innatedefenseregulatoridr-1018

Innate Defense Regulator IDR-1018 Activates Human Mast Cells Through G Protein-,

Phospholipase C-, MAPK- and NF-κB-Sensitive Pathways

KENSUKE YANASHIMA＊1), PANJIT CHIEOSILAPATHAM＊1) 2), ERI YOSHIMOTO＊1),

KO OKUMURA＊1), HIDEOKI OGAWA＊1), FRANÇOIS NIYONSABA＊1) 3)

＊1)Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan, ＊2)Department of

Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan, ＊3)Faculty of International

Liberal Arts, Juntendo University, Tokyo, Japan

Host defense (antimicrobial) peptides not only display antimicrobial activities against numerous pathogens but

also exert a broader spectrum of immune-modulating functions. Innate defense regulators (IDRs) are a class of

host defense peptides synthetically developed from natural or endogenous cationic host defense peptides. Of the

IDRs developed to date, IDR-1018 is more efficient not only in killing bacteria but also in regulating the various

functions of macrophages and neutrophils and accelerating the wound healing process. Because mast cells

intimately participate in wound healing and a number of host defense peptides involved in wound healing are also

known to activate mast cells, this study aimed to investigate the effects of IDR-1018 on mast cell activation. Here,

we showed that IDR-1018 induced the degranulation of LAD2 human mast cells and caused their production of

leukotrienes, prostaglandins and various cytokines and chemokines, including granulocyte-macrophage colony-

stimulating factor, interleukin-8, monocyte chemoattractant protein-1 and -3, macrophage-inflammatory protein-

1α and -1β, and tumor necrosis factor-α. Furthermore, IDR-1018 increased intracellular calcium mobilization and

induced mast cell chemotaxis. The mast cell activation was markedly suppressed by pertussis toxin, U-73122,

U0126, SB203580, JNK inhibitor II and NF-κB activation inhibitor II, suggesting the involvement of G-protein,

phospholipase C, ERK, p38, JNK and NF-κB pathways, respectively, in IDR-1018-induced mast cell activation.

Notably, we confirmed that IDR-1018 caused the phosphorylation of MAPKs and IκB. Altogether, the current

study suggests a novel immunomodulatory role of IDR-1018 through its ability to recruit and activate human mast

cells at the sites of inflammation and wounds.

Key words: Host defense peptide, immune system, mast cell, G-protein/PLC, MAPK/NF-κB, wound

healing

Abbreviations: CysLT; cysteinyl leukotriene, EIA;

enzyme immunoassay, ELISA; enzyme-linked

immunosorbent assay, ERK; extracellular signal-

regulated kinase, GM-CSF; granulocyte-macro-

phage colony-stimulating factor, IDR; innate

defense regulator, IL; interleukin, JNK; c-Jun N-

terminal kinase, LT; leukotriene, MAPK; mitogen-

activated protein kinase, MCP; monocyte chemoat-

tractant protein, MIP; macrophage-inflammatory

protein, NF-κB; nuclear factor-κB, PG; prostaglan-

din, PLC; phospholipase C, TNF; tumor necrosis

factor

43

Special Reviews

Juntendo Medical Journal2019. 65(1), 43-56

Corresponding author: François Niyonsaba

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine

2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

TEL: +81-3-5802-1591 FAX: +81-3-3813-5512 E-mail: [email protected]

345th Triannual Meeting of the Juntendo Medical Society: Medical Research Update〔Held on May 19, 2018〕

〔Received July 31, 2018〕〔Accepted Sep. 3, 2018〕

Copyright © 2019 The JuntendoMedical Society. This is an open access article distributed under the terms of Creative Commons Attribution Li-

cense (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited.

doi: 10.14789/jmj.2019.65.JMJ18-R12

https://creativecommons.org/licenses/by/4.0/

Introduction

Host defense peptides (HDPs), also termed

antimicrobial peptides, are a class of natural and

synthetic cationic amphipathic molecules that have

evolved to protect the host against pathogenic

microorganisms. Originally, HDPs were character-

ized by their broad-spectrum antimicrobial proper-

ties; however, recently, a plethora of studies have

demonstrated that these molecules contribute

enormously to the modulation of both innate and

adaptive immune responses 1)-3). The immunomodu-

latory activities of HDPs, such as human β-defen-

sins, cathelicidin LL-37, S100A7, etc., include, but

are not limited to, the stimulation of cytokine/

chemokine production by various cell types; the

induction of cell migration, survival, proliferation

and differentiation; the neutralization of lipopoly-

saccharide activities in macrophages; the stimula-

tion of macrophage and neutrophil phagocytosis;

the suppression of potentially harmful pro-inflam-

matory responses; and the inhibition of neutrophil

and epithelial cell apoptosis 1)-4). Furthermore, HDPs

are directly or indirectly involved in the wound

healing process by regulating inflammation, angio-

genesis, and tissue remodeling 1)-4). Therefore, HDPs

and their derivatives have recently captured

attention as potential candidates for novel anti-

infective and immunomodulatory therapies 5)6).

The important role of HDPs in protective

immunity has led to the development of innate

defense regulator (IDR) peptides that enhance the

efficiency of the immune response. IDRs are a novel

class of synthetic peptides with immunomodulatory

functions derived from natural or endogenous

cationic HDPs. Among these peptides, IDR-1, IDR-

1002, IDR-1018 and IDR-HH2, which are derived

conceptually from the bovine bactenecin, protect

against infections by selectively enhancing the

protective immune responses rather than by

exhibiting direct antimicrobial activity 7) 8). IDR-1 in

vivo protects against multi-drug resistant bacterial

infections by increasing chemokine production,

suppressing pro-inflammatory cytokines, and

recruiting phagocytes at the site of the infection 8).

