Six to seven week old male Balb/c mice were obtained from Charles River Laboratories and housed 3–5 mice per cage in a self-contained ventilated cage system (Innovive Inc., San Diego,CA) maintained at 40 air changes per hour. Both the intake and exhaust air were HEPA filtered. Mice were maintained in an environment of 22˚C with a 12 h light/12 h dark cycle. The mice were fed ad libitum a standard diet containing 22% crude protein and 5% fat (Harlan Teklad Laboratory, cat no 8640) and provided water ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Tampa Bay Research Institutional Animal Care and Use Committee (protocol number: 2011–001).
Preparation of PPC
Powdered PPC was kindly provided by Allera Health Products, Inc. (360 Central Avenue, Suite 1560, St. Petersburg, FL 33701). The pinecones used by Allera to produce PPC were obtained from a commercial supplier of Scotch pine (Pinus sylvestris) (identification of the supplier is regarded as proprietary information). Identification of the specific species of pine was performed by a qualified employee of the supplier. The pinecones were washed extensively in deionized water and then shredded. The pinecone shreds were then loaded into a stainless steel reaction vessel and extracted with water at elevated pH (12.0) and temperature (121°C, 30 min). The resulting extract was standardized by UV spectroscopy at a wavelength of 280 nm and by a series of biological assays. The liquid was tested extensively to ensure that it did not contain contaminants such as herbicides, pesticides, heavy metals, endotoxin, or microorganisms and was then spray-dried to form a dark brown, odorless, stable powder. The powdered extract was suspended in sterile water to a stock concentration of 25 mg/mL (w/v), centrifuged at 10,000 × g for 20 minutes to remove particulates and then filtered through a 0.2 μm nylon filter. The OD280 of the solution was measured and used to define the concentration of active ingredients:
where 22.8 L⋅gram-1⋅cm-1 is the extinction coefficient for polyphenylpropanoids. A working stock containing 200 μg/mL of polyphenylpropanoids diluted in sterile distilled water was prepared fresh each week.
In Vivo immune stimulation
On Day 0, three mice each in the SEB and SEB + PPC groups were injected intraperitoneally (i.p.) with 100 μg of Staphylococcal enterotoxin B (SEB, Sigma Chemical Company, St. Louis, MO) dissolved in 100 μL phosphate buffered saline (PBS), pH 7.4. Three mice in the Naive group were injected i.p. with 100 μL PBS, pH 7.4. The mice in the SEB + PPC group were gavaged with 100 μL of a 200 μg/mL solution of PPC three times daily on Day 0 and Day 1 for a total of 6 doses. At 48 or 96 hours post-SEB immunization, the mice were euthanized by CO2 inhalation. The set of inguinal lymph nodes from each mouse was pooled and used in each assay. Therefore, the results reported from lymph node cells represent the collection of data points from each mouse. The number of cell subsets per lymph node was obtained from the set of pooled inguinal lymph nodes and then dividing that number by two. To calculate the total number of cell subsets per lymph node, the percent of each subset was multiplied by the total number of cells isolated from the lymph nodes. The total number of cells isolated from the lymph nodes was determined using the Millipore Muse cell counter. Serum was collected for quantification of various cytokines and was stored at -80°C until analyzed.
Standard ELISA development kits specific for murine IL-2, IFNγ, and IL-12p70 were purchased from Peprotech (Rocky Hill, NJ). The detection of serum IL-2, IFNγ, and IL-12p70 was performed according to the manufacturer’s instructions. The absorbance of the final product was measured using BioTek’s μQuant plate spectrophotometer at a wavelength of 450 nm. Standard curves were prepared using Prism’s GraphPad software (GraphPad Software, Inc., La Jolla, CA).
Lymph node and spleen cell isolation
Isolated inguinal lymph nodes and spleens were homogenized in a sterile plastic bag containing 4 mL Hanks balanced salt solution (HBSS) pH 7.4, using a Seward Stomacher®80 with settings of medium speed for 60 seconds. The resulting cell suspensions were pelleted and then suspended in 2 mL HBSS (for lymph node cells) or 4 mL HBSS (for spleen cells). The yield of cells was determined by mixing 20 μL cells with 380 μL of Millipore’s Muse Count and Viability stain. The cell concentration and number of viable cells was determined using the Muse Cell Analyzer (Millipore). The cells were pelleted and then suspended in enough complete media (RPMI 1640 containing 10% fetal calf serum and 1x penicillin/streptomycin) to provide a final concentration of 4 × 106 cells/mL.
For each mouse, 1×106 lymph node cells were added to a 1.5 mL microfuge tube and then pelleted by centrifugation for 1 minute at 1000 × g. The pelleted cells were placed on ice and then suspended in 100 μL of FACS buffer (PBS containing 10% fetal bovine serum and 0.1% sodium azide) containing 1 μL anti-CD16/32 Fc blocking antibody (clone 93). Ten minutes later 1–5 μL of fluorescent antibodies specific for cell surface antigens were added. The fluorescent anti-mouse antibodies used to detect the SEB-activated T lymphocytes were as follows: CD3 (clone 17A2) labeled with allophycocyanin (APC), CD4 (clone GK1.5) labeled with fluorescein isothiocyanate (FITC), CD8 (clone SK1) labeled with phycoerythrin (PE), and Vβ8 (clone KJ16-133) labeled with a proprietary peridinin chlorophyll protein conjugated with eFluor710 (PerCP-eFluor®710). All of the antibodies were purchased from eBioscience, San Diego, CA. The cells were incubated on ice for 30 min and then washed by adding 500 μL FACS buffer and pelleting the cells at 1000 × g for 2 min. The pelleted cells were then suspended in 200 μL FACS buffer and analyzed by flow cytometry using a BD FACSCalibur (Becton Dickinson, San Jose, CA) equipped with CellQuest Pro software. The FACS data was further analyzed using FloJo software (TreeStar Inc, Ashland, CA).
