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Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 2, February 2006, pp 455–462
DOI 10.1002/art.21633
© 2006, American College of Rheumatology
Induction of Triggering Receptor Expressed on
Myeloid Cells 1 in Murine Resident Peritoneal Macrophages by
Monosodium Urate Monohydrate Crystals
Yousuke Murakami,1 Tohru Akahoshi,2 Izumi Hayashi,3 Hirahito Endo,2 Shinichi Kawai,4
Matsuhisa Inoue,2 Hirobumi Kondo,2 and Hidero Kitasato5
Objective. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface molecule that
was recently identified on monocytes and neutrophils.
TREM-1 has been implicated in the early inflammatory
responses induced by microbes, but its pathophysiologic
role in nonmicrobial inflammation remains unknown.
In the present study, we investigated the role of TREM-1
in acute inflammation induced by monosodium urate
monohydrate (MSU) crystals. Induction of TREM-1
expression by MSU crystal–stimulated murine resident
peritoneal macrophages and infiltrating leukocytes in a
murine air-pouch model of crystal-induced acute inflammation was determined. The biologic role of
TREM-1 in crystal-induced cytokine production by resident peritoneal macrophages was also investigated.
Methods. TREM-1 expression by resident peritoneal macrophages and infiltrating leukocytes in a mu-
rine air-pouch model was determined by quantitative
real-time polymerase chain reaction, Western blot analysis, and flow cytometry. Cytokine production by resident peritoneal macrophages after incubation with
MSU crystals in the presence or absence of an anti–
TREM-1 agonist antibody was determined by enzymelinked immunosorbent assay.
Results. TREM-1 expression by resident peritoneal macrophages was significantly induced after stimulation with the crystals. Maximum expression of
TREM-1 transcripts and protein occurred at 1 and 4
hours after exposure to the crystals, respectively. Costimulation of resident peritoneal macrophages with
MSU crystals and an anti–TREM-1 agonist antibody
synergistically increased the production of both
interleukin-1␤ and monocyte chemotactic protein 1
compared with stimulation with the crystals alone. MSU
crystals also induced TREM-1 expression in infiltrating
leukocytes in a murine air-pouch model of crystalinduced acute inflammation.
Conclusion. These findings suggest that rapid
induction of TREM-1 expression on resident peritoneal
macrophages and neutrophils by MSU crystals may
contribute to the development of acute gout through
enhancement of inflammatory responses.
Supported in part by a research grant from the Ministry of
Health, Labor, and Welfare of Japan, a project research grant from
Kitasato University Graduate School of Medical Sciences, and a grant
from Kitasato University School of Allied Health Sciences (grant-inaid for research project no. 2005-211).
1
Yousuke Murakami, PhD: Kitasato University School of
Medicine, Kanagawa, Japan, and the Japan Health Sciences Foundation, Tokyo, Japan; 2Tohru Akahoshi, MD, Hirahito Endo, MD,
Matsuhisa Inoue, PhD, Hirobumi Kondo, MD: Kitasato University
School of Medicine and Graduate School of Medical Sciences, Kanagawa, Japan; 3Izumi Hayashi, PhD: Kitasato University School of
Medicine, Kanagawa, Japan, and Nihon Pharmaceutical University,
Saitama, Japan; 4Shinichi Kawai, MD: Toho University School of
Medicine, Tokyo, Japan; 5Hidero Kitasato, PhD: Kitasato University
School of Allied Health Sciences and Graduate School of Medical
Sciences, Kanagawa, Japan.
Address correspondence and reprint requests to Hidero
Kitasato, PhD, Department of Environmental Microbiology, Kitasato
University Graduate School of Medical Sciences, 1-15-1 Kitasato,
Sagamihara, Kanagawa 228-8555, Japan. E-mail: hkita@kitasatou.ac.jp.
Submitted for publication March 7, 2005; accepted in revised
form November 10, 2005.