Similarly, IDR-1002 protects against bacterial

infections by inducing chemokine production and

recruiting neutrophils, monocytes and macro-

phages at the infection site 9). IDR-1002 also

augments monocyte migration and adhesion to

fibronectin 10) 11). In addition, IDR-HH2, IDR-1002

and IDR-1018 elevate human neutrophil adhesion

to endothelial cells; induce neutrophil migration,

chemokine production and the release of α-defen-

sins and LL-37; suppress pro-inflammatory cyto-

kine secretion; and increase neutrophil killing of

Escherichia coli12). Of the IDRs developed thus far,

IDR-1018 is the most potent inducer of chemokine

production 13), modulates macrophage differentia-

tion 14), protects against perinatal brain injury 15),

and demonstrates anti-infective and anti-inflamma-

tory activity in mouse models 13). Furthermore,

IDR-1018 stimulates the production of specific pro-

and anti-inflammatory mediators, increases the

phagocytosis of apoptotic cells, and elevates the

expression of wound healing-related genes 14).

Importantly, IDR-1018 is more efficient than

cathelicidin LL-37 in accelerating the wound

healing process in Staphylococcus aureus-infected

porcine and non-diabetic, but not in diabetic,

murine wounds 16).

Mast cells are normally distributed throughout

virtually all tissues of the body where they become

activated and generate a whole array of biologically

active products. Although they are classically

known for their famous role in allergic inflamma-

tion, it is currently evident that mast cells are

broadly involved in numerous physiological and

pathophysiological processes 17). For instance, mast

cells are tactically located at the sites of initial

antigen entry (such as skin, airways, and gastroin-

testinal tract), making these cells serve as watch-

dogs in the protective immune response 18)-20). In

fact, mast cells strategically protect against bacte-

rial, viral and parasitic infections and contribute to

the regulation of acquired immunity through the

activation and migration of dendritic cells and T

cells 21). Moreover, mast cells exhibit anti-inflamma-

tory and immunosuppressive properties and are

involved in the promotion of or protection against

cancer 20). Because of their presence in the vicinity

of blood vessels and lymphatics, mast cells are

capable of regulating homeostasis, for example, by

influencing flow, permeability and contraction 22).

This paper was initially published at “Immunol Res, 2017; 65 (4): 920-931”

Yanashima, et al: IDR-1018 activates human mast cells

44

Furthermore, mast cells produce a spectrum of

factors, such as proteases, cytokines, chemokines,

and growth factors, that stimulate endothelial cells

and fibroblasts, leading to the promotion of angio-

genesis, re-epithelialization, and tissue remodeling

and finally accelerating wound healing 20) 23) 24).

Considering the contributions of both IDR-1018

and mast cells in the wound healing process and

that a number of HDPs, such as defensins and

cathelicidin LL-37 that accelerate wound

healing 25)-28) are also known to activate mast cells,

this study aimed to investigate the effects of IDR-

1018 on mast cells. Here, we demonstrated that

IDR-1018 markedly stimulated human mast cells to

degranulate and produce leukotrienes (LTs),

prostaglandins (PGs), and several cytokines and

chemokines. Furthermore, IDR-1018 caused tre-

mendous increases in the intracellular Ca2+ mobili-

zation and induced mast cell chemotaxis. As

evidenced by specific inhibitors of various signaling

pathways, the IDR-1018-mediated mast cell activa-

tion was controlled by the G-protein, phospholipase

C (PLC), mitogen-activated protein kinase (MAPK)

and nuclear factor-κB (NF-κB) pathways. We indeed

confirmed that IDR-1018 caused the phosphoryla-

tion of MAPKs and IκB. Altogether, the results

observed in this study provide novel evidence of the

immunomodulatory role of IDR-1018 through the

recruitment and activation of human mast cells at

the sites of inflammation and wounds.

Materials and Methods

1. Reagents

The peptides IDR-1 (KSRIVPAIPVSLL-NH2),

IDR-1002 (VQRWLIVWRIRK-NH2), and IDR-

1018 (VRLIVAVRIWRR-NH2) were synthesized

using the solid-phase method with a peptide

synthesizer (Model PSSM-8; Shimadzu, Kyoto,

Japan) using fluorenylmethoxycarbonyl (FMOC)

chemistry. The inhibitors pertussis toxin, U-73122,

U0126, SB203580, JNK inhibitor II and NF-κB

activation inhibitor II were obtained from Calbio-

chem (La Jolla, CA). The antibodies against

phosphorylated and unphosphorylated ERK, p38,

JNK and IκB were purchased from Cell Signaling

Technology (Beverly, MA). The enzyme immuno-

assay (EIA) kits for cysteinyl leukotriene (CysLT),

PGD2 and PGE2 were purchased from Cayman

Chemical Company (Ann Arbor, MI), whereas the

cytokine, chemokine, and growth factor ELISA kits

were obtained from R&D Systems (Minneapolis,

MN). The calcium assay kit was obtained from

Molecular Devices (Sunnyvale, CA).

2. Mast cell culture

The human mast cell sarcoma cell line LAD2 was

a kind gift from Dr. A. Kirshenbaum at the National

Institutes of Health (Bethesda, MD) 29). These cells

were cultured in a Stem Pro-34 serum-free

medium (Invitrogen, Carlsbad, CA) supplemented

with nutrient supplements, 2 mM L-glutamine,

100 IU/ml penicillin, 100 μg/ml streptomycin, and

100 ng/ml recombinant stem cell factor (Wako,

Osaka, Japan). The culture medium was changed

weekly, and the cells were maintained at 1 × 105

cells/ml30). The cells were occasionally assessed for

their expression of the c-Kit and FcεRI receptors.

