Cutting Edge: TREM-2 Attenuates Macrophage Activation1
Isaiah R. Turnbull, Susan Gilfillan, Marina Cella, Taiki Aoshi, Mark Miller, Laura Piccio,
Maristela Hernandez, and Marco Colonna2
The triggering receptor expressed on myeloid cells 2
(TREM-2) delivers intracellular signals through the
adaptor DAP12 to regulate myeloid cell function both
within and outside the immune system. The role of
TREM-2 in immunity has been obscured by the failure to
detect expression of the TREM-2 protein in vivo. In this
study, we show that TREM-2 is expressed on macrophages
infiltrating the tissues from the circulation and that alter-
native activation with IL-4 can induce TREM-2.
TREM-2 expression is abrogated by macrophage matura-
tion with LPS of IFN-?. Using TREM-2?/?mice, we
find that TREM-2 functions to inhibit cytokine produc-
tion by macrophages in response to the TLR ligands LPS,
zymosan, and CpG. Furthermore, we find that TREM-2
completely accounts for the increased cytokine production
previously reported by DAP12?/?macrophages. Taken
together, these data show that TREM-2 is expressed on
newly differentiated and alternatively activated macro-
activation. The Journal of Immunology, 2006, 177:
and outside the immune system. The nonimmune function of
TREM-2 is demonstrated by the presentation of people defi-
cient for TREM-2. These patients develop Nasu-Hakola dis-
ease, which is characterized by bone cysts and demyelinating
lesions in the CNS that ultimately result in fatal presenile de-
mentia (3). A role for TREM-2 in the immune system is evi-
denced by the expression of TREM-2 on in vitro derived mac-
rophages (4) and dendritic cells (1) and by the identification of
TREM-2 transcript in macrophage cell lines (2). Additionally,
a TREM-2 transcript has been detected in the lungs during ex-
perimental mycobacterial infection (5). However, the cell type
expressing TREM-2 and its function in the immune system is
he triggering receptor expressed on myeloid cells-2
(TREM-2)3is a cell surface receptor and a member of
the Ig superfamily with one V-type extracellular do-
failure to detect the TREM-2 protein in the immune system
TREM-2 associates with the adaptor DAP12, which is re-
quired for surface expression and signaling by TREM-2 (2).
DAP12 mediates downstream signaling through a cytoplasmic
ITAM domain, which can recruit Syk and activate PI3K, phos-
pholipase C, and Vav signaling cascades (6). In NK cells,
DAP12-associated receptors are activating receptors and con-
DAP12 in NK cells, Hamerman et al. demonstrated that
DAP12?/?macrophages have increased cytokine production
as compared with wild type (WT) (8), suggesting an inhibitory
role for DAP12. Additional studies have validated this observa-
tion in type I IFN-producing cells mediated by NKp44 or Si-
glec-H (9). Several studies have addressed TREM-2-DAP12
signaling. The cross-linking of TREM-2 transfected into MT2
cells induced NO release (2), and in vitro cross-linking of
TREM-2 on dendritic cells caused partial maturation (1). Ad-
ditionally, blockade of TREM-2 inhibited dendritic cell-medi-
ated activation of NK cells (10). These data suggest that
TREM-2 has an activating role. In contrast, in microglial cell
culture the knockdown of TREM-2 with short hairpin RNA
led to decreased phagocytosis but increased levels of proinflam-
matory transcripts, suggesting that TREM-2 may inhibit the
inflammation (11). Although TREM-2 is reported to be ex-
pressed by in vitro cultured macrophages, the role of TREM-2
in macrophage function is unknown.
that have been recruited to the peripheral tissues but not on
myeloid progenitors, circulating cells, or tissue-resident macro-
phages. We also find that TREM-2 is induced on resident peri-
toneal cells by IL-4. We have generated TREM-2?/?mice and
find that on these cells TREM-2 functions to inhibit cytokine
production in response to microbial products. Further, we find
no difference between TREM-2?/?and DAP12?/?macro-
receptor operative in the increased macrophage cytokine pro-
duction previously reported for DAP12?/?cells (8).
