GLUCAN AND RESVERATROL COMPLEX – POSSIBLE SYNERGISTIC EFFECTS ON IMMUNE SYSTEM

BACKGROUND
Recent data showing that glucan elicited defense responses in grapevine and induced protection via induction of resveratrol production led us to evaluate the possible synergetic effects of glucan and resveratrol complex on immune reactions.


METHODS
We measured phagocytosis using HEMA particles, expression of cell surface markers via fl ow cytometry, expression of cytokines using ELISA, recovery after fluorouracil-induced leucopenia and effects on gene expression via RT-PCR.


RESULTS
Our results showed that both glucan and resveratrol complex stimulated phagocytosis of blood leukocytes, caused increase in surface expression of CD(+) splenocytes and showed higher restoration of spleen recovery after experimentally induced leucopenia. In all these cases, strong synergetic effects were observed. When we measured the effects of these substances on expression level of NF-kappaB2, Cdc42 and Bcl-2 in breast cancer cells, upregulation of Cdc42 expression was evident only using both immunomodulators in combination.


CONCLUSIONS
In conclusion, our data suggest significant synergy in stimulation of immune reactions and support further studies of these natural immunomodulators.


INTRODUCTION
β−1,3-Glucan's role as a biologically active immunomodulator has been well documented for over 40 years.Initial interest in the immunomodulatory properties of polysaccharides was raised after experiments showed that a crude yeast cell preparation stimulated macrophages via activation of the complement system 1 .Further work identified the immunomodulatory active component as β−1,3-glucan 2 .Numerous studies have subsequently shown that β-1,3-glucans, either particulate or soluble, exhibit immunostimulating properties, including antibacterial and anti-tumor activities 3 .At this time, glucans are considered to be one of the most effi cient biological response modifi ers.
More than 1,800 publications have reported that β−1,3-glucans, either soluble or particulate, exhibit signifi cant immunomodulatory properties.However, from this vast amount of data, only limited information about immunostimulation of human immune system is available.Browder et al. 4 described a strong decrease in septic morbidity.A well-documented multicenter blind study demonstrated that glucan-treated patients had signifi cantly lower infections 5 .The same type of glucan was also shown to reduce infection-stimulated periapical bone resorption 6 .Positive eff ects were found in patients after cardiopulmo-nary bypass and inhibition of antiviral activity has been found in HIV-infected patients 7 .Some β-glucans have been routinely used in patients for tumor immunotherapy 8 .In addition to the immunomodulatory eff ects, β-glucans were also shown to reduce the cholesterol levels of hypercholesterolemic patients 9 .
A variety of β-1,3 glucans have been shown to bind to glucan receptors on monocytes, macrophages, neutrophils and NK cells 10,11 .Despite the progress, it is not clear if there is a separate receptor for glucan 10 , only CR3 (CD11b/CD18) receptor 11 , dectin-1 receptor 12 , or a combination of all these receptors.As biological eff ects of glucans appear to be multifactorial, it is not surprising that glucans also infl uence the production and secretion of cytokines.
Resveratrol (trans-3,4',5-trihydroxystilbene) is nonfl avonoid polyphenol found in various fruits and vegetables and is abundant in grape skin.In addition to various biochemical, biological and pharmacological activities, resveratrol has been found to exhibit numerous immunomodulatory activities such as suppression of lymphocyte proliferation, changes in cell-mediated cytotoxicity, cytokine production 13 or induction of apoptosis 14 .In addition, resveratrol has been reported to exhibit a cancer-chemopreventive activity 15 .
Recent observation showing that seaweed-derived glucan elicited defense responses in grapevine and induced protection against Botrytis cinerea and Plasmopara viticola via induction of production of two phytoalexins including resveratrol 16 led us to evaluate the possible synergetic eff ects of glucan and resveratrol complex on immune reactions.

Animals
Female, 6-to-10 week old BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME).All animal work was done according to the University of Louisvile IACUC protocol.Animals were sacrifi ced by CO 2 asphyxiation.

Antibodies
For fl uorescence staining, the following antibodies have been employed: anti-mouse CD4, CD8, CD11b and CD19, conjugated with FITC were purchased from Biosource (Camarillo, CA), anti-mouse CD71 and CD122, also conjugated with FITC, were purchased from Pharmingen (San Diego, CA).

