J Periodont Res 2003; 38; 557–563 Copyright
Blackwell Munksgaard Ltd

Printed in the UK. All rights reserved

JOURNAL OF PERIODONTAL RESEARCH

Jun-ichi Kido1, Reiko Kido1,

Calprotectin release from Suryono1, Masatoshi Kataoka1,
Magne K. Fagerhol2, Toshihiko

human neutrophils is Nagata1

1
Department of Periodontology and
Endodontology, Tokushima University School of

induced by Porphyromonas Dentistry, Tokushima, Japan and 2Department of

Immunology and Transfusion Medicine, Ullevaal
University Hospital, Oslo, Norway

gingivalis lipopolysaccharide
via the CD-14–Toll-like
receptor–nuclear factor jB
pathway
Kido J, Kido R, Suryono, Kataoka M, Fagerhol MK, Nagata T. Calprotectin release
from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide
via the CD-14–Toll-like receptor–nuclear factor jB pathway. J Periodont Res 2003;
38; 557–563.
Blackwell Munksgaard, 2003

Objectives: Calprotectin is a cytosolic protein with antibacterial action in leuko-

cytes and its level increases in some inflammatory diseases, including periodontal
diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the
lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin
release from human neutrophils. P-LPS, a major virulence factor of periodontal
pathogens, is known to induce the production and release of inflammatory
cytokines through CD14, Toll-like receptor (TLR) and nuclear factor jB (NF-jB).
In the present study, we investigated whether calprotectin release by P-LPS is
induced via the CD14–TLR–NF-jB pathway and the cellular mechanism of
calprotectin release in human neutrophils.
Material and methods: Human neutrophils were isolated from the peripheral blood
of healthy donors and pre-incubated in medium containing antibodies against
CD14, TLR2 and TLR4, or several inhibitors of NF-jB, microtubules and
microfilaments, and then incubated with P-LPS. The calprotectin amount in the
culture medium was determined using ELISA, and the nuclear extracts from cells
were used for the examination of NF-jB binding activity using electrophoretic
mobility shift assays.

