B-cell proliferation activity of pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. and its structural requirement

Abstract

Pectic polysaccharide fraction (BR-2) containing pharmacologically active pectic polysaccharide, bupleuran 2IIc, which was prepared from a medicinal herb, the roots of Bupleurum falcatum L., was administered orally to C3H/HeJ mice for 7 consecutive days. Proliferative responses of spleen cells were enhanced in the presence of the purified pectic polysaccharide, bupleuran 2IIc, but another B-cell mitogen, lipopolysaccharide (LPS) did not give a similar effect. In vitro studies using spleen cells showed that bupleuran 2IIc also stimulated lymphocytes, depleted of adherent cells or T cells. Bupleuran 2IIc treatment increased subpopulation of CD25+ and surface immunoglobulin M-positive (sIgM+) lymphocytes. Non-specific immunoglobulin secretion of spleen cells treated with bupleuran 2IIc was increased according to the culture time, and coexistence of interleukin-6 (IL-6) enhanced the secretion more than that of bupleuran 2IIc alone. These results suggest that bupleuran 2IIc proliferates B cells in the absence of macrophages, and the resulting activated B cells are then induced into antibody-forming cells in the presence of IL-6. Among the structural region of bupleuran 2IIc, ramified region (PG-1), which consists of rhamnogalacturonan core rich in neutral sugar chain, showed the potent mitogenic activity suggesting it to be an active site. Mitogenic activity of bupleuran 2IIc was reduced in the presence of antipolysaccharide antibody (antibupleuran 2IIc/PG-1-IgG), which recognizes the ramified region of bupleuran 2IIc as the antigenic epitope. Mitogenic activity of bupleuran 2IIc was also reduced by the addition of beta-d-GlcpA-(1-->6)-beta-d-Galp-(1-->6)-d-Galp or beta-d-GlcpA-(1-->6)-d-Galp, which are a part of the epitopes of antibupleuran 2IIc/PG-1-IgG. These results suggest that the epitopes in bupleuran 2IIc act as active sites of the polysaccharide during mitogenic activity.

Figure 1

Effect of BR-2 on mitogenic activity of spleen cells. C3H/HeJ female mice were orally given BR-2 (250 mg/kg) for 7 days. Resulting spleen cells (5 × 10⁵ cells/200 μl/96-well plate) isolated from C3H/HeJ mice were cultured with various concentrations of water, LPS or bupleuran 2IIc on spleen cells for 3 days and the proliferative responses of cells were assessed by Alamar Blue™ reduction assay.

Figure 2

Mitogenic activity of bupleuran 2IIb and 2IIc on spleen cells (a) and Peyer’s patch cells (b). Cells (5 × 10⁵ cells/200 μl/96-well plate) isolated from C3H/HeJ mice were cultured with different concentrations (10 μg/ml, 100 μg/ml) of polysaccharides for 3 days. The proliferative responses of cells were assessed by Alamar Blue™ reduction assay.

Figure 3

Flow cytometric analysis of bupleuran 2IIc-induced proliferation of spleen cells. Spleen cells (1 × 10⁶ cells/ml) from C3H/HeJ mice were cultured with bupleuran 2IIc (50 μg/ml) for 3 days. The proliferated cells were incorporated BrdU for last 1 day of the cultivation. Cells were stained with FITC-labelled anti-BrdU, PE-labelled antimouse B220 and PE-labelled antimouse Thy-1.2. (a) PE-labelled antimouse B220; (b) PE-labelled antimouse Thy-1.2.

Figure 4

Effects of macrophage and T cells on bupleuran 2IIc induced proliferation of spleen cells. Spleen cells (1 × 10⁶ cells/ml) from C3H/HeJ mice were cultured with and without bupleuran 2IIc (50 μg/ml) for 3 days. The proliferative responses of cells were assessed by Alamar Blue™ reduction assay. (a) whole spleen cells, (b) macrophage-depleted cells, (c) T-cell-depleted cells.

Figure 5

Flow cytometric analysis of bupleuran 2IIc-induced differentiation of B cells. Spleen cells (1 × 10⁶ cells/ml) from C3H/HeJ mice were cultured with bupleuran 2IIc (50 μg/ml) for 3 days. Cells were stained with PE-labelled antimouse B220, FITC-labelled antimouse CD25 (a, b) and FITC-labelled antimouse sIgM (c, d). (a) and (c) control, (b) and (d) bupleuran 2IIc-treated.

Figure 6

Effect of bupleuran 2IIc on antibody production. Spleen cells (1 × 10⁶ cells/ml) from C3H/HeJ mice were cultured with bupleuran 2IIc (• 50 μg/ml), IL-6 (▵ 10 unit), bupleuran 2IIc+IL-6 (▴) or water (○) for 3 days. IgM and IgG in the culture supernatants were measured by ELISA. (a) IgM, (b) IgG.

Figure 7

Mitogenic activity of the digestion products of bupleuran 2IIc by endo-β-(1→4)-polygalacturonase treatment on spleen cells. Cells (5 × 10⁵ cells/200 μl/96-well plate) isolated from C3H/HeJ mice were cultured with various concentrations of polysaccharides for 3 days. The proliferative responses of cells were assessed by Alamar Blue™ reduction assay.

Figure 8

Proposed structure of the antigenic epitopes in the ramified region of bupleuran 2IIc for antibupleuran 2IIc/PG-1-IgG.

Figure 9

The effect of antibupleuran 2IIc/PG-1-IgG on mitogenic activity. Spleen cells (1 × 10⁶ cells/ml) from C3H/HeJ mice were cultured with (a) several concentrations of PG-1which was preincubated with and without antibupleuran 2IIc/PG-1-IgG (10 μg/well) (b) (i) control (ii) bupleuran 2IIc (50 μg/ml) (iii) bupleuran 2IIc (50 μg/ml) + non-immune-rabbit-F(ab′)₂ (10 μg/well) (iv) bupleuran 2IIc (50 μg/ml)+antibupleuran 2IIc/PG-1-F(ab′)₂ (10 μg/well). After incubation for 3 days, cell growth was measured by means of Alamar Blue™ reduction assay.