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ARTHRITIS & RHEUMATISM

Vol. 52, No. 9, September 2005, pp 29362946


DOI 10.1002/art.21238
2005, American College of Rheumatology

Innate Immunity Conferred by Toll-like Receptors 2 and 4 and


Myeloid Differentiation Factor 88 Expression Is Pivotal to
Monosodium Urate Monohydrate CrystalInduced Inflammation
Ru Liu-Bryan, Peter Scott, Anya Sydlaske, David M. Rose, and Robert Terkeltaub
, and transforming growth factor 1 also were significantly suppressed in TLR-2/ and TLR-4/ BMDMs
and were blunted in MyD88/ BMDMs in vitro. Neutrophil influx and local induction of IL-1 in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-2/ and TLR-4/ mice
and were attenuated in MyD88/ mice.
Conclusion. The murine host requires TLR-2,
TLR-4, and MyD88 for macrophage activation and
development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings
implicate innate immune cellular recognition of naked
MSU crystals by specific TLRs as a major factor in
determining the inflammatory potential of MSU crystal
deposits and the course of gouty arthritis.

Objective. In gout, incompletely defined molecular factors alter recognition of dormant articular and
bursal monosodium urate monohydrate (MSU) crystal
deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define
primary determinants of cellular recognition, uptake,
and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor 2
(TLR-2), TLR-4, and the cytosolic TLR adapter protein
myeloid differentiation factor 88 (MyD88), which are
centrally involved in innate immune recognition of
microbial pathogens.
Methods. We isolated bone marrowderived
macrophages (BMDMs) in TLR-2 / , TLR-4 / ,
MyD88/, and congenic wild-type mice, and assessed
phagocytosis and cytokine expression in response to
endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse
synovium-like subcutaneous air pouches.
Results. TLR-2/, TLR-4/, and MyD88/
BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystalinduced production of
interleukin-1 (IL-1), tumor necrosis factor ,
keratinocyte-derived cytokine/growth-related oncogene

The deposition of monosodium urate monohydrate (MSU) crystals in articular joints and bursal
tissues can be asymptomatic in gout or can erupt via
incompletely defined factors into acute, episodic, selflimiting joint inflammation largely dependent on
marked neutrophil influx (13). Because neutrophils
are absent from normal joint fluid, the interaction of
MSU crystals with resident cells in the joint is believed
to be the primary factor stimulating neutrophil
endothelial adhesion in the synovial microvasculature
(4), acute neutrophil ingress, and paroxysms of gouty
inflammation (1,4,5).
Cells that encounter MSU crystals express a
broad array of inflammatory mediators that contribute
to acute gouty inflammation, including cyclooxygenase
2, tumor necrosis factor (TNF), interleukin-1 (IL-1),
and IL-6 (59). Neutrophil chemotactic CXCR2-binding
chemokines, including keratinocyte-derived cytokine
(KC)/growth-related oncogene (GRO)/CXCL1 and
IL-8/CXCL4 (10), appear to be absolutely essential for
the neutrophil-dependent inflammation triggered by

Dr. Liu-Bryans work was supported by the NIH (grant


AR-049416). Dr. Roses work was supported by a VA Merit Review
Entry Program award, an Arthritis Foundation Arthritis Investigator
award, and NIH grant P30-AR-43360. Dr. Terkeltaubs work was
supported by a VA Merit Review award and NIH grant HL-077360.
Ru Liu-Bryan, PhD, Peter Scott, BS, Anya Sydlaske, BS,
David M. Rose, DVM, PhD, Robert Terkeltaub, MD: VA Medical
Center, University of California, San Diego.
Address correspondence and reprint requests to Ru LiuBryan, PhD, VA Medical Center, 111K, 3350 La Jolla Village Drive,
San Diego, CA 92161. E-mail: rliu@vapop.ucsd.edu.
Submitted for publication January 20, 2005; accepted in
revised form May 18, 2005.
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TLRs AND GOUTY INFLAMMATION