Department of Pathology and Immunology, Washington University School of Medicine,
St. Louis, MO 63110
Received for publication April 13, 2006. Accepted for publication July 19, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1I.R.T. was supported by National Institutes of Health Institutional Training Grant
2Address correspondence and reprint requests to Dr. Marco Colonna, Department of Pa-
thology and Immunology, Washington University School of Medicine, St. Louis, MO
63110. E-mail address: email@example.com
3Abbreviations used in this paper: TREM-2, triggering receptor expressed on myeloid
cells-2; BMDM, bone marrow-derived macrophage; MCSF, macrophage CSF; WT, wild
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
Materials and Methods
Committee (St. Louis, MO). B6.129P2-TYROBPtm1Ttkmice (hereafter re-
ferred to as DAP12?/?) were generously provided by T. Takai (Tohoku Uni-
versity, Sendai, Japan) (12) and backcrossed to C57BL6, with background de-
termined by simple sequence length polymorphism typing. STAT6 knockout
mice (strain B6.129S2(C)-Stat6tm1Gru/J) were from The Jackson Laboratory.
Generation of TREM-2?/?mice
The TREM-2 targeting construct was designed to delete a portion of the trans-
membrane and cytoplasmic domains encoded by exons 3 and 4. Two genomic
fragments (4.6 and 5.4 kb) flanking this region were amplified by PCR and
cloned into the targeting vector pMC1neo. The targeting construct was trans-
fected into E14.1 embryonic stem cells. Correctly targeted clones were injected
into C57BL/6 blastocysts, and chimeras were bred to transgenic mice express-
ing Cre under the CMV promoter to delete the neomycin resistance gene, re-
sulting in the strain of mice designated as B6.129P2-TREM2tm1cln(hereafter
referred to as TREM-2?/?). These mice were backcrossed to C57BL/6 until
?99% of the loci were derived from the C57BL6 strain by simple sequence
length polymorphism typing. We find that the TREM-2 mutation is transmit-
ted with the expected Mendelian ratios and that the mice have no gross
A Wistar rat was immunized with recombinant protein consisting of the extra-
cellular domain of TREM-2 fused to the human Ig constant domain. The
were selected by ELISA and screened against Jurkat cells that had been tran-
siently transfected with TREM-2.
To elicit primary macrophages, mice were treated with 1.5 ml of 2% thiogly-
collate medium i.p. injected; cells were isolated by peritoneal lavage. To gener-
ate bone marrow-derived macrophages (BMDM), total bone marrow was cul-
tured in DMEM supplemented with 10% bovine calf serum, 5% horse serum,
and 6 ng/ml recombinant human CSF-1 (R&D Systems). Cells were cultured
were stained with commercially available Abs: anti-CD11b, anti-CD40, anti-
GR1 (BD Pharmingen), and F4/80 (Caltag Laboratories).
Allergen-induced pulmonary inflammation
mice where challenged intranasally with 20 ?l of 1% OVA, and 48 h later the
recruited cells were isolated by bronchioalveolar lavage.
TLR stimulations and cytokine determination
BMDM were replated and allowed to adhere for 4 h at 37°C, and then TLR
agonists were added. LPS (Salmonella abortus-equi), zymosan (Saccharomyces
cerevisiae), and CpG 1826 DNA were from Sigma-Aldrich. Cell culture super-
were assayed by cytometric bead array (BD Biosciences mouse inflammation
Results and Discussion
TREM-2 is expressed on infiltrating (but not resident) macrophages
To establish the distribution of TREM-2, we generated a mAb
control cells transfected with the related receptor TREM-1
(data not shown). We were unable to find surface expression of
TREM-2 on peripheral blood cells or in the bone marrow. We
also measured TREM-2 in peripheral organs including liver,
spleen, and lung by flow cytometry; we found no expression of
TREM-2 in any tissue studied.
