Vol. 39, No. 2 199Biol. Pharm. Bull. 39, 199–206 (2016)

© 2016 The Pharmaceutical Society of Japan

Regular Article

Effects of Glycated Whey Protein Concentrate on Pro-inflammatory Cytokine Expression and Phagocytic Activity in RAW264.7 MacrophagesSu-Hyun Chun,a Hyun Ah Lee,a Keon Bong Lee,b Sae Hun Kim,a Kun-Young Park,c and Kwang-Won Lee*,a

a Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University; Seoul 136–713, Republic of Korea: b R&D Center, Seoul Dairy Cooperative; Ansan, Gyeonggi-do 425–839, Republic of Korea: and c Department of Food Science and Nutrition, Pusan National University; Busan 609–735, Republic of Korea.Received July 30, 2015; accepted November 6, 2015

The aim of this study was to determine the stimulatory effects of Maillard reaction, a non-enzymatic browning reaction on the expression of pro-inflammatory cytokines and phagocytic activity induced by whey protein concentrate (WPC). Glycated WPC (G-WPC) was prepared by a reaction between WPC and the lactose it contained. The fluorescence intensity of G-WPC dramatically increased after one day, and high molecular weight complexes formed via the Maillard reaction were also observed in the sodium dodecyl sul-fate-polyacrylamide gel electrophoresis profiles. G-WPC demonstrated immunomodulatory effects, including stimulation of increased nitric oxide production and cytokine expressions (i.e., tumor necrosis factor-α, in-terleukin (IL)-1β, and IL-6), compared to WPC. Furthermore, the phagocytic activity of RAW264.7 cells was significantly increased upon treatment with G-WPC, compared to WPC. Therefore, we suggest that G-WPC can be utilized as an improved dietary source for providing immune modulating activity.

Key words Maillard reaction; phagocytosis; RAW264.7 cell; immune enhancing activity; glycated whey protein concentrate

The immune system can largely be separated into innate and acquired immunities. Innate immunity is a defense mech-anism that evolved from unicellular organisms into multicellu-lar organisms. It involves the induction of immune responses through recognition of pathogen-associated molecular patterns or other complexes by host cells,1,2) to defend against the pathogens as well as for modulating acquired immunity.3) As pathogens invade the body, phagocytosis is activated, involv-ing neutrophils, monocytes and macrophages. Macrophages play a role as the first line of host defense systems, secreting nitric oxide (NO) and cytokines such as tumor necrosis fac-tor (TNF)-α, interleukin (IL)-1β and IL-6 upon activation.4) Several functions of NO have been reported, including vasodi-latation, neurotransmission, inhibition of platelet aggregation, transmission of inflammatory reactions and direct destruction of invading microbes and cancers.5)

With increase of the global cheese production, enormous amounts of whey protein are being produced.6) Occupying about 20% of the milk proteins, whey protein is composed of approximately 20% α-lactalbumin (α-LA), 53% β-lactoglobulin (β-LG), 7% bovine serum albumin (BSA), 13% immunoglobu-lins, 3% lactoferrin and 4% other proteins.7) Whey protein concentrate (WPC) is produced by condensing whey proteins via ultrafiltration or reverse osmosis.8) It was also reported that WPC improved the oxidative damage caused by alcohol,9) and exhibited anti-cancer10) and immunomodulatory effects.11) The major protein in WPC, β-LG has functional and nutri-tional characteristics that allow it to be used as a raw material in a variety of foods, and is known as a precursor of bioactive peptides which possess antioxidant, immunomodulatory, and cholesterol-lowering effects.12–14)

The Maillard reaction (also called glycation reaction) is a non-enzymatic browning reaction between a reducing sugar and proteins, during which Maillard reaction products (MRPs)

are formed.15) Several studies have already reported the ben-eficial effects of MRPs, such as antioxidant and anticancer activities.16–18) Recently, a few studies reported that MRPs are also associated with immunomodulatory effects, such as the improvement of immunogenicity of food allergens on T-cells via Maillard reaction,19,20) and the immune-enhancing effects of chitosan-MRPs.21) However, there has been limited study on the effects of Maillard-conjugated whey proteins on living cells. In the present study, the immune modulating effects of glycated WPC (G-WPC) on cytokine release and phagocytosis by RAW264.7 macrophage cells was investigated.

MATERIALS AND METHODS

Materials The sources of purchased materials were as follows: Two different lots of WPC samples were from DAVISCO Food International (Le Sueur, MN, U.S.A.), and WPC samples from the lots showed the similar results (Supplementary Fig. 1); 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), o-phtaldialdehyde (OPA), Nε-acetyl-L-lysine, dimethyl sulfoxide (DMSO), lipo-polysaccharide (LPS), N-(1-naphthyl) ethylene diamine dihy-drochloride (NEAD), and sodium nitrite were from Sigma (St. Louis, MO, U.S.A.); the GelCode® glycoprotein staining kit and Pierce® LAL Chromogenic Endotoxin Quantitation Kit were from Pierce Biotechnology (Rockford, IL, U.S.A.); Fluorescein-conjugated Escherichia coli (K-12) bioparticles (FITC-E. coli; E-2861) and Opsonizing Reagent (E-2870) were from Molecular Probe (Eugene, OR, U.S.A.); TRIzol was from Invitrogen (Carlsbad, CA, U.S.A.); LeGene 1st strand cDNA synthesis kit was from LeGene Bioscience (San Diego, CA, U.S.A.); Dream-Taq DNA polymerase was from Fermentas (Waltham, MA, U.S.A.); RAW264.7 cells (TIB-71) were from American Type Culture Collection (ATC C; Rockbille, MD,

