by using bichromatic microplate reader (Bio-rad, USA). The cytotoxicity was expressed using the following formula:

Statistical analyses

Data were presented as mean ± SD, and differences among the groups were evaluated by ANOVA for multiple groups and by the Student’s t-test for two groups by using Prism Graph Pad Software (San Diego, CA, USA). p values below 0.05 were considered statistically significant. The results will be discussed later.

Results

Tumor sizes and survival

The results showed that tumor sizes in the MENK group after 18 days were suppressed significantly compared with those in the control group (p < 0.01), as shown in Fig. 1a. The survival rate in the MENK group was significantly prolonged in comparison with that in the control group (p < 0.05), as shown in Fig. 1b. The result showed that MENK could delay tumor growth and significantly prolong the survival of mice bearing S180 tumors.

Fig. 1.

Tumoricidal effect of MENK in vivo. a The xenograft C57/BL6 mice (8/group) were treated with MENK (20 mg/kg) every other day. Tumor sizes were measured every 3 days, and survival rate in each group was monitored daily. Tumor sizes in the MENK group versus those in the vehicle control group at the 6th, 12th, and 18th day, respectively. The median is indicated by a horizontal line. A significant difference was observed by unpaired Student’s t-test at the 18th day (p < 0.01). b Survival rate in the MENK group versus that in the control. A significant difference was observed (p < 0.05). c AnnexinV/PI staining was performed for the apoptotic analysis of tumor cells and splenocytes by FCM. The percentage of early apoptotic tumor cells (AV⁺/PI⁻, p < 0.01) and late apoptotic tumor cells (AV⁺/PI⁺, p < 0.05) in the MENK group increased significantly versus that in the control group, and the percentage of viable tumor cells (AV⁻/PI⁻) in the MENK group decreased significantly (p < 0.05) versus that in the control group. The percentage of apoptotic splenocytes was very low in the MENK group, and no significant difference was observed (p > 0.05) versus that in the control group. d Morphology of histopathological sections of tumors. Large amount of tumor regression accompanied by massive apoptosis (black arrow) throughout the tumor (both in the periphery and in the center of the tumor) was shown in the MENK group. At same time, some discernible infiltration of inflammatory cells (gray arrow) and fibrous tissue (white arrow) were observed in the peripheral area of the tumors. The tumor in the control group showed only several punctate or scattered necrotic regions among isles of the tumor cells (black arrow)

Analysis of tumor cells/splenocytes apoptosis by FCM

Annexin V/PI staining was performed as described above. Viable cells are in the lower left quadrant (AV⁻/PI⁻). The lower right quadrant represents early apoptotic cells (AV⁺/PI⁻), and the upper right quadrant represents late apoptotic cells (AV⁺/PI⁺). The results indicated the percentage of early apoptotic tumor cells in the MENK group increased markedly versus that in the control group (p < 0.01). The percentage of late apoptotic tumor cells in the MENK group were much higher than that in the control group (p < 0.05), and the percentage of viable tumor cells decreased compared with that in the control group (p < 0.05). The percentage of apoptotic splenocytes was very low in the MENK group, and no significant difference was observed versus that in the control group (p > 0.05), as shown in Fig. 1c. These data demonstrated that MENK could effectively induce the apoptosis of tumor cells without damaging the normal cells.

Histopathologic morphology of tumors

The histopathologic morphology of tumors in the MENK group showed that there was a large portion of the necrosis area in the center of the tumors, accompanied by massive apoptosis. Also, some discernible infiltration of inflammatory cells and fibrous tissue were observed in the peripheral area of the tumors. However, the histopathologic morphology of tumors in the vehicle control group showed only several punctate or scattered necrotic regions among isles of the tumors. The neoplastic cells showed poorly differentiated with significant heterogeneity, characterized by large hyperchromatic nuclei, and relatively small amount of cytoplasm. The binuclear or multinuclear giant cells were observed in some tumors, as shown in Fig. 1d.