Flow cytometry
Cells were stained with monoclonal antibodies on ice in 12 x 75-mm glass tubes using standard techniques for 30 minutes on ice.After washing with cold PBS, the cells were resuspended in PBS containing 1% BSA and 10 mM sodium azide.Flow cytometry was performed with a FACScan (Becton Dickinson, San Jose, CA) fl ow cytometer and the data from over 10,000 cell/sample were analyzed.

Phagocytosis
The technique employing phagocytosis of synthetic polymeric microspheres was described earlier 17 .Briefl y: peritoneal cells were incubated with 0.05 ml of 2-hydroxyethyl methacrylate particles (HEMA; 5×10 8 /ml).The test tubes were incubated at 37 o C for 60 min with intermittent shaking.Smears were stained with Wright stain.The cells with three or more HEMA particles were considered positive.The same smears were also used for evaluation of cell types.The same technique was used for evaluation of phagocytosis of peripheral blood cells 17 .

5-Fluorouracil
Mice were injected i.v. with 0.1 ml of 5-fl uorouracil (3 mg/mouse).From day 0, mice were injected ip.daily with 100 μg of tested substance.At diff erent time intervals, individual mice were sacrifi ced and the cellularity in bone marrow and spleen was evaluated.
The numbers of cells in bone marrow were evaluated as follows: mice were sacrifi ced by cervical dislocation, the legs were separated from the body at the hip joint and the feet were removed.The legs were placed in a Petri dish obtaining RPMI 1640 medium.All muscle tissue from the femurs and tibia was removed and the bones were separated (only femurs were used).The epiphyses were cut off on both ends; the bone end was punctured with a 23G needle and fl ushed out with 3 ml of warm (22 o C) RPMI 1640 medium.The large debris and cell clumps were removed by layering the cell suspension over 3 ml of heat-inactivated FCS for 10 minutes on ice.The cells collected from the top of FCS were washed once by centrifugation at 300 × g for 10 minutes at 4 o C and kept in RMPI 1640 medium containing 5 % FCS.One drop of the fi nal solution was dropped into hemocytometer and counted under an optical microscope.The same technique was used for counting of isolated splenocytes.

Cytokine assay
BALB/c mice were intraperitoneally injected with 100 μg of tested samples.Control mice obtained PBS only.After 60 minutes, the mice were sacrifi ced and blood was collected in Eppendorf tubes.Subsequently, the serum was prepared, collected and stored at -80 o C for no more than 1 week.
The levels of IL-1β, IL-6 and TNF-α in serum samples were evaluated using a commercial kits OptEIA Mouse IL-1β, IL-6 or TNF-α Set, resp., (Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions.The optical density was determined using a STL ELISA reader (Tecan U.S., Research Triangle Park, NC) at 450 nm with a correction at 570 nm.Data shown in Figure 6 were calculated from the standard curve prepared by the automated data reduction using linear regression analysis.A standard curve was run with each assay.

RNA Extraction and Reverse Transcriptase-PCR
Total RNA was extracted from control and treated cells using Trizol reagent (Life Technologies, Inc.).RNA quality and quantity were determined by ultraviolet spec-Glucan and resveratrol complex -possible synergistic eff ects on immune system trophotometry and agarose gel electrophoresis.200ng of total RNA was reverse transcribed using SuperScript TM One-Step RT-PCR with Platinum Taq kit (Invitrogen Inc.) using gene-specifi c primers (Table1) with the following conditions: 30 min at 50 o C followed by 2 min at 95 o C and then 25 cycles of 30s at 95 o C, 45s at 52 o C for NF-κβ2 and β-Actin (68 o C for BCL-2, 43 o C for cdc42), 3 min at 72 o C for NF-κβ2 (45s for cdc42, BCL-2, 90s for β-Actin).PCR reaction was completed by 7min at 72 o C. RT-PCR products were then separated on a 1.0% agarose gel, visualized under UV light and photographed.β-Actin served as internal control.

STATISTICS
Student's t-test was used to statistically analyse the data.