* To whom correspondence should be addressed. e-mail: kwangwon@korea.ac.kr

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U.S.A.); High glucose Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Hyclone (Logan, UT, U.S.A.).

Sample Preparation　The chemical composition of the WPC (moisture, protein, fat, lactose and ash) was analyzed using the methods of the Korea Food Code, provided by the Ministry of Food and Drug Safety in Korea.22) Briefly, to prepare glycated WPC (G-WPC), WPC (10 g) was dissolved in 1 L of 0.1 M sodium phosphate buffer (pH 7.4), and then the solution was placed in a shaking water bath (60 rpm) at 55°C for up to 24 h under a cover to exclude light. G-WPC samples were dialyzed using dialysis tubing (MWCO 3500) against 0.1 M sodium phosphate buffer (pH 7.4) at 4°C, lyophilized, and stored at −70°C prior to further chemical and biological analysis. WPC in this study was also dialyzed, and lyophi-lized except heat treatment for sample preparation. Protein concentrations were measured by bicinchoninic acid (BCA) assay.23)

Determination of the Degree of Glycation The Maillard-like fluorescence24) of G-WPC was measured at the excitation and emission wavelengths of 370 nm and 440 nm, respec-tively, using a VICTOR3™ spectrofluorometer (PerkinElmer, Inc., Boston, MA, U.S.A.).25) Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli26) using 15% and 5% acrylamide separating and stacking gels, respectively. Samples were mixed with SDS sample buffer [25% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.025% (w/v) bro-mophenol blue and 60 mM Tris–HCl in pH 6.8], and boiled for 5 min. After electrophoresis, the gel sheets were stained with Coomassie brilliant blue G-250 for proteins, and with the GelCode® glycoprotein staining kit for carbohydrates.

Determination of Free Amino Acid Residues The amount of total free primary amino acids was determined by the modified fluorogenic OPA assay.27–29) The samples (100 µL) were mixed with 300 µL of OPA reagent [40 mg/mL OPA in methanol, 0.1 M sodium borate buffer in pH 10.0 (50 mL), 2-mercaptoethanol (0.2 mL), 20% (w/v) SDS solu-tion (1.5 mL) and 12% (w/v) SDS solution (52.7 mL)], mixed overnight at room temperature (RT, 20°C), and then incubated for 5 min at RT. Fluorescence was measured at the excitation and emission wavelengths of 360 and 460 nm, respectively, using a VICTOR3™ spectrofluorometer. A calibration curve was obtained by using Nε-acetyl-L-lysine at concentrations of 10–250 µM as a standard.

Cell Culture The murine macrophage cell line, RAW264.7 was cultured in high glucose DMEM containing 10% FBS, 100 units/mL penicillin and 100 units/mL strepto-mycin. The cells were grown in a humidified incubator con-taining 5% CO2 and 95% air at 37°C. The cells were grown in 96- (1×105 cells/well) or 12-well (9×105 cells/well) plates for 18 h and then serum-starved for 6 h.

Cell Viability Assay Cell viability was determined via the colorimetric MTT assay.30) After incubation of the RAW264.7 cells (1×105 cells/well) with samples for 24 h, MTT solution (5 mg/mL in phosphate buffered saline (PBS)) was added to each well (8 µL) with 40 µL of serum free DMEM. The plate was then incubated for an additional 3 h at 37°C, after which the formazan crystals were dissolved in 100 µL of DMSO. The optical density was determined at 540 nm by a multiplate reader (EL-808, Bio-Tek Instruments, Winooski,

VT, U.S.A.). Values of the untreated controls were set to 100% viability. The percentage of cell survival was determined by comparison with the control group.