Acute gouty arthritis is characterized by the
deposition of monosodium urate monohydrate (MSU)
crystals in articular and periarticular tissues (1). These
crystals have been shown to cause massive infiltration of
neutrophils into joints and to promote neutrophil activation, leading to tissue damage (2). MSU crystals have
a remarkable capacity to induce the release of various
inflammation mediators from synovial cells, macrophages, and infiltrating leukocytes (3,4). These mediators include interleukin-1␤ (IL-1␤), IL-8, and monocyte
455
456
chemotactic protein 1 (MCP-1), all of which have been
shown to play an important role in promoting the
infiltration and activation of inflammatory cells in acute
gout (5,6).
Triggering receptor expressed on myeloid cells 1
(TREM-1) is a recently identified cell surface molecule
that is found on neutrophils and monocytes (7–9).
TREM-1 is a member of the 30-kd immunoglobulin
superfamily, and its expression is up-regulated by lipoteichoic acid or lipopolysaccharide (LPS) (7,8,10,11).
TREM-1 activates monocytes through the transmembrane adaptor protein DAP12 (7–9). Targeting of
TREM-1 expressed by neutrophils and monocytes with
agonist monoclonal antibodies promotes the production
of various inflammatory cytokines, such as IL-8, MCP-1,
tumor necrosis factor ␣ (TNF ␣ ), granulocyte–
macrophage colony-stimulating factor, and IL-1␤
(8,10,11). In addition, marked enhancement of TNF␣,
IL-1␤, and MCP-1 production by monocytes incubated
with agonist monoclonal antibodies occurs when LPS is
added as a costimulant, indicating that TREM-1 can
amplify inflammatory responses initiated by Toll-like
receptors (TLRs) (8,10,11). Although natural ligands for
TREM-1 remain to be identified, its pathophysiologic
significance in acute inflammation has already been
demonstrated in murine models of septic shock, in which
competition for binding to TREM-1 by a recombinant
TREM-1 fusion protein or synthetic soluble TREM-1
(sTREM-1) was shown to protect mice against lethal
LPS challenge or bacterial sepsis (11,12).
Several lines of evidence indicate that TREM-1 is
strongly expressed in acute and chronic inflammatory
lesions caused by bacterial or fungal infection, while it
shows only weak expression in nonmicrobial inflammatory conditions, such as psoriasis, ulcerative colitis, and
immune complex–mediated vasculitis (11,13). However,
the mechanisms that regulate TREM-1 expression in
various inflammatory diseases remain unknown. An
essential role of TLR-mediated signaling in acute gout
was recently demonstrated (14). Because TREM-1 may
potentially amplify the inflammatory responses initiated
by TLRs, we hypothesized that cooperation between
TREM-1 and TLRs may occur during acute attacks of
gout. Therefore, we investigated the expression of
TREM-1 by MSU crystal–stimulated murine resident
peritoneal macrophages, and also assessed the effect of
a TREM-1 agonist antibody on cytokine production by
MSU crystal–stimulated resident peritoneal macrophages.
MURAKAMI ET AL
MATERIALS AND METHODS
Reagents. Uric acid, polymyxin B, and LPS were
obtained from Sigma (St. Louis, MO), and IL-1␤ and TNF␣
were obtained from PeproTech (London, UK). Specific
enzyme-linked immunosorbent assays (ELISAs) for murine
IL-1␤ and MCP-1 were obtained from Biosource International
(Camarillo, CA). Anti-mouse TREM-1 polyclonal antibody,
phycoerythrin (PE)–conjugated anti-mouse TREM-1 monoclonal antibody (mAb), and the Mouse TREM-1 DuoSet were
purchased from R&D Systems (Minneapolis, MN). Anti-actin
polyclonal antibody and goat IgG1 were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA), and fluorescein isothiocyanate (FITC)–conjugated anti-mouse Gr-1 mAb and allophycocyanin (APC)–conjugated anti-mouse CD11b mAb were
purchased from eBioscience (San Diego, CA). Horseradish
peroxidase (HRP)–conjugated rabbit anti-goat IgG antibody
was obtained from DakoCytomation (Kyoto, Japan).
