innatedefenseregulatoridr-1018
TRANSCRIPT
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
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
Yanashima, et al: IDR-1018 activates human mast cells
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
Juntendo Medical Journal 65(1), 2019
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
400
200
0
8006004002000
40,00030,00020,00010,000
****
Med
IDR-1002
IDR-1018(μg/ml)
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).
Yanashima, et al: IDR-1018 activates human mast cells
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
******
***
***
***
******
***
***
***
**
**
**
**
**
**
*
*
*
**** ****
****
**** ****
************
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml) Med
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml) Med
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml)
Med
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml) Med
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml) Med
IDR-1002
IDR-15 10 20 40
IDR-1018(μg/ml)
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
2,400
1,600
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
Juntendo Medical Journal 65(1), 2019
49
***
**
****
***
****
Med
IDR-1002
IDR-15
2.5 10 20
IDR-1018(μg/ml)
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.
Yanashima, et al: IDR-1018 activates human mast cells
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)
MIP-1α(pg/ml)
TNF-α(pg/ml)
MCP -1(pg/ml)
MIP-1β(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
Med IDR-1018 +PTx +U-73122 Med IDR-1018 +PTx +U-73122
Med 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
Juntendo Medical Journal 65(1), 2019
51
GM-CSF IL-8MCP-1
MIP-1α TNF-αMIP-1β
**** ****
****
*******
****
####
####
####
####
####
####
##
####
## ##
#####
###
###### ###
###
######## ####
########
####
GM-CSF(pg/ml)
MIP-1α(pg/ml)
TNF-α(pg/ml)
MCP-1(pg/ml)
MIP-1β(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,
Yanashima, et al: IDR-1018 activates human mast cells
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
Juntendo Medical Journal 65(1), 2019
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.
References
1) Niyonsaba F, Kiatsurayanon C, Ogawa H: The role of
human beta-defensins in allergic diseases. Clin Exp
Allergy, 2016; 46: 1522-1530.
2) Niyonsaba F, Nagaoka I, Ogawa H: Human defensins
and cathelicidins in the skin: beyond direct antimicro-
bial properties. Crit Rev Immunol, 2006; 26: 545-576.
3) Niyonsaba F, Nagaoka I, Ogawa H, Okumura K:
Multifunctional antimicrobial proteins and peptides:
natural activators of immune systems. Curr Pharm Des,
2009; 15: 2393-2413.
4) Oppenheim JJ, Yang D: Alarmins: chemotactic activa-
tors of immune responses. Curr Opin Immunol, 2005; 17:
359-365.
5) Hancock RE, Nijnik A, Philpott DJ: Modulating immun-
ity as a therapy for bacterial infections. Nat Rev
Microbiol, 2012; 10: 243-254.
6) Yeung AT, Gellatly SL, Hancock RE: Multifunctional
cationic host defence peptides and their clinical applica-
tions. Cell Mol Life Sci, 2011; 68: 2161-2176.
7) Easton DM, Nijnik A, Mayer ML, Hancock RE: Potential
of immunomodulatory host defense peptides as novel
anti-infectives. Trends Biotechnol, 2009; 27: 582-590.
8) Scott MG, Dullaghan E, Mookherjee N, et al: An anti-
infective peptide that selectively modulates the innate
immune response. Nat Biotechnol, 2007; 25: 465-472.
9) Nijnik A, Madera L, Ma S, et al: Synthetic cationic
peptide IDR-1002 provides protection against bacterial
infections through chemokine induction and enhanced
leukocyte recruitment. J Immunol, 2010; 184: 2539-
2550.
10) Madera L, Hancock RE: Synthetic immunomodulatory
peptide IDR-1002 enhances monocyte migration and
adhesion on fibronectin. J Innate Immun, 2012; 4: 553-
568.
11) Madera L, Hancock RE: Anti-infective peptide IDR-
1002 augments monocyte chemotaxis towards CCR5
chemokines. Biochem Biophys Res Commun, 2015; 464:
800-806.