Measurement of NO Production The concentration of NO produced by the RAW264.7 cells was determined using the colorimetric assay based on the Griess reaction.31) After treating RAW264.7 cells (1×105 cells/well) with sample for 24 h, 50 µL of the cell supernatant was mixed with 100 µL of Griess reagent [1 : 1 mixture (v/v) of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) NEAD in distilled water]. The mixture was incubated at RT for 10 min, and then measured by a multiplate reader at 540 nm. The level of NO was quantified using a standard curve generated with sodium nitrite in the range of 0 to 100 µM.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis To determine the effects of WPC and G-WPC on inducible NO synthase (iNOS) and the expres-sions of several cytokines, i.e., TNF-α, IL-1β and IL-6, the RAW264.7 cells were activated with sample treatment, and the levels of cDNA expression were compared using RT-PCR. RAW264.7 cells (9×105 cells/well) were incubated with the samples for 4 h (TNF-α, IL-1β and iNOS) and 12 h (IL-6), after which each well was washed twice with PBS. The TRIzol reagent and LeGene 1st strand cDNA synthe-sis kit were used for the extraction of total RNA and for cDNA synthesis, respectively. The reaction conditions were 65°C for 3 min, followed by quick chilling, 37°C for 50 min and 85°C for 5 min. The synthesized cDNA was ampli-fied by thermal cycler (Bio-Rad Laboratories Inc., Hercu-les, CA, U.S.A.) using Dream-Taq DNA polymerase. The forward and reverse primers for specific oligonucleotides were as follows: TNF-α, 5′-CCT GTA GCC CAC GTC GTA GC-3′ and 5′-AGC AAT GAC TCC AAA GTA GAC C-3′; IL-1β, 5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ and 5′-CAG GAC AGG TAT AGA TTC TTT CCT TT-3′; IL-6, 5′-GAT GCT ACC AAA CTG GAT ATA ATC-3′ and 5′-GGT CCT TAG CCA CTC CTT CTG TG-3′; iNOS, 5′-CTT CCG AAG TTT CTG GCA GCA GCG-3′ and 5′-GAG CCT CGT GGC TTT GGG CTC CTC-3′; β-actin, 5′-TGT GAT GGT GGG AAT GGG TCA G-3′ and 5′-TTT GAT GTC ACG CAC GAT TTC C-3′. The denaturation cycling conditions were followed by 20 cycles (β-actin), 23 cycles (TNF-α and IL-1β), 26 cycles (IL-6), or 28 cycles (iNOS) at 95°C for 30 s, and annealing at the optimum temperatures of 60°C for β-actin, 60.5°C for TNF-α, 62.5°C for IL-1β, 63.3°C for IL-6, and 64°C for iNOS. The amplified PCR products were separated electrophoretically on 1.5% agarose gels and visualized by UV illumination after staining with RedSafeTM (iNtRON Biotech. Inc., Seoul, South Korea).

Quantitative Real-Time RT-PCR (qRT-PCR) Analy-sis To quantify the mRNA expression levels of iNOS, TNF-α, IL-1β and IL-6 in RAW264.7 cells, the synthesized cDNA was mixed 2X SYBR® Green RT-PCR master mix (Invitrogen, Carlsbad, CA, U.S.A.) and amplified as follows: 95°C for 5 min, 40 cycles of 95°C for 10 s and annealing tem-perature of 56°C for iNOS and 63.6°C for TNF-α, IL-1β and IL-6. β-Actin was used a housekeeping gene. The forward and reverse primers for specific oligonucleotides were as follows: TNF-α, 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ and 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; IL-1β, 5′-CAA CCA ACA AGT GAT ATT CTC CAT-3′ and 5′-GAT CCA CAC TCT CCA GCT GCA-3′; IL-6, 5′-TCC AGT TGC CTT CTT GGG

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AC-3′ and 5′-GTG TAA TTA AGC CTC CGA CTT G-3′; iNOS, 5′-GGC AGC CTG TGA GAC CTT TG-3′ and 5′-GCA TTG GAA GTG AAG CGT TTC-3′; β-actin, 5′-AGA GGG AAA TCG TGC GTG AC-3′ and 5′-CAA TAG TGA TGA CCT GGC CGT-3′.

Phagocytosis Assay FITC-E. coli was dissolved in PBS at 20 mg/mL and then mixed with equal volumes of opsoniz-ing reagent. After incubation at 37°C for 1 h, the mixture was washed 2–3 times with PBS using centrifugation (10000 rpm for 3 min). The pellets were dissolved in PBS for use in the phagocytosis assay on RAW264.7 cells (9×105 cells/well). After incubation of the RAW264.7 cells with the samples (100 µg/mL) for 24 h, 100 µL of opsonized FITC-E. coli (5×106 colony forming unit (CFU)/well) was added to each well, and then incubated for 2 h at 37°C under a humidi-fied atmosphere of 5% CO2. Extracellular fluorescence was quenched by adding 100 µL of trypan blue. After 1 min, the cells were rinsed five times with PBS to remove non-ingested FITC-E. coli and then fluorescence was measured using a con-focal laser scanning microscope (LSM 5 Exciter, Carl Zeiss, Germany). Image quantification was performed using Image J software (National Institutes of Health, Bethesda, MD, U.S.A.). The relative phagocytic activity was calculated as the percentage of fluorescence intensity in the samples supple-mented with FITC-E. coli compared to the control with no supplementation.

Statistical Analysis Data were presented as the mean±standard deviation (S.D.). The difference between each experimental group and control was compared by Student’s t-test (* p<0.05, ** p<0.01, *** p<0.001) using SAS software version 9.4 (SAS Institute, Cary, NC, U.S.A.).