Results: P-LPS increased calprotectin release from neutrophils and its induction Dr Jun-ichi Kido, Department of Periodontology
was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 and Endodontology, Tokushima University
School of Dentistry, 3-18-15 Kuramoto,
antibodies. NF-jB inhibitors suppressed P-LPS-induced NF-jB binding activity Tokushima 770-8504, Japan
and calprotectin release. The inhibitors of microtubule and microfilament Tel: + 81 886 33 7344
polymerization significantly decreased P-LPS-induced calprotectin release. Fax: + 81 886 33 7345
e-mail: kido@dent.tokushima-u.ac.jp
Conclusion: These results suggest that calprotectin release is induced by P-LPS via Key words: calprotectin; Porphyromonas
the CD14–TLR2–NF-jB signal pathway in human neutrophils and may be gingivalis; lipopolysaccharide
dependent on microtubule and microfilament systems. Accepted for publication April 24, 2003
558 Kido et al.
Calprotectin is a calcium-binding pro-
tein contained in the cytosol of neu-
trophils, monocytes/macrophages and
epithelial cells, and is known as L1
antigen, macrophage migration inhibi-
tory factor-related protein 8 and 14
(MRP8 and MRP14), S100A8/
S100A9, calgranulin A/B and cystic
fibrosis antigen (1–3). This protein is a
heterogeneous complex composed of a
light subunit (MRP8, S100A8, cal-
granulin A) and a heavy subunit
(MRP14, S100A9, calgranulin B). The
calprotectin level increases in several
inflammatory diseases such as rheu-
matoid arthritis and ulcerative colitis,
and was suggested to be a marker for
these diseases (4–6). We previously
reported that the calprotectin level in
gingival crevicular fluid from perio-
dontal pockets with severe inflamma-
tion was higher than that from healthy
sites and positively correlated with the
known markers for periodontitis (7–
9). However, the origin of gingival
crevicular fluid calprotectin and the
regulation of calprotectin release from
cells around periodontal pockets were
unclear.
Recently, we found that calprotectin
existed in gingival tissues with an
infiltration of inflammatory cells and
lipopolysaccharide of P. gingivalis
(P-LPS) induced calprotectin release
from human neutrophils (10). It is
known that P. gingivalis is a major
pathogen of periodontitis and that
P-LPS affects the functions of poly-
morphonuclear leukocytes (PMN) and
stimulates the production and release
of inflammatory cytokines including
interleukin-1b (IL-1b), tumor necrosis
factor-a (TNF-a) and IL-6 from poly-
morphonuclear leukocytes and mono-
cytes (11–13). In the signal
transduction of LPS, LPS binds to
LPS-binding protein in serum and
interacts with CD14, the main LPS
receptor, then with Toll-like receptor
(TLR), a transmembrane receptor,
which then activates a transcription
factor, nuclear factor jB (NF-jB) (14,
15). Through this CD14–TLR–NF-jB
signaling pathway, P-LPS induces IL-1
production and the expression of IL-6
and IL-8 mRNAs in human neu-
trophils and gingival fibroblasts, and
P-LPS may regulate the viability,
apoptosis and physiological functions
of neutrophils via this signal pathway
(16–20).
In the present study, we investigated
whether calprotectin release was also
induced by P-LPS via the CD14–TLR–
NF-jB signal pathway in the same
manner as the cytokines release, using
antibodies against those receptors and
NF-jB inhibitors. Furthermore, we
examined the effect of cytoskeleton
change on calprotectin release using
tubulin polymerization inhibitors and
