For almost 2000 years it has been recognized that aqueous extracts from pine cones possess medicinal properties beneficial for the treatment of a broad variety of diseases and conditions. In this report, the ability of an orally administered poly phenylpropanoid-polysaccharide rich extract of pine cones (PPC) to suppress the generation of IgE and to significantly enhance antigen-specific cellular responses to a variety of vaccines was tested.
A variety of vaccine protocols were utilized to determine the affects of orally administered PPC on the Th1/Th2 cytokine balance, the production of IgE antibodies, and the generation of antigen-specific cytotoxic T cells. The effect of PPC on the Th1/Th2 balance in aged mice was also investigated.
Oral delivery of PPC was found to significantly suppress serum IgE levels in naïve mice and in mice sensitized to ovalbumin. PPC was also found to enhance the generation of antigen-specific CD8+ T cells in mice immunized with DNA, dendritic cell, and soluble protein vaccines. The suppression of IgE was associated with reduction of IL-4 secretion and the enhanced production of IL-12 and IFNγ by antigen-stimulated splenocytes from PPC treated mice. PPC also suppressed the Th2 response and enhanced the Th1 response of splenocytes from aged mice.
Oral delivery of PPC enhances the generation of an antigen-specific CD8+ T cell responses induced by soluble protein, DNA, and dendritic cell vaccines while at the same time suppressing the generation of a Th2 dominant IgE response. This effect on the Th1/Th2 balance was also observed in aged mice.
Almost 2000 years ago the knowledge that aqueous extracts of pine cones possessed medical properties was first recorded [
PPC is a poly-phenylpropanoid-polysaccharide complex prepared from a single species of pine cones [
In investigations where the toxicity of PPC has been examined in mice, oral administration at doses as high as 1.83 g/kg produce no noticeable toxic effects [
Tissue distribution of the orally administered extract (10 mg/kg) was examined by measuring the levels of 125iodine-labeled extract in a variety of murine tissues at intervals of 3, 24, or 48 h after delivery [
While the pine cone extract has been traditionally used to complement the treatment of a variety of illnesses, an increasing number of people using the commercial product have remarked that when taken on a daily basis it provides significant relief from, or even eliminates, their allergy symptoms. Since numerous studies have demonstrated a strong correlation between the reduction of serum IgE levels and noticeable improvement in the well being of people suffering from IgE-mediated allergies [
The results presented herein suggest that PPC might in fact be able to reduce IgE levels in patients with allergy. Oral administration of PPC was found to reduce serum IgE levels in mice and to significantly suppress the development of an allergen induced IgE response. Interestingly, the potential of PPC to suppress IgE levels appears to be associated with its ability to enhance the generation of a Th1 - associated cellular immune response, a response demonstrated to improve the activity of a variety of vaccine types.
Six week old female C57BL/6 and Balb/c mice were purchased from Charles River Laboratories. Aged male B6C3F1 mice (23-25 months old) were obtained from the National Institute of Aging. The mice were maintained in accord with the NIH
Pine cones from Scotch pine (
The powdered form of PPC, Proligna™, was obtained from Allera Health Products Inc. (St. Petersburg, FL), suspended in sterile water to a stock concentration of 25 mg/mL (w/v), centrifuged at 10,000 × g for 20 minutes and then filtered through a 0.2 μm nylon filter. The stock solution was diluted in sterile distilled water to prepare as drinking water for the mice.
Female C57BL/6 mice were injected subcutaneously with either 100 μg of ovalbumin (Grade V, Sigma Chemical Co, St. Louis, MO) or 500 μg ovalbumin along with either 100 μg CpG (ODN 1826) (provided by Mayo Clinic Molecular Core, Rochester MN) or 50 μg poly I:C (Sigma Chemical Co., St. Louis, MO) formulated in IFA on day 0 (priming) and then again on day 14 (boost). In one set of experiments female Balb/c mice were injected i.p. with 10 μg ovalbumin formulated in 2 mg Imject alum (Thermo Fisher Scientific, Rockford, IL) on day 0 (prime) and again on day 14 (boost).