Analysis of expression of surface molecules on macrophages by FCM and immunofluorescent staining

As shown in Fig. 2a, b, the expression of CD206 of splenic macrophages and TAMs in the MENK group was markedly lower than that in the control group (p < 0.01). Whereas the main M1 markers, the expression of CD64 and MHC-II on splenic macrophages, and TAMs in the MENK group were much higher than that in the control group (p < 0.01). This result was also confirmed by laser scan confocal analysis of the expressions of CD206 on F4/80⁺ TAMs with immunofluorescent staining, shown in Fig. 2c. Finally, the expression of CD206 was indicated by double staining with anti-CD206 (green) combined with anti-F4/80 (red) on TAMs. The percentage of TAMs expressing CD206 in the MENK group was obviously attenuated compared with that in the control group (p < 0.05). These data demonstrated that MENK in the situation of tumor in mice could reverse macrophages from M2 to M1.

Fig. 2.

Analysis of expression of surface molecules on macrophages by FCM and immunofluorescent staining. a–b The fluorescent intensity of CD206, CD64, and MHC-II on TAMs and splenic macrophages obtained with the gated population of F4/80⁺ by FCM. The statistical analysis of the expression of membranous molecules in the MENK group versus that in the control group by multiple-color flow cytometry. c The double staining of fluorescent images of TAMs with anti-CD206 (green) combined with anti-F4/80 (red) using a laser scanning confocal microscope. The double-staining cells to both antibodies (yellow) in the superimposed images. The density of specific immunoreactive cells measured using Image-Pro Plus image analysis software 6.0. Columns, the percentage of mean of three replicates; bars, SD. *p < 0.05, **p < 0.01 versus that of the vehicle controls using unpaired Student’s t-test

Analysis of Th lymphocyte subsets by FCM

The percentage of CD4⁺ IFN-γ⁺ (Th1) lymphocytes in the MENK group were markedly higher than that in the control group, while the percentage of CD4⁺ IL-4⁺ (Th2) lymphocytes in the MENK group was significantly reduced than that in the control group (p < 0.05), as reflected in Fig. 3a. These data demonstrated that MENK in the situation of tumor in mice could induce Th1 immunity.

Fig. 3.

Analysis of Th lymphocyte subsets and the expression of TNF-α, iNOS, Arg-1 in TAMs, and BMDMs. a Analysis of splenic Th lymphocyte subsets by FCM. The splenic Th lymphocytes were gated based on mean expression of the FSC and low SSC. The mean percentage for IFN-γ⁺ or IL-4⁺ cells analyzed within the Th lymphocyte gate (CD3⁺CD4⁺) were indicated. The mean percentage for CD4⁺IFN-γ⁺ (Th1)/CD4⁺ IL-4⁺ (Th2) cells in the MENK group compared to that in the controls. Columns, mean percentage of five replicates; bars, SD. *p < 0.05 versus that in the vehicle controls using unpaired Student’s t test. b The mRNA analysis of iNOS, Arg-1 and TNF-α in TAMs from the MENK group, and the controls in vivo. **p < 0.01 versus that in the controls using unpaired Student’s t-test. c The mRNA levels of iNOS, Arg-1 and TNF-α in the polarized BMDMs cultured for 48 h. Columns, mean percentage of three replicates; bars, SD. ^# p < 0.05 versus that in RPMI 1640, ^## p < 0.01 versus that in RPMI 1640, *p < 0.05 versus that in MENK, **p < 0.01 versus that in MENK, ^Δ p < 0.05 in LPS + IFN versus that in LPS + IFN + MENK, ^ΔΔ p < 0.01 in IL-4 versus that in IL-4 + MENK, using one-way ANOVA

mRNA analysis for TAMs by RT-PCR

The results of RT-PCR in TAMs in vivo showed the ratio of iNOS, and TNF-α (M1 markers) to β-actin in the MENK group were much higher (p < 0.01) than that in the control group. Whereas the levels of Arg-1 (M2 marker) were obviously reduced (p < 0.01) in the MENK group versus that in the control group, as shown in Fig. 3b.

mRNA analysis for BMDMs by RT-PCR

The results of RT-PCR showed the ratio of iNOS to β-actin in the MENK group increased (p < 0.05) versus that in the RPMI 1640 group. MENK could enhance the effect of producing TNF-α induced by LPS + IFN-γ (p < 0.05). There was no significant difference in the levels of Arg-1 between the MENK group and the RPMI 1640 group (p > 0.05), but it obviously increased in the MENK + IL-4 group versus that in the IL-4 group (p < 0.01), as shown in Fig. 3c. These data confirmed that MENK could up-regulate the expression of iNOS and TNF-α mRNA (M1 markers) effectively; simultaneously, it down-regulated the expression of Arg-1 mRNA (M2 markers).

