Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes

Abstract

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt₂ cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt₂ cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt₂ cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

INTRODUCTION

Food-derived peptides exhibit various biological activities, including an immunomodulatory function.¹⁾ For example, food-derived peptides can induce chemotactic ability of immune cells. Migration, a dynamic phenomenon of immune cells, is involved in the defense against infections.^2,3) Since ancient times, milk casein, a food protein, has been administered to the abdominal cavity of mice and used to collect migrated immune cells. In vitro experiments reported casein-induced macrophage and neutrophil migration.^4–6) Most proteins are digested into peptides and amino acids when a food protein is taken orally. Previously, it has been reported that YPVEP, produced by digesting β-casein with actinase, derived from actinomycetes, helps migrate mouse macrophages.⁷⁾ In recent years, it has been observed that circulating neutrophils accumulate in the intestine by administering wheat gliadin pepsin-tryptic peptides to mice.⁸⁾ The report suggests that peptides derived from wheat gliadin act on the formyl peptide receptor (FPR) 1 receptor in mice.⁸⁾ Several reports showed that the food peptide exerts its function through the receptors on the cell membrane.

Soybeans contain abundant nutrients and approximately 30% proteins, and are consumed worldwide as food. Soy protein isolate (SPI) is obtained by degreasing soybeans and isolating proteins. β-Conglycinin, one of the main constituent proteins of SPI, has beneficial effects, such as anti-obesity and metabolic improvement.^9,10) Many immune cells are present in the intestinal tract, showing different immune responses to foreign matter.^11,12) Previously, in soybean peptides, Yoshikawa reported MITLAIPVNKPGR and MITL as immunostimulating peptides and named those soymetide-13 and soymetide-4.¹³⁾ MITLAIPVNKPGR is produced by trypsin digestion of β-conglycinin, and MITL is produced by incubating pancreatic elastase and MITLAIPVNKPGR in vitro.¹³⁾ It may be produced in the human body. However, direct migratory function of MITLAIPVNKPGR and MITL on human immune cells is unknown.

Various endogenous digestive enzymes digest ingested food proteins in the body, and peptides are absorbed by the immune cells in the intestinal tract. Various actions are known for peptides derived from food proteins.^14,15) On the other hand, few reports demonstrate chemotactic ability and receptors against human immune cells by food peptides produced by endogenous digestive enzymes. We analyzed the effect of soybean β-conglycinin derived peptides on human polymorphonuclear leukocytes to identify the chemotactic activity of immune cells induced by food ingredients.

MATERIALS AND METHODS

Chemicals

Fmoc-amino acids were purchased from WATANABE CHEM. IND., Ltd. (Japan). Dimethyl sulfoxide (DMSO) and pertussis toxin (PTX) were procured from FUJIFILM WAKO (Japan). The FPR inhibitor, tert-butoxycarbonyl (Boc)-MLF, was obtained from Tocris bioscience. The FPR2-specific inhibitor, WRW4, was purchased from Alomone labs (Israel).

Solid-Phase Peptide Synthesis

Synthetic peptides were synthesized using 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase chemistry as described previously with minor modifications.¹⁶⁾ Briefly, the reaction was carried out using each Fmoc-amino acids (2 equivalents (equiv.)), 1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (2 equiv.), 1-hydroxybenzotriazole (2 equiv.), and N-methylmorpholine (1 equiv.) in dimethylformamide (DMF). The N-terminal Fmoc group was deblocked with 20% piperidine in DMF. The cleavage of the peptide and its protective groups from the resin was performed with 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 3% ethylmethylsulfide, 2.5% 1,2-ethanedithiol, and 2% thiophenol for 8 h. The crude peptides were purified by HPLC using a C18 column (Capcell Pak C18 ACR, 10 × 250 mm; Shiseido, Japan) with a linear gradient of 0–50% acetonitrile in the presence of 0.1% TFA. The purity of each peptide was confirmed to be >95% by analytical HPLC and mass spectrometry.

Cultured Cells

The human promyelocytic leukemia 60 (HL-60) cells were cultured at 37 °C in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum in 95% air and 5% CO₂ atmospheric conditions. The HL-60 cells were differentiated into neutrophil-like cells after treatment with 1 µM all-trans-retinoic acid (ATRA; WAKO)^17–20) and 0.5 mM dibutyryl cAMP (Bt₂ cAMP) for 3 d.^21–23) The DMSO concentration was aligned with the sample and control data. The cells were pretreated with PTX (100 ng/mL) for 16 h and added to the Transwell.