3. β-Hexosaminidase release assay

The mast cells were washed and suspended at

2×105 cells/100 μl in Tyrodeʼs buffer as reported

previously 31), followed by stimulation with IDR-

1018 for 40 min at 37℃. After the stimulation, the

cell culture supernatants were collected by centri-

fugation and then incubated with 1.3 mg/ml

4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-

Aldrich, St. Louis, MO) for 90 min at 37℃ to

measure β-hexosaminidase activity. The β-hexosa-

minidase release was calculated as reported previ-

ously as a percentage of the total β-hexosaminidase

content from cells stimulated with 1% Triton

X-100 31). In some experiments, the mast cells were

pre-treated with various inhibitors for indicated

time periods before the stimulation.

4. Enzyme immunoassay (EIA) and enzyme-linked

immunosorbent assay (ELISA)

The mast cells were seeded at a concentration of

1×106 cells/ml and then loaded with various doses

of IDR-1018 for 30 min and 3 h for the EIA and

ELISA, respectively, at 37℃. The cell cultures were

collected by centrifugation, and the cell-free

supernatants were assayed for CysLT, PGD2, and

PGE2 contents using an EIA, and for granulocyte-

macrophage colony-stimulating factor (GM-CSF),

interleukin (IL)-8, monocyte chemoattractant pro-

tein (MCP)-1, MCP-3, macrophage-inflammatory

Juntendo Medical Journal 65(1), 2019

45

protein (MIP)-1α, MIP-1β, and tumor necrosis factor

(TNF)-α amounts using the appropriate ELISA

kits according to the manufacturerʼs instructions. In

some experiments, the mast cells were pre-incu-

bated with specific inhibitors for indicated time

periods before the stimulation with IDR-1018, and

the EIA and ELISA assays were performed as

above.

5. Chemotaxis assay

An 8-μm pore-size polyvinylpyrrolidone-free

polycarbonate membrane (Neuro Probe, Cabin

John, MD) was used to separate the mast cells

treated with various doses of IDR-1018 and was

placed above the lower compartment of a 48-well

chemotaxis micro-chamber (Neuro Probe). Mast

cells, at the density of 1.5×105 cells/50 μl, were

loaded to the upper compartments. Following a 3 h-

incubation at 37℃ in an atmosphere of humidified

air, the membrane was removed and stained with

DiffQuick (Kokusai Shiyaku, Kobe, Japan). After

the membrane was mounted onto slides, the mast

cells that had migrated and adhered to the

underside of the membrane were counted under a

light microscope. In some experiments, the mast

cells were treated with various inhibitors prior to

the assay, and chemotaxis was evaluated as

described above.

6. Measurements of intracellular Ca2+ mobilization

Mast cells were seeded at a density of 2×105 cells/

100 μl into 96-well plates coated with poly-D-lysine

(Becton-Dickinson, NJ), and an equivalent volume

of HBSS containing 20 mM HEPES, 2.5 mM

probenecid, and calcium 3 reagent (Molecular

Devices, Menlo Park, CA) was added as previously

reported 31). Following a 1 h-incubation at 37℃, the

microplate was gently centrifuged to form a

uniform monolayer of cells on the bottom of the

wells. The plate was then placed into a FlexStation

II (Molecular Devices), and 50 μl of various doses of

IDR-1018 were added. The fluorescence was

quantified using SoftMax Pro (version 5) software.

7. Western blot analysis

The mast cells (1×106 cells) were stimulated

with IDR-1018 for indicated periods, and the lysates

obtained by lysing the cells in RIPA buffer (Cell

Signaling Technology) were loaded onto a 12.5%

SDS-PAGE gel for immunoblotting. The mem-

branes were blocked using ImmunoBlock (DS

Pharma Biomedical, Osaka, Japan) and then incu-

bated with polyclonal antibodies against phosphory-

lated or unphosphorylated ERK, JNK, p38 and IκB

overnight according to the manufacturerʼs instruc-

tions. For detection, the membranes were incubated

with the Luminata Forte Western HRP substrate

(Millipore, Billerica, MA) for 5 min, and the signals

were visualized using Fujifilm LAS-4000 Plus

(Fujifilm, Tokyo, Japan). To quantify the band

intensities, a densitometry analysis was performed

using the software program Image Gauge (LAS-

4000 Plus, Fujifilm) to correct for protein loading

discrepancies.

8. Statistical analysis

The statistical analysis was performed using

either one-way ANOVA followed by the appropri-

ate post hoc test or Studentʼs t-test (Prism 6,

GraphPad Software, San Diego, CA). Data are

expressed as the means ± standard deviation. A

value of p<0.05 was considered statistically

significant.

Results

1. IDR-1018 induces human mast cell degranulation

The fact that both mast cells and IDR-1018 are

intimately involved in the orchestration of wound

healing 16) 20) and that some HDPs, such as defensins

and cathelicidins, that influence the wound healing

process also trigger mast cell functions 30) 32)-36)

inspired us to speculate that IDR-1018 may also

activate mast cells. We observed that IDR-1018

markedly caused the degranulation of human mast

cells as assessed by β-hexosaminidase release.

β-Hexosaminidase is a marker of mast cell degranu-

lation and is released in combination with

histamine 37). IDR-1018-induced mast cell degranu-

lation was dose-dependent and strongly detected at

concentrations as low as 5 μg/ml (Figure-1A). We

also found that IDR-1002 (40 μg/ml) showed a

degranulating potency comparable to that of

IDR-1018, while IDR-1 failed to cause mast cell

degranulation at the same concentration. The

trypan blue dye exclusion assay complimented with

lactate dehydrogenase activity assay showed that

the doses of IDR-1018, IDR-1002, and IDR-1 used in


46

this study were not cytotoxic to mast cells

(Figure-S1).