RESULTS AND DISCUSSION

Degree of Glycation　The chemical composition of the WPC is shown in Table 1. The ratio of weight between lactose and protein was approximately 1 to 10. The extent of modifi-cations in G-WPC was assessed by measuring the Maillard-like fluorescence intensity, as well as by carrying out SDS-PAGE and OPA assay. The fluorescence intensity at 370 nm (excitation) and 440 nm (emission) was increased from the initial value of 4040±260 to 63600±3200 after 24 h of glyca-tion reaction at 55°C (Fig. 1). MRPs were reported to generate fluorescent compounds at the measured wavelengths of excita-tion and emission.32)

Based on the SDS-PAGE of WPC samples (Fig. 2A), the proteins showed three bands of α-LA (14.1 kDa), β-LG (18.4 kDa) and BSA (67 kDa), and these bands are major ones in whey protein.33) It should be pointed out that the WPC used in this experiment contained lactose (7.66±0.14%; Table 1). It has been known that Maillard reactions occurs in WPC

having heating process such as spray drying.34) When α-LA (14.1 kDa), β-LG and BSA, dispersed in whey permeate con-taining lactose were heated at 75°C, large aggregates were generated.35) Also, the higher molecular weight (HMW)-fraction was observed in whey protein powder, and it was presumed that the lactosylation of the whey proteins had oc-curred during the spray drying process.36,37) The WPC used in our experiment contained lactose, and was prepared with the spray-drying process. Thus, the thermal treatment of whey protein containing lactose could contribute to the formation of a HMW-fraction through the Maillard reaction, which ap-peared at the boundary near the separating gel (Fig. 2A). The HMW-fraction was further increased in the G-WPC compared to WPC after reacting at 55°C for up to 24 h. On the other hand, as shown in Fig. 2B, the band intensity of the GelCode® glycoprotein-stained HMW-fraction contained in the WPC in-dicated that it possessed proteins with sugar moieties, whereas the G-WPC showed slightly more GelCode® glycoprotein staining, reflecting the contribution of lactose-induced modifi-cation. In addition, G-WPC showed slightly decreased intensi-ties of α-LA and β-LG bands by 5.93 and 9.44%, respectively, compared with those of WPC (Supplementary Fig. 2). In contrast, BSA level in both WPC and G-WPC did not change. This observation is consistent with the reports that SDS-PAGE of glycated whey protein samples before and after for 10 min at 80°C showed that the intensity of β-LG band diminished more than that of α-LA,38) and intensity of BSA was observed no difference with whey protein isolate heated at 70°C for increasing time.39)

Loss of the available lysine residues occurs in the early stage of the Maillard reaction in dairy products.40,41) In this study, the amount of free primary amino acid residues during glycation was measured by OPA assay. After a sharp drop of the residues in WPC to 50.0±4.9% of the value of untreated WPC at 1 h of reaction, gradual decrease was observed (Fig. 3). The loss of lysine content was reported to be caused by the reaction of lactose with the protein amino acid groups.41) In the present study, the glycation reaction mixture contained sodium azide (0.02%) and 1 mM diethylene triamine pentaace-

Table 1. Composition of the Whey Protein Concentrate (WPC) Utilized

Composition WPC (%, w/w)

Moisture 5.17±0.04Total protein 73.6±0.2Total fat 3.41±0.25Lactose 7.66±0.14Ash 2.64±0.02

Values represent as the mean±S.D. of three independent experiments performed in triplicate.

Fig. 1. Time Course of Changes in the Fluorescence Intensity of Gly-cated Whey Protein Concentrate (G-WPC)

Whey protein concentrate (WPC), at a concentration of 10 mg/mL in 0.1 M sodium phosphate buffer (pH 7.4), was incubated with shaking (60 rpm) at 55°C for 24 h to prepare G-WPC. The fluorescence intensity was measured at 370 nm (excita-tion) and 440 nm (emission).

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tic acid (DTPA). Hence, the observed decrease of amino group content was not due to microbial growth or metal-induced degradation of the G-WPC.

Cytotoxicity of LPS, WPC, and G-WPC There has been concern that the physical, chemical or enzymatic modifications9–11,42) of whey protein for improvement of the functional properties might result in the production of toxic compounds which cannot be used in food processing.43) In the present glycation conditions (55°C, 24 h), the viability of RAW264.7 macrophages ranged between 95 and 119% com-pared to the control cells when treated with WPC and G-WPC at the levels of 25, 50 and 100 µg/mL, while LPS demonstrat-ed cytotoxicity at 1 µg/mL (Fig. 4). These results suggest that G-WPC may have no toxicity.

Effects of G-WPC on NO Production and Pro-inflam-matory Cytokine Expression NO is known as an important cellular signaling transmitter of the immune system in re-sponse to pathogens, and is generated from the transformation of L-arginine to L-citrulline through the actions of NOS.44) NOS has both a constitutive form, which is always present in the cytoplasm, and an inducible form, iNOS involved in im-mune response.45)

As seen in Fig. 5A, the results showed that macrophages activated by G-WPC displayed significant induction of NO

Fig. 3. Time Course Changes in the Free Primary Amino Acid Resi-dues of G-WPC

WPC, at a concentration of 10 mg/mL in 0.1 M sodium phosphate buffer (pH 7.4), was incubated with shaking (60 rpm) at 55°C for 24 h. The free primary amino acid residues were measured at 370 nm (excitation) and 440 nm (emission).

