Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production

Materials and Methods

Preparation of degalactosylated/desialylated bovine colostrum and GcMAF-containing human serum. Bovine colostrum was obtained from Jun Sei Co. Ltd. (Tokyo, Japan). One milligram of bovine colostrum powder was dissolved in 1 ml of 50 mM sodium phosphate buffer (pH 7.0) and incubated with 65 mU of β-D-galactosidase (from Escherichia coli; WAKO Pure Chemical Industries, Ltd., Osaka, Japan) either with or without 65 mU of neuraminidase (sialidase from Clostridium perfringens; Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 1 h. The reaction mixture was then heated at 60°C for 10 min to deactivate the enzymes. The protein concentrations were determined using a Pierce® BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). GcMAF-containing human serum was prepared as previously reported by Kuchiike et al. (11).

SDS-PAGE and western blotting. Degalactosylated/desialylated bovine colostrum was subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE; XV PANTERA GEL MP, 7.5-15%; DRC Co., Ltd., Tokyo, Japan) and, subsequently, electroblotted onto a nitrocellulose membrane. Non-specific binding was blocked by overnight incubation in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and 1% BSA at 4°C. The membranes were then probed with biotin-conjugated Helix pomatia agglutinin (HPA) lectin (Sigma-Aldrich) specific for GalNAc moiety. After membrane washing, the blots were incubated with horseradish peroxidase (HRP)-labeled streptavidin (GE Healthcare Life Sciences, Uppsala, Sweden) as a secondary antibody. The blots were developed using an ECL western blotting detection system (GE Healthcare). The visualization and quantification of the Western blot bands were achieved using an ECL Western blotting detection system (GE Healthcare), a LumiCube chemiluminescence analyzer and JustTLC image analysis software (Liponics, Tokyo, Japan).

In vitro phagocytosis assay. Mouse peritoneal adherent cells containing macrophages were collected from 8-week-old female ICR mice (Japan SLC, Hamamatsu, Japan), as previously reported by Uto et al. (13), and cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free RPMI 1640 (Life Technologies, Carlsbad, CA, USA) for 1 h. The cultured cells were then washed three times with serum-free RPMI 1640 to separate adherent macrophages from non-adherent cells, such as T and B cells. Mouse peritoneal macrophages were layered onto coverslips in a 24-well plate and cultured for 15 h. After 3 h of degalactosylated/desialylated bovine colostrum treatment, the cultures were assayed for phagocytic activity. Sheep red blood cells (SRBCs; Nippon Bio-Supp. Center, Tokyo, Japan) were opsonized by rabbit hemolytic serum (anti-sheep red blood cells, Cosmo Bio Co., Tokyo, Japan). Opsonized SRBCs (0.5%) in serum-free RPMI 1640 were overlaid onto each macrophage-coated coverslip and cultured for 1.5 h. The non-internalized erythrocytes were lysed by immersing the coverslip into a hypotonic solution (1/5-diluted phosphate-buffered saline). The macrophages were fixed with methanol, air-dried and stained with Giemsa stain. The number of phagocytosed erythrocytes per cell was determined microscopically; in total, 250 macrophages were counted for each data point. The data were expressed in terms of the phagocytosis index (PI), which was defined as the percentage of macrophages with ingested erythrocytes multiplied by the mean number of erythrocytes ingested per macrophage.