the MSU crystals, as demonstrated previously in


CXCR2/ mice (11). Release of the neutrophilexpressed calgranulin heterodimer S100A8/A9, one of
the most abundant protein constituents of the neutrophil
cytoplasm (12), appears to substantially amplify neutrophil recruitment in gouty inflammation (13). Importantly, the state of macrophage differentiation is a major
factor in the uptake of MSU crystals and sequelae,
including the capacity to express transforming growth
factor 1 (TGF1), a native suppressor of experimental
gouty inflammation (1416).
The naked MSU crystal has a highly negatively
charged, reactive surface that nonspecifically binds more
than 25 serum proteins (17) and also binds plasma
membrane proteins, including integrins (18,19). Significantly, MSU crystals activate both the classic and alternative complement pathways in vitro (2023). In studies
of C6-deficient rabbits, we recently discovered that local
assembly of the C5b9 membrane attack complex played
a major role in both intraarticular IL-8 expression and
acute neutrophilic inflammation in experimental MSU
crystalinduced arthritis of the knee joint (24). These
findings implicated the complement arm of innate immunity as one of the factors that controls acute gouty
inflammation. Furthermore, we observed that MSU
crystals activate the chondrocyte, a cell of mesenchymal
origin, in a manner that requires canonical signal transduction via Toll-like receptor 2 (TLR-2) (25). TLRs play
a vital role in host defense by initiating the innate
immune response against pathogens (26). Here, we
tested the hypothesis that innate immunemediated
cellular recognition of the naked MSU crystal through
certain TLRs is critical in the capacity of MSU crystals
to launch acute gouty inflammation.
There are more than 10 defined members in the
TLR group (26). TLRs are type I transmembrane receptors characterized by the presence of extracellular
leucine-rich repeat motifs that recognize pathogenassociated molecular patterns (26). Most TLRs have a
cytoplasmic Toll/IL-1 receptor (IL-1R) domain, which is
required for the activation of downstream signaling
pathways that lead to the activation of NF-B (26), a
transcription factor activated rapidly by MSU crystals
and centrally involved in MSU crystalinduced cell
activation (27,28). In response to pathogen-associated
molecular patterns, the canonical TLR signaling pathway recruits the cytosolic TLR adapter protein myeloid differentiation factor 88 (MyD88), IL-1R
activated kinase, and TNF receptorassociated factor
6 to activate IKKs, and the process culminates in

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NF-Bmediated expression of proinflammatory messenger RNA (26).


Ligation of certain TLRs also activates signal
transduction pathways, leading to phagocytosis, killing,
and clearance of pathogens by leukocytes (29). Recently,
we observed that TLR-2mediated and MyD88dependent signaling in chondrocytes played critical roles
in NF-B activation and nitric oxide generation in
response to MSU crystals (25). However, the direct
exposure of MSU crystals to chondrocytes is generally
limited in the joint, unlike the case with fibroblast-like
and macrophage-like synovium lining cells (1). Hence, in
this study, we sought to determine the roles of phagocyte
expression of TLR-2, MyD88, and TLR-4 (30) in inflammatory responses to MSU crystals by macrophages
in vitro, and in the synovium-like mouse subcutaneous
air pouch (11,31) in vivo.

MATERIALS AND METHODS


Reagents. All chemical reagents were obtained from
Sigma-Aldrich (St. Louis, MO), unless indicated otherwise.
Triclinic MSU crystals were prepared under pyrogen-free
conditions, using uric acid pretreated for 2 hours at 200C
prior to crystallization (10). The crystals were suspended
at 25 mg/ml in sterile, endotoxin-free phosphate buffered
saline (PBS), and verified to be free of detectable lipopolysaccharide contamination (0.025 endotoxin units/ml) by
the Limulus amebocyte cell lysate assay (BioWhittaker,
Walkersville, MD). MSU crystals (14C labeled) were prepared as described above, using as a starting material trace
14
C uric acid (Perkin Elmer, Boston, MA) added to 1 gm of
cold uric acid. The specific activity of the 14C-labeled MSU
crystals was 1.4 Ci/mg.
Isolation and culture of murine macrophages. All
animal experiments were performed humanely under institutionally approved protocols. TLR-2knockout (TLR-2/),
TLR-4/, and MyD88/ mice on a C57BL/6 background
(kindly provided by Dr. Shizuo Akira, University of Osaka,
Japan) were maintained under specific pathogenfree conditions and genotyped by polymerase chain reaction, as previously described (3234). Macrophages were prepared from
bone marrow obtained from 810-week-old homozygous TLR2/, TLR-4/, MyD88/, and congenic wild-type (WT)
control mice on the C57BL/6 background.
Briefly, bone marrow cells were isolated from the
femurs and tibias of the mice by flushing the medullary
cavity with PBS containing 2% fetal calf serum (FCS). After 1
wash in the same solution, cells were seeded in tissue culture
dishes in low-glucose Dulbeccos modified Eagles medium
supplemented with 10% FCS, 100 g/ml of streptomycin, 100
IU/ml of penicillin, and 40 ng/ml of recombinant granulocyte
macrophage colony-stimulating factor (BioSource International, Camarillo, CA) (35) at 37C for 79 days. Macrophages
were then assessed by flow cytometry using a FACScan
(Becton Dickinson Biosciences, San Jose, CA) by staining with