12) Niyonsaba F, Madera L, Afacan N, Okumura K, Ogawa
H, Hancock RE: The innate defense regulator peptides
IDR-HH2, IDR-1002, and IDR-1018 modulate human
neutrophil functions. J Leukoc Biol, 2013; 94: 159-170.
13) Wieczorek M, Jenssen H, Kindrachuk J, et al: Structural
studies of a peptide with immune modulating and direct
antimicrobial activity. Chem Biol, 2010; 17: 970-980.
14) Pena OM, Afacan N, Pistolic J, et al: Synthetic cationic
peptide IDR-1018 modulates human macrophage differ-
entiation. PLoS One, 2013; 8: e52449.
15) Bolouri H, Sävman K, Wang W, et al: Innate defense
regulator peptide 1018 protects against perinatal brain
injury. Ann Neurol, 2014; 75: 395-410.
16) Steinstraesser L, Hirsch T, Schulte M, et al: Innate
defense regulator peptide 1018 in wound healing and
wound infection. PLoS One, 2012; 7: e39373.
17) Rodewald HR, Feyerabend TB: Widespread immuno-
logical functions of mast cells: fact or fiction? Immunity,
2012; 37: 13-24.
18) Abraham SN, St John AL: Mast cell-orchestrated
immunity to pathogens. Nat Rev Immunol, 2010; 10:
440-452.
19) Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky
AM, Williams CM, Tsai M: Mast cells as“tunable”
effector and immunoregulatory cells: recent advances.
Annu Rev Immunol, 2005; 23: 749-786.
20) Moon TC, St Laurent CD, Morris KE, et al: Advances in
mast cell biology: new understanding of heterogeneity
and function. Mucosal Immunol, 2010; 3: 111-128.
21) Galli SJ, Tsai M, Piliponsky AM: The development of
allergic inflammation. Nature, 2008; 454: 445-454.
22) Maurer M, Theoharides T, Granstein RD, et al: What is
the physiological function of mast cells? Exp Dermatol,
2003; 12: 886-910.
23) Beghdadi W, Madjene LC, Benhamou M, et al: Mast cells
as cellular sensors in inflammation and immunity. Front
Immunol, 2011; 2: 37.
24) Wulff BC, Wilgus TA: Mast cell activity in the healing
Yanashima, et al: IDR-1018 activates human mast cells
54
wound: more than meets the eye? Exp Dermatol, 2013;
22: 507-510.
25) Gronberg A, Mahlapuu M, Stahle M, Whately-Smith C,
Rollman O: Treatment with LL-37 is safe and effective
in enhancing healing of hard-to-heal venous leg ulcers:
a randomized, placebo-controlled clinical trial. Wound
Repair Regen, 2014; 22: 613-621.
26) Hirsch T, Spielmann M, Zuhaili B, et al: Human beta-
defensin-3 promotes wound healing in infected diabetic
wounds. J Gene Med, 2009; 11: 220-228.
27) Mangoni ML, McDermott AM, Zasloff M: Antimicrobial
peptides and wound healing: biological and therapeutic
considerations. Exp Dermatol, 2016; 25: 167-173.
28) Niyonsaba F, Ushio H, Nakano N, et al: Antimicrobial
peptides human beta-defensins stimulate epidermal
keratinocyte migration, proliferation and production of
proinflammatory cytokines and chemokines. J Invest
Dermatol, 2007; 127: 594-604.
29) Kirshenbaum AS, Akin C, Wu Y, et al: Characterization
of novel stem cell factor responsive human mast cell
lines LAD 1 and 2 established from a patient with mast
cell sarcoma/leukemia; activation following aggregation
of FcepsilonRI or FcgammaRI. Leuk Res, 2003; 27: 677-
682.
30) Niyonsaba F, Ushio H, Hara M, et al: Antimicrobial
peptides human beta-defensins and cathelicidin LL-37
induce the secretion of a pruritogenic cytokine IL-31 by
human mast cells. J Immunol, 2010; 184: 3526-3534.
31) Aung G, Niyonsaba F, Ushio H, et al: Catestatin, a
neuroendocrine antimicrobial peptide, induces human
mast cell migration, degranulation and production of
cytokines and chemokines. Immunology, 2011; 132:
527-539.