2. IDR-1018 induces the production of LTs and

PGs

Upon activation, mast cells release various

products, including preformed mediators, newly

synthesized lipid mediators, cytokines and

chemokines 18) 19). Given the ability of IDR-1018 to

cause mast cell degranulation, we next examined

whether this peptide induces the production of

eicosanoids, such as LTs and PGs. The presence of

LTs in IDR-1018-stimulated mast cell supernatants

was assessed using the CysLT EIA kit, which

collectively measures the contents of LTC4 , LTD4 ,

and LTE4 . As shown in Figure-1B, we found that

IDR-1018 significantly enhanced the production of

CysLT in a dose-dependent manner. Furthermore,

IDR-1018 markedly increased the production of

PGD2 but not that of PGE2 . The IDR-1018-induced

CysLT production was elevated by approximately

250-fold, while the production of PGD2 was only

increased by 6-fold. IDR-1002 but not IDR-1 also

increased the production of CysLT and PGD2 but

not PGE2 . A longer stimulation, up to 12 h, did not


47

Figure-1 IDR-1018 induces mast cell degranulation and the release of LTs and PGs(A) Mast cells were incubated with 5 to 40 μg/ml IDR-1018, 40 μg/ml IDR-1002, 40 μg/ml IDR-1, or diluent 0.01% acetic acid (Med,

medium). Following 40 min of incubation, the β-hexosaminidase released into the supernatants was measured. Values are the mean±SD of

five separate experiments compared between stimulated and non-stimulated cells (Med, medium). (B) Mast cells were stimulated for 30 min

with 5 to 40 μg/ml IDR-1018, 40 μg/ml IDR-1002, 40 μg/ml IDR-1, or diluent 0.01% acetic acid (Med, medium). The amounts of CysLT, PGD2

and PGE2 into the culture supernatants were quantified by an enzyme immunoassay. Values are shown as the mean±SD of five separate

experiments compared between stimulated and non-stimulated cells (Med, medium). ＊p<0.05, ＊＊p<0.01, ＊＊＊p<0.001, ＊＊＊＊p<0.0001.

Aβ－Hexosaminidase release（％）

CysLT（pg/ml）

CysLT

PGD2（pg/ml）

PGD2

PGE 2（pg/ml）

PGE2

Med

＊＊＊＊

＊＊＊＊

＊＊＊＊＊＊＊

＊＊

＊＊＊＊＊＊＊＊

＊＊＊＊

＊＊＊＊

＊＊＊＊＊＊＊＊＊＊＊＊

＊＊＊＊

BIDR－1002

IDR－15 10 20 40

Med

IDR －1002

IDR－15 10 20 40

Med

IDR －1002

IDR －15 10 20 40

Med

IDR－1002

IDR－1018（μg/ml）IDR－1018（μg/ml）IDR－1018（μg/ml）

IDR－1018（μg/ml）

IDR－15 10 20 40

100

80

60

40

20

0

60

45

30

15

0

800

600

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200

0

8006004002000

40,00030,00020,00010,000

＊＊＊＊

Med

IDR－1002


IDR－15 10 20 40 AD

3.5

LDH release（OD at 490 nm）

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Figure-S1 Effects of IDRs on mast cell cytotoxicity andviability

The mast cells (1×106 cells) were treated with 5-40 μg/ml IDR-

1018, 40 μg/ml IDR-1002, 40 μg/ml IDR-1, 5 μg/ml actinomycin D

(AD, Sigma-Aldrich) used as a positive control, or the diluent

alone (Med) for 3 h. The level of lactate dehydrogenase (LDH)

released in the cell supernatants was assayed using a colorimetric

Cytotoxicity Detection Kit (Roche, Mannheim, Germany). The

values obtained from three separate experiments using stimulated

and non-stimulated cells (Med) were compared. ＊＊＊＊p< 0.0001.

further increase the amounts of PGE2 (data not

shown).

3. IDR-1018-activated mast cells produce various

cytokines and chemokines

Next, we investigated the ability of IDR-1018 to

stimulate mast cell cytokine and chemokine produc-

tion. Following a 3 h- to 6 h-stimulation of mast

cells with IDR-1018, a panel of 10 cytokines and

chemokines was examined. As shown in Figure-2,

IDR-1018 selectively induced the production of

GM-CSF, IL-8, MCP-1, MIP-1α, MIP-1β, and

TNF-α. The IDR-1018-induced cytokine/chemo-

kine production was dose-dependent, and only

higher doses of IDR-1018 (20-40 μg/ml) were able

to produce significant amounts of TNF-α. A longer

incubation period did not further enhance the

production of the tested cytokines and chemokines.

IDR-1002 but not IDR-1 also noticeably enhanced

the production of above cytokines and chemokines.

4. IDR-1018 enhances mast cell migration

IDR-1018 has been reported to promote human

neutrophil chemotaxis 12) and induce the production

of chemokines involved in immune cell

recruitment 13). Therefore, we hypothesized that

IDR-1018 would also influence human mast cell

migration. Corroborating our hypothesis, we found

that IDR-1018 considerably induced mast cell

migration in a bell-shaped concentration-depend-

ent curve, which is a feature common for chemotac-

tic migration. This chemotaxis was first observed

with IDR-1018 concentrations as low as 2.5 μg/ml

and peaked with approximately 10 μg/ml, display-

ing 5-fold increases compared to that in the control

cells. Higher concentrations (20 μg/ml) resulted in

the loss of cell migration (Figure-3A). We con-

firmed that IDR-1002 also markedly induced mast

cell chemotaxis.