Human Peripheral Blood Cells

Human polymorphonuclear leukocytes were separated from peripheral blood of young healthy donors as follows. Human peripheral blood was collected in VenojectII (heparinized, TERUMO, Japan). Heparinized blood was transferred to Polymorphprep (Alere Technologies AS, Oslo, Norway) in 15 mL falcon tubes and centrifuged at 500 × g for 35 min at room temperature. The polymorphonuclear leukocytes suspension was collected using a pipette and an aliquot of 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-buffered saline was diluted using with an equal volume of water and one volume of this suspension was mixed with half concentration of saline. The cells were harvested by centrifugation at 400 × g for 10 min. To remove any residual erythrocyte contamination of the polymorphonuclear leukocytes, the cell pellet was suspended in 3 mL of ammonium chloride lysis buffer and incubated at 37 °C for 7 min and centrifuged at 400 × g for 10 min at room temperature. The pellet was suspended in the migration medium at the density of 2 × 10⁶ cells/mL, and incubated in a CO₂ incubator.

Migration Assay

The migration of differentiated HL-60 cells and human polymorphonuclear leukocytes were assayed using Transwell inserts (pore size, 5 µm) and 24-well culture plates (Corning Incorporated costar).²⁴⁾ Briefly, the cells (10⁶ for the differentiated HL-60 cells and 2 × 10⁵ for human polymorphonuclear leukocytes) suspended in 0.1 mL of RPMI 1640 medium containing 0.1% bovine serum albumin were transferred to the Transwell insert (the upper compartment). MITLAIPVNKPGR and MITL were dissolved in DMSO, and 0.6 mL of RPMI 1640 medium containing 0.1% bovine serum albumin was added to each well of the culture plate (the lower compartment) (the final concentration of DMSO was 0.2%). After incubating at 37 °C (for 4 h for HL-60 cells and 2 h for human polymorphonuclear leukocytes) in 95% air and 5% CO₂ atmosphere, the number of cells that migrated from the upper compartment to the lower compartment was counted using a hemocytometer. Independent experiments were performed at least twice.

RNA Purification and Real-Time PCR Analysis

The total RNA from HL-60 cells was purified with Quick-RNA™ (Zymo Research Corporation, U.S.A.) according to the manufacturer’s instructions (for pelleted cells). The cDNAs were synthesized in a TaKaRa PCR Thermal Cycler Dice TP650 (TaKaRa Bio Inc., Japan). Two micrograms of total RNA were reverse-transcribed by using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, U.S.A.) in a final volume of 20 µL. The samples were incubated at 25 °C for 10 min followed by 37 °C for 120 min and 85 °C for 5 min. Aliquots of cDNA (1/20 of reverse transcription reactions) were used in real-time PCR experiments. SYBR Green-based real-time PCR was used to determine the cDNA levels. 7500 Fast Real-Time PCR System (Applied Biosystems) was used for real-time PCR. The sequence of the FPR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers was reported previously.²⁵⁾ FPR1 transcripts were amplified with the primers 5′-TACCCAGAGCAAGACCACA-3′ (forward) and 5′-AAAAGGCTGCTGCGACAA-3′ (reverse), FPR2 with 5′-TGCTGGTGCTGCTGGCAA-3′ (forward) and 5′-AATATCCCTGACCCCATCCTCA-3′ (reverse), FPR3 with 5′-AGTTGCTCCACAGGAATCCA-3′ (forward) and 5′-GCCAATAATGAAGTGGAGGATCAGA-3′ (reverse), and GAPDH with the primers 5′-GGCTGCTTTTAACTCTGG-3′ (forward) and 5′-GGAGGGATCTCGCTCC-3′ (reverse).²⁵⁾ Primer synthesis was commissioned by Fasmac Co., Ltd. (Japan). The expression levels were calculated relative to GAPDH as an endogenous control. Relative expression was calculated as 2^{(Ct test gene-Ct GAPDH)}. Independent experiments were performed at least three times.

