Concentration-dependent Activation of Inflammatory/Anti-inflammatory Functions of Macrophages by Hydrolyzed Whey Protein

Materials and Methods

Preparation of hydrolyzed whey protein. Three grams of whey protein (ProDiet 80S; Ingredia, France) were dissolved in 100 ml of 100 mM sodium phosphate buffer (SPB) (pH 7.0) and stirred for about 20 min to prepare a 3% whey protein solution. The mixture was centrifuged at 9,000 ×g at 4°C for 1 h, and the supernatant was collected. The collected supernatant was dialyzed in SnakeSkin Dialysis Tubing 3.5K (Thermo Fisher Scientific K.K., Tokyo, Japan) overnight. The dialyzed solution was filtered through a 0.2 μm filter (Advantec, Tokyo, Japan). Each protease (1.35 mg), namely, P Amano 3SD, A Amano SD, Proteax, or Protin NY 10, (Amano Enzyme Inc., Nagoya, Japan) was added to the whey protein (27 mg) and allowed to react at 37°C for 1 h (P Amano 3SD) or 50°C for 1 h, and the enzyme was inactivated by heat treatment at 60°C for 10 min (P Amano 3SD) or at 80°C for 5 min.

SDS-PAGE and western blotting. Hydrolyzed whey protein was subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE; XV PANTERA GEL MP, 7.5-15% or 15-25%; DRC Co., Ltd., Tokyo, Japan), and subsequently, electroblotted onto a nitrocellulose membrane. Non-specific binding was blocked by overnight incubation at 4°C in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and 1% BSA. The membranes were then probed with Wisteria floribunda lectin (WFA) Biotin Conjugate (Vector Laboratories Inc., Burlingame, CA, USA), specific for the GalNAc moiety. After washing of the membrane, the blots were incubated with enhanced chemiluminescence (ECL) streptavidin-horseradish peroxidase (HRP) conjugate (GE Healthcare Life Sciences, Uppsala, Sweden) The blots were visualized with a LumiCube chemiluminescence analyzer (Liponics, Tokyo, Japan).

Gel permeation chromatography (GPC) analysis. GPC analysis was performed using a Shimadzu-LC20AD system (Shimadzu Corporation, Kyoto, Japan), equipped with a SPD-M20A detector and CTO-20AC column oven. Chromatographic separation was achieved using a TSKgel G3000SWXL column (300×7.8 mm i.d.; 5 μm) (Tosoh Corporation, Tokyo, Japan). GPC assay was performed using an isocratic system with a flow rate of 1 ml/min, a column temperature of 25°C, and 50 mM phosphate buffer (pH 6.8) with 150 mM sodium chloride as the mobile phase. Ultraviolet (UV) detection was performed at 260 nm. The injection volume was 100 μl. The total run time was 20 min for each injection. Data were acquired and processed with LabSolution LC/GPC (Shimadzu Corporation, Kyoto, Japan).

In vitro phagocytosis assay. Mouse peritoneal adherent cells, including macrophages, were collected from 8-week-old female ICR mice (Japan SLC, Hamamatsu, Japan), as previously reported by Uto et al. (5), and cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free roswell park memorial institute medium (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 a 3-h treatment with hydrolyzed whey protein, 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 in 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 observed under a microscope; in total, 250 macrophages were counted for each data point. Data were expressed as the phagocytosis index (PI), which was defined as the percentage of macrophages with ingested erythrocytes multiplied by the average number of erythrocytes ingested per macrophage.

Enzyme-linked immunosorbent assay (ELISA) assay for cytokine production. RAW 264.7 cells (RIKEN BRC, Ibaragi, Japan) were cultured in Dulbecco's modified eagle's medium (DMEM) for 18 h and then washed with 1 × PBS. One milliliter of DMEM was added as the control; 1 μg of lipopolysaccharide (LPS; Sigma-Aldrich Co., St. Louis, MO, USA) and 10 ng of mouse IFN-γ recombinant protein (Thermo Fisher Scientific K.K.) was added as the positive control, or LPS (10 μg) was used. Untreated whey protein or various protease-treated whey protein 100-1000 ng (1-100 μg) was added to each sample and incubated at 37°C for 24 h, and the culture supernatant was analyzed using a TNF-α mouse uncoated ELISA kit and an interleukin 10 (IL-10) mouse uncoated ELISA kit (Thermo Fisher Scientific K.K.), according to the instructions for use. To investigate the effect of the protease-treated whey protein on inflammation-induced macrophages, LPS (1 μg) and IFN-γ (10 ng) were added simultaneously with the protease-treated whey protein. Curcumin (20 mM) served as the positive control.

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Figure 2.

GPC of protease-treated whey protein. (A) Whey protein, (B) Proteax, (C) P Amano 3SD, (D) Protin NY 10, and (E) A Amano SD.

Statistical analysis. Data are expressed as the mean with standard deviations of at least three independent experiments. The statistical significance of the differences between the results was analyzed using Student's t-test. p<0.05 was considered statistically significant.