Determination of optimal MENK concentration in vitro

After incubation for 7 days, more than 95 % of the adherent cells were PE-F4/80 + (Biolegend, California, USA) confirmed by FCM (Fig. 4a). The cells identified by the staining of non-specific esterase (NAE) and acid phosphatase (ACP) showed typical morphology (Fig. 4a). To determine the optimal MENK concentration, we tested concentrations of MENK ranging from 10⁻⁸ to 10⁻¹⁵ M on BMDMs for 24 or 48 h. The result showed that 10⁻¹² M was the best concentration for the cell proliferation at 48 h. Therefore, we chose 10⁻¹² M as the optimal concentration, shown in Fig. 4b. The morphology of the activated macrophages in each group was shown in Fig. 4c.

Fig. 4.

Morphology and the analysis of intracellular ROS of BMDMs. a Morphology of BMDMs under light microscope (a) morphology of BMDMs under inverted microscope (×200), (b) Wright-Giemsa staining, (c) NAE staining, (d) ACP staining (×1000), (e) Histogram of fluorescent intensity F4/80 + BMDMs by FCM. b Effect on the growth of BMDMs by a range of MENK from 10⁻¹⁵ to 10⁻⁸ M. BMDMs were incubated for 24 or 48 h. The cell vitality was assessed by MTT at 570 nm. Columns, mean of three replicates; bars, SD. *p < 0.05 and**p < 0.01 versus the RPMI 1640 control. c Morphology of polarized BMDMs in RPMI 1640 supplemented with 5 % FCS and LPS plus IFN-γ or IL-4 along with MENK (10⁻¹² M) for 48 h (a: RPMI1640, b: LPS + IFN, c: LPS + IFN +MENK, d: IL-4, e: IL-4 + MENK, f: MENK). d The analysis of intracellular ROS in BMDMs. The intracellular ROS was monitored (green) by fluorescence microscope. The mean of fluorescence intensity was measured at ×200 in three randomly fields of each replicated experiments (a: RPMI1640, b: LPS + IFN, c: LPS + IFN + MENK, d: IL-4, e: IL-4 + MENK, f: MENK, scale bar = 50 μm). e The histogram of intracellular fluorescent intensity of ROS by flow cytometry analysis in BMDMs. Columns, mean percentage of three replicates; bars, SD. Statistically significant differences (p < 0.05) obtained using one-way ANOVA. ^# p < 0.05 versus that in RPMI 1640, ^## p < 0.01 versus that in RPMI 1640,*p < 0.05 versus that in MENK, **p < 0.01 versus that in MENK, ^ΔΔ p < 0.01 in IL-4 or LPS + IFN versus that in IL-4 + MENK or LPS + IFN + MENK, using one-way ANOVA

Measurement of intracellular ROS in BMDMs

ROS oxydase produced by phagocyte is the major mediator for anti-microbial and tumoricidal activity [28]. Our result in vitro showed no significant difference of the mean fluorescence intensity monitored by fluorescence microscope between the MENK group and the RPMI 1640 group (p > 0.05), while it was much higher in the MENK + IL-4 group (p < 0.05) than that in the IL-4 group, as shown in Fig. 4d. Correspondingly, the analysis of intracellular ROS by FCM showed that MENK enhanced the intracellular ROS significantly (p < 0.01) while combined with LPS + IFN-γ or IL-4, as indicated in Fig. 4e.

Analysis of expression of surface molecules on BMDMs by FCM

The expression of CD64 by FCM analysis was much higher in the MENK group (p < 0.01) than that in the RPMI 1640 group, although the expression of CD64 had no difference (p > 0.05) between MENK + LPS + IFN-γ group and LPS + IFN-γ group. It increased in the MENK + IL-4 group versus that in the IL-4 group (p < 0.01). Furthermore, the expression of CD206 decreased obviously in the MENK + IL-4 group versus that in the IL-4 group (p < 0.01), shown in Fig. 5a, b. This result was also confirmed by laser scan confocal analysis of the expression of CD206 on F4/80⁺ BMDMs, shown in Fig. 5c. The percentage of CD206⁺ cells in the MENK group reduced compared with that in the RPMI 1640 group, and MENK could decrease the expression of CD206 induced by IL-4 significantly (p < 0.01). These data demonstrated that MENK could reverse immunophenotype of BMDMs from M2 to M1.