In vivo phagocytosis assay. Seven-week-old female C57BL/6 mice under anesthesia were injected with 300 μl of degalactosylated/desialylated bovine colostrum directly into the small intestine. After 1 h, 300 μl of AF488-labeled ovalbumin (OVA) protein (Invitrogen, Tokyo, Japan) was injected into the small intestine. After 1 h, mice were sacrificed and the small intestine was extracted. Fat and a Peyer's patch were removed, washed by PBS and, then, stirred at 37°C in 20 ml of FACS buffer for 20 min. Collagenase (Roche Diagnostics K.K., Tokyo, Japan) was also added and then cut the small intestine finely. After stirring at 37°C for 1 h, EDTA was added and stirred for an additional 5-min period. The supernatant was filtered with a cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and the residue was suspended in 10 ml of FACS buffer. The supernatant was removed by centrifugation. The cell pellet was suspended in 10 ml of 40% percoll (GE Healthcare), followed by addition of 5 ml of 75% percoll to the bottom and centrifugation. After the supernatant was removed, the residue was suspended in 9 ml of FACS buffer and then centrifuged again. After the supernatant was removed, anti-mouse F4/80, anti-mouse/human CD11b and anti-mouse/human CD16/32 (BioLegend, Inc., San Diego, CA, USA) antibodies were added and reacted for 15 min. After the supernatant was removed, wash buffer was added and the mixture subjected to a final centrifugation. 7-Aminoactinomycin D (Sigma-Aldrich) was added and analyzed by FACSCantoII (Becton, Dickinson and Company).

Flow cytometry assay. Mouse peritoneal macrophages were cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free RPMI 1640 for 15 h. The cultured cells were washed two times with serum-free RPMI 1640 and then treated with degalactosylated/desialylated bovine colostrum for 24 h. The supernatant (50 μl) was added to a mixture of capture bead diluent (48 μl), mouse IL-1β capture bead E5 (1 μl) and mouse TNF-α capture bead C8 (1 μl) (Becton, Dickinson and Company). After the solution containing beads was incubated at room temperature for 1 h, the mixture of capture bead diluent (48 μl), mouse IL-1β PE detection reagent (1 μl) and mouse TNF-α PE detection reagent (1 μl) were added. The solution containing beads was incubated at room temperature for 1 h, the beads were washed with 1 ml of wash buffer (Becton, Dickinson and Company) and centrifuged at 200 × g for 5 min. The supernatant was removed and then 300 μl of wash buffer was added and analyzed using FACSVerse.

Statistical analysis. Data are expressed as mean and standard deviation. The statistical significance of the differences between the results of the independent experiments was analyzed using the Student's t-test. A p-value of <0.05 was considered statistically significant.

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Figure 1.

SDS-PAGE of degalactosylated and degalactosylated/desialylated bovine colostrum. (A) CBB-stain and (B) western blots probed with anti-human Gc globulin and Helix pomatia agglutinin (HPA) lectin. M, Marker; lane 1, bovine colostrum; lane 2, degalactosylated bovine colostrum; lane 3, degalactosylated/ desialylated bovine colostrum.

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Figure 2.

In vitro phagocytic activity of mouse peritoneal macrophages observed using degalactosylated/desialylated bovine colostrum and GcMAF-containing human serum. C, Control; 1, 1 μg of LPS; 2, 10 ng of non-treated bovine colostrum; 3, 10 ng of degalactosylated bovine colostrum; 4, 10 ng of degalactosylated/desialylated bovine colostrum. All experiments were performed in triplicate. Each error bar represents the standard deviation. The number on each bar indicates the mean value. *p<0.05.

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Figure 3.

In vivo phagocytic activity of mouse intestinal macrophages observed using degalactosylated/desialylated bovine colostrum and GcMAF-containing human serum. The macrophage cells gated by expression of F4/80 and CD11b. Q2 area express the phagocytic macrophage. (A) 1 mg/kg of LPS; (B) 1 mg/kg of bovine colostrum; (C) 1 mg/kg of degalactosylated/desialylated bovine colostrum; (D) 1 mg/kg of GcMAF-containing human serum.

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Figure 4.

Induction of inflammatory cytokines by mouse peritoneal macrophage treated with degalactosylated/desialylated bovine colostrum or GcMAF-containing human serum. (A) IL-1β and (B) TNF-α. C, Control; 1, 1 μg of LPS and 10 ng of interferon-γ; 2, 10 ng of non-treated bovine colostrum; 3, 10 ng of degalactosylated bovine colostrum; 4, 10 ng of degalactosylated/desialylated bovine colostrum; 5, 10 ng of non-treated human serum; 6, 10 ng of degalactosylated/desialylated human serum. All experiments were performed in triplicate. Each error bar represents the standard deviation. The number on each bar indicates mean value. ***p<0.005.