Detection of apoptosis
Annexin V staining was performed according to the protocol provided with eBioscience’s Annexin V Apoptosis Detection Kit. Isolated lymph node cells (1 × 106/mouse) were suspended in Annexin V staining buffer containing 1 μL APC-labeled anti-CD3, 5 μL PerCP-eFluor®710-labeled anti-Vβ8, and 1 μL FITC-labeled Annexin V to identify T cells undergoing apoptosis. The cells were incubated at room temperature for 10 min and then washed in binding buffer. The cells were suspended in binding buffer and then analyzed by flow cytometry.
Detection of reactive oxygen species
To detect the presence of reactive oxygen species in activated T cells, isolated lymph node cells from each mouse were exposed to Invitrogen’s CM-H2DCFDA (5-(and-6)-chloromethyl-2’ ,7’-dichlorodihydrofluorescein diacetate, acetyl ester). CM-H2DCFDA passively diffuses into cells where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Oxidation then produces a fluorescent adduct that cannot escape the cell. The resulting fluorescence was then measured by flow cytometry. Isolated lymph node cells (1 × 106/mouse) were exposed to 100 μL FACS buffer containing 1 μL APC-labeled anti-CD3 antibody, 5 μL PerCPeFluor®710-labeled anti-Vβ8 antibody and 2 μL of a 50 μM solution of CM-H2DCFA for 15 min at 37˚C. The cells were then washed with 500 μL FACS buffer, pelleted and suspended in 200 μL FACS buffer. The levels of fluorescence within the various cell samples were measured by flow cytometry. The resulting data is shown as the percentage of ROS+ lymph node T cells (CD3+) and as the frequency distribution (histogram) of ROS levels in lymph node CD3+ T cells in SEB and SEB + PPC treated mice.
Detection of intracellular Bcl-2
To detect the intracellular levels of the anti-apoptosis related protein, Bcl-2, cells previously stained with APC-labeled anti-CD3 and PerCPeFluor®710-labeled anti-Vβ8 antibodies were washed in 0.03% saponin dissolved in HBSS (HBSS-SAP) and then pelleted by centrifugation at 1000 × g for 2 minutes. The cell pellets were suspended in 100 μL HBSS-SAP containing 1 μL FITC-labeled anti-Bcl-2 antibody (clone 10C4, eBioscience), and incubated on ice for 60 minutes. The cells were washed twice with 500 μL HBSS-SAP buffer and then once with HBSS only. The cells were then suspended in 200 μL HBSS and analyzed by flow cytometery.
Analysis of gene expression
Total RNA was isolated from lymph node cells using Qiagen’s RNeasy Plus Mini Kit following the manufacturer’s instructions. The resulting RNA was eluted in 30 μL sterile RNase-free water and the yield of RNA was determined by spectrophotometry at an optical density of 260 nm.
One microgram of total RNA was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription kit from Qiagen (Valencia, CA) and by following the manufacturer’s instructions. SABiosciences’ (Valencia, CA) RT2 Profiler qPCR SYBR green master mix kit was used for the real time quantitative PCR (qPCR). One microliter of the cDNA per reaction was added to a 1.5 mL microfuge tube containing 12.5 μL 2x SYBR green/fluorescein master mix and 10.5 μL H2O per reaction needed. Validated primer pairs from SABiosciences' RT2 qPCR Primer Assays specific for murine Bcl-2, Bcl-3, Bcl-xL, Bax, and Bim were utilized. These primers are pre-mixed at a concentration of 10 μM. One microliter of each primer pair was placed in duplicate wells of a 96 well PCR plate and then 24 μL of the cDNA/SYBR green master mix was added to the appropriate wells. Once all the samples had been added to the plate the plate was covered with clear plastic film and then centrifuged at 200 × g for 1 min to mix and collect all of the solutions in the bottom of the wells. The plate was then placed in a BioRad (Hercules, CA) iCycler programmed to run one cycle of 10 min at 95˚C to activate the polymerase and then 40 cycles of [95˚C × 15 sec → 60˚C × 1 min] to amplify the gene-specific cDNA. The relative levels of mRNA expression were normalized to the mouse β-actin housekeeping gene expressed in lymph node cells from naive (untreated) mice and determined by the ∆∆-Ct method .
The significance of responses to the multiple treatments was determined by ANOVA using Newman-Keuls multiple comparison test provided in GraphPad’s Prism® software (GraphPad Software, Inc, La Jolla, CA). All experiments were performed at least 3 times. Error bars on the graphs represent the standard deviation and demonstrate the ability to detect statistical significance when using only 3 mice per group.