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allophycocyanin (APC)conjugated anti-F4/80 (Caltag, Burlingame, CA), a marker preferentially expressed by mature
macrophages (36). Double staining was performed using APCconjugated anti-F4/80 and phycoerythrin-conjugated anti
TLR-2 or antiTLR-4 (eBioscience, San Diego, CA) for
TLR-2 or TLR-4 expression, or APC-anti-F4/80 and MyD88
polyclonal primary antibodies (eBioscience, San Diego, CA)
and fluorescein isothiocyanateconjugated goat anti-rabbit
IgG (Jackson ImmunoResearch, West Grove, PA) as secondary antibodies for MyD88 expression. For double-staining
studies, cells were permeabilized in Cytofix/Cytoperm (Becton
Dickinson), following the manufacturers protocol.
Assays of phagocytosis and cytokine production.
BMDMs of individual genotypes were treated with MSU
crystals (0.5 mg/ml) for 2 hours at 4C or 37C and then washed
3 times with cold PBS containing 5 mM EDTA and harvested
in the same buffer. The proportion of macrophages taking up
MSU crystals was assessed by flow cytometry based on increased
side scatter properties (36). The amount of MSU crystals ingested
by the macrophages was determined under the same conditions using 14C-labeled MSU crystals by measuring 14C radioactivity in each sample of cells after they were washed 3 times
in cold PBS containing 5 mM EDTA.
The generation of IL-1, TNF, KC/GRO, and
TGF1 was evaluated by DuoSet enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN),
following the manufacturers protocol, by testing conditioned
media collected from BMDMs of each genotype (5 105/well)
stimulated with MSU crystals (0.5 mg/ml) for 24 hours.
Studies of synovium-like subcutaneous air pouches.
Subcutaneous pouches were generated by the injection of
sterile, filtered air to create an accessible space that developed
a synovium-like membrane within 7 days, as previously described (10). Briefly, anesthetized 1012-week-old WT, TLR2/, TLR-4/, and MyD88/ mice were injected with 5 ml
of sterile air into the subcutaneous tissue of the back, followed
by a second injection of 3 ml of sterile air into the pouch 3
days later. MSU crystals (3 mg), in 1 ml of sterile, endotoxinfree PBS, were injected into the pouch 7 days after the first
injection of air. Mice were killed and pouch fluids were
harvested at specific time points by injecting 5 ml of PBS
containing 5 mM EDTA, and cells infiltrating the air pouch
were counted manually using a hemocytometer. Smears of
cells from the air pouches were prepared by centrifugation
of either 50 l of the pouch exudates or 105 of cells in
cytofunnels (ThermoShandon, Pittsburgh, PA) in a Cytospin
4 centrifuge (ThermoShandon) at 110g for 2 minutes. Differential leukocyte subpopulation counts were measured by
Wright-Giemsa staining of cytospin slides. IL-1 in supernatants of air pouch exudates was measured by ELISA, as
described above.
For histologic analysis of the air pouches, frozen
sagittal sections of the air pouches were fixed in 80% ethanol
and then stained with hematoxylin for 25 minutes. Sections
were washed 3 times with water and incubated in ammonium
hydroxide for 30 seconds, and then incubated with acid alcohol
(0.5 % HCl in 70% ethanol) for 30 seconds. Sections were then
washed 3 times again and counterstained with eosin for 35
minutes.