RESULTS
The eff ects of various glucans and resveratrol on macrophages are well established.However, in order to demonstrate that a new combination of immunomodulators really exhibits an immunomodulatory characteristic, an evaluation of phagocytosis is necessary.We measured the eff ects of glucan and/or resveratrol complex on phagocytosis of synthetic HEMA microspheres (Fig. 1).Both glucan and resveratrol complex (RC) stimulated the internalization of synthetic particles; however, the combined preparation exhibited signifi cant synergetic eff ect both on monocytes and neutrophils.
We then evaluated the eff ects of our substances on expression of several membrane markers.Twenty-four hrs after an ip.injection, spleen cells were isolated and the surface expression of CD4 (T helper lymphocytes), CD8 (T suppressor lymphocytes), and CD19 (B lymphocytes) was evaluated.The results summarized in Fig. 2 show that the tested substances aff ected only CD4 + lymphocytes.Again, glucan and RC showed signifi cant synergetic effects.Conversely, the total numbers of peritoneal cells was infl uenced only slightly (data not shown).
We then compared the eff ects of a single intraperitoneal injection of the tested substances on systemic in vivo release of three cytokines, IL-1 , IL-6 and TNF-α.Peripheral blood was isolated 60 min after the injection and the serum obtained was stored at -80 o C for no longer than 1 week.The dose and time interval have been established previously 18 .The data summarized in Fig. 3 show signifi cant elevation in levels of all three tested cytokines after the combined injection of glucan and RC complex.RC singularly stimulated release of IL-6 and IL-1, since the glucan had no eff ect.
Being similar to cyclophosphamide, cancerostatic drug 5-fl uorouracil is well known for signifi cant depression of immune system.We evaluated the eff ects of orally-given glucan or RC on fl uorouracil-induced leucopenia.The data showed that whereas glucan strongly increases the recovery of bone marrow, the resveratrol complex alone showed signifi cant stimulation but only after 10 days of application.When combined, glucan and resveratrol had small, albeit insignifi cant, synergetic eff ects (Fig. 4).A diff erent situation was discovered in the spleen.Even if glucan again demonstrated more activity than RC, its eff ects showed signifi cantly higher restoration of spleen cellularity from day 4. Combined glucan/resveratrol complex substances showed stronger synergetic eff ect, which was signifi cant from day 11 (Fig. 5).
This RT-PCR Figure (Fig. 6) shows the eff ect of rested glucan and/or RC complex on expression level of NF-κB2, Cdc42 and Bcl-2 in ZR-75-1 cells.A distinct upregulation in expression levels of NF-kB2 and Bcl-2 was evident in response to either glucan or RC, with slightly more eff ect with RC as compared to control cells.In addition, the eff ect of the combined sample was similar to the eff ect shown by RC on expression of these genes.However, upregulation of Cdc42 expression was evident only when both immnunomodulators were used in combination.