actin polymerization inhibitor to elu-
cidate the mechanism of calprotectin
release from neutrophils.
Materials and methods
Neutrophil preparation and culture
Peripheral blood was collected from 16
healthy donors (13 males and three
females, age 28–44 years), after the
donors gave their informed consent to
participation, and incubated in glass
tubes containing EDTA for 5 min.
Neutrophils were isolated from EDTA
blood by centrifugation using Mono-
Poly Resolving Medium (Dainippon
Pharmaceutical Co., Osaka, Japan)
according to the manufacturer’s
instructions and suspended in RPMI
1640 (ICN Biomedicals, OH, USA)
containing 5% fetal calf serum (JRH
Biosciences, Lenexa, KS, USA) within
1–1.5 h after blood collection. The cells
(1.25 · 106 cells/ml) were pre-incuba-
ted with 2 lg/ml anti-TLR2 antibody
(Santa Cruz Biotechnology, Inc.,
Heidelberg, Germany), 20 lg/ml anti-
TLR4 monoclonal antibodies (HTA125
and HTA1216), 1 lM pyrrolidine
dithiocarbamate (Sigma Chemical Co.,
St. Louis, USA), 10 mM N-acetyl-
L-cysteine (Sigma), 0.1 mM caffeic acid
phenethyl ester (Biomol Research
Laboratories, Inc., Plymouth Meeting,
PA, USA), 3 lM MG-132 z-Leu-
Leu-Leu-CHO (MG-132, Alexis Bio-
chemicals, San Diego, CA, USA) or
18 lM NF-jB SN 50, cell-permeable
inhibitory peptide (NF-jB SN 50 pep-
tide, Biomol Research Laboratories)
for 1 h at 37C, and then incubated
with 1 lg/ml P-LPS for 30 min at
37C. Anti-TLR4 monoclonal anti-
bodies were supplied by Dr Kensuke
Miyake (Institute of Medical Science,
University of Tokyo, Japan). In some
experiments, neutrophils were pre-
incubated with anti-CD14 monoclonal
antibody (Coulter Clone MY4, 1/200
dilution, Coulter Corporation, Miami,
FL, USA) for 30 min at 4C and
incubated with 1 lg/ml P-LPS for
30 min at 37C, or incubated with
microtubule polymerization inhibitors
(25 lM colchicine, 10 lM nocodazole
and 2 lM demecolcine) and actin
polymerization inhibitor (2 lM cyto-
chalasin B) in the absence or presence
of P-LPS (1 lg/ml) for 30 min at 37C.
These four inhibitors were purchased
from Sigma-Aldrich Co. (St. Louis,
MO, USA). For the analysis of NF-jB
binding activity using electrophoretic
mobility shift assays (EMSA), neu-
trophils (2 · 106 cells/ml) were pre-
incubated with five NF-jB inhibitors
(1 lM pyrrolidine dithiocarbamate,
10 mM N-acetyl-L-cysteine, 0.1 mM
caffeic acid phenethyl ester, 3 lM MG-
132 or 18 lM NF-jB SN 50 peptide)
for 1 h at 37C and incubated with
P-LPS (1 lg/ml) for 10 min at 37C.
The specificities of the antibodies used
in the present study were investigated
using each negative control IgG
(mouse IgG2b, IgG2a, IgG1 or goat
IgG), and the concentrations of anti-
bodies and inhibitors were decided by
the preliminary experiments.
The cell viability was examined using
the trypan blue exclusion test and was
greater than 98.2% after isolation and
maintained at greater than 93.2% after
cell incubation with or without P-LPS,
several antibodies and inhibitors. After
the incubation of neutrophils, the con-
ditioned medium was collected and
mixed with protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 1 lg/ml
N-q-tosyl-L-phenylalanine chlorome-
thyl ketone, 1 lg/ml Na-q-tosyl-L-
lysine chloromethyl ketone and 1 lg/ml
pepstatin from Sigma-Aldrich) and used
for the determination of the released
calprotectin amount, and the cells were
prepared for the sample of EMSA.
Preparation of P-LPS
P-LPS was prepared from P. gingivalis
381 strain according to a modification
of the method of Kumada et al. (21).