Measurement of Intracellular Ca²⁺ Concentrations

Measurement of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) was performed using the previous method with minor modifications.^26,27) The harvested cells were suspended by gentle pipetting in 25 mM Hepes-bufferd Tyrode’s solution (Ca²⁺ free) (pH 7.4) containing 5 µM Fura-2/AM and further incubated at 37 °C for 60 min. The cells were then centrifuged (180 × g for 7 min), washed twice with Hepes-Tyrode’s solution (Ca²⁺ free), and resuspended in Hepes-Tyrode’s solution (Ca²⁺ free) containing 0.1% bovine serum albumin (10⁶ for the differentiated HL-60 cells and 4 × 10⁵ for human polymorphonuclear leukocytes). CaCl₂ was added 4–5 min before the measurement (final Ca²⁺ concentration in the cuvette, 1 mM) and incubated at 37 °C. MITLAIPVNKPGR and fMLP were dissolved in DMSO, and aliquots (1 µL each) were added to the cuvette (final DMSO concentration, 0.2%). After the addition of ligands, changes in [Ca²⁺]_i were analyzed using a CAF-110 Ca²⁺ analyzer (JASCO, Tokyo, Japan). DMSO did not markedly affect the [Ca²⁺]_i.

Statistical Analysis

The experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using JMP Pro. 15 (SAS Institute Inc., NC, U.S.A.), Dunnett’s test (Figs. 1, 2, 6), and Tukey’s test (Figs. 3–5.). The significance level was set at less than 0.05.

Ethical Considerations

This study was carried out with the approval of the Teikyo University Medical Research Ethics Committee (No. 17-094-2).

RESULTS

MITLAIPVNKPGR Induced the Migration of Human Peripheral Polymorphonuclear Leukocytes and Bt₂ cAMP-Differentiated HL-60 Cells

First, we examined the effect of MITLAIPVNKPGR on the motility of human peripheral polymorphonuclear leukocytes. Approximately 0.1 µM MITLAIPVNKPGR exerted effects on the migration of human peripheral polymorphonuclear leukocytes. The proportion of the migrated cells was 5.6% for the control and 48.6% for the 3 µM MITLAIPVNKPGR-stimulated cells (Fig. 1A). The addition of 1 µM MITLAIPVNKPGR markedly accelerated the migration. The effect was observed from 0.1 µM MITLAIPVNKPGR (12.1%) and peaked at 3 µM (48.6%). The number of migrated cells increased in a dose-dependent manner in human peripheral polymorphonuclear leukocytes (Fig. 1A).

Fig. 1. The Migration of Differentiated HL-60 Cells and Human Polymorphonuclear Leukocytes for Each Condition

(A) The effect of MITLAIPVNKPGR on the migration of human polymorphonuclear leukocytes. The incubation was carried out for 2h. ** p < 0.0001 (compared with the control (open circle)) (B) The HL-60 cells were differentiated by treatment with 1 µM ATRA (●) and by treatment with 0.5 M Bt₂ cAMP (■) for 3 d. Differentiated HL-60 cells were added to the Transwell (the upper compartment), and various concentrations of MITLAIPVNKPGR or control (DMSO alone) were added to the well of the culture plate (the lower compartment). The incubation was carried out for 4h. (C) The HL-60 cells were differentiated by treatment with 1 µM all trans retinoic acid (ATRA) (□) and by treatment with 0.5 M dibutyryl cyclic AMP (Bt₂ cAMP) (■) for 3 d. The cells were added to the Transwell (the upper compartment), and 3 µM MITLAIPVNKPGR or vehicle (DMSO) was added to the well of the culture plate (the lower compartment). The incubation was carried out for the indicated periods of time. Closed circle (●), closed square (■) and open square (□): 3 µM MITLAIPVNKPGR; open circle (○): vehicle (DMSO) alone. The data are the means ± S.D. of four determinations.