Fig. 4. Effect of G-WPC on Viability of RAW264.7 CellsRAW264.7 cells (1×105 cells/well) were incubated with LPS (1 µg/mL) or differ-

ent doses of sample (25, 50, 100 µg/mL) for 24 h. Cell viability was determined by MTT assay, and was expressed as a percentage of the control. Values represent as the mean±S.D. of three independent experiments performed in triplicate. The dif-ference between each experimental group and control was compared by Student’s t-test (* p<0.05, ** p<0.01). CON, control; LPS, lipopolysaccharide; WPC, whey protein concentrate.

Fig. 2. SDS-PAGE Gel Showing Proteins of WPC and G-WPCMolecular weights (kDa) are shown on the left of gel, and correspond to the markers loaded. (A) Coomassie brilliant blue G-250 staining of proteins; single protein (6 µg

per lane) of α-LA, β-LG and BSA was loaded, respectively. Samples (20 µg per lane) of WPC and GWPC were loaded, respectively. (B) Glycoprotein staining of proteins; samples (40 µg per lane) of WPC and GWPC were loaded, respectively. HMW, high molecular weight bands; BSA, bovine serum albumin; β-LG, β-lactoglobulin; α-LA, α-lactalbumin.

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synthesis, in a dose-dependent manner (p<0.05). When RAW264.7 cells were incubated with 100 µg/mL of G-WPC, the NO production was dramatically increased (37.4±3.6 µM), and was comparable to the levels observed for treatment with 1 µg/mL of LPS, the positive control (38.4±4.4 µM). In addi-tion, the expression of iNOS mRNA upon G-WPC treatment (100 µg/mL) was also significantly (p<0.001) increased to 2.4-fold more than that obtained by treatment with intact WPC (Fig. 5B). Because these NO-enhancing activities of WPC and G-WPC might be due to the potential endotoxin contamina-tion of sample preparation, we evaluated possible contribution of endotoxin to the biological activity of samples using LPS-neutralizing agent polymyxin B (PMB). In NO assay with WPC, G-WPC or LPS in the presence and absence of PMB, the inhibitor almost completely suppressed NO production induced by LPS, while it did not reduce responses induced by WPC or G-WPC indicating that the observed immuno-modulatory response of G-WPC is independent of potential endotoxin contamination. (Supplementary Fig. 3). In addition, we measured the endotoxin unit (EU) as an endotoxin concen-tration of sample. The endotoxin concentrations of WPC and G-WPC were determined to be 7.24±0.56 EU/100 µg proteins and 7.80±1.01 EU/100 µg proteins, respectively using Pierce® LAL Chromogenic Endotoxin Quantitation Kit according to the manufacturer’s protocol. Based on product information of LPS used in this experiment, LPS contains approximately 10000 EU/1 µg of LPS. These results show that WPC and G-WPC used in our experiment contained very low endotoxin level.

Next, the expressions of pro-inflammatory cytokines were determined (Fig. 6). TNF-α is one of the major systemic inflammatory cytokines secreted by macrophages upon the acute phase stimulation of pro-inflammatory molecules.46) Compared to WPC treatment, G-WPC was found to stimulate the expression of TNF-α from 4.0±2.3 to 13.3±5.4 based on qRT-PCR analysis of TNF-α expression (Fig. 6B). The IL-1β generated by activated macrophages is known to enhance

the activity of T, NK and B cells, while IL-6 is involved in the differentiation of T and B cells, stimulation of antibody secretion, and induction of immunoglobulin generation.47,48) The induction of both IL-1β and IL-6 mRNA expression in macrophages was dramatically increased upon treatment with G-WPC by 4.2- and 110-fold, respectively, compared to the levels obtained via WPC treatment (Figs. 6C, D). WPC treat-ment of macrophage seems to induce TNF but not IL-1 β and IL-6 mRNA expression compared to control, but based on the qRT-PCR data, the mRNA expression of these cytokines between control and WPC-treated cells was not significantly different.

Although many studies on the immunomodulatory effects of WPC prepared by enzymatic hydrolysis or membrane fil-tration have been reported,49) few studies have reported the stimulation of pro-inflammatory cytokines via the Maillard reaction.21,50) It was reported that WPC itself induces TNF-α production in RAW cells and murine splenocytes.51) These results suggest that the Maillard reaction may contribute to induction of the above cytokines produced by macrophages against pathogenic bacteria in the gut mucosa. Upon phago-cytizing a bacterial particle, activated macrophages release a series of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α.52) This immune response serves as the body’s first line of defense against invasion, and inflammation by these inflam-matory cytokines accompanies a wide range of effects, includ-ing (1) induction of vascular endothelial receptors required for moving of immune cells out of the circulation and into the local site of inflammation, and (2) drawing and activating additional leukocytes to assist in the destruction.53) However, it should be noted that imbalances due to over-production of pro-inflammatory cytokines can cause pathological condition including rheumatoid arthritis.54)