Briefly, the bacterial cells were extrac-
ted with hot-phenol-water and treated
with Cetavlon (Nacalai Tesque, Kyoto,
Japan). Crude P-LPS was digested with
RNase A, DNase I and proteinase K
(Sigma-Aldrich) and purified by cen-
trifugation (105,000 · g, 12 h, 4C)
two times and further washed with
phenol–chloroform–petroleum ether
(2 : 5 : 8 vol.) and acetone.
Calprotectin determination
The amount of calprotectin in condi-
tioned medium was determined by
ELISA according to the previous
method (5, 7–9, 22–24). In brief the
microtiter plates were pre-coated with
anti-human calprotectin rabbit anti-
body at 4C overnight. The antibody
was purified by immunoaffinity chro-
matography and reacted with calpro-
tectin components consisted of light
and heavy subunits. In immunoblotting
analysis using anti-calprotectin anti-
body, the light (8 kDa) and heavy
(14 kDa) subunits of calprotectin were
detected in the medium fraction of
neutrophils (10). The medium samples
or calprotectin standard derived from
human neutrophils were added to the
pre-coated plates and incubated for
45 min at room temperature. After
washing the plates with 50 mM Tris
HCl buffer (pH 8.0) containing 0.05%
Tween 20, alkaline phosphatase-con-
jugated anti-calprotectin was added to
the wells and incubated for 45 min at
room temperature. After washing, wells
were incubated with 1 mg/ml p-nitro-
phenol phosphate for 20 min at 37C
and the absorbance was determined
at 405 nm using a microplate reader.
The calprotectin amount in the sam-
ples was calculated from a standard
curve.
EMSA
The preparation of nuclear extracts
from neutrophils was performed
according to the method of Madan
et al. (25). Briefly, the cells treated with
P-LPS or NF-jB inhibitors were
washed in phosphate-buffered saline
and suspended in cell lysis buffer (10 mM
HEPES, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 0.1 mM EGTA, 0.1 mM
Calprotectin release by P. gingivalis LPS
EDTA, 1 mM dithiothreitol and 0.5%
Nonidet P-40) containing protease
inhibitors, and disrupted by sonication
for 5 s in ice water, then centrifuged at
3500 · g for 15 min at 4C. The pre-
cipitate (nuclear extract) was resu-
spended in nuclear extraction buffer
(20 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 420 mM NaCl, 0.1 mM EGTA,
0.1 mM EDTA, 1 mM dithiothreitol
and 25% glycerol) containing protease
inhibitors, incubated for 30 min at 4C
and centrifuged at 20,400 · g for
20 min at 4C. The supernatant was
used as nuclear extract. EMSA was
performed using a DIG Gel Shift kit
(Roche Molecular Biochemicals,
Mannheim, Germany) according to the
method of Carballo et al. (26). Briefly,
double-stranded NF-jB oligonucleo-
tide (5¢-AGTTGAGGGGACTTTCC-
CAGGC-3¢) was labeled with
digoxigenin. The nuclear extract (10 lg
protein) was incubated with digoxige-
nin-labeled NF-jB oligonucleotide (60
fmol), 1 lg poly [d(I-C)], 0.1 lg poly
L-lysine in the binding buffer contained
in the kit for 30 min at room tem-
perature. For the competitive binding
assay, the reaction was performed with
excess unlabeled NF-jB oligonucleo-
tide (6 pmol). The NF-jB binding
complex was electrophoresed on 5%
native polyacrylamide gel in 0.4 · TBE
buffer (pH 8.0), transferred to a posi-
tively charged nylon membrane and
reacted with alkaline phosphatase-
conjugated anti-digoxigenin antibody
and Disodium 3-(4-methoxyspiro
{1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo
[3.3.1.13,7]decan}-4-yl) phenyl phos-
phate (Tropix Inc., Bedford, MA,
USA).
Results
Effect of anti-CD14 monoclonal
antibody and anti-TLR antibodies
on calprotectin release
P-LPS increased calprotectin release
from human neutrophils to about
seven-fold that of the control
(non-treatment) level after 30 min
treatment. When the cells were pre-
incubated with anti-CD14 monoclonal
antibody, P-LPS-induced calprotectin
release was significantly inhibited and
559
Fig. 1. Effect of anti-CD14 monoclonal
antibody on P-LPS-induced calprotectin
release. Neutrophils (1.25 · 106 cells/ml)
were pre-incubated with anti-CD14 mono-
clonal antibody (1/200 dilution) for 30 min,
and then incubated with P-LPS (1 lg/ml) for
30 min. The amount of calprotectin released
from neutrophils was determined by ELISA.
Values are means ± SD for five donors’
samples. *, Significantly different from con-
trol (*p < 0.01) or P-LPS ( p < 0.01).
its level decreased almost to the level of
the culture treated with anti-CD14
monoclonal antibody alone (Fig. 1).
The anti-CD14 monoclonal antibody
alone did not significantly affect cal-
protectin release. HTA125 and
HTA1216, the TLP4 monoclonal
antibodies, slightly decreased calpro-
tectin release to 87% and 82% of the
P-LPS-induced level, respectively, but
did not significantly inhibit the induc-
tion by P-LPS (Fig. 2). However, anti-
TLR2 antibody markedly inhibited