Fig. 5.

The expression of surface molecules on BMDMs and analysis of the levels of IL-12p40, TNF-α, and IL-10. a, b The histogram of fluorescent intensity of CD206/CD64 on BMDMs by FCM (a: 1640, b: LPS + IFN, c: LPS + IFN +MENK, d: Il-4, e: Il-4 + MENK, f: MENK). The statistical analysis of the expression of CD64 and CD206 on BMDMs using one-way ANOVA. Columns, mean of three replicates; bars, SD. c The double staining of fluorescent images of BMDMs with anti-CD206 (green) combined with anti-F4/80 (red) using a laser scanning confocal microscope. The double-staining cells to both antibodies (yellow) in the superimposed images. The density of specific immunoreactive cells measured using Image-Pro Plus image analysis software 6.0. The statistical analysis of the expression of CD206 on BMDMs was obtained using one-way ANOVA. d The statistical analysis of the levels of IL-12p40, TNF-α, and IL-10 in supernatants of treated BMDMs in each group using one-way ANOVA. e The statistical analysis of the tumoricidal activities of treated BMDMs in each group using one-way ANOVA. Columns, mean percentage of three replicates; bars, SD. ^# p < 0.05 versus that in RPMI 1640, ^## p < 0.01 versus that in RPMI 1640, *p < 0.05 versus that in MENK, **p < 0.01 versus that in MENK, ^Δ p < 0.05 in LPS + IFN versus that in LPS + IFN + MENK, ^ΔΔ p < 0.01 in IL-4 versus that in IL-4 + MENK, using one-way ANOVA

The production of inflammatory cytokines

IL-12p40, TNF-α, and IL-10 are indicative of the inflammatory function of macrophages. They are produced at high levels (with the exception of IL-10) by cells of the M1 phenotype. The cytokine production in vitro assayed by ELISA showed that the levels of IL-12p40 and TNF-α in the MENK group increased significantly (p < 0.01) compared with those in the RPMI 1640 group, and the level of IL-12p40 in the MENK group was higher as compared with that either in LPS + IFN-γ group (p < 0.05) or IL-4 group (p < 0.01). The level of TNF-α in the MENK + LPS + IFN-γ group showed no difference as compared with that in the LPS + IFN-γ group (p > 0.05), while level of TNF-α in the MENK + IL-4 group was higher than that in the IL-4 group (p < 0.01). The level of IL-10 decreased in the MENK group (p < 0.01) versus that in the RPMI 1640 group and showed no significance in the MENK + LPS + IFN-γ group compared with that in the LPS + IFN-γ group (p > 0.05), while MENK could increased the level of IL-10 production by the cells induced by IL-4 (p < 0.01), as shown in Fig. 5d. These data showed that the productions of cytokines IL-12p40 and TNF-α by BMDMs induced by LPS plus IFN-γ were remarkably promoted in the presence of MENK. Meanwhile, the production of cytokine IL-10 by the cells induced by IL-4 was down-regulated by MENK.

BMDM-mediated cytotoxicity

B16 melanoma cells were sensitive to TNF-α or NO. To determine the effect of MENK on macrophages to kill the tumor cells, BMDMs stimulated with LPS/IFN-γ or IL-4 with or without MENK were incubated with target cells (B16 melanoma cells). The results showed that the tumoricidal activity of BMDMs increased in the MENK group (p < 0.01) versus that in the RPMI 1640 group, similarly in the MENK + IL-4 group (p < 0.01) versus that in the IL-4 group, although MENK + LPS/IFN-γ could not increase the tumoricidal activity (p > 0.05), as shown in Fig. 5e.

Discussion

Immune system is a very complicated and diverse entity, which is connected with endocrine system via secreting signal agent. The coordinating effect between immune system and endocrine system maintains the stability of body functions. Moreover, there is interaction among immune cells, which are secreting a variety of cytokines to regulate the relationships each other.