LIU-BRYAN ET AL

Statistical analysis. Data are expressed as the mean


SD. Statistical analyses were performed using Students
2-tailed t-test. P values less than 0.05 were considered significant.

RESULTS
Roles of TLR-2 and MyD88 in macrophage responsiveness to MSU crystals in vitro. We first tested
the hypothesis that both TLR-2 and MyD88 mediated
the capacity of MSU crystals to induce macrophage
expression of selected cytokines previously implicated in regulating gouty inflammation and known to
be directly and rapidly induced in cells via simple
exposure to MSU crystals (IL-1, TNF, KC/GRO,
and TGF1, as cited above). To do so, we generated
BMDMs from TLR-2/, MyD88/, and congenic WT
mice. The differentiation state of isolated BMDMs
was confirmed by flow cytometry using F4/80 as a
mature, macrophage-specific marker (36). In all experiments and for all genotypes studied, 85% of the
isolated BMDMs were F4/80 positive. In F4/80-positive
WT BMDMs, 95% expressed TLR-2, 85% expressed
TLR-4, and 90% expressed MyD88, as assessed by
flow cytometry of permeabilized cells.
To avoid potential masking effects of both serum
protein opsonization of the crystals (1) and crystalinduced complement activation (24), we treated BMDMs
with endotoxin-free MSU crystals at a concentration of
0.5 mg/ml under entirely serum-free conditions, based
on our previous studies (27). At 24 hours, MSU crystals
induced the production of several cytokines, including
IL-1, TNF, KC/GRO, and TGF1 in WT BMDMs
(Figure 1A), as determined by ELISA of conditioned
media. However, production of each of these cytokines
was partially, but substantially, reduced in TLR-2/
BMDMs compared with WT BMDMs (Figure 1B).
Blunting of production of each of these cytokines in
response to MSU crystals was observed in MyD88/
BMDMs (Figure 1).
Mediation of MSU crystal phagocytosis in the
macrophage by TLR-2 and MyD88. Ingestion of MSU
crystals promotes cell activation in phagocytes
(12,37,38), and TLR-2 signaling is known to mediate a
phagocytic program (29) and to physically cooperate
with other receptors in promoting phagocytosis of
specific microbial pathogens (39). Therefore, we hypothesized that there was impaired uptake of MSU crystals
in TLR-2/ cells. First, we validated that our primary
assay system discerned crystal uptake rather than nonspecific crystalcell association by treating murine

TLRs AND GOUTY INFLAMMATION

Figure 1. Effects of Toll-like receptor 2 (TLR-2) and myeloid differentiation factor 88 (MyD88) deficiency on cytokine production in bone
marrowderived macrophages (BMDMs) in response to monosodium
urate monohydrate (MSU) crystals in vitro. BMDMs prepared from
wild-type (WT), TLR-2/, and MyD88/ mice were incubated with
endotoxin-free MSU crystals (0.5 mg/ml) for 24 hours under serumfree conditions, as described in Materials and Methods. Levels of
interleukin-1 (IL-1), tumor necrosis factor (TNF), keratinocytederived cytokine (KC)/growth-related oncogene (GRO), and transforming growth factor 1 (TGF1) in conditioned media were measured by enzyme-linked immunosorbent assay. A, Cytokine production
in WT BMDMs. B, Percentage of cytokine production in TLR-2/
and MyD88/ BMDMs relative to WT BMDMs. Values are the mean
and SEM of 5 individual experiments, using cells from 5 different
mice of each genotype. P 0.05 versus WT mice.