calprotectin release to 33% of the
P-LPS-induced level. These results
suggest that calprotectin release is
induced by P-LPS via CD14 and
TLR2. Mouse IgG2b, IgG2a, IgG1
and goat IgG (polyclonal), isotype
controls against anti-CD14 monoclo-
nal antibody, anti-TLR4 monoclonal
antibodies (HTA125, HTA1216) and
anti-TLR2 antibody, did not signifi-
cantly inhibit LPS-induced calprotectin
release.
Effect of NF-jB inhibitors
on calprotectin release
We examined the effect of NF-jB
inhibitors, including pyrrolidine dithio-
carbamate, N-acetyl-L-cysteine, caffeic
560 Kido et al.
Fig. 2. Effect of anti-TLR antibodies on
P-LPS-induced calprotectin release. Neu-
trophils (1.25 · 106 cells/ml) were pre-incu-
bated with HTA125 (20 lg/ml), HTA1216
(20 lg/ml) or anti-TLR2 antibody (2 lg/ml)
for 1 h, and then incubated with P-LPS
(1 lg/ml) for 30 min. The calprotectin
amount was determined by ELISA. Values
are means ± SD for 12 donors’ samples.
*, Significantly different from control
(*p < 0.01) or P-LPS ( p < 0.01) alone.
acid phenethyl ester, MG-132 and NF-
jB SN50 peptide, on P-LPS-
induced calprotectin release. In EMSA
of nuclear extracts from human
neutrophils, P-LPS stimulated NF-jB
DNA-binding activity, but NF-jB
complex was not observed in the
competition binding assays using
excess unlabeled oligonucleotide
(Fig. 3A). P-LPS-stimulated NF-jB
activation was completely blocked by
the pre-treatment with pyrrolidine
dithiocarbamate, N-acetyl-L-cysteine,
caffeic acid phenethyl ester, MG-132
and NF-jB SN50 peptide. These
NF-jB inhibitors clearly suppressed
LPS-induced calprotectin release from
neutrophils (Fig. 3B). Pyrrolidine
dithiocarbamate, caffeic acid phenethyl
ester, MG-132 and NF-jB SN50 pep-
tide markedly inhibited the release to
15%, 10%, 12% and 7% of the P-LPS-
induced calprotectin level, respect-
ively, whereas N-acetyl-L-cysteine was
slightly weaker than the other four
inhibitors but still significantly
decreased the release to 26% of the
P-LPS-induced levels. These results
suggest that P-LPS-induced calprotec-
tin release is regulated via the CD14–
TLR2–NF-jB pathway.
B
Fig. 3. Effects of NF-jB inhibitors on
P-LPS-induced NF-jB activation and cal-
protectin release. Neutrophils (2 · 106 cells/
ml) were pre-incubated with 1 lM pyrroli-
dine dithiocarbamate (a), 10 mM N-acetyl-
L-cysteine (b), 0.1 mM caffeic acid phenethyl
ester (c), 3 lM MG-132 (d) or 18 lM NF-jB
SN50 peptide (e) for 1 h, and then incuba-
ted with P-LPS (1 lg/ml) for 10 min
(EMSA) and 30 min (calprotectin deter-
mination). (A) The nuclear extract from
neutrophils (10 lg protein) was used for
EMSA of NF-jB DNA-binding activity.
Lane 1: control; 2: P-LPS; 3: P-LPS +
pyrrolidine dithiocarbamate; 4: P-LPS +
N-acetyl-L-cysteine; 5: P-LPS + caffeic acid
phenethyl ester; 6: P-LPS + MG-132; 7:
P-LPS + NF-jB SN50 peptide; 8: P-LPS
and an excess of unlabeled oligonucleotide
for the competition binding assay; 9: nuc-
lear extract free. (B) The amount of calpro-
tectin released from neutrophils that were
incubated with pyrrolidine dithiocarbamate,
N-acetyl-L-cysteine, caffeic acid phenethyl
ester, MG-132, NF-jB SN50 peptide or
P-LPS was determined by ELISA. Values
are means ± SD for five to nine donors’
samples. *,**, Significantly different from
control (*p < 0.01, **p < 0.05) or P-LPS
( p < 0.01).
Fig. 4. Effect of cytoskeleton-related inhibi-
tors on P-LPS-induced calprotectin release.
Neutrophils (1.25 · 106 cells/ml) were incu-
bated with colchine (25 lM), nocodazole
(10 lM), demecolcine (2 lM) or cytochalasin
B (2 lM) in the absence (open column)
or presence (hatched column) of P-LPS
(1 lg/ml) for 30 min. The calprotectin
amount was determined by ELISA. Values
are means ± SD for seven donors’ samples.
*,**Significantly different from P-LPS alone
(*p < 0.05, **p < 0.01).
Effect of cytoskeleton-related
inhibitors on calprotectin release
In the first step to elucidate the mech-
anism of calprotectin release, the
effects of tubulin polymerization
inhibitors (colchicine, nocodazole and
demecolcine) and actin polymerization
inhibitor (cytochalasin B) on calpro-
tectin release were investigated. Col-
chicine, nocodazole and demecolcine
significantly suppressed P-LPS-induced
calprotectin release and decreased the
calprotectin level to 46%, 25% and
45% of the P-LPS-induced level,
respectively (Fig. 4). Furthermore,
cytochalasin B markedly inhibited cal-
protectin release greater than three
tubulin polymerization inhibitors
(19% of the P-LPS-induced level).
However, each inhibitor alone did not
demonstrate a significant effect on
calprotectin release.
Discussion
Several factors are known to regulate
calprotectin release from neutrophils
and monocytes/macrophages. In hu-
man neutrophils, chemotaxis C5a and
fMLP increased calprotectin release
after 20 min treatment (24) and P-LPS