Preparation of MSU crystals. MSU crystals were
prepared according to the method described by Seegmiller et
al (15). Briefly, 8 gm of uric acid was dissolved in 1,600 ml of
boiling distilled water containing 49 ml of 1N NaOH. After the
pH of the solution was adjusted to 7.2 by addition of HCl, it
was gradually cooled with stirring at room temperature and
then stored overnight at 4°C. The crystals that formed were
sterilized by heating at 180°C for 2 hours and were suspended
in phosphate buffered saline (PBS) at a concentration of 10
mg/ml. The crystals obtained by this method were rod-shaped
and fairly uniform in size (5–25 ␮m long). A Limulus amebocyte cell lysate assay verified the absence of endotoxin in the
crystal preparation.
Resident peritoneal macrophages. Resident peritoneal
macrophages were isolated from male ICR mice (ages 6–8
weeks), as reported elsewhere (16). After washing with PBS,
the cells were suspended in RPMI 1640 medium (Sigma)
supplemented with 5% heat-inactivated fetal calf serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 ␮g/ml
streptomycin (Invitrogen, Carlsbad, CA). Next, the resident
peritoneal macrophages were incubated in the presence or
absence of various concentrations of MSU crystals for the
indicated periods at 37°C in a humidified incubator with an
atmosphere of 5% CO2 and 95% air. The cells were also
incubated with or without polymyxin B (5 ␮g/ml) for 1 hour
and then were incubated for 1 hour in the presence or absence
of various inflammatory agents, such as IL-1␤, TNF␣, LPS,
and MSU crystals. Expression of TREM-1 was determined by
quantitative real-time PCR and Western blot analysis. Production of sTREM-1 in a culture supernatant was determined
using the TREM-1 DuoSet (R&D Systems).
Quantitative real-time PCR and reverse
transcription–PCR. Total RNA was extracted from resident
peritoneal macrophages, infiltrating cells, and soft tissues of
murine air pouches by using an RNeasy Mini Kit (Qiagen,
Tokyo, Japan) and was treated with DNase I (Qiagen). Complementary DNA (cDNA) was synthesized from 2 ␮g of
random-primed total RNA in a total volume of 20 ␮l using
Omniscript reverse transcriptase (Qiagen). Murine TREM-1
expression was assessed by quantitative real-time PCR using
the oligonucleotide primers 5⬘-CCAGAAGGCTTGGCAGAGACT-3⬘ and 5⬘-ACTTCCCCATGTGGACTTCACT-3⬘.
Murine GAPDH was used as the internal control, being
RAPID INDUCTION OF TREM-1 IN ACUTE GOUT
amplified with the primers 5⬘-TGCAGTGGCAAAGTGGAGATT-3⬘ and 5⬘-CCATCAACGACCCCTTCATTGACCTC-3⬘. The expected sizes of the PCR products for
TREM-1 and GAPDH were 101 bp and 97 bp, respectively.
PCR was performed in duplicate with a 25-␮l reaction
mixture containing 1 ␮l of cDNA, 12.5 ␮l of QuantiTect SYBR
Green PCR Kit (Qiagen), and 300 nM of the forward and
reverse primers. The PCR mixture was incubated for 15
minutes at 95°C to activate HotStarTaq DNA polymerase
(Qiagen). Subsequently, amplification was performed for 40
cycles of denaturation at 94°C for 15 seconds, annealing at
58°C for 30 seconds, and extension at 72°C for 30 seconds.
During the extension step, the ABI Prism 7700 Sequence
Detection System (Applied Biosystems, Tokyo, Japan) monitored real-time amplification by quantitative analysis of the
emitted fluorescence. The amount of sample messenger RNA
(mRNA) was estimated relative to the control sample, which
was assigned a value of 1 arbitrary unit.
Western blot analysis. Resident peritoneal macrophages (1.0 ⫻ 106 cells) were solubilized in 200 ␮l of sample
buffer (350 mM Tris [pH 6.8], 10% sodium dodecyl sulfate
[SDS], 30% glycerol, 600 mM dithiothreitol [DTT], and 0.05%
bromophenol blue), loaded onto 10% SDS–polyacrylamide gel
electrophoresis gel, and run at 20 milliampere for 1.5 hours.
