Enhancement of umbilical cord blood cell hematopoiesis by maitake beta-glucan is mediated by granulocyte colony-stimulating factor production

Abstract

Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 microg/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33+ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF.

FIG. 1.

Effect of MBG on CFU-GM responses of umbilical cord blood cells. Cord blood cells from eight healthy infants were prepared by ammonium chloride lysis of red cells, treated with MBG at different doses, as indicated, and then cultured in methylcellulose medium for 14 days. Cultures without MBG were used as controls. Data are presented as the mean percentage of colony counts ± SD scored in control cultures without MBG. *, P < 0.05 versus the values for the controls; **, P < 0.001 versus the values for the controls.

FIG. 2.

Protective effect of MBG on CFU-GM formation of cord blood cells in the presence of DOX. Cord blood cells were prepared as described in the legend to Fig. 1, treated with serial doses of DOX in the absence or the presence of MBG (100 μg/ml), and cultured in methylcellulose medium for 14 days. (A) Data are presented as the mean percentage of colony counts ± SD scored in control dishes (which did not receive drug). Data represent the combined results of four separate experiments. *, P < 0.05 versus the data for DOX alone. (B) Data are presented as the mean percentage of colony counts ± SD scored for dishes with 100 μg/ml MBG alone with no DOX. *, P < 0.05 versus the results for MBG alone.

FIG. 3.

Effect of MBG on CFU-GM formation in the absence of rhG-CSF. Cord blood cells were prepared as described in the legend to Fig. 1, treated with MBG at different doses, as indicated, and then cultured in the absence of rhG-CSF in methylcellulose medium for 14 days. The results were compared to those for control cultures without MBG but with rhG-CSF at 500 ng/ml and rhIL-3 at 10 ng/ml. Data show the mean percentage of colony counts ± SD scored for control dishes (in the presence of both rhG-CSF and rhIL-3). Data represent the combined results for four different umbilical cord blood samples.

FIG. 4.

MBG induction of intracellular G-CSF in CD33⁺ cord blood cells. Umbilical cord blood was incubated with MBG at 100 μg/ml or LPS at 1 μg/ml for 4 h. Intracellular cytokine assays were carried out as described in Materials and Methods with anti-CD33 FITC for cell surface staining and anti-G-CSF monoclonal antibody to detect intracellular G-CSF. Data present the combined results for umbilical cord blood samples from five healthy full-term infants. *, P < 0.05 versus the values for the controls with no stimuli.

FIG. 5.

MBG stimulates intracellular G-CSF production in CD33⁺ cells of CB but not adult peripheral blood. Umbilical cord blood or peripheral blood samples were incubated with MBG at 100 μg/ml or LPS at 1 μg/ml for 18 h. Then the intracellular cytokine assay was carried out as described in Materials and Methods by using anti-CD33 FITC for cell surface staining. After the cells were treated with permeabilization solution, anti-G-CSF antibodies were added to detect the intracellular production of G-CSF. Data show the means ± SDs of the combined results for nine cord blood samples from healthy full-term infants and six peripheral blood samples from healthy adult donors. *, P = 0.006 for the values for cord blood for the MBG group versus the values for the controls; *, P = 0.005 for the values for cord blood for the LPS group versus the values for the controls; P = 0.137 for the values for cord blood for the MBG group versus the values for the LPS group; P = 0.44 for the values for adult peripheral blood for the MBG group versus the values for the controls; *, P = 0.04 for the values for adult peripheral blood for the LPS group versus the values for the controls. unstim, unstimulated; NC, normal control peripheral blood.

FIG. 6.

MBG induces secretion of G-CSF by cord blood cells. Umbilical cord blood or peripheral blood was incubated with MBG at 100 μg/ml or LPS at 1 μg/ml for 18 h, and the supernatants were collected. Secretion of G-CSF in the supernatant was detected by a high-sensitivity human G-CSF ELISA. Data show the means ± SDs of the combined results for cord blood from seven healthy infants or adult peripheral blood from five healthy donors. *, P = 0.02 for the values for cord blood for the MBG group versus the values for the controls with no stimulus; **, P < 0.001 for the values for cord blood for the LPS group versus the values for the controls; P = 0.11 for the values for cord blood for the MBG group versus the values for the LPS group; P = 0.791 for the values for adult peripheral blood for the MBG group versus the values for the controls; **, P < 0.001 for the values for adult peripheral blood for the LPS group versus the values for the controls; **, P < 0.001 for the values for adult peripheral blood for the MBG group versus the values for the LPS group. unstim, unstimulated; NC, normal control peripheral blood.