Natural Immunomodulators and their Stimulation of Immune Reaction: True or False?

Materials and Methods

Animals. Female, 8-week-old BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments were performed in accordance with the University of Louisville IACUC protocol. Animals were sacrificed by CO₂ asphyxiation.

Materials. Individual immunostimulators were purchased as follows: Astragalus (A. membranaceous) from Solaway (Park City, UT, USA), resveratrol from Amax (Eugene, OR, USA), curcumin from Natural Remedies (Bangalore, India), pterostilbene from Pteropure (Irvine, CA, USA), ellagic acid (Sigma. St. Louis. MO, USA), vitamin C (ascorbic acid) from Sigma (St. Louis, MO, USA), Chlorella from Sun Chlorella USA (Torrance, CA, USA) Cat's claw from Piping Rock Health Products (Ronkonkoma, NY, USA), Glucan #300 from Transfer Point (Columbia, SC, USA), ginseng (Panax ginseng) from Puritan's Pride (Oakdale, NY, USA), Echinacea (E. purpurea) from Nature's Way Products (Green Bay, WI, USA). Ovalbumin, sodium citrate, antibiotics, Freund's adjuvant, gelatin, cytochrome c, concanavalin A, PMA, RPMI 1640 medium and cyclosphorine were purchased from Sigma, fetal calf serum (FCS) from Hyclone Laboratories (Logan, UT, USA).

Cells. Murine tumor cell line YAC-1 was provided by Dr. Julie Djeu of the Moffitt Cancer Research Center, Tampa, FL. The human neutrophil HL-60 cell line was obtained from the ATCC (Manassas, VA, USA). Each cell line was maintained in RMPI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and antibiotics, in plastic disposable flasks at 37°C in a 5% CO₂/95% air incubator.

Treatment. The samples were collected after a 14-day feeding with supplement-containing diet (100 μg/day), except for in vitro experiments. All diets (Laboratory Rodent Diet 5001 alone or enhanced with of supplement) were formulated and prepared by Purina (Richmond, IN, USA). Diet ingredients for all groups were identical except for the addition of supplement.

Phagocytosis. The technique employing phagocytosis of synthetic polymeric microspheres was described earlier (16). Briefly: peripheral blood cells or peritoneal exudate cells were incubated in vitro with 0.05 ml of 2-hydroxyethyl methacrylate particles (HEMA; 5×10⁸/ml). The test tubes were incubated at 37°C for 60 min, with intermittent shaking. Smears were stained with Wright stain. The cells with three or more HEMA particles were considered positive. Mice were injected with individual substances or their combinations (or PBS as a control). All experiments were performed in triplicates. At least 200 cells in 60 high-power fields were examined in each experiment.

IL-2. Purified spleen cells (2×10⁶/ml in RPMI 1640 medium with 5% FCS) from control or treated animals were added into wells of a 24-well tissue culture plate. After the addition of 5 μg of Concanavalin A into positive control wells; cells were incubated for 72 h in a humidified incubator (37°C, 5% CO₂). At the end-point of incubation, supernatants were collected, filtered through 0.45-μm filters, and tested for the presence of IL-6 using a Quantikine mouse kits (R&D Systems, Minneapolis, MN, USA).

Antibody formation. Formation of antibodies was evaluated using ovalbumin as an antigen. Mice were injected twice (two weeks apart) with 100 μg of albumin and serum was collected 7 days after last injection. Levels of specific antibodies against ovalbumin were detected by ELISA., Combination of ovalbumin and Freund's adjuvant was used as a positive control.

Lewis lung carcinoma therapy. Mice were injected i.m. with 5×10⁶ Lewis lung carcinoma cells. Cyclophosphamide (150 mg/kg) was used i.p. at day 10 after tumor application, Phycarine was used either i.p. (250 μg/mouse) or orally (200 μg/mouse) from day 0 do day 14 after tumor application. The control group of mice received daily i.p. PBS. Each group held a minimum of 5 mice. At the conclusion of the experiment, mice were euthanized, lungs removed, fixed in 10% formalin and the number of hematogenic metastases in lung tissue was estimated using a binocular lens at 8× magnification.

Natural killler cell assay. Spleen cells were isolated from the spleen of mice by standard methods. Cell suspension was generated by pressing minced spleen against the bottom of a petri dish containing PBS. After elimination of erythrocytes by 10-s incubation in distilled water, and five washes in cold PBS, the cells were resuspended in PBS and counted. The viability was determined by the trypan blue exclusion assay. Only cells with viability better than 95% were used in subsequent experiments. Splenocytes (10⁶/ml; 0.1 ml/well) in V-shaped 96-well microplates were incubated with supplements (2 μg/ml) for 120 min at 37°C and then washed three times with RPMI 1640 medium. After washing, 50 μl of target cell line YAC-1 (three different concentrations of target cells were used so the final effector-target ratio was 10:1, 50:1 and 100:1). After spinning the plates at 250 x g for 5 min, the plates were incubated for 4 hr at 37°C. The cytotoxic activity of cells was determined by the use of CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, 10 μl of lysis solution was added into appropriate control wells 45 min before the end of incubation. The next step was to spin the plates at 250× g for 5 min, followed by transferring 50 μl of supernatant into flat-bottomed, 96-well microplates. After 50 μl of reconstituted substrate was added into each well, plates were covered and incubated for 30 min at room temperature at dark. The optical density was determined by using a STL ELISA reader (Tecan U.S., Research Triangle Park, NC) at 492 nm. Specific cell-mediated cytotoxicity was calculated using the formula:

Percent-specific killing (% cytotoxicity)=100×[(OD₄₉₂ experimental - OD₄₉₂ spontaneous) divided (OD₄₉₂ maximum - OD₄₉₂ spontaneous)] as described in the manufacturer's instructions, where spontaneous release was target cells incubated with medium alone and maximum release was that obtained from target cells lysed with the solution provided in the kit.

Superoxide production. Firstly, cells were incubated with 1 μg/ml of tested material for 24 h. Next, cells were incubated in a final volume of 200 μl of medium containing 0.1% gelatin and 100 μM cytochrome C. The reaction was initialized by the addition of 5 ng/ml PMA. After gentle mixing, the absorbance was measured after 30 min incubation at 37°C using multiwall spectrophotometer at 550 nm. Results are expressed as nanomoles of cytochrome C reduced/2.5×10⁵ cells/30 min, after subtraction of the SOD and spontaneous release controls (17).

Statistics. A test of the null hypothesis that the difference between two responses measured on the same statistical unit has a mean value of zero (Student's t-test) was used to statistically analyze the data.