Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum

Abstract

1 The polysaccharide from Ganoderma lucidum (PS-G) has been reported to enhance immune responses and to elicit antitumor effects. In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis when cultured in vitro. Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS-G on these two cell functions, and the molecular events leading to these actions. 2 Using latex beads and heat-inactive Escherichia coli serving as particles for neutrophil engulfment, we found that PS-G is able to enhance phagocytic activity of human primary neutrophils and neutrophilic-phenotype cells differentiated from all trans retinoic acid-treated HL-60 cells. 3 Chemotactic assay using Boyden chamber also revealed the ability of PS-G to increase neutrophil migration. 4 Exposure of neutrophils to PS-G time dependently caused increases in protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), Hck, and Lyn activities. 5 Results with specific kinase inhibitors indicate that phagocytic action of PS-G was reduced by the presence of wortmannin (Phosphatidylinositol 3-kinase, PI3K inhibitor), pyrazolpyrimidine 2 (Src-family tyrosine kinase inhibitor), Ro318220 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), but not by PD98059 (mitogen-activated protein/ERK kinase inhibitor). Moreover, chemotactic action of PS-G requires the activities of PI3K, p38 MAPK, Src tyrosine kinases and PKC. 6 All these results demonstrate the abilities of PS-G to enhance neutrophil function in phagocytosis and chemotaxis, and further provide evidence to strengthen the beneficial remedy of G. lucidum in human to enhance defense system.

Figure 1

Effects of PS-G on phagocytosis of latex beads in neutrophils. Isolated neutrophils were preincubated with vehicle, PS-G (at indicated concentrations), GM-CSF (10 ng ml⁻¹), or zymosan (100 μg ml⁻¹) for 30 min. Following incubation, cells were challenged with latex particles and stood at 37°C for different time periods. Phagocytosis of latex particles by neutrophils was then analyzed using flow cytometry. The data in (a) indicate the time-dependent phagocytotic ability of neutrophils. The mean fluorescent intensity after drug treatment was compared with control and represents the phagocytosis index (b). Each column represents the mean±s.e.m. of at least three independent experiments. ^*P<0.05 as compared to the control group without PS-G, GM-CSF, or zymosan treatment.

Figure 2

Effects of PS-G on phagocytosis of heat-inactivated E. coli particle in neutrophils. Isolated neutrophils were preincubated with vehicle, PS-G (at indicated concentrations), GM-CSF (10 ng ml⁻¹), or zymosan (100 μg ml⁻¹) for 30 min. Following incubation, cells were challenged with E. coli particles and stood at 37°C for another 15 min (a) or different time periods (b). Phagocytosis of E. coli particles by neutrophils was then examined using flow cytometry. Each column represents the mean±s.e.m. of at least three independent experiments. ^*P<0.05 as compared to the control group without PS-G, GM-CSF, or zymosan treatment.

Figure 3

Assessment of HL-60 cells differentiation and phagocytosis. HL-60 cells were incubated with vehicle or different inducers of differentiation (1 μM ATRA, 200 μM dBcAMP, and 1.25% DMSO) for 96 h, except otherwise indicated. (a) Cells were then incubated with antibody against CD11b antigen and counted by flow cytometry. Shaded areas in top panel represented undifferentiated HL-60 cells. Bottom panel-showed the level of CD11b expression in HL-60 cells, incubated with or without ATRA for 1–4 days. (b) HL-60 cells by 4-day induction culture with ATRA or not was examined for phagocytic ability, using latex beads for engulfment. (c) ATRA-differentiated HL-60 cells were preincubated with vehicle, PS-G (100 μg ml⁻¹), GM-CSF (10 ng ml⁻¹), or zymosan (100 μg ml⁻¹) for 30 min. Following incubation, cells were treated with latex particles and stood at 37°C for different time periods. Phagocytosis of latex beads was then analyzed using flow cytometry. Each column represents the mean±s.e.m. of at least three independent experiments. ^*P<0.05 as compared to the control group without stimulant treatment.

Figure 4

Effects of inhibitors on enhanced phagocytic functions of PS-G, GM-CSF, and zymosan in neutrophils. Neutrophils were preincubated with vehicle, 30–300 nMwortmannin, 1–10 μM SB203580, 30 μM PD98059, 10 μM Ro318220, 10 μM Bis X, or 100 nM PP2 for 30 min and then incubated with GM-CSF (10 ng ml⁻¹), PS-G (100 μg ml⁻¹), and/or zymosan (100 μg ml⁻¹) for an additional 30 min. Following incubation, cells were challenged with latex beads (a, b) or E. coli particles (c) and stood at 37°C for another 15 min. Phagocytosis was then determined using FACScan. ^*P<0.05 as compared to the enhanced phagocytic effect of PS-G, GM-CSF, or zymosan alone.

Figure 5

Effects of PS-G on PKC, Src-family tyrosine kinases and p38 MAPK activities in neutrophils. (a) Cells following incubation with PS-G (100 μg ml⁻¹) for different time periods as indicated were harvested. PKC activity was then determined using PKC activity assay kit, according to the manufacturer's instruction. Each column represents the mean±s.e.m. of at least three independent experiments. Hck or Lyn tyrosine kinases activity (b) or p38 MAPK activity (c) of cells treated with PS-G for different time periods was assessed by immune complex kinase assays (KA) using enolase or MBP as substrates as described in ‘Methods.' After SDS–PAGE, ³²P-labeled enolase or MBP was visualized by autoradiography and normalized for the amount of immunoprecipitated Hck, Lyn and p38 MAPK by immunoblotting (IB) with specific antibodies. Results are representative of three independent experiments.

Figure 6

Effects of PS-G on neutrophil migration. Neutrophils were preincubated with vehicle or inhibitors (100 or 300 nM wortmannin, 100 nM PP2, 10 μM Ro318220, 30 μM PD98059, or 1–10 μM SB203580) for 30 min, and then allowed to migrate for 30 min toward fMLP (100 ng ml⁻¹)- or PS-G (100 μg ml⁻¹)-containing medium as indicated. Neutrophils that had migrated through the membrane were stained with phalloidin–FITC and shown under fluorescence microscopy (a). In (b), migrated neutrophils were stained with crystal violet and data were expressed as the absorbance at 550 nm. ^*P<0.05 as compared to the control group with PS-G treatment alone.