We next examined the effect of MITLAIPVNKPGR on the motility of HL-60 cells. Undifferentiated HL-60 cells did not migrate to 3 µM MITLAIPVNKPGR for 4 h compared to vehicle (DMSO) (data not shown). The addition of 3 µM MITLAIPVNKPGR enhanced the migration of ATRA-differentiated HL-60 cells to some extent, although the number of migrated cells was low; the percentages of the migrated cells were 0.8 and 2.1% for the control and 3 µM MITLAIPVNKPGR-stimulated cells, respectively (Fig. 1B closed circle). On the other hand, MITLAIPVNKPGR exerted effects on the migration of the HL-60 cells differentiated by treatment with Bt₂ cAMP for 3 d (Fig. 1B closed square). The addition of 1 µM MITLAIPVNKPGR markedly accelerated the migration (13.4%). The effect was observed from 0.1 µM MITLAIPVNKPGR (3%) and reached a peak at 10 µM (26.2%). Further, compared to ATRA-differentiated HL-60 cells, migration rate was found to be greater in Bt₂ cAMP-differentiated HL-60 cells. The number of migrated cells increased dose-dependently.

Furthermore, we examined the course of MITLAIPVNKPGR-induced cell migration (Fig. 1C). In ATRA-differentiated and Bt₂ cAMP-differentiated HL-60 cells, 3 and 20% of cells migrated toward 3 µM MITLAIPVNKPGR respectively (Fig. 1C open square and closed square). The number of migrated cells were augmented with time. These data indicate that this migration increases in a time-dependent manner.

Expression Analysis of FPR by Real-Time PCR

ATRA and Bt₂ cAMP are known to induce the differentiation of HL-60 cells.^17–20) On the other hand, mRNA expression of the FPR was elevated in Bt₂ cAMP-differentiated HL-60 cells but not upon ATRA differentiation.^21–23,28) However, the details of the FPR subtype have yet to be discovered in previous reports. Real-time PCR was performed to examine the induction of FPR1, FPR2, and FPR3 expression by ATRA and Bt₂ cAMP for HL-60 cells (Fig. 2). mRNA was extracted from the cultured cells, native HL-60 cells, ATRA-differentiated HL-60 cells, and Bt₂ cAMP-differentiated HL-60 cells. The expression of FPR1 in Bt₂ cAMP-differentiated HL-60 cells significantly increased (71.5-fold) compared to the controls (p < 0.05). Similarly, ATRA-differentiated HL-60 cells showed a 1.5-fold increase in FPR1 expression compared to the controls. On the other hand, in Bt₂ cAMP-differentiated HL-60 cells, the expression of FPR2 was 1.5-fold higher than that of the controls, and ATRA differentiation was unchanged. Furthermore, FPR3 expression was 1.2-fold higher in ATRA-differentiated HL-60 cells compared to the controls, and no change was observed in Bt₂ cAMP-differentiated HL-60 cells.

Fig. 2. Expression of FPRs Isoforms in Native, ATRA-Differentiated and Bt₂ cAMP-Differentiated HL-60 Cells

Total RNA was isolated from native HL-60, ATRA-differentiated HL-60 and Bt₂ cAMP-differentiated HL-60 cells. cDNAs analysis was performed by real time-PCR. The fold of increase of FPRs isoforms. The graphic shows the relative gene expression of FPRs isoforms as native HL-60 is 1.0. The data are the means ± S.D. of four determinations. * p < 0.05.

Mechanism Underlying MITLAIPVNKPGR-Induced Cell Migration

Transwell is a powerful tool to investigate cell motility.^24,29) We then investigated whether the MITLAIPVNKPGR-induced migration resulted from chemotaxis (directional movement along a concentration gradient) or chemokinesis (stimulated movement in no specific direction). The migration of the cells from the upper to the lower compartment in the absence of MITLAIPVNKPGR was 1.3%, which served as a control (Fig. 3A). The proportion of migrated cells was elevated to 15.6% when MITLAIPVNKPGR (3 µM) was added to the lower compartment. The migration was reduced when MITLAIPVNKPGR (3 µM) was added to both the upper compartment (with cells) and lower compartment (6.4%). On the other hand, the presence of MITLAIPVNKPGR (3 µM) in the upper compartment alone (with cells) did not evoke cell migration (0.8%). These data suggest that the migration might be induced via chemotaxis.

Fig. 3. The Mechanism Underlying MITLAIPVNKPGR-Induced Cell Migration

(A) Bt₂ cAMP-differentiated HL-60 cells were incubated with or without MITLAIPVNKPGR (3 µM) in the upper compartment of the Transwell. The migration of the cells from the upper compartment to the lower compartment in response to MITLAIPVNKPGR (3 µM). (B) Bt₂ cAMP-differentiated HL-60 cells were pretreated with PTX (100 ng/mL) for 16 h and then added to the Transwell. ** p < 0.0001. The data are the means ± S.D. of four determinations.