also induced the marked release of
calprotectin, about 12- and 20-fold
that of the control levels, after 30 min
treatment (10). LPS of Escherichia coli,
interferon-c and TNF-a slightly
increase MRP8 release from macro-
phages after long-term (12–96 h)
treatment (27) and LPS, IL-1b and
phorbol myristate acetate significantly
elevate calprotectin (MRP8/MRP14)
release after 4 h treatment (28). How-
ever, the regulatory mechanisms of
calprotectin release by these factors
remain unclear. It is known that LPS
stimulates the release of inflammatory
cytokines including IL-1b, TNF-a and
IL-6 via the CD14–TLR–NF-jB
pathway in polymorphonuclear leuko-
cytes and monocytes (11–13). Sugita
et al. (16) demonstrated that anti-
CD14 monoclonal antibody (MY4)
partially blocked P-LPS-induced
NF-jB activation in human neu-
trophils and CD14 was closely related
to P-LPS signal transduction. From
those findings and our results that
P-LPS-induced calprotectin was
markedly suppressed by anti-CD14
monoclonal antibody (MY4) and
NF-jB inhibitors, it is suggested that
P-LPS stimulates calprotectin release
from neutrophils via the LPS signal
pathway including CD14 and NF-jB.
TLR2 and TLR4 are transmem-
brane receptors and transmit LPS sig-
nal via CD14 to intracellular
components in the signal transduction,
and play important roles in the innate
immune system (29). TLR2 is asso-
ciated with the recognition of Gram-
positive bacteria (30) and TLR4 is
related to the recognition of Gram-
negative bacteria (31). Tabeta et al.
(17) and Wang et al. (18) reported that
anti-TLR4 monoclonal antibody
inhibited P-LPS-induced IL-6 gene
expression and IL-1 production in
human gingival fibroblasts. In con-
trast, we found that anti-TLR2 anti-
body, but not anti-TLR4 monoclonal
antibodies, significantly inhibited
P-LPS-induced calprotectin release
from neutrophils. P-LPS stimulates
TNF-a production in macrophages
derived from C3H/HeJ mice that
have a point mutation in TLR4 gene
(32). Furthermore, Martin et al. (33)
reported that P-LPS-induced TNF-a
Calprotectin release by P. gingivalis LPS
production in human macrophages
was blocked by anti-TLR2 antibody,
but not anti-TLR4 antibody, and
Hirschfeld et al. (34) showed that
P-LPS induced IL-6 production in the
human astrocytoma cell line that
overexpresses TLR2, but not TLR4,
and suggested that P-LPSs do not sig-
nal through TLR4. The signals of most
LPSs are transmitted via TLR4, but
the P-LPS signal is recognized by
TLR2 in macrophages because this
LPS has a different structure from
many Gram-negative LPSs (29, 33–35).
The present findings and previous
studies suggest that P-LPS acts not
only via TLR4 but also via TLR2 or
other signal transducers in neutrophils
and macrophages. Therefore, the
P-LPS signal for calprotectin release
from human neutrophils may not be
mediated through TLR4, but through
TLR2.
With regard to the mechanism of
calprotectin release, Voganatsi et al.
(36) reported that calprotectin was
released from polymorphonuclear leu-
kocytes only by cell death. However,
the present experiment demonstrated
that cell viability was maintained at
greater than 93.2% after incubation
with P-LPS, antibodies and inhibitors
for 30 min, and that MRP8 and
MRP14 mRNAs are constitutively
expressed in neutrophils that were
incubated with P-LPS for 20 min-
2.5 h (data not shown), suggesting
that neutrophils were alive after the
incubation with P-LPS, antibodies and
inhibitors. From these results, it is
suggested that calprotectin is released
by another cellular physiological
mechanism as well as cell death.
Rammes et al. (28) reported that the
secretion of calprotectin (MRP8/
MRP14) from monocytes stimulated
by phorbol myristate acetate was
blocked by tubulin polymeralization
inhibitors and suggested that calpro-
tectin secretion was dependent on an
intact microtubule network. In the
present study, tubulin polymeralization
inhibitors and actin polymeralization
inhibitor significantly suppressed
P-LPS-induced calprotectin release
from neutrophils. These results sup-
ported the existence of a calprotectin
release mechanism in addition to that
561
of cell death. It is suggested that sev-
eral NF-jB inhibitors suppressed
P-LPS-induced calprotectin release and
there is an association of NF-jB acti-
vation and calprotectin release, but the
exact mechanism between these two
responses caused by P-LPS is not elu-
cidated. It is known that LPS regulates
protein production and secretion by its
effects on microtubules and microfila-
ments depolymerization (37, 38), and
that NF-jB activation is modulated by
IjB degradation with ubiquitin, a
component of the microtubule network
and further that microtubule organ-
ization induces NF-jB activation (39–
41). The present findings and those of
other studies suggest that calprotectin
may be released by LPS stimulation via
a microtubule-dependent pathway with
NF-jB activation or a microfilament-
dependent pathway.
LPSs from several periodontopathic
bacteria (Prevotella intermedia, Fuso-
bacterium nucleatum, Actinobacillus
actinomycetemcomitans) in addition to
P. gingivalis induced calprotectin
release from neutrophils (10). Calpro-
tectin affects the growth and cell
adhesion in neutrophils and macro-
phages (2, 3), and protects the adhe-
sion of P. gingivalis to gingival
epithelium (42) and inhibits the growth
of P. gingivalis (unpublished data).
LPS-induced calprotectin has an anti-
bacterial action against periodonto-
pathic bacteria and may play
significant roles in immune reactions of
periodontal diseases. To investigate the
mechanism of calprotectin release will
contribute to the elucidation of the
host defence mechanism by calprotec-
tin in periodontal diseases.
Acknowledgements
We are very grateful to Dr Kensuke
Miyake and Dr Sachiko Akashi
(Department of Microbiology and
Immunology, The Institute of Medical
Science, The University of Tokyo,
Japan) for providing anti-TLR4 anti-
bodies and helpful suggestions.
This study was supported by two
Grants-in-Aid for Scientific Research
(No. 13357018 and 13672188) from the
Japan Society for the Promotion of
Science.
562 Kido et al.
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