BMDMs with MSU crystals for 2 hours at 4C versus


37C, again under serum-free conditions. The proportion of macrophages containing MSU crystals was measured by flow cytometry, based on an increase in side
scatter profile, as previously described (36). As seen in
Figure 2A, there was an absence of detectable uptake of
MSU crystals by either WT or TLR-2/ BMDMs at
4C. At 37C, the proportion of TLR-2/ BMDMs that
took up MSU crystals was reduced by 50% relative to
WT BMDMs.
To ascertain the effects of TLR-2 deficiency on
the amount of crystals taken up by the BMDM population as a whole, we treated cells with 14C-labeled MSU
crystals. The amount of 14C-labeled MSU crystals ingested by BMDMs of TLR-2/ mice was decreased by
70% compared with WT BMDMs (Figure 2B). Similarly, the proportion of MyD88/ BMDMs that took up
MSU crystals was reduced by 50% compared with WT

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BMDMs, with inhibitory effects of MyD88 deficiency


observed as early as 15 minutes after stimulation with
MSU crystals (Figure 2C). Moreover, the amount of
14
C-labeled MSU crystals ingested by MyD88/
BMDMs was decreased by 70% at 15 minutes, and by
80% 2 hours after stimulation relative to WT BMDMs.
Effects of TLR-2 and MyD88 deficiency on MSU
crystalinduced inflammation in the mouse synoviumlike subcutaneous air pouch. Next, we tested the roles
of TLR-2 and MyD88 in MSU crystalinduced inflammation in vivo. To do so, we injected endotoxin-free
MSU crystals into subcutaneous air pouches of TLR2/, MyD88/, and WT mice, a model system characterized by generation of a synovium-like lining cell
layer containing fibroblastic and phagocytic cells (31). In
order to screen for inhibitory effects on a submaximal
inflammatory response, we chose a dose of MSU crystals
(3 mg in 1 ml of PBS) that was 70% lower than that
used in our previous studies of MSU crystalinduced air
pouch inflammation (11). At 0, 6, and 24 hours post
MSU crystal injection, mice were killed, and we measured both IL-1 production and leukocyte influx in the
air pouch exudates. IL-1 expression (Figure 3C) and
leukocyte ingress (Figure 3A) were robust in WT mice 6
hours after MSU crystals were injected into the air
pouch, with neutrophils accounting for the majority of
infiltrated leukocytes (Figure 3B). Both cytokine induction and neutrophilic inflammation were self-limiting by
24 hours postinjection (Figure 3). Six hours after MSU
crystal injection, IL-1 expression and the total number
of infiltrated leukocytes and neutrophils were partially,
but significantly, suppressed in TLR-2/ mice relative
to WT mice (Figure 3). Furthermore, there was a virtual
absence of IL- induction as well as leukocyte ingress in
the air pouch of MyD88/ mice in response to MSU
crystals (Figure 3).
In comparisons of TLR-2/ and WT mice, we
analyzed the histologic features of the air pouch following MSU crystal injection. The resting air pouches
possessed a thin synovium-like lining and a thicker
subcutaneous layer of vascularized fibrous and adipose
tissue beneath the synovium-like lining (Figure 4). Six
hours after the injection of MSU crystals, the architecture of the WT synovium-like lining layer became disrupted, and there was marked swelling of the tissues
beneath the air pouch lining layer and massive infiltration of leukocytes throughout the air pouch lining and
tissue immediately surrounding it (Figure 4). By comparison, at the same time point following MSU crystal
injection in the TLR-2/ air pouch, there was markedly
less swelling of the synovium-like layer (Figure 4). There

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LIU-BRYAN ET AL

Figure 2. Decreased uptake of MSU crystals by TLR-2/ and MyD88/ BMDMs in vitro. BMDMs
were incubated with either unlabeled or 14C-labeled MSU crystals (0.5 mg/ml) under the conditions
indicated. A and C, The percentage of BMDMs that took up the unlabeled MSU crystals was analyzed
by flow cytometry based on the increase in the side scatter profile. B and D, The amount of 14C-labeled
MSU crystals ingested by BMDMs was determined by measuring the radioactivity of 14C associated
with washed cells. In A and B, TLR-2/ BMDMs were incubated with unlabeled MSU crystals for 2
hours at 4C or 37C, or with 14C-labeled MSU crystals for 2 hours at 37C. In C and D, MyD88/
BMDMs were incubated with unlabeled or 14C-labeled MSU crystals for 15 minutes and 2 hours at
37C. Values in B and D are the mean and SEM of 3 different experiments on 3 different mice of each
genotype. P 0.05 versus WT mice. SSC-H side scatter height; FSC-H forward scatter height;
R1 region 1; R2 region 2 (see Figure 1 for other definitions).