32) Chen X, Niyonsaba F, Ushio H, et al: Antimicrobial
peptides human beta-defensin (hBD)-3 and hBD-4
activate mast cells and increase skin vascular permeabil-
ity. Eur J Immunol, 2007; 37: 434-444.
33) Niyonsaba F, Iwabuchi K, Someya A, et al: A cathelici-
din family of human antibacterial peptide LL-37 induces
mast cell chemotaxis. Immunology, 2002; 106: 20-26.
34) Niyonsaba F, Someya A, Hirata M, Ogawa H, Nagaoka I:
Evaluation of the effects of peptide antibiotics human
beta-defensins-1/-2 and LL-37 on histamine release
and prostaglandin D(2) production from mast cells. Eur
J Immunol, 2001; 31: 1066-1075.
35) Subramanian H, Gupta K, Guo Q, Price R, Ali H: Mas-
related gene X2 (MrgX2) is a novel G protein-coupled
receptor for the antimicrobial peptide LL-37 in human
mast cells: resistance to receptor phosphorylation,
desensitization, and internalization. J Biol Chem, 2011;
286: 44739-44749.
36) Subramanian H, Gupta K, Lee D, Bayir AK, Ahn H, Ali H:
beta-Defensins activate human mast cells via Mas-
related gene X2. J Immunol, 2013; 191: 345-352.
37) Schwartz LB, Austen KF, Wasserman SI: Immunologic
release of beta-hexosaminidase and beta-glucuronidase
from purified rat serosal mast cells. J Immunol, 1979;
123: 1445-1450.
38) Lee J, Veatch SL, Baird B, Holowka D: Molecular
mechanisms of spontaneous and directed mast cell
motility. J Leukoc Biol, 2012; 92: 1029-1041.
39) Pundir P, Kulka M: The role of G protein-coupled
receptors in mast cell activation by antimicrobial
peptides: is there a connection? Immunol Cell Biol, 2010;
88: 632-640.
40) Coneely J, Kennelly R, Bouchier-Hayes D, Winter DC:
Mast cell degranulation is essential for anastomotic
healing in well perfused and poorly perfused rat colon. J
Surg Res, 2010; 164: e73-76.
41) Noli C, Miolo A: The mast cell in wound healing. Vet
Dermatol, 2001; 12: 303-313.
42) Rivas-Santiago B, Serrano CJ, Enciso-Moreno JA:
Susceptibility to infectious diseases based on antimicro-
bial peptide production. Infect Immun, 2009; 77: 4690-
4695.
43) Turner-Brannen E, Choi KY, Lippert DN, et al: Modula-
tion of interleukin-1beta-induced inflammatory responses
by a synthetic cationic innate defence regulator peptide,
IDR-1002, in synovial fibroblasts. Arthritis Res Ther,
2011; 13: R129.
44) Haney EF, Mansour SC, Hilchie AL, de la Fuente-Núñez
C, Hancock RE: High throughput screening methods for
assessing antibiofilm and immunomodulatory activities
of synthetic peptides. Peptides, 2015; 71: 276-285.
45) Huante-Mendoza A, Silva-García O, Oviedo-Boyso J,
Hancock RE, Baizabal-Aguirre VM: Peptide IDR-1002
inhibits NF-kappaB nuclear translocation by inhibition
of IkappaBalpha degradation and activates p38/ERK1/
2-MSK1-dependent CREB phosphorylation in macro-
phages stimulated with lipopolysaccharide. Front Immu-
nol, 2016; 7: 533.
46) Mann A, Niekisch K, Schirmacher P, Blessing M:
Granulocyte-macrophage colony-stimulating factor is
essential for normal wound healing. J Investig Dermatol
Symp Proc, 2006; 11: 87-92.
47) Williams CM, Galli SJ: Mast cells can amplify airway
reactivity and features of chronic inflammation in an
asthma model in mice. J Exp Med, 2000; 192: 455-462.
48) Gillitzer R, Goebeler M: Chemokines in cutaneous
wound healing. J Leukoc Biol, 2001; 69: 513-521.