Next, cellular proteins were transferred to a polyvinylidene
difluoride membrane (Roche Diagnostics, Mannheim, Germany) for 1.5 hours at 200 mA by the semi-dry blot method.
The membrane was blocked with 5% skim milk in Tris
buffered saline (TBS) containing 0.05% Tween 20 for 1 hour at
37°C, washed with TBS containing 0.1% Tween 20, and
incubated with anti-mouse TREM-1 polyclonal antibody or
anti-actin polyclonal antibody overnight at 4°C. The blots were
washed 4 times with TBS and incubated for 30 minutes with
HRP-conjugated rabbit anti-goat IgG antibody. Immunoreactive bands were developed using a chemiluminescent substrate
(ECL Plus; Amersham Biosciences, Piscataway, NJ).
Cytokine production. Flat-bottomed plates were precoated overnight at 4°C with 5 ␮g/ml anti–TREM-1 polyclonal
antibody or an isotype-matched control (goat IgG1). After
washing with PBS, resident peritoneal macrophages (1.0 ⫻ 105
cells) were added to the wells, and then incubated in the
presence or absence of MSU crystals (100 ␮g/ml) for 24 hours.
Culture supernatant was obtained by centrifugation and was
stored at ⫺20°C until the IL-1␤ and MCP-1 levels were
determined using specific ELISAs.
Murine air-pouch model. Subcutaneous air pouches
were created by the injection of sterile filtered air, as described
previously (17). Briefly, C57/BL6 mice (6–8 weeks old) were
anesthetized, and 5 ml of air was injected into the subcutaneous tissue of the back, followed by reinjection of an additional
3 ml of air after 3 days. On day 7 after the first injection, the
air pouches thus created were used for these experiments.
First, MSU crystals (3 mg in 1 ml of sterile PBS) were injected
into the air pouches. After the indicated time periods, pouch
fluid was harvested by injecting 3 ml of cold PBS. Next, the
cells that had accumulated inside the air pouches were counted
using a hemocytometer and were stained with Wright Giemsa
solution to determine the differential leukocyte count. Expression of TREM-1 by the infiltrating cells and soft tissues of the
457
air pouches was investigated by quantitative real-time PCR.
Soluble TREM-1 in the pouch fluid was determined using the
TREM-1 DuoSet (R&D Systems).
Flow cytometric analysis. Cells infiltrating into the air
pouches were harvested 8 hours after stimulation.
Thioglycolate-elicited peritoneal neutrophils were harvested
20 hours after peritoneal injection of 2 ml of 3% thioglycolate,
and cells were used as a negative control. Cells were incubated
with an Fc blocking agent for 5 minutes at room temperature
to block Fc receptors. Subsequently, cells were incubated with
PE-labeled anti–TREM-1 mAb, FITC-labeled anti–Gr-1 mAb,
or APC-labeled anti-CD11b mAb for 20 minutes on ice.
Gating on neutrophils was based on characteristic forward and
side scatter parameters as well as the binding of anti–Gr-1
mAb. In order to ensure specific staining, appropriate isotype
control mAb were used. Samples were analyzed by flow
cytometry.
RESULTS
MSU crystal–induced TREM-1 expression by
resident peritoneal macrophages. In order to investigate
the role of MSU crystals in TREM-1 expression, resident peritoneal macrophages were stimulated with crystals (100 ␮g/ml), and TREM-1 expression was evaluated
by quantitative real-time PCR and Western blot analysis.
As shown in Figure 1A, exposure to MSU crystals
rapidly induced TREM-1 mRNA expression by resident
peritoneal macrophages in a time-dependent manner.
The maximum induction of TREM-1 mRNA occurred 1
hour after stimulation, with a return to the basal level at
4 hours. Expression of TREM-1 protein was also induced 2–8 hours after stimulation with the crystals
(Figure 1B). Maximum induction occurred at 4 hours,
and expression returned to the basal level by 12 hours
after stimulation. Actin (used as a control) was also
detected in all of the samples. Resident peritoneal
macrophages were also incubated with various concentrations of MSU crystals for the indicated periods of
time, and TREM-1 expression was evaluated. The crystals enhanced TREM-1 mRNA expression at 1 hour and
TREM-1 protein expression at 4 hours, in a
concentration-dependent manner; maximum expression
occurred after stimulation with 100 ␮g/ml of the crystals
(Figures 1C and D).