DISCUSSION
It is important to note that despite the fact we used a concentrated RC, the levels of emodin or piceid never reached bioactive levels (data not shown).
Various types of immunomodulators, glucans in particular, well are known to stimulate phagocytosis 19 .Therefore, the evaluation of this basic type of immune reaction is important to determine the eff ectiveness of any biologically active immunomodulator.We tested the peripheral blood leukocytes for changes in phagocytosis.Using synthetic microspheres based on 2-hydroxyethyl methacrylate, we found that both tested substances caused a signifi cant increase in phagocytosis, but the combined preparation showed significant synergetic effect both on monocytes and neutrophils.The data shown refl ects the eff ects of a single injection of either glucan or RC.However, our preliminary experiments showed that these eff ects last up to 3 days after treatment (data not shown).These data were in agreement with previously published data using diff erent types of glucan 18 .
Observations of the eff ects on expression of cell surface markers present on spleen cells demonstrated that only the numbers of CD4+ lymphocytes were infl uenced.Again, the combined preparation of glucan and RC showed synergetic eff ects.Three days after application, the increased numbers returned to normal.A similar increase in the number of CD4-positive cells after glucan application has been described for lentinan 20 and Phycarine 18 .
In addition to the direct eff ect on various cells of the immune system, the immunostimulating action of β-glucans is caused by potentiation of a synthesis and release of several cytokines such as TNFα, IFNγ, and IL-1.This cytokine-stimulating activity was found to be dependent on the triple helix conformation 21 .Most glucans have been shown to stimulate TNF-α both in vivo and in vitro 22 .It is hypothesized that glucans enhance leukocyte functions     through increased TNF-α secretion, particularly during early stages of infection.The elevated levels of TNF-α and IL-1 after Phycarine injection 18 correlated with fi ndings of eff ects of lentinan in the treatment of human cancer 23 and stimulation caused by Paramylon.
The potential eff ect of resveratrol on individual cytokines is much less clear.Some studies suggest that resveratrol can inhibit some IL-2 or TNF-α mediated functions 24 represent only indirect proofs of the resveratrol-cytokine interaction.Therefore, the evaluation of the potential systemic eff ect on three model cytokines was particularly interesting.Using the previously established dose and time interval 18 , we observed the resveratrol complex-induced release of IL-1 and IL-6.In addition, the combined preparation of glucan and RC not only showed synergetic eff ects, but also stimulated release of TNF-α, which was not aff ected by RC alone.
The major side-eff ects of both traditional chemotherapy and/or irradiation are leucopenia and the noteworthy suppression of the immune system, which is a signifi cant problem in most cancer patients.These negative eff ects both limit the dosage and frequency of treatment 25 .The eff ects of injected glucan on enhanced recovery after experimental leucopenia have been documented 26 and similar data were found after irradiation.Recent observation showed that seaweed-derived glucan can restore the leukopedia when used both intraperitoneally or orally 27 .However, nothing is known about the resveratrol.Considering the strong synergistic eff ects we observed in previous experiments, it was therefore important to know if RC can similarly improve the cell recovery.Using a 5-fl uorouracil model with well-established destruction of immune cells, we found a strong prophylactic eff ect of orally-given glucan.These eff ects are most probably caused by CR3-mediated eff ects of glucan on hematopoietic progenitor cells 28 .In bone marrow, RC alone had only limited aff ects and the synergistic eff ects were not signifi cant.In spleen we observed diff erent situation.Even if glucan was again signifi cantly more active that resveratrol, its eff ects showed a much higher restoration of spleen cellularity from day 4. Combined glucan/resveratrol substances showed stronger synergetic eff ect Upregulation of NF-κB2 gene expression is considered signifi cant as members of this family are important regulators of cell cycle progression, cell survival, cell adhesion/angiogenesis, invasion and infl ammatory responses 29 .They are also known to exert strong anti-apoptotic activity by inducing expression of several anti-apoptotic proteins like Bcl-2 family proteins and interfering with expression or activity of pro-apoptotic proteins 29 .Studies have shown the positive role of NF-κB family proteins in regulating the expression of adhesion molecules, matrix metalloproteinases and angiogenic factors which are known to increase the invasion and metastasis of cancer cells.Cdc42 belongs to the RHO-family proteins (a branch of the RAS family) and is implicated in the regulation of cell growth 30 .It has been reported that levels of Cdc42 are elevated in many diff erent human cancers, including human breast cancer 31 and that they contribute to enhanced mitogenic signaling.In addition, another recent study has suggested that Cdc42 can activate NF-kb by a distinct pathway 32 .

Fig. 1 .
Fig. 1.Potentiation of phagocytosis of synthetic microspheres (HEMA particles) by i.p. injected glucans and/or resveratrol complex.Peripheral blood neutrophils with three and more HEMA particles were considered positive.Each value represents the mean ± SD. *represents signifi cant diff erences at P < 0.05 level.

Fig. 2 .
Fig. 2. Eff ect of ip.injection of 100 μg of tested substances on the expression of CD4, CD8, and CD19 markers by spleen cells.The cells from three donors/sample were examined and the results given represent the means ± SD. *Represents significant diff erences between PBS control and tested substances at P ≥ 0.05 level.

Fig. 3 .
Fig. 3. Eff ect of diff erent doses of injected samples on levels of TNF-α, IL-6 and IL-1 β in peripheral blood.For details, see Material and Methods section.Each value represents mean ± SD.As the control values (PBS) and glucan were always zero, each value represents signifi cant diff erences at P ≥ 0.05 level.

Fig. 4 .Fig. 6 .
Fig. 4. Induced cell recovery in bone marrow after 5fl urouracil-induced leucopenia.Starting at day 0 (injection of 5-fl uorouracil), mice were orally fed, on a daily basis, with samples tested in water.Subsequently, individual mice were sacrifi ced and the cellularity in bone marrow was evaluated.The results represent a mean of 10 mice in 3 independent experiments.

Fig. 5 .
Fig. 5. Induced cell recovery in spleen after 5-fl urouracilinduced leucopenia.Starting at day 0 (injection of 5-fl uorouracil), mice were orally fed, on a daily basis, with samples tested in water.Subsequently, individual mice were sacrifi ced and the cellularity in spleen was evaluated.The results represent mean of 10 mice in 3 independent experiments.