Novel analysis of maturation of murine bone-marrow-derived dendritic cells induced by Ginkgo Seed Polysaccharides

Abstract

Our understanding of the mechanisms of effect of Ginkgo Seed Polysaccharides (GSPs) on the immune system remains unclear. The aim of this work was to investigate the effect of GSPs on the maturation and function of bone-marrow-derived dendritic cells (BMDCs). The results demonstrate that GSP could exert positive immune modulation on the maturation and functions of BMDCs. This effect was evidenced by decreased changes of phagosome number inside BMDCs, decreased activity of acidic phosphatase (ACP), decreased phagocytosis of BMDCs, and increased changes of key membrane molecules on BMDCs. Upregulated production of cytokines IL-12 and TNF-α also was confirmed. Therefore, it can be concluded that GSPs can efficiently induce the maturation of BMDCs. Our exploration provides direct data and a rationale for potential application of GSPs as an immune enhancer in improving immunity and as a potent adjuvant in the design of DC-based vaccines.

Keywords:

• bone-marrow derived dendritic cells
• Gingko seed polysaccharides
• immune system
• modulation
• maturation

Abbreviations:

• BMC-Bone marrow cell
• BMDCs-Bone marrow derived dendritic cells
• DCs-dendritic cells
• GSPs-Gingko seed polysaccharides
• IL-4-interleukin-4
• GM-CSF-Granulocyte macrophage colony stimulating factor
• FCM-Flow cytometry
• MACS-Magnetic activated cell sorting
• PBS-Phosphate buffer solution
• ACP-Acidic phosphatase
• APCs-Antigen presenting cells
• LPS-Lipopolysaccharide
• TEM-Transmission electron microscopy
• MTS-methyl tolyl sulfide
• ELISA-Enzyme linked immunosorbent assay

Introduction

Ginkgo or Gingko (Ginkgo biloba) is a large tree, normally reaching a height of 20–35 m, and some specimens in China are over 50 m. Ginkgo, also known as the maidenhair tree, is a unique species of tree with no close living relatives. The tree was introduced to human history about 270 million years ago, and is cultivated in many countries. It has various uses as a traditional medicine and as a source of food.¹

With more investigations, many Ginkgo ingredients have been identified, and fractions of Ginkgo extracts have shown high medicinal value. For example, flavonol glycosides and terpene lactones in Ginkgo can exert the pharmacological effects of anti-oxidant, anti-platelet aggregation, memory improvement, enhancement of immune function and anti-cancer.^2-8

GSPs are purified polysaccharides from Gingko seed, composed of rhamnose, arabinose, galactose, glucose and mannose at the ratio of 3.5: 8.5: 3.4: 1.8: 1.The molecule structure is formed through 1–4 or 1–6 binding, and molecular weight is 1.6 × 10⁶ D. GSPs are water soluble.^9–11

Previous data indicated that GSPs could improve immune function of animals by enhancing secretion of a series of cytokines such as IL-2 and TNF-α, activating NK cells and macrophages, increasing lymphocyte transformation in tumor-bearing mice, inhibiting growth of tumors, and inducing apoptosis of cancer cells. Many Chinese journals have reported that GSPs are used to treat several types of cancers clinically, such as gastric cancer and melanoma, and other situations of infection.^12,13

With the advent of the discovery and understanding of dendritic cells (DCs), immunology has developed a new epoch, and profound changes in the field of immunology are affecting both basic and clinical medicine. DCs are now recognized as the most powerful antigen presenting cell (APC) in initiating specific T-cell responses. While seeking foreign invaders such as bacteria, viruses, or toxins, DCs capture antigens and develop into mature DCs with stronger potential to display the antigenic fragments on their cell surface with higher expression of key membrane molecules to be recognized by T cells, resulting in the initiation of T-cell responses.¹⁴ Matured DCs also show decreased phagocytosis and ACP enzyme activity.¹⁵

There is no known report on the effect of GSPs on DCs, and related mechanisms remain elusive. Due to the importance of DCs in their interaction with both the innate and adaptive immune systems and the ever-increasing application of GSPs, we conducted the following study.