The FPR is a Gi-protein-coupled receptor,^30,31) and to understand the mechanisms underlying MITLAIPVNKPGR-induced cell migration, we examined the effects of pretreatment on the cells with PTX (100 ng/mL for 16 h). As shown in Fig. 3B, MITLAIPVNKPGR (3 µM) had an activation effect on the migration, whereas pretreatment of cells with PTX reduced the migration induced by MITLAIPVNKPGR. These results suggest that MITLAIPVNKPGR induces the migration in Bt₂ cAMP-differentiated HL-60 cells via Gi protein-coupled receptors.

Effects of Boc-MLF and WRW4 Treatment on MITLAIPVNKPGR-Induced Migration of Human Polymorphonuclear Leukocytes

As shown in Figs. 1 and 3B, the MITLAIPVNKPGR-induced cell migration might involve the FPR mechanism. Different subtypes of FPR, FPR1, FPR2, and FPR3 have been identified in humans.^32,33) It is well known that human neutrophils express FPR1 and FPR2.³²⁾ To understand the mechanisms underlying MITLAIPVNKPGR-induced cell migration, we examined the effects of treatment of Boc-MLF and WRW4 (Boc-MLP and WRW4 are inhibitors of the FPR 1 and 2.^34,35) Administration of Boc-MLF (100 µM) and WRW4 (20 µM) alone did not affect migration. As shown in Fig. 4, MITLAIPVNKPGR (3 µM) had an activation effect on the migration, whereas, upon treatment with 100 µM Boc-MLF or 20 µM WRW4, the migration of cells induced by MITLAIPVNKPGR was reduced (Fig. 4). These results suggest that MITLAIPVNKPGR induces the migration in differentiated HL-60 cells and human polymorphonuclear leukocytes via FPR.

Fig. 4. Effects of FPR Antagonists Boc-MLF and WRW4 on MITLAIPVNKPGR-Induced Migration

The effects of MITLAIPVNKPGR (3 µM) on the migration of human polymorphonuclear leukocytes in the presence or absence of Boc-MLF (100 µM) and WRW4 (20 µM) were examined using the Transwell. MITLAIPVNKPGR or vehicle (DMSO) was added to the lower compartment. WRW4 and Boc-MLF were added to the cell suspension in the upper compartment and the lower compartment. ** p < 0.0001. The data are the means ± S.D. of four determinations.

Dose-Dependency and Effects of Boc-MLF and WRW4 Treatment on MITL-Induced Migration of Human Peripheral Polymorphonuclear Leukocytes

MITL is produced by incubating pancreatic elastase and MITLAIPVNKPGR in vitro.¹³⁾ It has been proposed that intake of soybean β-conglycinin might lead to the generation of MITL in humans. Next, we examined the effect of MITL on the motility of human peripheral polymorphonuclear leukocytes. Approximately 1 µM of MITL affected the migration of human peripheral polymorphonuclear leukocytes. The proportion of the migrated cells was 5.7% for the control and 31.5% for the 30 µM MITL-stimulated cells (Fig. 5A). The addition of 10 µM MITL markedly accelerated the migration. The number of migrated cells also increased in a dose-dependent manner. The effects were observed upon administration of 1 µM MITL (7.3%) and reached a peak at 30 µM (31.5%). The number of migrated cells increased in a dose-dependent manner in human peripheral polymorphonuclear leukocytes by MITL.

Fig. 5. Effect of MITL on the Migration of Human Polymorphonuclear Leukocytes, and Effects of FPR Antagonists Boc-MLF and WRW4 on MITL-Induced Migration

(A) The effect of MITL on the migration of human polymorphonuclear leukocytes. The cells were added to the Transwell (the upper compartment), and various concentrations of MITL or control (DMSO alone) were added to the well of the culture plate (the lower compartment). The incubation was carried out for 2h. ** p < 0.0001 (compared with the control (open circle)) (B) The effects of MITL (30 µM) on the migration of human polymorphonuclear leukocytes in the presence or absence of Boc-MLF (100 µM) and WRW4 (20 µM) were examined using the Transwell. MITL or vehicle (DMSO) was added to the lower compartment. WRW4 and Boc-MLF were added to the cell suspension in the upper compartment and the lower compartment. ** p < 0.0001. The data are the means ± S.D. of four determinations.