Hind limbs of C57BL/6 mice were harvested and bone marrow was isolated. Bone marrow derived dendritic cells (BM-DC) were generated by plating bone marrow cells at 1 × 106 cells/mL in RPMI media (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen) for 7 days with murine GM-CSF (10 ng/mL) and murine IL-4 (5 ng/mL) (Peprotech, Rocky Hill, NJ). Media was replaced periodically and fresh GM-CSF and IL-4 was added to the cells. At day 7, BM-DC were harvested and replated at 2 × 106 cells/mL with 1 μg/mL of LPS to induce maturation overnight. On day 8, BM-DC were harvested and pulsed with 10 μg/mL of peptide for 2 hours at 37°C and 5% CO2.
C57BL/6 mice (3 per group) were immunized by i.v. injections with 2 × 106 LPS-matured BM-DC, pulsed with either the MHC-Class I (H-2Kb) restricted OVA257-264 (SIINFEKL) or Trp2180-188 (SVYDFFVWL) peptides (A&A Labs., San Diego, CA). Each mouse received the DC vaccine injections on days 0, 7, and 14. For some groups, PPC (200 μg/mL) was added in the drinking water, beginning 24 hours before the first vaccination and continued throughout the period of the study.
Mice (3 per group) were immunized with a total of 100 μg of plasmid DNA suspended in 100 μL normal saline. A total of 50 μL of the DNA solution was then injected into each of 2 sites in the
Serum from mice was analyzed by ELISA using the antibody kit from Bethyl Laboratories (Montgomery, TX) and following the manufacturer's instructions. The sensitivity of the assay was 3.9 ng/mL for murine IgE. Assays were performed in 96 well flat bottom Maxisorp plates (Nunc; Thermo Fisher Scientitific, Rochester, NY) using serum diluted 1:10 with 50 mM Tris, 0.14 M NaCl, 1% BSA, and 0.05% Tween 20.
Maxisorp 96 well flat-bottom plates were coated with 100 μg/mL ovalbumin in carbonate-bicarbonate buffer (Sigma) overnight at 10°C. The plates were washed with PBS containing 0.05% Tween-20 (PBST) and then blocked by adding 300 μL StartingBlock (Pierce Scientific, Rockford, IL) to each well for 10 min. This was removed and 2-fold serial dilutions of the serum samples, ranging from 1:250 to 1:32,000, in PBS containing 1% bovine serum albumin were added in triplicate to appropriate wells and incubated overnight at 10°C. After the overnight incubation the wells were washed 4 times with PBST and then horseradish peroxidase-conjugated goat anti-mouse IgG2a antibody (cat no. M32307, Invitrogen, Carlsbad, CA), diluted 1:2000, was added to each well for 1 h at room temperature. The wells were washed with PBST and then 100 μl of the SureBlue substrate (KPL, Inc., Gaithersburg, MD) was added. The color reaction was stopped by adding 50 μL of 2 M H2SO4 or 1 M HCl and the absorbance was read at 450 nm.
To prepare single cell suspensions of splenocytes the spleens were isolated using aseptic technique, placed in sterile polypropylene bags containing 5 mL of incomplete media (RPMI 1640), and homogenized in a Stomacher 80 Laboratory Blender (Seward Medical, London, England) at high setting for 60 seconds. The resulting cell suspension was filtered through a 70 μm nylon and then mixed with 5 mL of 2× ACK lysis buffer (1× ACK lysis buffer is 8.29 g/L NH4Cl, 1 g/L KHCO3, and 37.2 mg/L Na2EDTA, pH 7.2) for 5 minutes at room temperature to remove the RBC. The splenocytes were pelleted and then suspended in complete media (RPMI 1640 containing 10% fetal calf serum, 4 mM L-glutamine, and 50 μM β-2 mercaptoethanol) to a final concentration of 1 × 106 per mL. The cell count and viability was performed using the trypan blue dye exclusion method. The viability of the cells was typically >95%.