Results

Immature DCs are a relatively primitive cell type, showing a relatively smooth surface, less expression of surface molecules (receptors), weak phagocytosis, and limited secretion of cytokines. However, upon maturation, DCs undergo marked changes both phenotypically and functionally to fit the activation of T cell. For example, matured DCs show a rougher surface, elevated expression of key surface makers, and increased secretion of cytokines. According to our deduction from reported data, GSPs should exert a positive modulation on BMDCs.

Dose responses of BMDCs

After treatment with a range of GSP concentrations, BMDCs grow into differential colonies. An optimal concentration of 75 μg/mL and incubation time of 48 hours to stimulate BMDCs are obtained (Fig. 1).

Figure 1. Dose response of BMDCs to GSPs. The number of expanded BMDCs was determined from triplicate samples across time points in both media with 75 μg/mL GSP (Panel A) and GSP concentrations (Panel B) with use of MTS method. P-values. ** <0.01 for (A) GSP vs RPMI 1640 and (B) GSP concentration vs. RPMI 1640.

Observation of changes of BMDCs induced by GSPs under Transmission electron microscopy (TEM)

There usually are more phagosomes inside immature BMDCs with a powerful ability to phagocytose antigen, and with the process of maturation, the number of phagosomes would be reduced, accompanied by increased expression of key surface molecules as co-signals to fit antigen presentation. Figure 2 shows markedly reduced phagosome number in BMDCs treated with GSP compared with that in untreated BMDC, accompanied by an unclear cytoplasm with long and slender cellular processes, indicating maturation.

Figure 2. Intracellular changes of BMDCs treated with GSPs under TEM. BMDCs were incubated for 48 hr and harvested by trypsinization, fixed in 2.5% gluteraldehyde/4% paraformaldehyde in 0.1 mol/L cacodylate buffer, and post-fixed in 1% osmium tetroxide buffer. After acetone dehydration, cells were embedded in spur resin. Thin sections were cut on a Reichert Ultracut E microtome, and stained with saturated uranylacetate and lead citrate solution. Sections were examined with TEM. Representative micrographs are shown for cells incubated in each of the 3 media.

Expression of key surface molecules on BMDC confirmed by Flow cytometry (FCM)

BMDCs treated with 75 μg/mL GSP for 48 h matured, as identified by the up-regulated expression of key surface molecules CD80, CD86, CD83, CD40, and MHC-II, which work with antigen as combinational signals to activate T cells (Fig. 3). These molecules will be helping antigen presentation of BMDCs in a coordinating way to initiate T cell responses.

Figure 3. Elevation of key surface molecules on BMDCs. (A) Histograms of percent positive cells in the 3 different groups following incubation for 48 hr for each of the 5 indicated surface markers as analyzed by FACS. (B) Graphs representing frequency of percent positive cells for CD40, CD80, CD86, CD83 and MHC-II markers. Each value represents mean ± SEM. p-values: LPS × RPMI 1640 (CD80: <0.001; CD86: <0.05; CD83: <0.00001; CD40: <0.00001; MHC- II: <0.00001); GSP × RPMI 1640(CD80: <0.001; CD86: <0.05; CD83: <0.00001; CD40: <0.001; MHC- II: <0.05).

Confirmation of phagocytosis by FCM

Antigen capture by BMDCs were further analyzed using FITC-dextran (labeled antigen particles). The function of immature BMDCs to phagocyte antigen is downregulated with the process of maturation. BMDCs would phagocytose more FITC-labeled Dextran. However when BMDCs mature, means of G would decline. GSP downregulated phagocytosis by BMDCs to antigen as shown in Figure 4. The result indicate that matured BMDCs become weaker in antigen phagocytosis.

Figure 4. Phagocytosis of BMDCs confirmed by FCM. The % phagocytosis for BMDCs cultured in each of 3 media for 48 hr as shown in (A) distribution plots; and (B) Mean ± SE from three samples per group (RPMI 1640 vs. LPS: <0.00001; RPMI 1640 vs. GSPs: <0.00001).

Acidic phosphatase (ACP is a key enzyme for degrading antigen in BDMCs. Its activity measurement is used to probe phagocytic activity. After treatment with 75 μg/mL GSP for 48 h, BDMCs developed into mature BMDCs corresponding to decreased phagocytosis ACP activity, showing the gradual termination of phagocytosis. The amount of ACP activity of BMDCs responded to a value of OD 250 nm (A₅₂₀) as shown in Figure 5.