To understand the mechanisms underlying MITL-induced cell migration, we examined the treatment of Boc-MLF and WRW4. Administration of Boc-MLF (100 µM) and WRW4 (20 µM) alone did not affect migration. As shown in Fig. 5B, MITL (30 µM) showed an activation effect on the migration, whereas, with the treatment of 100 µM Boc-MLF or 20 µM WRW4, the cells reduced the migration induced by MITL (Fig. 5B). These results suggest that MITL also induces the migration in human polymorphonuclear leukocytes via the FPR.

Comparison of Migration Ratio by MITLAIPVNKPGR and fMLP (in Human Polymorphonuclear Leukocytes and Bt₂ cAMP-Differentiated HL-60 Cells)

fMLP is known as a ligand of FPR receptor.³²⁾ The degree of potency of MITLAIPVNKPGR was verified using fMLP as a positive control (Fig. 6). First, we compared the ability of fMLP and MITLAIPVNKPGR to induce migration (Figs. 6A, B). In Bt₂ cAMP-differentiated HL-60 cells, approximately 40% of the cells migrated to 10 nM fMLP, and approximately 50% of cells migrated to 1 µM MITLAIPVNKPGR. Furthermore, in human peripheral polymorphonuclear leukocytes, approximately 80% of cells migrated to 10 nM fMLP and 1 µM MITLAIPVNKPGR.

Fig. 6. Comparison of fMLP with MITLAIPVNKPGR Using Migration Assay and Measurement of Intracellular Free [Ca²⁺] Levels

The migration of Bt₂ cAMP-differentiated HL-60 cells (A) and human polymorphonuclear leukocytes (B). The intracellular free [Ca²⁺] levels of Bt₂ cAMP-differentiated HL-60 cells (C) and human polymorphonuclear leukocytes (D). 10 nM fMLP and 3 µM MITLAIPVNKPGR induced increases in intracellular free [Ca²⁺] levels on human polymorphonuclear leukocytes equally (E). ** p < 0.0001, * p < 0.05 (compared with the control). The data are the means ± S.D. of four determinations.

Comparison of the Intracellular Ca²⁺ Concentrations ([Ca²⁺]_i) by MITLAIPVNKPGR and fMLP (in Human Polymorphonuclear Leukocytes and Bt₂ cAMP-Differentiated HL-60 Cells)

Increase in Ca²⁺ cytosolic levels is considered to be a necessary condition for neutrophil responses to formylpeptides. Next, we verified MITLAIPVNKPGR and fMLP-induced [Ca²⁺]_i (Figs. 6C–E). In Bt₂ cAMP-differentiated HL-60 cells, Δ [Ca²⁺]_i was approximately 500 nM at 10 nM fMLP and Δ [Ca²⁺]_i was approximately 300 nM at 3 µM MITLAIPVNKPGR. Furthermore, in human peripheral polymorphonuclear leukocytes, Δ [Ca²⁺]_i was approximately 40 nM at 10 nM fMLP and 3 µM MITLAIPVNKPGR. Three micromolar MITLAIPVNKPGR induced an intracellular calcium response comparable to 10 nM fMLP in human peripheral polymorphonuclear leukocytes. In each agonist, the potency of the induction of Δ [Ca²⁺]_i are corresponding to the potency of cell migration.

Furthermore, when Bt₂ cAMP-differentiated HL-60 cells were challenged by fMLP, MITLAIPVNKPGR-induced calcium responses were remarkably reduced, suggesting that desensitization was observed (Supplementary Fig. S1.). Pretreatment with MITLAIPVNKPGR also desensitized the fMLP-induced calcium response in the reverse order of treatment in human peripheral polymorphonuclear leukocytes (Supplementary Fig. S2.). These observations strongly indicate that fMLP and MITLAIPVNKPGR acted on the same receptor.