Phagocytic Activity of RAW264.7 Cells Treated with G-WPC One of the critical activities of macrophages is phagocytosis, which serves as a primary immune defense against pathogens. As such, the measurement of phagocytosis

Fig. 5. Nitric Oxide (NO) Production and Expression of Inducible Form-Nitric Oxide Synthase (iNOS) mRNA by RAW264.7 Cells in Response to G-WPC

(A) After 24 h, level of nitrite, a stable and accumulated oxidation product of NO was measured in culture supernatant by Griess reaction as described. (B) iNOS mRNA expression was measured using quantitative real-time RT-PCR (qRT-PCR) after 4 h of sample treatment. Values represent as the mean±S.D. of three independent experi-ments performed in triplicate. The difference between each experimental group and control was compared by Student’s t-test (*** p<0.001). CON, control; LPS, lipopoly-saccharide; WPC, whey protein concentrate.

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Fig. 7. Intracellular Distribution of E. coli Phagocytosed by RAW264.7 MacrophagesAfter incubation of the RAW264.7 cells (9×105 cells/well) with no treatment (A), 1 µg/mL of LPS (B), 100 µg/mL of WPC (C) or G-WPC (D) for 24 h, opsonized FITC-

E. coli (5×106 CFU/well) was added to each well, and incubated for 2 h. Extracellular fluorescence was measured using a confocal laser scanning microscope. Quantifica-tion of image A–D was performed using Image J software (E). Values represent as the mean±S.D. of three independent experiments performed in triplicate (*** p<0.001).

Fig. 6. Expression of Tumor Necrosis Factor (TNF)-α, Interleukin (IL)-1β and IL-6 mRNA in RAW264.7 CellsTNF-α, IL-1β and IL-6 mRNA expressions were measured using reverse transcription PCR (RT-PCR) (A) and qRT-PCR (B–D) after 4, 4, and 12 h of sample treatment,

respectively. Values represent as the mean±S.D. of three independent experiments performed in triplicate. The difference between each experimental group and control was compared by Student’s t-test (** p<0.01, *** p<0.001). CON, control; LPS, lipopolysaccharide; WPC, whey protein concentrate; G-WPC, glycated whey protein con-centrate.

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provides a useful method for evaluating macrophage function. Phagocytosis was reported to induce transcriptional activation of cytokine genes.55,56) The density of RAW264.7 macrophage cells which ingested FITC-E. coli, reflecting phagocytosis, was compared among the sample treatments. Confocal laser scanning images of the ingested FITC-E. coli in RAW264.7 macrophage cells are shown in Figs. 7A–D, for which the re-sults were quantified in Fig. 7E. The fluorescence intensity of the G-WPC treatment (188.1±20.8%) was significantly higher (p<0.05) than the WPC treatment (122.5±5.6%), suggesting that G-WPC may provide a new material for enhancing the phagocytic activity of RAW264.7 macrophages.

CONCLUSION

In conclusion, G-WPC prepared via a non-enzymatic Mail-lard reaction had no cytotoxicity on RAW264.7 macrophage cells, caused increased expression of various cytokines (i.e., TNF-α, IL-1β and IL-6 mRNA), and showed ability to en-hance phagocytosis. Accordingly, G-WPC could be used as a potential dietary protein source for providing immune modu-lating properties.

Acknowledgment This research was supported by the High Value-Added Food Technology Development Program (111137-03-HD120), funded through the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (iPET).

Conflict of Interest The authors declare no conflict of interest.

Supplementary Materials The online version of this ar-ticle contains supplementary materials.

REFERENCES

1) An H, Yu Y, Zhang M, Xu H, Qi R, Yan X, Liu S, Wang W, Guo Z, Guo J, Qin Z, Cao X. Involvement of ERK, p38 and NF-κB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expres-sion induced by lipopolysaccharide in mouse dendritic cells. Immu-nology, 106, 38–45 (2002).

2) Ahmad-Nejad P, Häcker H, Rutz M, Bauer S, Vabulas RM, Wagner H. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol., 32, 1958–1968 (2002).

3) Oriss TB, Ostroukhova M, Seguin-Devaux C, Dixon-McCarthy B, Stolz DB, Watkins SC, Pillemer B, Ray P, Ray A. Dynamics of den-dritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance. J. Immunol., 174, 854–863 (2005).

4) Uthaisangsook S, Day NK, Bahna SL, Good RA, Haraguchi S. In-nate immunity and its role against infections. Ann. Allergy Asthma Immunol., 88, 253–264, quiz, 265–266, 318 (2002).

5) Lowenstein CJ, Snyder SH. Nitric-oxide, a novel biologic messen-ger. Cell, 70, 705–707 (1992).

6) McIntosh GH, Royle PJ, Le Leu RK, Regester GO, Johnson MA, Grinsted RL, Kenward RS, Smithers GW. Whey proteins as func-tional food ingredients? Int. Dairy J., 8, 425–434 (1998).

