For many centuries, velvet antler (VA) has been popular as a remedy for treatment of various conditions in a number of Asian cultures. In this study, we investigated the effect of aqueous extract of VA on proliferation of human T cells to a superantigen, staphylococcal enterotoxin B (SEB) in vitro. CFSE-labeled peripheral blood mononuclear cells from healthy donors were incubated with SEB in the presence of the extract, and their proliferation was analyzed by flow cytometry 72 h later. VA increased proliferation of both CD4+ and CD8+ T cells to SEB in a dose-dependent manner, but was not mitogenic in the absence of SEB within a concentration range studied (1–500 µg/mL). Heat inactivation did not affect the co-stimulatory activity. This activity was more pronounced in individuals with weaker responses to SEB. Our findings suggest that VA extract may modulate human T cell immune responses to antigens.
VA has been used in China, Russia, Korea, and other countries for more than 10 centuries as an “adaptogen” that helps recovery from various illnesses, including trauma, postoperative stress, infectious diseases, and fatigue.1 As organs that grow very quickly (up to 2.75 cm per day),2 deer antlers have been considered to be driven by, and serve as a source of, factors mediating tissue regeneration; however, the nature of the factors responsible for the medicinal effects ascribed to it remained unclear. Recently growing interest in “natural” or complementary medicine encouraged research on the antlers themselves that resulted in demonstration of a number of growth factors expressed in the growing antlers, such as insulin-like growth factors (IGF)-1 and -2, transforming growth factor b (TFGb), epidermal growth factor, bone morphogenetic protein-4, neurotrophin-3, fibroblast growth factor-2, vascular endothelial growth factor, and nerve growth factor.3–8 These factors may be responsible for the regenerative effects of antler preparations observed in animal or in vitro models,9–11 and some of them are known players in immune responses.12–14 Yet, the effects of VA on inflammation and immune responses, although frequently claimed by practitioners, have been difficult to demonstrate directly, and there is a limited literature that addresses the role of VA in immunity.15
Immune responses are complex and involve general recognition of “danger signals” and selective responses to specific targets, antigens. The first is generally a function of the innate immune system (macrophages), whereas the second is the responsibility of the adaptive immune system (T and B lymphocytes). If the immune response is too weak, the body cannot clear the target (often, a pathogen); if it is too strong, the immune cells may harm the cells and tissues of the body due to an excessive inflammation. Usually, the immune system is capable of fine-tuning itself, and failure to do so results in inflammatory diseases, chronic infections, or severe autoimmunity, hence the importance of the search for drugs with abilities to reduce or to strengthen immune responses. Western pharmacopoeia has a number of potent drugs that act in either direction, such as non-specific immunosuppressive ones (eg, corticosteroids, cyclosporine, tacrolimus), immunostimulatory ones (eg, interferons, other cytokines), and novel and more selective blockers of cytokines or cytokine receptors (eg, infliximab, anakinra, daclizumab). The clinical use of such drugs is often associated with marked side effects, some of which may be serious (eg, hepato- and nephrotoxicity, activation of latent infections, high fever, rashes, arthralgias). This, along with the high cost of these medications, limits clinical indications to the most serious conditions (eg, allograft rejection, resistant infections, septic shock) and necessitates further search for safer modulators of immune responses. This leads many researchers to the field of complementary medicine and its focus on the direct clinical use of natural preparations (of plant and animal origin). Complementary medicine research has potential for discoveries of active molecular components with known and novel therapeutic properties. Direct demonstration of the pharmacological effects of complex compounds, as they are presented in the natural formulations, in defined biological systems, such as in vitro cell cultures, could serve as an important link.
We were interested in studying the effects of VA on human T cell responses to antigens. Here, we used an aqueous extract of finely dispersed antlers of Siberian deer in an in vitro system, in which normal human T cells respond to staphylococcal enterotoxin B (SEB). We measured T cell proliferation (clonal expansion) in response to SEB with and without VA as a readout of the responsiveness, and report that VA increased T cell proliferation in a dose-dependent manner.
Material and Methods
Cell cultures and proliferation assay
All protocols were approved by the Yale University Institutional Review Board. Normal human blood enriched for leukocytes was obtained from the Central Laboratory Facility, New York Blood Center (Long Island City, NY), and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) gradient centrifugation. They were labeled with CFSE using Cell Trace Kit (Invitrogen, Grand Island, NY) and cultured in AIM-V medium (Invitrogen), at 1x106/mL with SEB (1μg/ml, Sigma, St. Louis, MO) at 37oC, in an atmosphere of 5% CO2.