Figure 5. Measurement of ACP activity. BMDCs are cultured in each of the 3 media for 48 h, and ACP activity was measured (RPMI 1640 vs. LPS: <0.01; RPMI 1640 vs. GSPs: <0.05).

BMDCs drive T-cell proliferation

Immature BMDCs have higher activity for antigen capture and processing, but with lowered ability to stimulate T-cell proliferation. When BMDCs mature, they will down-regulate antigen phagocytotic activity with increased T-cell proliferation. BMDC treated with GSPs for 48 h significantly upregulated their ability to drive T-cell responses. The level of BMDCs for driving allogeneic lymphocyte proliferation correspond to a value of OD 492 nm (A₄₉₂) as shown in Figure 6.

Figure 6. CD4⁺ T-cell proliferation driven by BMDCs treated by GSPs. The purified CD4+T cells were labeled with CFSE to quantify the number of cell divisions relative to T-cell proliferation. The purified CD4+T cells were resuspended in pre-warmed PBS containing 5 M CFSE and incubated at 37°C for 8 min. After washing twice with RPMI 1640+FCS10%, the cells were counted and further plated in plates. At the same time the BMDCs from different groups were seeded at different ratios in the plates. After 48 hr of co-culture, T cells were harvested, washed and stained with anti-CD4 MAb. Finally the net increased percentages of CD4⁺ T cells driven by BMDCs were determined using the CFSE technique by flow cytometry. Panel A shows statistical results of proliferation of CD4+T cells driven by BMDCs post treatment with GSPs. Panel B is an original profile of flow cytometry to prove proliferation of CD4+T cells.

Determination of IL-12, TNF-α and IL-10 by ELISA

After the immature BMDCs were treated with 75 μg/mL GSP for 48 h, the BMDCs maturate. Besides structural and morphological maturation, BMDCs also underwent functional maturation with secretion of higher levels of IL-12 and TNF-α and lower levels of IL-10 (Table 1). These cytokines are also involved in a chain of immune responses through interaction with other immune cells, as shown in

Table 1. Levels of cytokines (ng/mL) secreted by BDMCs following incubation with various media

	RPMI 1640	LPS	GSP
IL-12p70	80.65	432.3	103.6
TNF-α	523.3	853.8	674.8
IL-10	285.0	111.0	131.8

p-values: LPS × RPMI 1640 (IL-12p70: <0.0001; TNF-α: <0.001; IL-10: <0.0001), GSP × RPMI 1640 (IL-12p70: <0.01; TNF-α: <0.05; IL-10: <0.001).

BDMCs were incubated in the indicated medium for 48 hr, and levels of cytokines in the media were quantified by ELISA.

Discussion

The number of studies on Ginkgo have rapidly increased in recent years, and a variety of medical effects of GSPs have been identified. Besides its actions as an antiviral, anti-infectious and antitumor agent, the role of Ginkgo as an immunological modulator has been proven to be functional.^16-21 Recent studies have underscored some new modulatory effects of GSPs, which mainly include: enhancing Concanavalin A (ConA) induced T-cell proliferation, and enhancing LPS-induced B-cell proliferation, cytokine secretion and activity of cytotoxic T cells, which may provide a mechanism to the understanding of tumor immunity induced by GSPs.

The trial on immune correlation in cancer patients responding to GSPs treatment provided additional data supporting the importance of the interactions of GSPs with cells in both innate and adaptive immune systems for the generation of long-lasting antitumor responses.^22,23

In the current study, we have proven that GSPs could efficiently induce BMDC maturation. The findings are evidenced by the following data: (1) Ultra-structural study of BMDCs induced by GSPs reveal reduced numbers of phagosomes inside BMDCs, which would decrease phagocytosis accordingly; (2) Simultaneously, BMDCs upregulate the expression of key surface molecules of CD83, CD40, MHC II, CD80, and CD86, which would play a pivotal role in antigen presentation by BMDCs to initiate T-cell responses; (3) A phagocytosis test in response to labeled dextran antigen indicates that decreased phagocytosis is consistent with ACP activity and the number of phagosomes; (4) Down-regulation of ACP activity is confirmed, which corresponds to maturation of BMDCs in a reverse relationship; (5) The potential of T cells driven by BMDCs is proven; and (6) Functional maturation of BMDCs is represented by higher levels of IL-12 and TNF-α production, which would be involved in interlinking a chain of immune responses. These findings provide direct evidence to support GSPs working as a cellular immune enhancer or as an adjuvant to initiate specific T-cell responses.