It has been demonstrated that LPS could enhance the production of sTREM-1 and expression of
TREM-1 on the cell surface. Therefore, we investigated
the production of sTREM-1 from MSU crystal–
stimulated resident peritoneal macrophages. We could
not detect sTREM-1 in a culture supernatant of MSU
crystal–stimulated resident peritoneal macrophages.
458
MURAKAMI ET AL
Figure 1. Rapid induction of triggering receptor expressed on myeloid cells 1
(TREM-1) expression in monosodium urate monohydrate (MSU) crystal–
stimulated resident peritoneal macrophages. A, TREM-1 mRNA level as determined by quantitative real-time polymerase chain reaction (PCR). Resident
peritoneal macrophages were incubated with MSU crystals (100 ␮g/ml) for the
indicated time periods. Murine GAPDH was used as the internal control. The
relative amount of TREM-1 mRNA was evaluated by comparison with the level in
vehicle-treated resident peritoneal macrophages, which was defined as 1 arbitrary
unit. B, Western blot analysis of the TREM-1 protein level. Murine actin was used
as the loading control. C, TREM-1 mRNA level as determined by quantitative
real-time PCR in resident peritoneal macrophages incubated with or without
various concentrations of MSU crystals for 1 hour. D, Western blot analysis of
TREM-1 protein level 8 hours after stimulation. Data are expressed as the mean
and SD results of triplicate determinations.
Expression of TREM-1 in a murine air-pouch
model of MSU crystal–induced acute inflammation. In
order to determine whether MSU crystals could promote TREM-1 expression in vivo, a murine air-pouch
model of MSU crystal–induced acute inflammation was
used. Injection of MSU crystals (3 mg) caused infiltration of cells into the air pouches (Figure 2), and expression of TREM-1 mRNA by the infiltrating cells increased in a time-dependent manner (Figure 2).
Maximum induction occurred at 4 hours, and expression
subsequently declined 8 hours after stimulation. We
previously demonstrated increased expression of IL-1␤,
macrophage inflammatory protein 2, and KC in infiltrating cells in this air-pouch model (18,19). The time course
of TREM-1 mRNA expression closely resembles gene
expression of inflammatory cytokines and chemokines.
Gene expression of TREM-1 in soft tissue around air
pouches was also evaluated, and it increased ⬃4-fold
relative to the control pouch (data not shown). However,
the TREM-1 expression level in soft tissues was lower
than that in infiltrating cells.
Neutrophils are a predominant cell type among
the accumulated cells in MSU crystal–induced acute
inflammation. In order to evaluate TREM-1 expression
on neutrophils, flow cytometric analysis was performed
on the infiltrating cells. As shown in Figure 2B, 8 hours
after stimulation, infiltrating neutrophils expressed
TREM-1 at an increased level in comparison with
thioglycolate-elicited neutrophils. We failed to determine TREM-1 expression in infiltrating neutrophils at
different time points because of the limited number of
cells. This result indicated that MSU crystals could also
up-regulate TREM-1 expression in infiltrating neutrophils. Furthermore, sTREM-1 was also detected in
pouch fluid 8 hours (but not 4 hours) after stimulation
(Figure 2C). Overall, these findings clearly demon-
RAPID INDUCTION OF TREM-1 IN ACUTE GOUT
459
Figure 2. TREM-1 expression in a murine air-pouch model of MSU crystal–induced
acute inflammation. A, MSU crystals (3 mg in 1 ml of sterile phosphate buffered
saline) were injected into subcutaneous air pouches created in mice. Infiltrating cells
in the air pouches were harvested after the indicated periods of time. Expression of
TREM-1 by infiltrating cells was determined by quantitative real-time PCR. Murine
GAPDH was used as the internal control. Results are the mean and SD values from
3–6 mice. (Similar results were obtained in 2 independent experiments.) B, Infiltrating cells into the air pouches were harvested 8 hours after stimulation. Thioglycolateelicited neutrophils were used as a negative control. Cellular expression of TREM-1
was determined by flow cytometry. Representative histogram illustrates TREM-1
expression by infiltrating neutrophils (bold solid line) and control neutrophils (solid
line). Broken line indicates background staining of infiltrating cells with an isotypematched control monoclonal antibody. C, Pouch fluid was harvested 8 hours after
stimulation. Soluble TREM-1 levels were determined by enzyme-linked immunosorbent assay. Results are the mean and SD. See Figure 1 for definitions.