One milliliter of splenocytes, at a concentration of 1 × 106 cells/mL (from aged B6C3F1 mice) was plated in each of six wells of a 24 well plate. To enhance cytokine production in the splenocyte culture, 50 μL of a 100 μg/mL solution of concanavalin A (ConA) was added to 3 of the 6 wells. After 48 hours of incubation at 37°C in an atmosphere containing 5% CO2, the culture media was removed and placed in a sterile 1.5 mL microcentrifuge. The media was cleared of cells and debris by centrifugation at 16,000 × g for 2 minutes. The cleared media was transferred to a sterile 1.5 microfuge tube and stored at -80°C until it was analyzed by ELISA for cytokine levels.
Splenocytes from Balb/c mice that had been immunized in the presence or absence of PPC were incubated in 24 well plates at a concentration of 4 × 106/mL for 48 h in the presence of media alone, 100 μg/mL ovalbumin (Fraction V, Sigma Chemical Company), or 10 ng/mL PMA + 100 ng/mL ionomycin. The 48 h supernatants were cleared of cells and then analyzed by ELISA for the presence of appropriate cytokines.
For detection of CD8+ T cells secreting IFNγ, enzyme linked immunosorbent spot (ELISPOT) assays were performed. Briefly, Millipore Multiscreen Plates were coated by overnight incubation at 4-10°C with 100 μl of 5 μg/mL anti-mouse IFNγ (clone AN-18, eBioscience, Inc., San Diego, CA) in PBS and then blocked by incubation for 3 h at 37°C with 200 μL RPMI-10 (RPMI 1640 with 10% fetal bovine serum). Stimulator cells were washed once with complete media and then suspended at a concentration of 3-5 × 106/mL. The appropriate peptide was then added at a concentration of 20 μg/mL and the cells were incubated for a minimum of 3 hours in a 5% CO2 atmosphere at 37°C. The stimulator cells were then irradiated at 10,000-20,000 Rads, depending on the cell line being used. The stimulator cells were washed twice in RPMI-10 and then suspended in RPMI-10 to a concentration of 1 × 106/mL. For the IFNγ ELISPOT assay, irradiated stimulator cells (EL4 or EG7) were used to present endogenous OVA or were pulsed with the Class I OVA peptide (SIINFEKL).
CD8+ T cells were purified from splenocytes of vaccinated mice by positive selection using antibody-coated magnetic beads (Miltenyi Biotec, Auburn, CA). Responder (CD8 purified) cells were incubated at different concentrations per well, together with 5 × 104 stimulator cells (peptide pulsed, unpulsed, or expressing tumor cells). Cultures were incubated at 37°C for 20 h. After the incubation the cells were removed from the plate and the wells washed 6 times with PBST. Then, 100 μL of biotinylated anti-IFNγ at 1 μg/mL in PBST was added to each well, the plate was covered and incubated 2-3 hours at room temperature. The plate was then washed with PBST and 100 μL/well of avidin-HRP was added at a 1:1000 dilution in PBST followed by incubated at room temperature for 1 hour. The plate was washed 6 times with PBST and then 3 times with PBS only. Freshly prepared 3-amino-9-ethylcarbazole (AEC) substrate (100 μL) was added to each well and incubated for 10-30 minutes at room temperature. The plate was flooded with tap water to stop the color development and then submerged in a tank of tap water to assure low background. The plates were allowed to dry overnight. Spot counting was performed with an AID EliSpot Reader System (Autoimmune Diagnostika GmbH).
The detection of the cytokines, IL-12p40, IFNγ, IL-4, and IL-10 was performed using the murine cytokine development kits from Peprotech (Rocky Hill, NJ). The IL-5 ELISA was performed using the Ready-SET-Go kit from eBioscience (San Diego, CA). The assays were performed following the manufacturer's instructions. The sensitivities for the IL-12p40, IFNγ, IL-4, IL-5 and IL-10 ELISAs were, 32, 16, 16, 4, and 47 pg/mL, respectively.