We then measured TREM-2 expression on peritoneal mac-
rophages. Mice were injected with thioglycollate, and perito-
neal exudate cells were harvested either before or 1, 2, or 3 days
ident peritoneal macrophages. However, macrophages re-
injection of thioglycollate (Fig. 1A). These cells were character-
ized for other markers expressed on infiltrating macrophages.
The TREM-2?cells uniformly expressed the macrophage
markers CD11b, F4/80, and CD115.
Based on the fraction of cells expressing TREM-2 (19% by
24 h), the effect we observe cannot be due simply to the induc-
tion of TREM-2 on the resident cells, because even if TREM-2
was induced on all resident peritoneal cells, that could not ac-
count for the fraction of TREM-2?cells, as thioglycollate in-
duces a ?5-fold increase in the number of macrophages recov-
ered from the peritoneum. Although we cannot exclude the
possibility that the injection of thioglycollate induces expres-
sion of TREM-2 on resident cells, TREM-2 must also be ex-
pressed on recently infiltrated cells.
TREM-2 is expressed during allergen- induced pulmonary inflammation
Considering that TREM-2 is expressed on macrophages re-
cruited to the peritoneum by thioglycollate, we hypothesized
that TREM-2 may be generally expressed on macrophages that
are infiltrating tissue. We tested this hypothesis in a second
model involving macrophage recruitment during allergen-in-
duced pulmonary inflammation. WT mice were sensitized to
OVA by immunization in the presence of alum. We then mea-
sured the expression of TREM-2 on macrophages recruited to
the air space after intranasal challenge. Consistent with prior
studies (13), challenge with OVA caused significant increase in
macrophages in the airspaces of sensitized mice. We found that
the cells that were recruited to the alveolar spaces of the lung
after challenge in the sensitized mice expressed TREM-2 (Fig.
1B). Importantly, we noted that the TREM-2?cells were also
phages were isolated before (day 1) or after (days 2–4) injection of thioglycol-
sitized to OVA by immunization and then challenged intranasally with OVA.
Macrophages recruited to the alveolar space in the sensitized mouse express
TREM-2. C, The murine macrophage cell line RAW264 express TREM-2,
which is down-regulated after overnight activation by IFN-? or LPS at indi-
cated doses. D, TREM-2 is highly expressed on BMDM from WT mice.
TREM-2 is expressed on macrophages. A, Peritoneal macro-
3521The Journal of Immunology
CD11c?CD11blow(data not shown). These markers are spe-
cific for alveolar macrophages (13), suggesting that TREM-2 is
induced on resident macrophages.
In pursuit of these studies we have also assayed TREM-2 ex-
pression during several other models of immune function. We
or EBV and splenocytes after infection with murine CMV; we
have also assayed the draining lymph nodes after peripheral in-
jection of mycobacteria or CFA plus OVA. In none of these
conditions was TREM-2 observed. Consistent with prior stud-
ies, we found that TREM-2 is expressed on the murine macro-
idly down-regulated in response to activation with LPS or
IFN-? (Fig. 1C). This activation-induced down-regulation
may explain our failure to detect TREM-2 n the other models.
In all of these experimental systems microbial products are
present to activate macrophages, which would rapidly down-
These data show that TREM-2 is expressed on macrophages
recruited to the peritoneum (by thioglycollate) or into the air
spaces (by OVA in sensitized mice). Importantly, we did not
find TREM-2 expression in the blood or bone marrow after
administration of thioglycollate, suggesting that the expression
of TREM-2 is not induced centrally (e.g., in the bone-marrow)
culation to the extravascular space or by cytokine-mediated
pulmonary inflammation, we hypothesized that alternative
macrophage activation by IL-4 or IL-13 may up-regulate
TREM-2. To test this hypothesis, we isolated resident perito-
mice. These cells were cultured with or without IL-4 for 48 h.
rophages with IL-4 induced TREM-2 (Fig. 2A).