strated that MSU crystals are capable of promoting
TREM-1 expression in soft tissue as well as neutrophils
in vivo.
Regulation of MSU crystal–induced TREM-1
expression. Because MSU crystals significantly induced
TREM-1 expression by resident peritoneal macrophages, we investigated the mechanism regulating this
change in TREM-1 expression. Because MSU crystals
are well known to promote cytokine production by
macrophages, the effects of inflammatory cytokines such
as IL-1␤ and TNF␣ on TREM-1 expression by resident
peritoneal macrophages were investigated. After resident peritoneal macrophages were incubated with IL-1␤
(20 ng/ml) or TNF␣ (20 ng/ml) for 1 hour, gene expression was determined by quantitative real-time PCR. As
shown in Figures 3A and B, these cytokines enhanced
the expression of both TREM-1 mRNA and protein, but
the potency of their effect on TREM-1 appeared to be
lower than that of LPS.
Enhancement of TREM-1 expression by MSU
crystals at a concentration of 100 ␮g/ml appeared to be
equivalent to that caused by LPS at a concentration of 20
ng/ml. A Limulus amebocyte cell lysate assay indicated
the absence of LPS in the crystal preparation. However,
we also used polymyxin B to eliminate possible contamination of the crystals by LPS. Polymyxin B is a cationic,
cyclic peptide antibiotic that can inhibit the activity of
LPS (20). Treatment with polymyxin B at a concentration of 5 ␮g/ml for 1 hour completely abolished LPSinduced TREM-1 expression by resident peritoneal
macrophages but failed to block crystal-induced
TREM-1 expression (Figure 3A). These findings indicated that MSU crystal–induced up-regulation of
TREM-1 expression was caused by direct interaction of
460
resident peritoneal macrophages with the crystals rather
than by induction of proinflammatory cytokines or LPS.
Synergistic effect of agonistic TREM-1 antibodies on MSU crystal–induced cytokine production. It was
previously shown that expression of TREM-1 on macrophages is induced by LPS, and that treatment of macrophages with LPS and an agonist antibody for TREM-1
synergistically enhances the production of proinflammatory cytokines. In order to evaluate whether antibody
binding to TREM-1 also had a synergistic effect on
cytokine production by crystal-stimulated macrophages,
resident peritoneal macrophages were incubated with
MSU crystals in the presence or absence of anti–
TREM-1 agonist polyclonal antibodies for 24 hours, and
cytokine production was determined by ELISA. As
shown in Figure 4, IL-1␤ production by resident peritoneal macrophages incubated with both MSU crystals
and anti–TREM-1 agonist polyclonal antibodies was
MURAKAMI ET AL
Figure 4. Synergistically enhanced production of proinflammatory
cytokines by macrophages, by binding of TREM-1 and MSU crystals.
Resident peritoneal macrophages were incubated with an isotype
control antibody (goat IgG1) or polyclonal anti–TREM-1 agonist
monoclonal antibodies in the presence of MSU crystals for 24 hours.
Production of IL-␤ (A) and macrophage chemotactic protein 1
(MCP-1) (B) was determined using specific enzyme-linked immunosorbent assays. Values are the mean and SD of triplicate determinations. PBS ⫽ phosphate buffered saline (see Figure 3 for other
definitions).
significantly increased, with production being 18-fold
greater than that by cells stimulated with crystals alone
(Figure 4A). Costimulation of resident peritoneal macrophages with crystals and anti–TREM-1 polyclonal antibodies also promoted an 8-fold increase of MCP-1
production in comparison with that by crystal-stimulated
cells (Figure 4B). These results clearly indicated that
antibody binding of TREM-1 and exposure to MSU
crystals synergistically up-regulated the production of
proinflammatory cytokines by resident peritoneal macrophages.