TLRs AND GOUTY INFLAMMATION

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Figure 3. Suppressed infiltration of leukocytes and induction of IL-1 in response to MSU crystals
in subcutaneous air pouches of TLR-2/ and MyD88/ mice. Subcutaneous air pouches were
created on the backs of mice of the indicated genotypes via injections of sterile air, as described in
Materials and Methods, and 7 days after initial generation of the air pouches, a 1-ml suspension of
3 mg MSU crystals in phosphate buffered saline (PBS) was injected into the air pouches. Mice were
killed at the indicated times, and the air pouch exudates were harvested by washing with 5 ml of
PBS containing 5 mM EDTA. A and B, The leukocytes in the pouch exudates were counted using
a hemocytometer, and the fraction of neutrophils was determined using Wright-Giemsa staining.
C, Supernatants of air pouch exudates were collected by centrifugation and IL-1 production was
measured by enzyme-linked immunosorbent assay. Under these conditions, injection of PBS
control alone was associated with a background of only 0.15 106 leukocytes per air pouch in WT
mice, 0.13 106 leukocytes per air pouch in TLR-2/ mice, and 0.05 106 leukocytes per air
pouch in MyD88/ mice throughout the time course. Values shown are the mean SEM of 8 WT
mice, 9 TLR-2/ mice, and 8 MyD88/ mice. P 0.05 versus WT mice. See Figure 1 for
other definitions.

also was less intense infiltration of leukocytes in the


TLR-2/ air pouch, a result that correlated well with
gross analyses of Wright-Giemsastained smears of
cells in the air pouch exudates (Figure 4) and with the
aforementioned counts of infiltrated leukocytes (Figures
3A and B).
Mediation of MSU crystalinduced macrophage
activation and inflammation by TLR-4. Results to this
point indicated that TLR-2 deficiency partially impaired
MSU crystalinduced inflammatory responses, as opposed to the nearly complete attenuation of these same
responses in macrophages and air pouches of mice
deficient in MyD88. Hence, we hypothesized that 1 or
more TLRs other than TLR-2 also contributed significantly to the triggering of MSU crystalinduced inflammation. We elected to assess TLR-4, which, like TLR-2,
is constitutively expressed in macrophages (30) and can
modulate recognition and phagocytosis of microbial

pathogens (29,40). TLR-4/ BMDMs showed marked


impairment of production of TNF, KC/GRO, and
TGF1 in response to MSU crystals (Figure 5A) as well
as impaired uptake of MSU crystals (Figures 5B and C).
Finally, the capacity of MSU crystals to induce IL-1
expression, leukocyte ingress, and neutrophilic inflammation in the air pouch was markedly suppressed in
TLR-4/ mice (Figures 6A and B).
DISCUSSION
In this study, we demonstrated that host expression of TLR-2, TLR-4, and their shared adapter protein
MyD88 was a major determinant of the capacity of
endotoxin-free MSU crystals to turn on the macrophage
in vitro and to trigger acute neutrophilic inflammation in
vivo. To avoid confounding effects of serum opsonins on
the in vitro results, we limited this study to an evaluation

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LIU-BRYAN ET AL

Figure 4. Comparison of histologic features of MSU crystalinduced inflammation in synovium-like


subcutaneous air pouches in WT and TLR-2/ mice. Top, frozen sections of air pouches stained with
hematoxylin and eosin. Bottom, Wright-Giemsastained smears of cells from the same air pouches,
following centrifugation of 50 l of air pouch exudates at 110g for 2 minutes. Results shown are
representative of 3 different experiments on 3 different mice of each genotype for each condition and
time point shown. See Figure 1 for definitions.