To determine whether the expression of TREM-2 that we
had previously observed on thioglycollate recruited macro-
phages was the result of endogenous type-II cytokines, we mea-
sured TREM-2 expression on peritoneal exudate cells recruited
rophages (15). We find that TREM-2 was well expressed on
thioglycollate-elicited cells independently of STAT6, demon-
strating that type II activation is sufficient but not necessary for
TREM-2 expression by macrophages (Fig. 2B).
Generation of TREM-2?/?mice and expression of TREM-2 on bone
marrow-derived macrophages (BMDM)
To address the biological function of TREM-2, we established
TREM-2 knockout mice (Fig. 3A). We generated BMDM
from WT and TREM-2?/?mice and measured the expression
of TREM-2 and other markers of macrophage differentiation.
We found that TREM-2 is expressed on WT BMDM but not
on TREM-2-knockout BMDM (Fig. 3B). We found no differ-
comparing WT and TREM-2?/?cells (data not shown).
Whereas Humphery et al. recently reported that TREM-2 ex-
pression is induced during in vitro osteoclastogenesis induced
by culture BMDM with macrophage CSF (MCSF) and the re-
ceptor activator of NF-?B ligand (4), we find that differentia-
tion of macrophages in MCSF alone leads to expression of
TREM-2 skews cytokine production in response to TLR agonists
To determine the function of TREM-2 in macrophages, WT
and TREM-2?/?BMDM were treated with the TLR agonists
zymosan (TLR2/6), LPS (TLR4), CpG DNA (TLR9),
poly(I:C) (TLR3), and imiquimod (TLR7/8) (16). After 24 h,
cytokines levels were measured. We found that TREM-2?/?
mosan, and CpG (Fig. 4A). However, there was no difference
when the cells were stimulated with imiquimod or poly(I:C)
TREM-2 expression. A, Resident peritoneal cells were isolated from WT mice
and cultured for 48 h in the presence of MCSF with or without IL-4. Culture
with IL-4 induced TREM-2 expression of resident peritoneal macrophages. B,
Thioglycollate-recruited peritoneal cells were isolated from WT and
STAT6?/?mice, and TREM-2 expression was assayed by FACS. TREM-2 is
expressed independently of STAT6 expression.
Alternative activation is sufficient but not necessary for
locus was deleted by homologous recombination with a construct eliminating
were derived from WT or TREM2?/?and stained with mAb 178. TREM-2
was well expressed on WT cells but absent on the TREM2?/?cells.
Generation of TREM-2?/?mice. A, The TREM-2 genomic
3522CUTTING EDGE: TREM-2 ATTENUATES THE TLR RESPONSE
(data not shown). We also find a significant but more modest
increased in IL-6 production in response to LPS, zymosan, and
ducible difference between WT and TREM-2?/?cells with re-
gard to IL-10 production, although there was a trend toward
increased IL-10 in the TREM-2?/?cells (data not shown); we
found no IL-12p70 or IFN-? produced by the cells in our
TREM-2 completely accounts for the skewed cytokine production seen in
studies have shown that DAP12?/?cells produce increased cy-
tokines in response to TLR agonists. To determine whether
pared TNF-? production by WT, TREM2?/?, and
DAP12?/?macrophages. We found no difference between
TREM2?/?and DAP12?/?macrophages in response to zy-
mosan, CpG, and LPS (Fig. 4B), demonstrating that TREM-2
alone accounts for the inhibition of TLR activation previously
TREM-2 inhibits the response to LPS by primary macrophages
To establish the role of TREM-2 on primary macrophages, we
measured cytokines produced by peritoneal macrophages in re-
sponse to TLR agonists. Cells were recruited by the injection of
thioglycollate and assayed for cytokine production in response to
LPS and zymosan. We found that, consistent with results from
BMDM, peritoneal macrophages from TREM-2?/?mice pro-
duced increased TNF-? and IL-6 in response to LPS (Fig. 4C), but
there was no difference in response to zymosan (data not shown).