DISCUSSION
Figure 3. Induction of triggering receptor expressed on myeloid cells
1 (TREM-1) expression in resident peritoneal macrophages by various
inflammatory agents. Resident peritoneal macrophages were incubated with or without polymyxin B (polyB; 5 ␮g/ml) for 1 hour, and
were subsequently stimulated with interleukin-1␤ (IL-1␤), tumor
necrosis factor ␣ (TNF␣), monosodium urate monohydrate (MSU)
crystals, or lipopolysaccharide (LPS) for 1 hour. A, TREM-1 mRNA
levels were determined by quantitative real-time polymerase chain
reaction. Murine GAPDH was used as the internal control. Values are
the mean and SD of triplicate determinations. B, TREM-1 protein
levels were determined by Western blot analysis.
The results of this study provide the first evidence
that a nonmicrobial agent, MSU crystals, can induce
TREM-1 expression by macrophages in vitro. In addition, TREM-1 expression was significantly up-regulated
in an in vivo (murine air-pouch) model of MSU crystal–
induced acute inflammation. Activation of TREM-1 in
combination with exposure to MSU crystals synergistically increased the production of IL-1␤ and MCP-1 by
macrophages.
Deposition of MSU crystals in articular and
periarticular tissues is an essential pathologic finding in
acute and chronic gouty arthritis (1,2). MSU crystals
stimulate various types of cells, including monocytes,
macrophages, neutrophils, and synovial cells, resulting in
a rapid increase in the production of proinflammatory
RAPID INDUCTION OF TREM-1 IN ACUTE GOUT
cytokines and chemokines. Several lines of evidence
indicate that release of these inflammatory mediators
plays an important role in the infiltration and activation
of inflammatory cells in acute gout (5,6,21). Therefore,
activation of inflammatory cells seems to be essential for
the initiation of MSU crystal–induced acute inflammation. It has previously been shown that direct contact
with or phagocytosis of these crystals by phagocytes may
promote cellular activation in acute gout, but the precise
mechanisms of cellular activation by the crystals remain
unknown. In this study, we demonstrated that rapid
induction of TREM-1 by MSU crystals may be involved
in the crystal-induced inflammatory response that occurs during acute gout attacks.
TREM-1 is a recently discovered cellular receptor that belongs to the immunoglobulin superfamily and
is expressed on monocytes and neutrophils. It promotes
cellular activation mediated by an associated signal
transduction molecule, DAP12, and a role of TREM-1
in both the innate and adaptive immune responses has
recently been documented (10). A pathophysiologic role
of TREM-1 as an amplifier of inflammation has also
been confirmed by in vivo studies (11,12). In animal
models of severe bacterial infection, blockade of signaling via TREM-1 with an sTREM-1 immunoglobulin
fusion protein or synthetic sTREM-1 was able to protect
mice against septic shock and death (11,12). It was
recently reported that TREM-1 expression is induced by
stimulation with microbial products, but not by nonmicrobial agents (11,22). We observed that proinflammatory cytokines (e.g., IL-1␤ and TNF␣) produced by
crystal-stimulated macrophages caused only slight induction of TREM-1 expression, whereas exposure to
MSU crystals led to a marked increase of TREM-1
expression even in the presence of polymyxin B.
This may be the first report of TREM-1 expression being caused by a nonmicrobial agent, i.e., MSU
crystals. MSU crystals induced TREM-1 expression of
infiltrating cells and sTREM-1 production in a murine
air-pouch model of crystal-induced acute inflammation.