DISCUSSION

In this study, we found that the peptides MITLAIPVNKPGR and MITL, derived from soybean β-conglycinin, induce the migration of Bt₂ cAMP-differentiated HL-60 cells and human peripheral blood polymorphonuclear leukocytes. These peptides were generated from soybean β-conglycinin via endogenous digestive enzymes.¹³⁾ In addition, the migration was suppressed by FPR inhibitors, WRW4, Boc-MLP and PTX. Moreover, in Bt₂ AMP-differentiated HL-60 cells with strong migration, the expression of FPR1 increased significantly (71.5-fold) compared to the controls. However, there was no change in the expression of FPR2 or FPR3. From the above, it was illustrated that cell migration by MITLAIPVNKPGR and MITL was mediated via FPR1. It was found that 10 nM fMLP and 3 µM MITLAIPVNKPGR had similar migration intensity to human polymorphonuclear leukocytes. Furthermore, MITLAIPVNKPGR induced an intracellular calcium response, and the strength of the intracellular calcium response to human polymorphonuclear leukocytes was found to be similar between 10 nM fMLP and 1 or 3 µM MITLAIPVNKPGR.

Food is an essential source of nutrition. Nutrients in foods have various physiological activities, most of which benefit humans. Soybeans have gained attention for their antioxidant effects, serum lipid-lowering effects, potential as enteral nutrition, and prevention of liver dysfunction in long-term total parenteral nutrition (TPN), and are eaten and utilized worldwide^9,36–39) Migration, a dynamic phenomenon of immune cells, is involved in defense against infections. For example, neutrophils migrate toward the target to fight infection, inflammation, and human tissue injury.³⁾ Most chemoattractants secreted by immune cells exert their effects via G protein-coupled receptor (GPCR).^24,40,41) Immune cells in contact with food digests are particularly essential in the intestinal tract immune system located in the mucosa of the gastrointestinal tract. In the intestinal tract, the immune cells migrate from the blood into the intestinal mucosa.⁴²⁾ Several studies reported that peptides derived from food proteins digested by endogenous enzymes invade the intestinal mucosa via M cells and/or gap junctions.^43–46) Recently, studies in the mFPR1 and 2 double knockout mice reported that the accumulation of neutrophils via mFPR1 and 2 by unknown host chemoattractants play an essential role in the wound healing effect in the sterile skin damage in mice.⁴⁷⁾ From the above, this is considered to be one of the health benefits of soybeans, as preparation against the invasion of foreign substances, there is a possibility of introducing intestinal immune cells into the intestinal mucosa by soybean peptides via FPRs.

In this study, we found that MITLAIPVNKPGR and MITL induce the migration of human polymorphonuclear leukocytes via FPR. The FPR is expressed in various immune cells.^32,33) Pretreatment with fMLP resulted in desensitization in the calcium response of MITLAIPVNKPGR, with similar results when the order of treatment was reversed. Previous reports have shown that MITLAIPVNKPGR and MITL bind to FPR1 like fMLP.¹³⁾ In addition, RT-PCR detected increased expression of FPR1, and FPR blockers inhibited the migration. Migration assays suggested that migration of cells by MITLAIPVNKPGR was due to chemotaxis. Based on the above, the mechanism of this migration is thought to be the concentration gradient of MITLAIPVNKPGR and its binding to FPR1. A previous study has reported that MITLAIPVNKPGR and MITL increase the phagocytic ability of neutrophils,³¹⁾ and the migration mechanism was consistent with this study. Some reports demonstrated that the oral administration of MITL increased the serum tumor necrosis factor (TNF)-α in Streptococcal-treated mice¹³⁾ and the effects of anti-alopecia in etoposide-treated neonatal rats.⁴⁸⁾ Our data suggested that MITLAIPVNKPGR or MITL are food peptides with immunomodulatory effects. Previous reports show MITL has a low peroxide release effect on neutrophils.¹³⁾ In addition, MITL is readily absorbed from the intestinal gap junctions due to its molecular weight and is thought to affect the intestinal immune cells.¹³⁾ Therefore, soybean-derived MITL may have a health effect by safely summoning immune cells through migratory action and preparing immune cells against the invasion of foreign substances from the intestinal tract. One of the limitations of this study is that the function of peptides in humans was only verified in vitro.

CONCLUSION

Our study suggested that MITLAIPVNKPGR and MITL induce the migration of human polymorphonuclear leukocytes via FPR1.

Acknowledgments

We thank the nurses at Teikyo University Hospital for their cooperation in collecting blood samples.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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