The results are expressed as means ± SD (standard deviation). All assays were perform in triplicate and repeated at least three times. The statistical difference between two groups was determined by Students t test. When multiple groups were being analyzed the statistical difference was determined using the one-way analysis of variance (ANOVA). A
To determine if oral administration of PPC affected IgE levels in mice, we administered a group of naive female Balb/c mice (n = 4 per group) with 200 μg/mL of PPC in their drinking water
C57BL/6 mice (4 per group) were injected s.c. on days 0 and 14 with 100 μg chicken egg ovalbumin (OVA) (Figure
When the C57BL/6 mice were immunized with 500 μg OVA formulated in IFA (Figure
Since it appeared that PPC was capable of suppressing an IgE response, a response known to be highly dependent on the Th2-associated cytokine, IL-4, we sought to determine if PPC might be suppressing the Th2 response by inducing the mutually inhibitory Th1 response. To do this we measured the serum levels of OVA-specific IgG2a, an isotype strongly associated with the presence of a Th1 response. Sera obtained from the C57BL/6 mice immunized with OVA + IFA were examined by ELISA (Figure
To determine if the observed suppression of IgE and enhancement of allergen-specific IgG2a were associated with overall enhancement of a Th1-type response, we determined whether PPC affected development of an OVA-specific CD8+ T cell response. Splenic CD8+ T cells were isolated on day 21 from C57BL/6 following immunization with 500 μg OVA formulated in IFA. The TLR ligands, CpG (100 μg) and poly IC (50 μg) were included in the formulation to serve as positive controls for immune modulators. Mice in three other groups (n = 4 per group) were provided PPC (20, 200, or 2000 μg/mL) in their drinking water beginning on day -1 and lasting until day 21 when an IFNγ ELISPOT analysis was performed (Figure
Combined with the finding that PPC suppresses IgE levels in mice, and that in mice this has been associated with elevation of antigen-specific IgG2a and a doubling of the number of antigen-specific CD8+/IFNγ T cells, these results suggest that oral administration of PPC is capable of significantly boosting a Th1-like response and that this response, via the mutual inhibitory feedback mechanism, could be responsible for suppressing the generation of a Th2 response and the production of IgE.
To demonstrate that PPC could be affecting the Th1/Th2 balance, we examined cytokine production by female Balb/c mice exposed to OVA. OVA (10 μg) formulated in alum was injected i.p. on days 0 and 14. One group of mice (OVA + PPC) received 200 μg/mL PPC in their drinking water beginning 7 days prior to the first injection. On day 28, fourteen days after the last OVA injection, the serum levels of IgE were determined and the
To determine if PPC could affect the Th1/Th2 balance in a very different immunologic model, we administered aged B6C3F1 mice (23-25 month old males) with relatively low levels of PPC (5 or 50 μg/mL) in their drinking water
These results demonstrate that the oral delivery of PPC can modulate an immune response away from one that is predominately Th2-mediated towards one that is Th1-mediated. If this were the case, we would expect to find that oral delivery of PPC would enhance the antigen-specific CD8+ T cell response to a variety of vaccine types and suppress the development of IgE antibodies.
Female C57BL/6 mice (4 per group) were immunized by electroporation with plasmid DNA encoding full length ovalbumin and then analyzed for the generation of IFNγ CD8+ T cells specific for the dominant Class I epitope (OVA257, SIINFEKEL).