Because intracellular Ca2+ mobilization has been

implicated in the cell migration of various cell types,

including mast cells 38), we next explored the

possibility that IDR-1018 would mobilize intracellu-

lar Ca2+ in mast cells. Consistent with our specula-

tion, the results revealed substantial increases in

intracellular Ca2+ mobilization in the mast cells

stimulated with IDR-1018 compared to that in the

control cells. The pattern of intracellular Ca2+

mobilization elicited by IDR-1018 displayed a

concentration-dependent response. Following the

addition of increasing doses of IDR-1018, the

response occurred rapidly with an initial peak

observed at approximately 50 s, followed by a

sustained and stable plateau (Figure-3B).


48

Med

GM－CSF（pg/ml）

MIP－1α（pg/ml）

MIP－1β（pg/ml）

TNF －α（pg/ml）

IL－8（pg/ml）

MCP －1（pg/ml）

GM－CSF

MIP－1α MIP－1β TNF－α

IL－8 MCP－1

＊＊＊＊＊＊

＊＊＊

＊＊＊

＊＊＊

＊＊＊＊＊＊

＊＊＊

＊＊＊

＊＊＊

＊＊

＊＊

＊＊

＊＊

＊＊

＊＊

＊

＊

＊

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＊＊＊＊

＊＊＊＊＊＊＊＊

＊＊＊＊＊＊＊＊＊＊＊＊

IDR－1002

IDR－15 10 20 40

IDR－1018（μg/ml） Med

IDR－1002

IDR－15 10 20 40


IDR－1002

IDR－15 10 20 40


Med

IDR－1002

IDR－15 10 20 40


IDR－1002

IDR－15 10 20 40


IDR－1002

IDR－15 10 20 40


1,000

800

600

400

200

0

800

600

400

200

0

4,000

3,200

2,400

1,600

800

0

4,000

3,200

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800

0

500

400

300

200

100

0

150

100

50

0

Figure-2 IDR-1018 increases the production of cytokines and chemokines in mast cellsCells were incubated with 5 to 40 μg/ml IDR-1018, 40 μg/ml IDR-1002, 40 μg/ml IDR-1, or diluent 0.01% acetic acid (Med,

medium) for 3 h. After the stimulation, the concentrations of GM-CSF, IL-8, MCP-1, MIP-1α, MIP-1β and TNF-α released into the

culture supernatants were quantified by ELISA. The values are shown as the mean±SD of six separate experiments compared

between the stimulated and non-stimulated cells (Med, medium). ＊p<0.05, ＊＊p<0.01, ＊＊＊p<0.001, ＊＊＊＊p<0.0001.

5. IDR-1018 activates pathways that involve G-

proteins and PLC in mast cells

A number of HDPs has been shown to activate

mast cells through the G-protein and PLC

pathways 32) 34) 39). Therefore, to characterize the

molecular mechanism for IDR-1018-induced mast

cell activation, the cells were pre-treated with

pertussis toxin or U-73122, specific inhibitors of

G-protein and PLC, respectively. We observed that

both pertussis toxin and U-73122 noticeably

suppressed the mast cell degranulation (Figure-

4A), the production of CysLT and PGD2 (Figure-

4B), and the secretion of GM-CSF, IL-8, MCP-1,

MIP-1α, MIP-1β, and TNF-α (Figure-4C). Fur-

thermore, pertussis toxin and U-73122 remarkably

inhibited IDR-1018-elicited intracellular Ca 2+ mobi-

lization (Figure-4D) and chemotaxis (Figure-4E).

These findings demonstrate that IDR-1018 acts via

G-protein and PLC pathways to stimulate human

mast cells.

6. MAPK and NF-κB signaling pathways are

required for the IDR-1018-induced mast cell

stimulation

We next attempted to clarify the downstream

signal transduction mechanism by which IDR-1018

activates mast cells. We focused on the MAPK and

NF-κB pathways because these signaling pathways

are implicated in HDP-mediated cell activation,

including mast cells 30) 32) 39). Given the contribution

of the MAPK and NF-κB pathways to cytokine/

chemokine production, we pre-treated mast cells

with specific inhibitors of MAPKs and NF-κB prior

to the stimulation with IDR-1018 and analyzed their

effect on the cytokine/chemokine production. As

shown in Figure-5, U0126 (ERK inhibitor),

SB203580 (p38 inhibitor), JNK inhibitor II, and

NF-κB activation inhibitor II strikingly reduced the

IDR-1018-induced production of GM-CSF, IL-8,

MCP-1, MIP-1α, MIP-1β, and TNF-α. Of these

inhibitors, U0126 and NF-κB activation inhibitor II

were apparently stronger than the others. Alto-

gether, these data suggest that the activation of the

MAPK and NF-κB pathways is indispensable for

the IDR-1018-mediated mast cell activation.

To confirm whether IDR-1018 indeed activates

the MAPK and NF-κB pathways in mast cells, the

effects of IDR-1018 on MAPK and IκB phosphoryla-

tion were examined. Figure-6 shows that IDR-1018

rapidly and remarkably enhanced the phosphoryla-

tion of ERK, JNK, p38, and IκB. The increased

phosphorylation of MAPK ERK, JNK and p38 was

detected as early as 5 min post stimulation, whereas

the IκB phosphorylation was transiently noticed at

15 min following the mast cell stimulation with IDR-

1018. We confirmed that IDR-1002 also similarly


49

＊＊＊

＊＊

＊＊＊＊

＊＊＊

＊＊＊＊

Med

IDR－1002

IDR－15

2.5 10 20


200

A

B

Migrated cell number

Fluorescence counts（×1000）

150

100

50

0

200

150

100

50

00 30

1018

60 90

Time（sec）

120 150 180

Med5μg/ml10μg/ml20μg/ml

Figure-3 IDR-1018 mediates mast cell chemotaxis andincreases intracellular Ca2+ mobilization

(A) Cells were seeded in the upper wells of a chemotaxis

micro-chamber and allowed to migrate towards 5 to 40 μg/ml

IDR-1018, 10 μg/ml IDR-1002, 10 μg/ml IDR-1, or diluent 0.01%

acetic acid (Med, medium) for 3 h. Cells that migrated through

the polycarbonate membrane were counting under a light

microscope in three randomly chosen high-power fields (HPF).