Preparation of the aqueous VA extract
VA powder (a gift from SpectrAcoustics Co., Saratov, Russian Federation) was aseptically added to sterile PBS (Invitrogen) at 1.5g/30 mL, incubated 24h at 4oC, filter-sterilized, and stored in frozen aliquots at -20oC. Protein concentration was determined using BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Freshly thawed aliquots were serially diluted with AIM-V medium to obtain final concentrations of 1–500 µg/mL when added to human PBMC. Bovine serum albumin (BSA) from Sigma was used as a negative control. In separate experiments, VA extract was incubated at 56oC in a water bath for 30 min prior to use in cell cultures. The effect of VA on proliferation was calculated as [1-(%divided with added VA/%divided without VA)] x100. Immunoreactive cytokine content was measured using Milliplex kits (Millipore Corporation, Billerica, MA) and Bioplex analyzer (Bio-Rad, Hercules, CA).
Data are expressed as means ± standard error. Statistical analysis was performed using GraphPad Prizm© Version 5 software (GraphPad Software Inc., San Diego, CA). One way analysis of variance (ANOVA) was applied for comparison between the groups. Linear regression analysis was used to evaluate correlation.
Effect of VA on proliferation of PBMC to SEB
SEB is recognized by a large proportion of T cells, which carry T cell receptors made of particular V beta chains. Since the usage of these V beta chains varies in different individuals, the proportion of T cells that respond to the superantigen varies, too. In this study, mean percent of divided cells at the end of 72h incubation was 66.8±5.6 (range 38.1–90.8%; n=8). Addition of VA extract increased cell division in a dose-dependent manner. Figure 1A shows an example of T cell proliferation measured as CFSE dilution in daughter cells and its increase at higher concentrations of VA. There was no proliferation in the absence of SEB, even at the highest concentrations of VA used (data not shown). The costimulatory effect was not due to a non-specific action of a xenogeneic protein, since addition of BSA at the same dose was ineffective (Figure 1B). A summary of 8 separate experiments shows that VA extract’s costimulatory effect was statistically significant compared to VA-free controls at concentrations of 100 and 500 μg/mL (Figure 1C). Costimulatory activity of the extract was resistant to heating at 56oC for 30 min, suggesting it is not within the most thermo-labile group of proteins, such as complement (data not shown).
Effects of aqueous VA extract on proliferation of human T lymphocytes to SEB.
(A) Human PBMC were labeled with CFSE and incubated with SEB in the presence of various concentrations of VA extract for 72 h, after which proliferation was analyzed by flow cytometry (a representative individual experiment showing % divided cells as daughter peaks). No cell division was seen in the absence of SEB. (B) The results of an experiment showing that costimulation of proliferation by VA (right panel) compared to SEB alone (left panel) is not due to the presence of a xenogeneic protein, because BSA addition (central panel) was ineffective even at the highest concentration. (C) A summary of 8 independent experiments, each representing a separate PBMC donor, showing % of increased proliferation at different concentrations of VA extract, as described in Materials and Methods. *P<0.05 by ANOVA.
VA extract costimulates proliferation of both CD4+ and CD8+ T cells, but not T cell lymphoma
To test whether VA preferentially costimulates CD4+ or CD8+ T cells, we labeled these subpopulations with fluorescent antibodies and analyzed their proliferation to SEB by flow cytometry. As shown on Figure 2A, both CD4+ and CD8+ T cells responded to SEB, and their responses were augmented by VA extract in a dose-dependent manner with a peak at 100 μg/mL.
The ability of VA extract to increase proliferation of human T cells might raise a safety concern, namely a potential for stimulation of T cell malignancies. To test that, we cultured Jurkat cells, which are a human T cell lymphoma line, with increasing concentrations of VA. No increase in proliferation of Jurkat cells was observed, suggesting that VA is not mitogenic to an autonomously dividing lymphoma (Figure 2B).
Since the costimulatory activity could be due to the presence of some cross-reactive cytokines, we measured the following human cytokines in the extract: IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17, IFNg, and TNF; none of the cytokines listed was detected in VA extract at total protein concentration up to 1.6 mg/mL.
Costimulatory activity of VA extract inversely correlates with T cell responsiveness
To test whether VA extract costimulates T cell responses regardless of the degree of the response to superantigen, we correlated the percent of divided cells in the absence of VA to the percent of increase of proliferation at 100 μg /mL. As shown on Figure 2C, a significant negative correlation was found (P=0.011; R2=0.57), suggesting that the effect of VA was stronger in individuals who were weaker responders to SEB.