While untreated mice produced 800 IFNγ spots per 106 isolated CD8+ T cells, co-administration of 200 μg/mL of PPC more than doubled that number (1750). This effect was significant (
Female C57BL/6 mice (4 per group) were immunized weekly for three weeks by intravenous injection of 2 × 106 LPS-matured bone marrow derived dendritic cells that had been pulsed with the melanoma-derived Trp2180-188 peptide (SVYDFFVWL). For some groups, PPC (200 μg/mL) was added in the drinking water beginning 24 hours before vaccination and continued throughout the period of the study. Seven days following the last injection, CD8+ T cells were isolated from the spleen and analyzed via ELISPOT for the antigen-specific production of IFNγ. As shown in Figure
For centuries people have utilized a tea prepared from pine cones to provide relief from a variety of ailments, including cancer [
Upon learning that consumers of the commercial pine cone extract (Immune Extra™) had experienced a significant reduction of their allergy symptoms we sought to determine if the product affected the generation of IgE in mice. It was observed that oral delivery of PPC was able to reduce the basal levels of IgE in naive mice Balb/c mice (Figure
It has been recognized that as the human population ages the incidence of disease increases and the ability of our immune systems to protect us from infectious diseases and cancer, or to respond to vaccines wanes [
To determine if PPC can influence the Th1/Th2 balance in the aged, we provided aged mice with PPC in their drinking water for 25 days and then activated their splenocytes
To demonstrate the biological relevance of PPC's ability to enhance a Th1 response in younger mice, we examined its influence on the development of antigen-specific CD8+ T cell responses induced by DNA and dendritic cell vaccines. In both vaccine models it was discovered that oral delivery of PPC more than doubled the number of antigen-specific IFNγ+/CD8+ T cells generated by the vaccine (Figures
Our results have provided significant evidence that prolonged oral administration of PPC leads to the reduction of serum IgE levels and the enhancement of cellular immune responses. To determine how this might take place we must consider the mechanisms that lead to IgE production. The first step consists of signals that favor the differentiation of naive Th0 cells to a Th2 phenotype, while the second step comprises the action of cytokines and co-stimulatory signals from Th2 cells that stimulate B cells to switch to the production of IgE antibodies. The differentiation of naive Th0 CD4 T cells is determined by the cytokines they are exposed to before and during this response, and by the intrinsic properties of the antigen, antigen dose, and route of presentation. Naïve CD4 T cells being primed by their first encounter with antigen presenting cells (APC) secreting IL-4 tend to develop into Th2 cells, while those encountering APC secreting IL-12 tend to develop into Th1 cells. Interestingly, development of the Th1 and Th2 phenotypes tend to be mutually exclusive [
It is well recognized that in the presence of IL-4 and co-stimulatory signals from Th2 cells, B lymphocytes are stimulated to switch to the production of IgE antibodies, while in the presence of IFNγ, the switch to the production of IgE is suppressed [
Based on the critical nature of IL-4 and IFNγ for the induction and suppression of IgE, respectively, we hypothesize that orally administered PPC is inducing the production of IFNγ which in turn suppresses IL-4 production and consequently IgE production. We believe that PPC's induction of IFNγ production is associated with activation of the Th1 pathway by macrophage and/or dendritic cells stimulated by PPC to secrete IL-12 and other pro-Th1 factors. This release of IL-12 and the interaction with Th0 CD4 cells during antigen encounter could lead to the generation of Th1 CD4 T cells, activation of NK cells, and the generation of antigen-specific CD8 effector cells, all of which can then release IFNγ and block the release and the effects of IL-4. Our finding of enhanced IL-12 and IFNγ and reduced IL-4 release by PPC treated mice (Figures
Oral delivery of PPC enhances the generation of an antigen-specific CD8+ T cell responses induced by soluble protein, DNA, and dendritic cell vaccines while at the same time suppressing the generation of a Th2 dominant IgE response. This effect on the Th1/Th2 balance was also observed in aged mice.
The authors, DA, and EC declare that they have no competing interests. Authors AT and WGB are listed as inventors on patents related to production of PPC and hold stock in the company (Allera Health Products, Inc) selling the commercial product, Immune Extra™. Author MB was employed by Allera during part of the time spent on this research. Author FT is CEO of Allera Health Products, Inc.
Authors EC, AT, FT, and WGB designed the experiments and interpreted the experimental results. WGB and MB performed all animal work and ELISA assays associated with several of the ovalbumin vaccine studies, and the aged mice studies. DA performed work associated with one of the ovalbumin vaccine studies and ELISPOTS and the DNA and dendritic cell vaccine studies. All authors contributed to the manuscript preparation and approved its submission.
The pre-publication history for this paper can be accessed here:
We wish to thank Norma Bradley for her excellent technical assistance in establishing the dendritic cell cultures and vaccines and in developing the ELISPOT assays utilized in this study. This research was partially supported by grant 1R21AT002883 from the National Center for Complementary and Alternative Medicine.