Values are shown as the mean±SD of four separate experiments

compared between stimulated and non-stimulated cells (Med,

medium). ＊＊p < 0.01, ＊＊＊p < 0.001, ＊＊＊＊p < 0.0001. (B) Cells

were incubated for 1 h in Hankʼs balanced salt solution containing

HEPES, probenecid and a calcium 3 reagent. Cells were then

loaded with 5 to 20 μg/ml IDR-1018, or diluent 0.01% acetic acid

(Med, medium). The results show one representative experiment

from five independent experiments yielding similar results and

are shown as changes in fluorescence.


50

＊＊＊＊

＊＊＊＊CysLT

GM－CSF IL－8 MCP－1

MIP－1α TNF－αMIP－1β

PGD2

＊＊＊＊

＊＊＊＊

＊＊＊＊

＊＊＊＊

＊＊＊＊

＊＊＊

＊＊＊

＊＊＊＊

＃＃＃＃＃＃＃＃

＃＃＃＃

＃＃＃＃

＃＃＃＃＃＃＃

＃＃＃＃＃＃＃

＃＃＃＃＃＃＃

＃＃＃＃＃＃＃＃

＃＃＃＃

＃＃＃＃＃＃＃＃

＃＃

＃＃＃＃

＃＃＃＃＃＃＃＃

Med

100A

B

C

D

E

β－Hexosaminidase release（％）

CysLT（pg/ml）

GM－CSF（pg/ml）


TNF－α（pg/ml）

MCP －1（pg/ml）


IL－8（pg/ml）

PGD2（pg/ml）

Fluorescence counts（×1000）

Migrated cell number

Med IDR－1018 ＋PTx ＋U－73122

Med IDR－1018 ＋PTx ＋U－73122 Med IDR－1018 ＋PTx ＋U－73122

Med IDR－1018 ＋PTx ＋U－73122

Med

IDR－1018

IDR－1018 ＋PTx ＋U－73122

Med IDR－1018 ＋PTx ＋U－73122



80

60

40

20

0

20,00017,50015,00012,50010,0002,0001,5001,0005000

800

600

400

200

0

800

600

400

200

0

800

600

400

200

0

5,000

4,000

3,000

2,000

1,000

0

4,000

3,000

2,000

1,000

0

600

450

300

150

0

250

200

150

100

50

0

200

150

100

50

0

200

150

100

50

0

0 30 60 90Time（sec）

120 150 180

1018＋PTx＋U73122

Figure-4 Pertussis toxin and U-73122 inhibit IDR-1018-induced mast cell activationMast cells were pre-treated with 200 ng/ml pertussis toxin (PTx), 20 μM U-73122 or 0.1% DMSO for 2 h. Pre-treated cells were

stimulated with 20 μg/ml IDR-1018 or diluent 0.01% acetic acid (Med, medium) for 40 min for β-hexosaminidase release (A) or

stimulated for 30 min for CysLT and PGD2 release (B). Pre-treated cells were also evaluated for cytokine and chemokine production

following 3 h of stimulation with 40 μg/ml IDR-1018 or diluent 0.01% acetic acid (Med, medium), and the levels of GM-CSF, IL-8,

MCP-1, MIP-1α, MIP-1β and TNF-α released into the supernatants were determined by ELISA (C). Furthermore, the pre-treated

cells were stimulated with 10 μg/ml IDR-1018 or diluent 0.01% acetic acid (Med, medium), and the intracellular Ca2+ mobilization

(D) and chemotaxis (E) assays were then performed. Values are the mean±SD of four to six separate experiments. ＊＊＊p<0.001

and ＊＊＊＊p<0.0001 for comparisons between the untreated cells (Med, medium) and the stimulated groups without the inhibitors

(IDR-1018). ##p<0.01, ###p<0.001, and ####p<0.0001 for comparisons between the presence and absence of the inhibitors.

increased MAPK and IκB phosphorylation (data

not shown).

Discussion

In addition to participating in allergic and

inflammatory reactions, mast cells also play a

pivotal role in immune regulation, angiogenesis and

wound healing 30) 32) 39). Because the immunoregula-

tory peptide IDR-1018 is also involved in the

acceleration of the wound healing process 16), we

hypothesize that it could also activate mast cells.

Consistent with this hypothesis, we herein demon-

strated that IDR-1018 induced mast cell degranula-

tion, the production of eicosanoids, cytokines and

chemokines, and enhanced cell migration. Further-

more, the IDR-1018-induced mast cell activation

was mediated by G-protein-, PLC-, MAPK- and

NF-κB-sensitive pathways. Therefore, the current

study provides evidence that IDR-1018 may

enhance the recruitment and activation of mast

cells at the sites of inflammation and wounds.

Mast cells are preponderantly distributed in the

skin, airways and gut and are closely associated

with blood vessels and nerve endings where they

act individually or together with other immune cells

to mount specific responses after inflammation and

or injury 18)-20). Upon being activated, mast cells

release a plethora of products that are either

preformed mediators stored in the granules (ex.

histamine) or de novo synthesized and secreted

mediators (ex. lipids, cytokines and chemokines).

Histamine, LTs and PGs serve to promote inflam-

mation, vasodilation and wound healing 20) 40) 41).