7) de Wit JN. Nutritional and functional characteristics of whey pro-teins in food products. J. Dairy Sci., 81, 597–608 (1998).

8) Wong DWS, Camirand WM, Pavlath AE, Parris N, Friedman M. Structures and functionalities of milk proteins. Crit. Rev. Food Sci. Nutr., 36, 807–844 (1996).

9) Tseng YM, Chen SY, Chen CH, Jin YR, Tsai SM, Chen IJ, Lee JH, Chiu CC, Tsai LY. Effects of alcohol-induced human periph-eral blood mononuclear cell (PBMC) pretreated whey protein con-centrate (WPC) on oxidative damage. J. Agric. Food Chem., 56, 8141–8147 (2008).

10) Bounous G, Batist G, Gold P. Whey proteins in cancer prevention. Cancer Lett., 57, 91–94 (1991).

11) Wong CW, Watson DL. Immunomodulatory effects of dietary whey proteins in mice. J. Dairy Res., 62, 359–368 (1995).

12) Gauthier SF, Pouliot Y, Saint-Sauveur D. Immunomodulatory pep-tides obtained by the enzymatic hydrolysis of whey proteins. Int. Dairy J., 16, 1315–1323 (2006).

13) Korhonen H, Pihlanto A. Food-derived bioactive peptides-opportu-nities for designing future foods. Curr. Pharm. Des., 9, 1297–1308 (2003).

14) Saito T. Antihypertensive peptides derived from bovine casein and whey proteins. Adv. Exp. Med. Biol., 606, 295–317 (2008).

15) Lee KG, Shibamoto T. Toxicology and antioxidant activities of non-enzymatic browning reaction products. Food Rev. Int., 18, 151–175 (2002), review.

16) Jing H, Kitts DD. Chemical and biochemical properties of casein-sugar Maillard reaction products. Food Chem. Toxicol., 40, 1007–1015 (2002).

17) Prabhu KS, Zamamiri-Davis F, Stewart JB, Thompson JT, Sordillo LM, Reddy CC. Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW264.7 macrophages: role of nuclear factor-κB in up-regulation. Biochem. J., 366, 203–209 (2002).

18) Marko D, Habermeyer M, Kemény M, Weyand U, Niederberger E, Frank O, Hofmann T. Maillard reaction products modulating the growth of human tumor cells in vitro. Chem. Res. Toxicol., 16, 48–55 (2003).

19) Ilchmann A, Burgdorf S, Scheurer S, Waibler Z, Nagai R, Well-ner A, Yamamoto Y, Yamamoto H, Henle T, Kurts C, Kalinke U, Vieths S, Toda M. Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A types I and II. J. Allergy Clin. Immunol., 125, 175–183. e11 (2010).

20) Yang SY, Kim SW, Kim Y, Lee SH, Jeon H, Lee KW. Optimization of Maillard reaction with ribose for enhancing anti-allergy effect of fish protein hydrolysates using response surface methodology. Food Chem., 176, 420–425 (2015).

21) Song S, Zhou F, Nordquist RE, Carubelli R, Liu H, Chen WR. Glycated chitosan as a new non-toxic immunological stimulant. Im-munopharmacol. Immunotoxicol., 31, 202–208 (2009).

22) Korean food standards code. Ministry of Food and Drug Safety (MFDS), Chucheongbuk-do, Republic of Korea (2013).

23) Walker JM. The bicinchoninic acid (BCA) assay for protein quan-titation. The Protein Protocols Handbook, Human Press Inc., N.J., pp. 11–15 (2009).

24) Monnier VM, Vishwanath V, Frank KE, Elmets CA, Dauchot P, Kohn RR. Relation between complications of type I diabetes mellitus and collagen-linked fluorescence. N. Engl. J. Med., 314, 403–408 (1986).

25) Nagaraj RH, Monnier VM. Protein modification by the degradation products of ascorbate: formation of a novel pyrrole from the Mail-lard reaction of L-threose with proteins. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1253, 75–84 (1995).

26) Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685 (1970).

27) van de Lagemaat J, Silván JM, Moreno FJ, Olano A, del Castillo MD. In vitro glycation and antigenicity of soy proteins. Food Res. Int., 40, 153–160 (2007).

28) Goodno CC, Swaisgood HE, Catignani GL. A fluorimetric assay for available lysine in proteins. Anal. Biochem., 115, 203–211 (1981).

29) Ramírez-Jiménez A, García-Villanova B, Guerra-Hernández E.

206 Vol. 39, No. 2 (2016)Biol. Pharm. Bull.

Loss of o-phthaldialdehyde reactivity during storage of infant cere-als. Int. J. Food Sci. Nutr., 55, 143–148 (2004).

30) Mosmann T. Rapid colorimetric assay for cellular growth and sur-vival: application to proliferation and cytotoxicity assays. J. Immu-nol. Methods, 65, 55–63 (1983).

31) Schulz K, Kerber S, Kelm M. Reevaluation of the griess method for determining NO/NO2

− in aqueous and protein-containing samples. Nitric Oxide, 3, 225–234 (1999).