of the effects of uncoated MSU crystals under serumfree conditions, including an analysis of cultured macrophage uptake of MSU crystals and cytokine release. But
it was notable that the decreases in inflammation induced by injected MSU crystals in vivo in the air
pouches of mice deficient in TLR-2, TLR-4, and MyD88
paralleled the decreases in cytokine production observed in MSU crystalstimulated macrophages under
serum-free conditions in vitro.
The inert MSU crystal has the capacity to avidly
bind at least 20 different plasma proteins (1,14,15).
Although such MSU crystal binding interactions are
nonspecific via hydrogen and electrostatic bonds,
MSU does associate preferentially with selected proteins in complex mixtures, as illustrated by apoB and
complement pathway protein binding in crystals exposed to human plasma (41,42). Clearly, naked MSU
crystals can directly promote inflammation by activating complement and the contact coagulation system
(1,24). But our results support a model for triggering
gouty inflammation in which the capacity of the MSU
crystal to directly activate resident cells in the joint is
pivotal (1).
MSU crystals bind IgG, which can promote rec-

ognition by phagocytes and enhanced cellular responses


to the crystals (1,43). However, the predominant effect
of coating MSU crystals with plasma or serum is marked
inhibition of cell activation via apoB binding to the
crystal surface, which physically suppresses MSU
crystalcell binding (44). Synovial MSU crystals deposited in microscopic tophi have been observed to be
tightly packed in a core contained by a protein-rich wall
that includes fibrinogen (45). As such, our results here,
and the known association of acute gouty attacks with
rapid rises and falls in the levels of serum urate, suggest
that gouty inflammation is triggered either by the de
novo formation of uncoated MSU crystals in the joint or
by the release from synovial tophi of MSU crystals
liberated from coating proteins by factors including
partial crystal dissolution.
MSU crystals preferentially bind to certain cell
membrane proteins, such as integrins and the Fc receptor CD16 (18,19). In this context, both macrophages and
synovium lining cells express TLR-2 and TLR-4
(30,46,47). Although expression of both TLR-2 and
TLR-4 is relatively low in normal synovium, TLR-2 and
TLR-4 expression is subject to regulation, as illustrated
by cytokine-inductive effects in vitro and up-regulated

TLRs AND GOUTY INFLAMMATION

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Figure 5. Effects of TLR-4 deficiency on MSU crystal uptake and MSU crystalinduced cytokine
expression in BMDMs in vitro. BMDMs prepared from WT and TLR-4/ mice were incubated
with MSU crystals (0.5 mg/ml) for 24 hours to determine TNF, KC/GRO, and TGF1
expression by enzyme-linked immunosorbent assay (A), or for 2 hours to assess crystal uptake by
flow cytometry (B) and levels of cell-associated 14C-labeled MSU crystals (C). Data shown in A,
which are presented as a percentage of cytokine production relative to that in WT mice, were
pooled from 5 different experiments on 5 WT and TLR-4/ mice. Data shown in B and C are
representative of 3 different experiments on 3 different mice of each genotype. Values in A and C
are the mean and SEM. P 0.05 versus WT mice. SSC-H side scatter height; FSC-H
forward scatter height; R1 region 1; R2 region 2 (see Figure 1 for other definitions).

expression in RA synovium in vivo (46,47). We speculate


that TLR-2 and TLR-4 or closely interacting proteins
are among the plasma membrane proteins directly engaged by MSU crystals. Articular chondrocytes have
been particularly advantageous in testing this notion,
because we observed that normal articular chondrocytes
constitutively expressed TLR-2 but not several other
TLRs, including TLR-4, in vitro (25). Moreover, direct
up-regulation and down-regulation of MSU crystal
induced chondrocyte nitric oxide production was inducible by specific gain-of-function of TLR-2 expression
versus loss-of-function of TLR-2dependent signaling
(25). It will be interesting to determine if TLRs other
than TLR-2 and TLR-4 expressed by macrophages and
synovium lining cells (48) also mediate MSU crystal
induced inflammation. We speculate that MSU crystals
also mediate the activation of infiltrating neutrophils via
TLRs to amplify synovitis once the acute gouty inflammatory process has been initiated.
The markedly reduced capacity of both TLR-2

deficient and TLR-4deficient macrophages to ingest


and respond to MSU crystals is compelling, because
deficiencies of TLR-2 and TLR-4 are associated with
selective rather than generalized phagocytosis defects
(29), mediated partly by divergent modes of TLRdependent and TLR-independent phagosome maturation (40). For example, in TLR-2/ macrophages,
phagocytosis of inert latex beads is intact (30,40), as is
ingestion of zymosan particles, although TLR-2/ macrophages demonstrate decreased activation by zymosan
(49,50). Furthermore, macrophages from TLR-2/TLR-4
double-knockout mice and MyD88/ mice demonstrate no differences in phagocytosis of apoptotic cells
relative to WT cells under conditions in which phagocytosis of bacteria is impaired (40). Although our results
suggest that TLR-2 and TLR-4 recognize the inert MSU
crystal surface as a pathogen-associated molecular pattern to directly promote phagocytosis, the current study
did not unequivocally prove this. One alternative scenario is that TLR-2 ligands promote phagocytosis