In view of a direct influence on the growth of neural and non-neural cells, opioid peptides and opioid receptors have been found [29]. Recently, studies have shown that neoplasia of several variety of tumors can be prevented or delayed by daily administration of MENK (OGF) in vivo, and the effects are mediated by OGF receptor [30–32]. In our previous study, we had demonstrated that that MENK alone or in combination with IL-2 or IFN-γ, respectively, strengthened the pathway between dendritic cells (DCs) and CD4⁺T cell and induced maturation of dendritic cells (DCs) with higher production of IL-12 [12].

It is well known that M2 macrophages play a key role in tumor progression and metastasis [33]. Therefore, targeting of these cells represents a novel antitumor strategy. It is widely accepted that TAMs are the major components of the leukocyte infiltrating in tumors, and it has been confirmed that TAMs have various functions, such as participating in antitumor or promoting metastasis and dissemination of tumor cells. Evidence currently available suggests that TAMs in progressively growing solid tumors play the main role in inducing immune suppression of host defenses in situ, through release of specific cytokines and immune active metabolites, and in turn inhibit the antitumor cell-mediated immune response [33]. Simultaneously, TAMs participate in the reactions that can promote tumor progression, stroma formation, and neovascularization tissue remodeling. This effect of TAMs is believed to be resulted in the modulation of the Th2 polarization of the host immune system [34]. TAMs undergo longtime interactions with tumor-infiltrating lymphocytes in vivo [35] and supply various immunosuppressive signals to enervate the activity of CTL cells [36, 37]. Clinical studies also suggest that high numbers of TAMs are correlated with tumor progression. But what determines the outcome of these competing interactions and subsequent defined macrophage functions during tumor growth is still concerned.

In view of the complex regulatory mechanisms, to control functional phenotype of macrophage is crucial in tumor therapy by achieving improvement of patient’s well being and prevention of the malignant process. However, the precise mechanisms to regulate M1/M2 balance by MENK remain unclear.

From the results obtained above, we found that (1) MENK could delay tumor growth and induce the apoptosis of tumor cells, with prolonging survival significantly in mice. (2) MENK could reverse the immunophenotype of either splenic macrophages or TAMs from M2 to M1 type. (3) MENK could markedly promote the production of Th1 cytokines, such as IL-12 and TNF-α of BMDMs, while down-regulate the production of Th2 cytokine IL-10 of BMDMs.(4) MENK could markedly up-regulate the levels of both ROS and iNOS in BMDMs, simultaneously, promote the macrophage-mediated cytotoxicity.

It should also be noted that macrophages have emerged as important antigen presenting cells that possess the ability to stimulate and initiate T-cell responses, acting as messengers between the innate and adaptive immunities. Thus, MENK can potentially be used in cancer immunotherapy and for other threatening diseases through modulation of macrophage activity.

In spite of fruitful results above, there are still quite a few detailed approaches to pursue in depth, such as the concrete signal pathway via which MENK modulates macrophage polarization and at which stage of differentiation MENK exerts modulation during process of macrophages polarization.

Conclusion

We believe this is first ever time that we publish data to provide evidence that MENK could induce tumoricidal regulation through reversing macrophages polarization from M2 to M1 to kill tumor cells. MENK could also enhance the tumoricidal responses in vivo and in vitro through up-regulating the expression of Th1 cytokine and ROS, as well as NO, produced by macrophages.

This study can, therefore, contribute to a broader understanding of MENK as positive modulating effectors on the immune system. Furthermore, this study also provides a meaningful mechanism of action for MENK and highlights the clinical significance of a cancer immunotherapy.

Abbreviations

MENK

Methionine enkephalin

OGF

Opioid growth factor

TAM

Tumor-associated macrophage

BMDMs

Bone marrow-derived macrophages

GM-CSF

Granulocyte macrophage colony stimulating factor

IL-4

Interleukin-4

IL-12p40

Interleukin-12p40

IL-10

Interleukin-10

IFN-γ

Interferon-γ

TNF-α

Tumor necrosis factor-α

iNOS

Induced nitric oxide synthase

Arg-1

Arginase-1

ROS

Reactive oxidant species

FCM

Flow cytometry

RT-PCR

Reverse transcriptase-polymerase chain reaction

ELISA

Enzyme-linked immunosorbent assay