The up-regulation of IL-12 and TNF-α levels not only would intensify BMDCs themselves via autocrine or paracrine pathway, but also would work as a message to amplify CD4⁺ T-cell responses. CD4⁺ T cells in turn would secret more cytokines such as IFN-γ or TNF-β, thus activating BMDCs, thus forming a feedback loop. Downregulation of IL-10 levels would help this process.

The immune network is a very complicated entity in which a variety of immune cells are entangled, forming precise regulation through secretion of a variety of cytokines as part of a dynamic immune system. When BMDCs are activated by GSPs, they secrete IL-12 and TNF-α, which will activate CD4⁺ T cells. CD4⁺ T cells will activate macrophages through secreting IFN-. Macrophages can secrete a variety of cytokines such as IL-12, TNF-α, and IL-6, and are involved in a series of immune responses, such as activation of the complement system. IFN-γ can activate BMDCs and NK cells, which also can secrete IFN-γ. As a result of this, more IL-12 is secreted to form an immune network maintaining balanced immunity. GSPs can thus potentially be used in regulating cellular immune system.

DC, originally identified by Steinman in 1970s, is marker of progress in immunology. There have been an ever-increasing number of publications on the understanding and mechanisms of DCs. DCs are the most powerful APCs for initiating naïve T cells and thus are crucial for T-cell-mediated immunity.^24-26 GSPs therefore provide a new mode of action for regulating cellular immune responses.

Despite the meaningful results obtained above, there are still more in depth investigations to be done on GSPs such as when GSPs act on BMDCs and what is the signaling pathway that GSPs trigger inside BMDCs. The level of purity and stability of GSPs are issues that may limit this work, and obtaining stable GSPs for further work with reproducible results is an important objective.

Finally, the present exploration could be of significance to help the understanding of GSP effects on the immune system and detailed working mechanisms of GSPs at the subcellular or molecular level. This work also highlights the major therapeutic value of GSPs in the treatment of cancer and other infectious diseases. It is also possible to use GSPs as an adjuvant in DC-based vaccine preparations.

Conclusion

This is the first known study demonstrating that GSPs at a defined concentration can induce both phenotypic and functional maturation of BMDCs. The study provides data to identify structural and functional changes of BMDCs following treatment with GSPs. This positive regulation could also supply additional IL-12, TNF-α, and other key surface molecules to help antigen presentation of BMDCs, resulting in the enhancement of T-cell responses.

Materials and Methods

Chemicals

GSPs (purity>98%) was prepared by the Liaoning Institute of Microbiological Sciences. LPS (from Escherichia coli, serotype 055:B5) used as positive control in this work was a Sigma product by St. Louis, MO.

The monoclonal antibodies used in this study include FITC-conjugated PE-anti-MHC-II, PE-anti-CD40, PE-anti-CD83, PE-anti-CD80, and PE-anti-CD86, which were all bought from eBioscience or BD PharMingen. ELISA kits for IL-12, TNF-α and IL-10 were made in eBioscience, USA (Mouse IL-12 p70 ELISA Ready-SET-Go!; Mouse TNF-α ELISA Ready-SET-Go!; Mouse IL-10 ELISA Ready-SET-Go!). Recombinant murine cytokines interleukin-4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF) were products of PeproTech Inc. Other frequently used chemicals were obtained from Sigma-Aldrich or BD PharMingen.

Preparation of BMDCs

BMDC induction from mice bone marrow cells (BMCs) was performed based on a reported method.²⁷ All animals were treated according to the Guide for the care and use of laboratory animals of China Medical University. BM from femurs and tibias of female C57BL/6 mice (4–6 weeks old) were depleted of red cells with lysis solution. About 10⁷ cells/mL cells obtained from this process were placed in 24-well culture plates containing RPMI 1640 media, enriched with 10% fetal bovine serum (FBS), 10 ng/mL GM-CSF, 10 ng/mL IL-4, 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine. After incubation for 4 h, the non-adherent cell was discarded and the culture containing DCs was kept for 6 continuous days. CD11c⁺ BMDCs were purified with magnetic sorting, using anti-CD11c-coated magnetic beads and the auto- Magnetic activated cell sorting (MACS) system per the manufacturer's instruction (Miltenyi Biotech, CA, USA). FCM analysis revealed that >90% of the purified cells expressed CD11c. All CD11c⁺ cells were counted and seeded for subsequent study.