Effects of aqueous VA extract on proliferation of human T cells, T cell subsets, and Jurkat lymphoma.
(A) Normal human PBMC were labeled with CFSE and incubated with SEB in the presence of various concentrations of VA extract for 72 h, after which labeled with anti-CD4 and anti-CD8 antibodies. Proliferation was analyzed by flow cytometry. Data are pooled from two separate experiments that gave similar results (NS, two way ANOVA). (B) Jurkat lymphoma cells were incubated for 96 h with different concentrations of VA and % change in proliferation was calculated, as described. (C) Linear regression analysis of relation between total T cell proliferation to SEB and % co-stimulation at 100 μg/mL of VA extract (P=0.011; R2=0.57).
In our experiments, we used cold aqueous extract of VA to preserve potential biological activity of proteins and found that, at a dose of 100 μg/mL of total protein content, VA increased T cell proliferation, which is a standard readout of immune responsiveness, and that both CD4+ and CD8+ T cells were involved; however the extract did not induce proliferation in the absence of antigenic stimulation, nor did it activate division of a lymphoma cell line. Taken together, these observations suggest that VA preparations may be suitable as a food supplement for immune modulation. Interestingly, in half of the individuals tested, costimulatory effect of VA at 500 μg/mL was less than at 100 μg/mL, suggesting that the preparation also contains antiproliferative factors that start to dominate at higher concentrations of the extract. Another possible explanation may be that certain individuals respond to high-dose costimulatory signal(s) paradoxically. Additional experiments are needed to further address this question.
Our data indicate that, when activated with a superantigen, human T cells respond by proliferation, which is increased in a dose-dependent manner by the addition of an aqueous extract of VA. This costimulatory activity was 1) not due to a non-specific effect of a protein added, since BSA was ineffective; 2) resistant to heat inactivation at 56oC, indicating that it was not due to circulating deer complement; and 3) not due to a direct mitogenic effect of an unknown origin, because there was no proliferation without SEB regardless of VA concentration.
The content of VA is very complex, and it remains unclear which factors are responsible for the costimulatory effect we report. Circulating and matrix-bound cytokines, such as TGFb, TNF, IGF-1 and some others could provide this activity.16–19 We therefore attempted to detect a number of immunoreactive cytokines using reagents for human factors; although none of the cytokines tested was detected in VA extract, we cannot rule out that they are present and may contribute to costimulation, because we do not have information on cross-reactivity of our reagents with deer cytokines.
Being a skin derivative, velvet antler contains melanin, the major pigment in the skin, and aqueous extract retains its dark brown color.
Being a skin derivative, VA contains melanin, the major pigment in the skin, and aqueous extract retains its dark brown color. Melanin has been reported as an activator of T cell responses20–21 and may be important in pathogenesis of autoimmune diseases such as uveitis and vitiligo. One of the mechanisms by which melanin could costimulate T cell proliferation is by quenching reactive oxygen species22 usually generated during lymphocyte activation23 and thus reducing oxidative stress and apoptosis in a way similar to the effects of polyphenols.24
Another candidate component that could be responsible for costimulation is phosphatidyl choline. An earlier report by Kim et al showed that responses of mouse spleen cells to a lectin, Concanavalin A, could be augmented by phosphatidyl choline isolated from deer VA.25 Interestingly, similarly to our observations, Kim et al found phosphatidyl choline was not costimulatory in the absence of lectin.
Our study did not aim to dissect which of the VA components is the major contributor to the observed effect. We used only aqueous extracts and therefore could have lost some hydrophobic components, such as lipid-soluble vitamins and hormones during extraction, although these can be solubilized by naturally occurring emulsifiers and by binding to proteins.
We detected the costimulatory effect of VA on T cell proliferation in the range between 10 and 500 µg/mL. It is unlikely that such a concentration can be achieved systemically after intake of 1–2 g orally, keeping in mind partial digestion of the components in the gut; however, we speculate that upon ingestion, VA might reach the upper small bowel at such amounts and, if so, may contribute to the role the mucosal immune system plays in protecting against pathogens and priming systemic responses.
The costimulatory effect was stronger in poor responders and weaker in strong responders (Figure 2), which suggests that the action of VA can be described as regulatory rather than stimulatory. If these results can be extended to humans, individuals with low immune reactivity may be better responders to VA supplementation than those with normal or high responses.
Our data indicate that VA has a costimulatory effect on T cell proliferation in response to superantigen SEB in vitro. These results provide evidence to support the traditional use of VA to enhance immune responses; however, our results require verification in vivo using other models, such as humanized mice, and direct clinical trials to study the effect of VA on immune function in humans.
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