49) Johnatty RN, Taub DD, Reeder SP, et al: Cytokine and
chemokine regulation of proMMP-9 and TIMP-1
production by human peripheral blood lymphocytes. J
Immunol, 1997; 158: 2327-2333.
50) Caley MP, Martins VL, OʼToole EA: Metalloproteinases
and wound healing. Adv Wound Care (New Rochelle),
2015; 4: 225-234.
51) Engelhardt E, Toksoy A, Goebeler M, Debus S, Bröcker
EB, Gillitzer R: Chemokines IL-8, GROalpha, MCP-1,
IP-10, and Mig are sequentially and differentially
expressed during phase-specific infiltration of leukocyte
subsets in human wound healing. Am J Pathol, 1998;
153: 1849-1860.
52) Barrientos S, Stojadinovic O, Golinko MS, Brem H,
Juntendo Medical Journal 65(1), 2019
55
Tomic-Canic M: Growth factors and cytokines in wound
healing. Wound Repair Regen, 2008; 16: 585-601.
53) Bousquenaud M, Schwartz C, Léonard F, Rolland-
Turner M, Wagner D, Devaux Y: Monocyte chemotactic
protein 3 is a homing factor for circulating angiogenic
cells. Cardiovasc Res, 2012; 94: 519-525.
54) Werry TD, Wilkinson GF, Willars GB: Mechanisms of
cross-talk between G-protein-coupled receptors result-
ing in enhanced release of intracellular Ca2+. Biochem J,
2003; 374 (Pt 2): 281-296.
55) Kehrl JH: Heterotrimeric G protein signaling: roles in
immune function and fine-tuning by RGS proteins.
Immunity, 1998; 8: 1-10.
56) Knall C, Johnson GL: G-protein regulatory pathways:
rocketing into the twenty-first century. J Cell Biochem
Suppl, 1998; 30-31: 137-146.
57) Clapham DE: Calcium signaling. Cell, 1995; 80: 259-268.
58) Pundir P, Catalli A, Leggiadro C, Douglas SE, Kulka M:
Pleurocidin, a novel antimicrobial peptide, induces
human mast cell activation through the FPRL1 receptor.
Mucosal Immunol, 2014; 7: 177-187.
59) Oskeritzian CA: Mast cells and wound healing. Adv
Wound Care (New Rochelle), 2012; 1: 23-28.
60) Tatemoto K, Nozaki Y, Tsuda R, et al: Immunoglobulin
E-independent activation of mast cell is mediated by
Mrg receptors. Biochem Biophys Res Commun, 2006;
349: 1322-1328.
61) Lembo PM, Grazzini E, Groblewski T, et al: Proenkepha-
lin A gene products activate a new family of sensory
neuron--specific GPCRs. Nat Neurosci, 2002; 5: 201-
209.
62) Kiatsurayanon C, Niyonsaba F, Chieosilapatham P,
Okumura K, Ikeda S, Ogawa H: Angiogenic peptide
(AG)-30/5C activates human keratinocytes to produce
cytokines/chemokines and to migrate and proliferate via
MrgX receptors. J Dermatol Sci, 2016; 83: 190-199.
63) Ferry X, Brehin S, Kamel R, Landry Y: G protein-
dependent activation of mast cell by peptides and basic
secretagogues. Peptides, 2002; 23: 1507-1515.
64) Ballif BA, Blenis J: Molecular mechanisms mediating
mammalian mitogen-activated protein kinase (MAPK)
kinase (MEK)-MAPK cell survival signals. Cell Growth
Differ, 2001; 12: 397-408.
65) Chang L, Karin M: Mammalian MAP kinase signalling
cascades. Nature, 2001; 410: 37-40.
66) Chen X, Niyonsaba F, Ushio H, et al: Human cathelicidin
LL-37 increases vascular permeability in the skin via
mast cell activation, and phosphorylates MAP kinases
p38 and ERK in mast cells. J Dermatol Sci, 2006; 43:
63-66.
Yanashima, et al: IDR-1018 activates human mast cells
56