Cell viability

In a preliminary study, the impact of a range of GSPs on BMDCs was tested in vitro. The cell vitality was measured using the MTS assay, which is a rapid colorimetric assay for cellular growth and survival based on the conversion of MTS to formazan crystals. The optimal concentration was found to be 75 μg/mL. The current work was carried out with this GSP concentration.

Structural changes of BMDCs induced by GSP

The BMDCs cultured in RPMI 1640 with 1 μg/mL LPS (medium called ‘LPS’ in the Figures and Table) or 75 μg/mL GSPs (medium called ‘GSP’ in the Figures and Table) for 48 hr were collected for structural analysis of maturation. The treated BMDCs were centrifuged, resuspended in 0.5 mL 0.05 M pH 7.2 PBS, fixed overnight in 2.5% glutaraldehyde, treated with 1% osmium tetroxide, dehydrated in ethanol, and embedded in epon. The sections were prepared using a Reiehert-Jung Ultracut E, stained with uranyl acetate, followed by lead citrate. The sample was observed under TEM for confirmation of structural changes.

Upregulation of expression of key surface molecules checked by FCM

BMDCs cultured with 75 μg/mL GSPs for 48 h were rinsed 3 times with PBS and incubated with anti-CD40, anti-CD83, anti-CD80, anti-CD86, and anti-MHC II antibodies for 20 min at 4°C. After extensive rinsing, the stained cells were analyzed using FACS Calibur (Becton Dickinson, San Diego, CA) for the expression of key surface molecules.

Decreased phagocytosis process by FCM

100 μl FITC-Dextran (40,000 D) 28–30 was added to BMDC cultures treated with 75 μg/mL GSP for 48 h, left standing for 2 h at 4°C, and subsequently for another 1 h at 37°C. The sample was localized for phagocytosis process using the FACS Calibur (Becton Dickinson, San Diego, CA).

ACP activity detection

BMDCs were adjusted to 1 × 10⁶/mL. ACP activity in the BMDCs treated with 75 μg/mL GSP for 48 h was tested with ACP testing kit (Jiancheng Bio-engineering Institute of South, China), combined with phenol-4-AAP (amino anti-pyrine) as described in the instruction manual in ACP testing kit. OD at 520 nm (A₅₂₀) was measured to reflect ACP activity.²⁸

T-cell testing driven by BMDCs

For driving the T-cell test, BMDCs post-treatment with75 μg/mL GSPs were harvested on 7 d. The CD4⁺ T cells from splenocytes were separated and purified with magnetic beads by following the instruction manualthen the purified CD4⁺ T cells were grown with 10 μg/mL ConA for 48 h to a substantial number. The purified CD4⁺ T cells (5 × 10⁵/well) were incubated with graded numbers of BMDCs in 96-well culture plates (Corning-Costar) for 5 d. T-cell proliferation was monitored by determining by monitoring A₄₉₂ using a bichromatic microplate reader. The results were expressed as mean ± sd from triplicate wells.

Cytokine assay of IL-12, TNF-α and IL-10

The supernatant of 10⁵/mL BMDCs cultured with 75 μg/mL GSPs for 48 h was collected for the study of cytokines secretion. IL-12, TNF-αand IL-10 were determined according to the instruction manual included in the enzyme linked immunosorbent assay (ELISA) kit (eBioscience) separately.²⁹ A₄₅₀ was measured using a bichromatic microplate reader (BIO-TEK, USA) to represent amount of cytokine production.

Statistical analysis

The data were processed statistically with the use of program Statistical Package for Social Sciences, Version (SPSS) 16.0 for Windows. The variables were shown as mean ± SE. The differences with P < 0.05, evaluated by ANOVA, indicated significance.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by the funding from project for construction of major discipline platform in universities of Liaoning province and funding from Liaoning science foundation (2009225008-7, 2012225016).