Other mast cell products, such as proteases,

cytokines, chemokines and growth factors, also

orchestrate the inflammatory response, tissue

remodeling, angiogenesis and wound

healing 20) 23) 40) 41). In addition, mast cells are

required for the resolution of inflammation and the

maintenance of tissue homeostasis. For example,

mast cells can degrade certain types of toxins and

venoms 17), participate in the prevention of tissue

damage induced by ultraviolet radiation, and

promote allograft tolerance by interacting with

regulatory T cells 23). The observation that

IDR-1018 induced mast cell degranulation and the

secretion of lipids, cytokines and chemokines


51

GM－CSF IL－8MCP－1

MIP－1α TNF－αMIP－1β

＊＊＊＊＊＊＊＊

＊＊＊＊

＊＊＊＊＊＊＊

＊＊＊＊

＃＃＃＃

＃＃＃＃

＃＃＃＃

＃＃＃＃

＃＃＃＃

＃＃＃＃

＃＃

＃＃＃＃

＃＃＃＃

＃＃＃＃＃

＃＃＃

＃＃＃＃＃＃＃＃＃

＃＃＃

＃＃＃＃＃＃＃＃＃＃＃＃

＃＃＃＃＃＃＃＃

＃＃＃＃

GM－CSF（pg/ml）


TNF－α（pg/ml）

MCP－1（pg/ml）


IL－8（pg/ml）

Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR－1018

Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR －1018

Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR －1018

Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR－1018Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR－1018

Med

＋U0126

＋SB203580

＋JBK inh Ⅱ

＋NFkBAⅢ

IDR －1018

600

400

200

0

8,000

6,000

4,000

2,000

0

800

600

400

200

0

4,000

3,000

2,000

1,000

0

600

450

300

150

0

250

200

150

100

50

0

Figure-5 IDR-1018-induced mast cell cytokine and chemokine production is mediated by the MAPK and NF-κB pathwaysMast cells were pre-treated with 10 μMU0126, SB203580, JNK inhibitor II (JNK inh II), NF-κB activation inhibitor II (NFκBAII) or

0.1% DMSO for 2 h. The cells were then stimulated with 40 μg/ml IDR-1018 or diluent 0.01% acetic acid (Med, medium) for 3 h, and

the amounts of GM-CSF, IL-8, MCP-1, MIP-1α, MIP-1β and TNF-α released into the supernatants were determined by ELISA.

Values are the mean±SD of five separate experiments. ＊＊＊p<0.001 and ＊＊＊＊p<0.0001 for comparisons between the untreated

cells (Med, medium) and the stimulated groups without inhibitors (IDR-1018). ##p<0.01, ###p<0.001, and ####p<0.0001 for

comparisons between the presence and absence of inhibitors.

suggests that this peptide plays a key role in

immune regulation via mast cell activation. In this

study, IDR-1002 appeared to be similar to or

slightly less potent than IDR-1018 in activating

mast cells. These data are consistent with results of

previous studies showing that both IDR-1002 and

IDR-1018 similarly exhibit antimicrobial activity

against Mycobacterium tuberculosis42), modulate

inflammation 43) 44), and regulate neutrophil

functions 12) through the same signaling

pathways 43) 45). In contrast, although IDR-1 enhan-

ces the levels of chemokines while reducing pro-

inflammatory cytokine production by monocytes 8),

this peptide lacks immunomodulatory activity in

fibroblasts 43). In the current study, IDR-1 also failed

to activate mast cells, suggesting that IDRs act

differently depending upon the cell type.

A number of reports have demonstrated that

mast cells are involved in multiple stages of wound

healing by enhancing acute inflammation, promot-

ing re-epithelialization and angiogenesis, and stimu-

lating scarring 24). Among the angiogenic factors

produced by mast cells, we observed that IDR-1018

significantly induced the production of vascular

endothelial growth factor and fibroblast growth

factor (data not shown). In addition, IDR-1018

evoked the secretion of various cytokines and

chemokines, such as GM-CSF, IL-8, MCP-1, MIP-

1α, MIP-1β and TNF-α, which play important roles

in many inflammatory responses and wound

healing. For example, the cytokines GM-CSF and

TNF-α are involved in neovascularization, tissue

remodeling and re-epithelialization, which are

imperative stages of the wound healing

process 46) 47). Furthermore, the observations that

the chemokines IL-8, MCP-1, MIP-1α and MIP-1β

promote the recruitment of neutrophils, macro-

phages, lymphocytes, and mast cells at the wound

sites have led to the conclusion that these molecules

contribute to epithelialization, tissue remodeling,


52

＊＊

＊＊

＊

＊＊＊

1.5

Ratio（p－ERK/ERK）

Ratio（p－p38/p38）

Ratio（p－JNK/JNK）

Ratio（p －lκB/lκB）

1.2

0.9

0.6

0.3

0.0

1.5

1.2

0.9

0.6

0.3

0.0

1.5

1.2

0.9

0.6

0.3

0.0

1.5

1.2

0.9

0.6

0.3

0.0

ERK JNK

p38 lκB

p－lκBp－p38

p－JNKp－ERK

Med 5 15 30 60（min） Med 5 15 30 60（min）

Figure-6 IDR-1018 induces the phosphorylation of MAPKs and IκBMast cells were incubated with 20 μg/ml of IDR-1018 or diluent 0.01% acetic acid (Med, medium) for 5 to 60 min, lysed, and then

equal amounts of protein were subjected to 12.5% SDS-PAGE. The membranes were stained with antibodies against

phosphorylated or unphosphorylated ERK (p-ERK and ERK), JNK (p-JNK and JNK), p38 (p-p38 and p38) and IκB (p-IκB and

IκB). The results show one representative experiment of four separate experiments yielding similar results. Bands were quantified

by densitometry to correct for protein loading discrepancies. The data represent the ratio of the intensity of phosphorylated protein

(p-ERK, p-JNK, p-p38 or p-IκB) divided by total protein (ERK, JNK, p38 or IκB). Values are the mean±SD of four independent

experiments. ＊p<0.05 as compared between stimulated and non-stimulated cells.

and angiogenesis 48). In fact, IL-8, MCP-1, MIP-1α

and MIP-1β regulate the expression of

metalloproteinases 49), which play a critical role in

regulating the extracellular matrix degradation and

deposition that are essential for wound

re-epithelialization 50). Furthermore, IL-8 promotes

re-epithelialization and tissue remodeling through

the induction of leukocyte migration and

proliferation 51). MCP-1-deficient mice show

delayed wound angiogenesis and re-epithelializa-

tion, confirming the importance of this chemokine in

wound healing 52), and MCP-3 targets neutrophils

and other immune cells to promote angiogenesis 53).