The phenotype observed in primary cells is thus much less dra-
matic than that of BMDM derived in vitro. In contrast to the vir-
tually uniform expression of TREM-2 observed on BMDM, only
one of three peritoneal macrophages express TREM-2 (Fig. 1, A vs
D). This likely accounts for the increased parity in the responses of
WT and TREM-2?/?peritoneal cells.
In this study, we found that TREM-2 is expressed by recently
differentiated macrophages and that it functions to attenuate
that type II inflammation induces TREM-2, both in vivo dur-
ing allergen-induced pulmonary inflammation and ex vivo by
the treatment of resident peritoneal cells with IL-4. We do not
find any expression of TREM-2 on other cell populations, in-
cluding progenitors in the bone marrow, circulating mono-
cytes, or tissue-resident macrophages. TREM-2 is rapidly
down-regulated in response to either IFN-? or LPS. We found
that similar to DAP12?/?cells, TREM-2?/?macrophages
and the levels of TNF-? and IL-6 in the supernatant were measured 24 h later. A, WT vs TREM-2?/?BMDM. TREM-2?/?cells produce increased cytokine as
compared with WT cells. ?, p ? 0.05; data are representative of at least three independent experiments. B, TREM-2 accounts for the decreased cytokine response
inhibits the primary macrophage response to LPS. Peritoneal macrophages were isolated from WT or TREM-2?/?mice and stimulated with LPS at the indicated
doses for 24 h. TREM-2?/?cells produced increased TNF-? and IL-6 in response to LPS. ?, p ? 0.05.
TREM-2 attenuates macrophage cytokine production. Macrophages were stimulated with the indicated concentration of LPS, zymosan, or CpG,
3523 The Journal of Immunology
produce increased levels of inflammatory cytokines when ex-
posed to TLR agonists. Importantly, we see no difference be-
tween DAP12?/?and TREM-2?/?, demonstrating that
TREM-2 completely accounts for the increased cytokine re-
sponse previously reported (8).
The mechanisms by which TREM-2 attenuates macrophage
cytokine production (or, generally, by which the ITAM motif
of DAP12 inhibits cells) remains unclear. Hamerman et al. re-
ported increased phosphorylation of ERK by DAP12?/?cells
in response to LPS as compared with WT cells (8). In contrast
to these results, we observe no increase of ERK activation by
LPS in the TREM2?/?cells (data not shown), suggesting that
there may other receptors (in addition to TREM-2) contribut-
ing to the difference in MAPK activation observed when WT
and DAP12?/?macrophages were previously compared. Pre-
vious studies have demonstrated that PI3K can inhibit TLR ac-
tivation through activation of the inhibitory apoptosis signal-
regulating kinase ASK (17), and DAP12 is known to activate
PI3K (6). It is possible that DAP12-mediated activation of
studies we were unable to detect any defects in PI3K activity;
however, this pathway merits further study.
What is the origin of the TREM-2?cells? In the context of
type II inflammatory responses, alternative activation by IL-4
or IL-13 likely up-regulates TREM-2 on resident cells such as
on infiltrating macrophages independent of alternative activa-
tion. Transmigration has been shown to induce differentiation
of monocytes, with circulating cells becoming macrophages as
they transmigrate across an endothelial layer (18), which could
provide a stimulus for induction of TREM-2. Overall, we con-
clude that TREM-2 is expressed in two contexts: 1) in the pres-
ident macrophages; and 2) as monocytes leave the circulation
and differentiate into macrophages, TREM-2 is induced.
M. Colonna and Washington University have a financial interest in BioXell,
which retains the rights to TREM-2, but did not support this work.
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3524CUTTING EDGE: TREM-2 ATTENUATES THE TLR RESPONSE