Effect of the Gc-derived Macrophage-activating Factor Precursor (preGcMAF) on Phagocytic Activation of Mouse Peritoneal Macrophages

Materials and Methods

Materials. Lipopolysaccharide (LPS; from Escherichia coli 0128:B12) was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). 25-Hydroxyvitamin D₃ (25(OH)D₃) and other chemicals of biochemical grade were purchased from WAKO Pure Chemical Industries Co. Ltd. (Osaka, Japan).

Preparation of human serum Gc_1f1f protein. The Gc_1f1f protein was prepared as reported by Link et al. (20). Briefly, healthy human serum (containing Gc_1f1f, 3.5 ml) was diluted 1:1 with a column buffer (50 mM Tris-HCl, 1.5 mM EDTA, 150 mM NaCl, 0.1% Triton X-100; pH 7.4) and applied to a 25(OH)D₃-Sepharose column, which was prepared according to the method reported by Link et al. (20). The column was washed with 300 ml of column buffer. The protein remaining on the matrix was eluted with 6 M guanidine-HCl, and 1 ml fractions were collected. Fractions with the protein peak of the guanidine-eluted fraction were pooled and dialyzed with 10 mM sodium phosphate (pH 7.0). A hydroxyapatite column with a volume of 5 ml (Bio-Scale Mini CHT Type II Cartridge; Bio-Rad Laboratories, Tokyo, Japan) was equilibrated in 10 mM sodium phosphate (pH 7.0). The dialyzed sample obtained from 25(OH)D₃-Sepharose column chromatography was then applied to the column. A linear gradient elution from 10 mM sodium phosphate to 200 mM sodium phosphate (pH 7.0) was used for column chromatography. Fractions were collected and concentrated with centricon (30,000 MVCO; Nihon Millipore Co., Tokyo, Japan), and their protein concentrations were determined using the bicinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL, USA). Bovine serum albumin (BSA) was used as a standard. The amount of purified Gc_1f1f protein was 743 μg.

Western blotting. The Gc_1f1f protein and preGc_1f1fMAF were subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and subsequently to electroblotting onto a polyvinylidene fluoride (PVDF) 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. In Western blotting, the blots were probed with anti-human Gc globulin (DakoCytomation, Glostrup, Denmark) for the Gc_1f1f protein, and with PNA lectin (Arachis hypogaea, biotin-conjugated; Sigma-Aldrich Japan Co.) and HPA lectin (Helix pomatia, biotin-conjugated; Sigma-Aldrich Japan Co.) for preGc_1f1fMAF. After extensive washing, the blots were incubated with secondary antibody (horseradish peroxidase (HRP)-labeled anti-rabbit IgG and electrochemiluminescence (ECL) streptavidin-HRP conjugate; GE Healthcare Japan Co., Tokyo, Japan). The blots were developed using an ECL Western blotting detection system (GE Healthcare Japan Co.).

Preparation of preGc_1f1fMAF. The purified Gc_1f1f protein (25 μg) was incubated with 250 mU of β-galactosidase (Grade III from bovine liver; Sigma-Aldrich) in 100 mM sodium phosphate buffer (pH 7.0) in an Eppendorf tube at 37.5°C for 1 hour. The reaction mixture was mixed with 25(OH)D₃-Sepharose beads and was subjected to stirring at 4°C using a vortex mixer for 60 minutes to separate the β-galactosidase. The solution was concentrated by microcon (10,000 MVCO; Nihon Millipore Co.). The protein concentrations were determined using the BCA method, and the amount of preGc_1f1fMAF was 17 μg.

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Figure 1.

SDS-PAGE and Western blot analysis of preGc_1f1fMAF. A: SDS-PAGE with CBB staining and a Western blot with anti-human Gc globulin of purified Gc_1f1f protein. B: Western blot of preGc_1f1fMAF using PNA lectin (affinity for Gal-GalNAc disaccharide) and HPA lectin (affinity for GalNAc moiety).

Isolation and culture of mouse peritoneal macrophages. Resident mouse peritoneal macrophages were collected from 8-week-old female Institute for Cancer Research (ICR) mice. After centrifugation at 1,000×rpm at 4°C for 15 minutes, the collected macrophages were cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free RPMI-1640 for 1 hour. The cultured cells were then washed 3 times with serum-free RPMI-1640 to separate adherent macrophages from non-adherent cells such as T- and B-cells. The macrophages were cultured in serum-free RPMI-1640 for 15 hours, and then the phagocytosis assay was performed as described below.

Phagocytosis assay. The macrophages were layered onto coverslips in a 24-well plate, and then treated with Gc_1f1fMAF, preGc_1f1fMAF, Gc_1f1fprotein, LSP, peritoneal fluid, or peritoneal fluid treated preGc_1f1fMAF (pre-incubated with peritoneal fluid at 37°C for 1 hour) for 3 hours. Sheep red blood cells (SRBCs) 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 on each macrophage-coated coverslip and were cultured for 1.5 hours. The non-internalized erythrocytes were lysed by immersing the coverslip in a hypotonic solution (1/5 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; 250 macrophages were counted for each data point. The data are expressed in terms of the phagocytic index, which is defined as the percentage of macrophages with ingested erythrocytes multiplied by the average number of erythrocytes ingested per macrophage.

Statistical analysis. Data are expressed as the mean (±SD). The significance in the differences between the results of the independent experiments was analyzed using Student's t-test. A p-value of <0.05 was considered to be significant.

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Figure 2.

Phagocytic activity of mouse peritoneal macrophages observed using preGc_1f1fMAF treated or not treated with mouse peritoneal fluid. All experiments were performed in triplicate. Each bar represents the mean (±SD). **p<0.01.