Grape seed extract activates Th1 cells in vitro

Abstract

Although flavonoids manifest a diverse range of biological activities, including antitumor and antiviral effects, the molecular mechanisms underlying these activities await elucidation. We hypothesize that the flavonoid constituents of a proprietary grape seed extract (GSE) that contains procyandins exert significant antiviral and antitumor effects, by inducing production of the Th1-derived cytokine gamma interferon (IFN-gamma) by peripheral blood mononuclear cells) from healthy donors. Our results show that GSE significantly induced the transcription of IFN-gamma mRNA as demonstrated by reverse transcription-PCR but had no effect on the Th2-derived cytokine interleukin-6. The enhancing effect of GSE on IFN-gamma expression was further supported by a concomitant increase in the number of cells with intracytoplasmic IFN-gamma as well as the synthesis and secretion of IFN-gamma. Our results demonstrate that the potentially beneficial immunostimulatory effects of GSE may be mediated through the induction of IFN-gamma.

FIG. 1.

GSE induces IFN-γ gene expression by PBMC as measured by RT-PCR. PBMC (3 × 10⁶ cells/ml) were cultured in RPMI 1640 medium alone or with GSE at concentrations of 0.01 (lanes 2), 0.1 (lanes 3), and 0.5 (lanes 4) mg/ml for 48 h. Cytoplasmic RNA was extracted, reverse transcribed, and amplified with specific primers for housekeeping gene G3PDH (A) and IFN-γ (B). Additional lanes: MW, molecular weight markers; 1, control; 5, PMA plus ionophore (50 ng/ml each). (C) Quantitation of changes in IFN-γ gene expression. The statistical significance of the difference between control and treated samples was calculated by Student's t test. These data represent the means ± SD (error bars) of three experiments using PBMC from three different subjects.

FIG. 2.

FACS analysis of PBMC treated with GSE. Percentage of cells positive for intracytoplasmic IFN-γ determined by using a carboxyfluorescein-conjugated antibody specific to IFN-γ. The lymphocyte population was gated by labeling the population with CD45 conjugated to cytochrome c. (A) Isotype control. Immunoglobulin G2b was used as a negative control to set the quadrants. (B) Untreated control. The FL1 axis represents FITC-labeled IFN-γ positive cells. Cells treated with GSE at concentrations of 0.05 (C) and 0.1 (D) mg/ml, respectively. (E) Data are presented as percentages (means ± SD [error bars]) of IFN-γ-positive cells from three separate experimental results.

FIG. 3.

GSE induces IFN-γ synthesis and secretion by normal PBMC. Supernatants from GSE-treated and control cultures (24 to 72 h) were assayed for IFN-γ by a quantitative sandwich ELISA technique using the Quantikine kit (R & D Systems) as described by the manufacturer. Data are means ± SD (error bars) of four experiments performed in triplicate using PBMC from four different healthy subjects. The statistical significance of the differences between control and GSE-treated cultures was evaluated by Student's t test. The cultures treated with PMA plus ionophore (50 ng/ml each) used as a positive stimulant produced a mean of 2,780 ± 189 pg of IFN-γ/ml.

FIG. 4.

GSE does not modulate IL-6-specific gene expression by normal PBMC. PBMC (3 × 10⁶ cells/ml) were cultured alone or with different concentrations of GSE (0.01 [lanes 2], 0.1 [lanes 3], and 0.5 [lanes 4] mg/ml) for 48 h. Cytoplasmic RNA was extracted, reverse transcribed, and amplified with specific primers for housekeeping gene β-actin (A) and IL-6 and IFN-γ (B). Additional lanes: MW, molecular weight markers; 1, control. Also shown are quantitation of changes in IL-6 (C) and IFN-γ (D) gene expression. Data are means ± SD (error bars) of four experiments performed in triplicate using PBMC from four different subjects. Results showed that GSE upregulated IFN-γ gene expression, while IL-6 gene expression was not modulated in the same RNA samples. The statistical significance of the differences between control and treated samples was calculated by Student's t test.

FIG. 5.

GSE does not induce IL-6 synthesis and secretion by normal PBMC. Supernatants from GSE-treated and control cultures (24 to 72 h) were assayed for IL-6 by a quantitative sandwich ELISA technique using the Quantikine kit (R & D Systems) as described by the manufacturer. Data are means ± SD (error bars) of four experiments performed in triplicate using PBMC from four different healthy subjects. Statistical significance of the differences between control and GSE-treated cultures was evaluated by Student's t test. These same culture supernatants were also quantitated for IFN-γ levels, and GSE at 0.1 and 0.5 mg/ml produced significant levels of IFN-γ compared to untreated control cultures, similar to results reported in Fig. 3 (these data are not included in Fig. 5 for clarity).