32) Reynolds TM. Chemistry of nonenzymic browning. I. The reaction between aldoses and amines. Adv. Food Res., 12, 1–52 (1963).

33) Havea P, Singh H, Creamer LK, Campanella OH. Electrophoretic characterization of the protein products formed during heat treat-ment of whey protein concentrate solutions. J. Dairy Res., 65, 79–91 (1998).

34) Walstra P, Wouters JTM, Geurts TJ. Dairy Science and Technology. Second Edition, CRC Press, F.L. (2006).

35) Havea P, Singh H, Creamer LK. Characterization of heat-induced aggregates of β-lactoglobulin, α-lactalbumin and bovine serum albumin in a whey protein concentrate environment. J. Dairy Res., 68, 483–497 (2001).

36) Nursten HE. The Maillard Reaction: Chemistry, Biochemistry, and Implications, Royal Society of Chemistry, Cambridge (2005).

37) Lillard JS, Clare DA, Daubert CR. Glycosylation and expanded util-ity of a modified whey protein ingredient via carbohydrate conjuga-tion at low pH. J. Dairy Sci., 92, 35–48 (2009).

38) Mulsow BB, Jacob M, Henle T. Studies on the impact of glycation on the denaturation of whey proteins. Eur. Food Res. Technol., 228, 643–649 (2009).

39) Bu G, Luo Y, Zheng Z, Zheng H. Effect of heat treatment on the antigenicity of bovine α-lactalbumin and β-lactoglobulin in whey protein isolate. Food Agric. Immunol., 20, 195–206 (2009).

40) Kilshaw PJ, Heppell LM, Ford JE. Effects of heat treatment of cow’s milk and whey on the nutritional quality and antigenic prop-erties. Arch. Dis. Child., 57, 842–847 (1982).

41) Matsuda T, Kato Y, Nakamura R. Lysine loss and polymerization of bovine β-lactoglobulin by amino carbonyl reaction with lactulose (4-O-β-D-galactopyranosyl-D-fructose). J. Agric. Food Chem., 39, 1201–1204 (1991).

42) Hakkak R, Korourian S, Ronis M, Irby D, Kechclana S, Rowland C. Dietary prevention of mammary cancer in multiparous female rats by whey protein, but not soy protein isolate. Proc. Am. Assoc. Cancer Res., 40, 2010 (1999).

43) Friedman M. Food browning and its prevention: an overview. J.

Agric. Food Chem., 44, 631–653 (1996).44) Nathan CF, Hibbs JB Jr. Role of nitric oxide synthesis in macro-

phage antimicrobial activity. Curr. Opin. Immunol., 3, 65–70 (1991).45) Vane JR, Mitchell JA, Appleton I, Tomlinson A, Bishop-Bailey D,

Croxtall J, Willoughby DA. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc. Natl. Acad. Sci. U.S.A., 91, 2046–2050 (1994).

46) Idriss HT, Naismith JH. TNFα and the TNF receptor superfamily: structure–function relationship(s). Microsc. Res. Tech., 50, 184–195 (2000).

47) Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-κB in cell growth regulation. Am. J. Pathol., 159, 387–397 (2001).

48) Delgado AV, McManus AT, Chambers JP. Production of tumor ne-crosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P. Neuropeptides, 37, 355–361 (2003).

49) Eriksen EK, Vegarud GE, Langsrud T, Almaas H, Lea T. Effect of milk proteins and their hydrolysates on in vitro immune responses. Small Rumin. Res., 79, 29–37 (2008).

50) Muscat S, Pelka J, Hegele J, Weigle B, Münch G, Pischetsrieder M. Coffee and maillard products activate NF-κB in macrophages via H2O2 production. Mol. Nutr. Food Res., 51, 525–535 (2007).

51) Huang SM, Chen KN, Chen YP, Hong WS, Chen MJ. Immunomod-ulatory properties of the milk whey products obtained by enzymatic and microbial hydrolysis. Int. J. Food Sci. Technol., 45, 1061–1067 (2010).

52) Helm O, Held-Feindt J, Schäfer H, Sebens S. M1 and M2: there is no “good” and “bad”—How macrophages promote malignancy-associated features in tumorigenesis. OncoImmunology, 3, e946818 (2014).

53) Watkins LR, Maier SF, Goehler LE. Immune activation: the role of pro-inflammatory cytokines in inflammation, illness responses and pathological pain states. Pain, 63, 289–302 (1995).

54) Ramani T, Auletta CS, Weinstock D, Mounho-Zamora B, Ryan PC, Salcedo TW, Bannish G. Cytokines the good, the bad, and the deadly. Int. J. Toxicol., 34, 355–365 (2015).

55) Fouqueray B, Philippe C, Amrani A, Perez J, Baud L. Heat shock prevents lipopolysaccharide-induced tumor necrosis factor-α syn-thesis by rat mononuclear phagocytes. Eur. J. Immunol., 22, 2983–2987 (1992).

56) Cavaillon JM. Cytokines and macrophages. Biomed. Pharmaco-ther., 48, 445–453 (1994).