Rapid induction of TREM-1 mRNA expression in infiltrating cells was observed, and maximal gene expression
of TREM-1 was found 4 hours after stimulation. Flow
cytometric analysis clearly demonstrated increased expression of TREM-1 in infiltrating neutrophils. In a
murine air-pouch model, expression of TREM-1 in
monocytes and macrophages among infiltrating cells
could not be identified because of the limited number of
cells. However, TREM-1 mRNA expression was detected in the soft tissue. These findings indicated that
MSU crystals could induce TREM-1 expression in both
461
macrophages and neutrophils in the setting of crystalinduced acute inflammation.
Furthermore, the addition of agonistic TREM-1
antibodies significantly enhanced cytokine production by
crystal-stimulated macrophages, indicating that the
TREM-1 molecules induced by crystal stimulation were
functional. Although natural ligand(s) for TREM-1 have
not yet been identified, our findings indicate that endogenous MSU crystals may promote an inflammatory
response, at least partly via TREM-1–mediated signaling. Thus, it is possible to hypothesize that natural
ligands are capable of amplifying the acute inflammatory
response in patients with acute gout after being recognized by TREM-1 expressed on crystal-stimulated
phagocytes.
TREM-1 and TLRs are both recently identified
cellular receptors that regulate innate immune responses, and an interaction between these 2 kinds of
receptors has been demonstrated. For example,
TREM-1 is significantly up-regulated by various ligands
for TLRs, including lipoteichoic acid (TLR-2),
polyinosinic–polycytidylic acid (TLR-3), and LPS
(TLR-4) (10). In addition, binding of agonistic mAb to
TREM-1 on monocytes in combination with the ligands
for TLR-2, TLR-3, or TLR-4 synergistically amplified
the cellular production of proinflammatory cytokines
(8,10,11). Liu-Bryan et al recently demonstrated an
essential role of TLR-2 in MSU crystal–induced acute
inflammation (14). This indicates that MSU crystals are
potent ligands for TLR-2, and recognition of the crystals
by TLR-2–expressing inflammatory cells promotes rapid
induction of various inflammation mediators. However,
because it is not known whether MSU crystal–induced
up-regulation of TREM-1 expression is mediated by
TLR-2, further investigations should be conducted to
elucidate the biologic interactions between TLRs and
TREM-1 in MSU crystal–induced acute inflammation.
Mechanisms for the synergistic effect of TREM-1
and TLRs have been postulated. For example, NF-␬B is
activated by the TLR signaling pathway. TREM-1–
mediated tyrosine phosphorylation, activation of
MAPK, and mobilization of Ca2⫹ might also lead to the
activation of transcription complexes, which could have
a synergistic effect with NF-␬B in promoting the expression of proinflammatory genes. It has also been shown
that MSU crystals potentially activate the NF-␬B signaling pathway (23). Overall, these findings suggest that
TREM-1 may act synergistically with MSU crystals in
promoting the production of proinflammatory cytokines
through activation of NF-␬B and MAPK, tyrosine phosphorylation, and mobilization of Ca2⫹.
462
MURAKAMI ET AL
Crosslinking of TREM-1 not only induces cytokine production but also up-regulates the expression of
various cell surface molecules, including CD40 and
intercellular adhesion molecule (ICAM) (8). Activation
of CD40 by binding to CD40 ligand amplifies the
production of proinflammatory cytokines by macrophages (24), while ICAM is an adhesion molecule that
facilitates recruitment of neutrophils and macrophages
to inflammation foci. Thus, induction of these molecules
by TREM-1 may lead to initiation of the inflammatory
response in acute gout (25).
Based on the present findings, we hypothesize
that rapid induction of TREM-1 may contribute to the
enhancement of MSU crystal–induced acute inflammation through recognition of a natural ligand for
TREM-1. Further investigation is needed to evaluate
the pathologic role of TREM-1 in increasing MSU
crystal–induced acute inflammation in vivo by using an
sTREM-1–immunoglobulin fusion protein. Such studies
may define the precise role of TREM-1 in acute gouty
arthritis and could also provide a novel therapeutic
target for the management of acute gout.
ACKNOWLEDGMENTS
9.
10.
11.
12.
13.
14.
15.
16.
17.
We thank Mrs. Rie Hasegawa and Terumi Mizuno for
excellent technical support.
18.
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