Altogether, these observations suggest that by

recruiting mast cells to the wounds and activating

these cells to release histamine, eicosanoids, cyto-

kines and chemokines, IDR-1018 may participate in

the acceleration of wound healing.

Mast cells are equipped with a large repertoire of

cell surface molecules that facilitate their interac-

tion with various stimulants. To understand the

mechanisms underlying IDR-1018-mediated mast

cell stimulation, we examined the role of the G-

protein and PLC pathways, because some HDPs,

such as human β-defensins and cathelicidin LL-37,

have been shown to activate mast cells through

these pathways 34) 39). We observed that specific

inhibitors of G-protein and PLCβ, pertussis toxin

and U-73122, respectively, abolished the IDR-1018-

mediated mast cell degranulation, production of

lipids, cytokines and chemokines, intracellular Ca2+

mobilization and chemotaxis. Following activation

of a G protein-coupled receptor that is coupled to a

Gq, the α-subunit of Gq induces activity in the

PLCβ, which catalyzes the generation of inositol

1,4,5-triphosphate, resulting in intracellular Ca2+

mobilization 54). Although PLCβ is typically acti-

vated by Gqα, it has been demonstrated that the βγ

subunits of G proteins dissociated from Gi/oα also

activate PLCβ 55) 56). Therefore, given that pertussis

toxin inhibits Gi/oα but not Gqα 57), we can conclude

that Giα and/or Goα are implicated in IDR-1018-

induced activation of PLCβ, leading to intracellular

Ca2+ mobilization in mast cells. Intracellular Ca2+ is

critical for mast cell degranulation, the release of

lipid mediators and migration 58). In this study, IDR-

1018 induced intracellular Ca2+ mobilization and

enhanced chemotaxis of mast cells. Mast cells

accumulate at the sites of inflammation and wounds,

and this accumulation requires directed migration

(chemotaxis) of the cells 59).

The involvement of the G-protein and PLCβ

pathways in IDR-1018-induced mast cell activation

suggests that IDR-1018 likely stimulates mast cells

via a receptor signaling pathway. IDRs, including

IDR-1018, belong to a new class of HDPs, and to

date, it is unknown whether they have specific

receptor(s). Thus, further studies are required to

clarify the receptor(s) through which IDRs activate

mast cells. Among candidate receptors, G pro-

tein-coupled Mas-related gene X (MrgX) recep-

tors are of particular interest. In fact, although

MrgX receptors are predominantly detected in

human neurons, recent reports have shown that

they are also expressed in mast cells 60), where they

modulate cytokine/chemokine production and cell

migration 35) 36). Interestingly, MrgXs bind to vari-

ous ligands, including mast cell stimulants such as

bovine adrenal medulla 8-22 peptide (for

MrgX1) 61); substance P, cortistatin, vasoactive

intestinal peptide and compound 48/80 (for

MrgX2) 60), and HDPs such as hBDs, LL-37 and

angiogenic antimicrobial peptide AG-30/5C (for

MrgX2-X4), which also trigger mast cell

activation 35) 36) 62). Nevertheless, we cannot exclude

that IDRs may activate mast cells in a non-receptor

signaling pathway as is the case with certain mast

cell secretagogues 63).

Our evaluation of the downstream signaling

pathway of the IDR-1018-mediated mast cell

activation demonstrated that both the MAPK and

NF-κB pathways are involved. When activated, the

MAPK and NF-κB pathways are capable of

mediating various effector functions, such as the

generation of cytokines and chemokines, expression

of adhesion molecules, promotion of cell growth and

differentiation 64) 65). We found that IDR-1018

evoked a rapid phosphorylation of MAPK ERK,

JNK and p38, and IκB, and this phosphorylation was

necessary for the mast cell activation as specific

inhibitors of MAPK and NF-κB markedly sup-

pressed the IDR-1018-induced mast cell production

of cytokines and chemokines. Defensins and LL-37

also stimulate the production of cytokines and

chemokines by mast cells via MAPK- and NF-κB-

sensitive pathways 30) 32) 66).

In conclusion, our study shows that IDR-1018

could effectively induce the recruitment and


53

activation of mast cells. IDR-1018-mediated mast

cell activation included degranulation and the

release of numerous lipid mediators, cytokines, and

chemokines. Given the contribution of mast cells to

the regulation of innate and acquired immunity and

wound healing, our study suggests that IDR-1018

may be useful for boosting immune responses and

accelerating the wound healing process by accumu-

lating and activating mast cells at inflammatory and

wound sites.

Acknowledgements

Wewould like to express our deepest gratitude to

all members of the Atopy (Allergy) Research

Center, Juntendo University Graduate School of

Medicine for their comments and Michiyo Matsu-

moto for secretarial assistance. This work was

partially supported by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Culture,

Sports, Science and Technology of Japan (Grant

number: 26461703 to F. N.) and the Atopy

(Allergy) Research Center, Juntendo University,

Tokyo, Japan.

Conflicts of interest

The authors declare that they have no conflicts of

interest.

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