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LIU-BRYAN ET AL

Figure 6. Suppressed infiltration of leukocytes and induction of IL-1 in response to MSU crystals in
subcutaneous air pouches of TLR-4/ mice. A suspension of 3 mg of MSU crystals in 1 ml of
phosphate buffered saline (PBS) was injected into the air pouches, and MSU crystalinduced leukocyte
infiltration (A), the number of neutrophils in the exudates (B), and IL-1 induction in the air pouch in
vivo (C) (measured by enzyme-linked immunosorbent assay in supernatants of air pouch exudates
collected by centrifugation) were determined. Under these conditions, injection of PBS control alone
was associated with a background of only 0.15 106 leukocytes per air pouch in WT mice and 0.12
106 leukocytes per air pouch in TLR-4/ mice. Values are the mean SEM of 8 WT mice and 9
TLR-4/ mice. P 0.05 versus WT mice. See Figure 1 for other definitions.

through induction of a function-specific gene expression


program that includes up-regulation of scavenger receptor A expression, an activity shared by ligands of certain
other TLRs (29). Given this, it is possible that deficient
TLR-2 and/or TLR-4mediated induction of other
plasma membrane proteins that recognize the inert
MSU crystal may have contributed to our findings.
We limited our analysis of cultured cells to
unfractionated bone marrow macrophage preparations.
The state of differentiation of macrophages clearly
mediates the ability of these cells to not only take up
MSU crystals (14), but to also respond in a proinflammatory or antiinflammatory manner to the crystals
(14,15). Hence, the observed effects of host TLR-2 and
TLR-4 expression on the capacity of macrophages to
ingest and react to MSU crystals were possibly at least
partially mediated by indirect effects on macrophage
differentiation. Previously described effects of expression of certain TLRs on bacterial phagocytosis by macrophages mediated by regulation of signal transduction

and gene expression (29) might provide an analogy to


the situation for MSU crystalmacrophage interactions.
However, further elucidation of direct and indirect
effects of TLR-2 and TLR-4 on true recognition of the
MSU crystal by the macrophage will clearly require
specific TLR loss-of-function and gain-of-function
studies in mature macrophages.
Striking suppression of MSU crystal uptake in
TLR-2/ and TLR-4/ macrophages was mirrored in
across-the-board suppression of the capacity of MSU
crystals to induce expression by macrophages of proinflammatory cytokines and of TGF1 in the same cells in
this study. Importantly, fully differentiated macrophages
that clear MSU crystals express TGF1, thereby promoting resolution of acute MSU crystalinduced inflammation (4,15). Therefore, our results suggest that under
some conditions, TLR-2 and TLR-4 expression might
not only promote the triggering of acute gout, but also
contribute to the spontaneous self-limitation so characteristic of gouty inflammation (1,4).

TLRs AND GOUTY INFLAMMATION

Limitations of this study include the fact that that


we did not assess whether MSU crystals regulate inflammation indirectly through TLR and MyD88 signaling via
induced cellular release of endogenous ligands of TLR-2
and TLR-4, such as Hsp70 and HMGB-1 (51,52).
MyD88 is one of several cytosolic adapter proteins for
TLR-2 and TLR-4 (26), and MyD88 also transduces
IL-1Rmediated responses (53). These are points to be
considered because we did not test to determine whether
the blunting effects of MyD88 deficiency on MSU
crystalinduced macrophage activation and inflammation were mediated partly by impaired IL-1 signaling.
Moreover, we have not yet evaluated the potential roles
of the extracellular TLR-2 and/or TLR-4 adapter molecules CD14 and myeloid differentiation protein 2 (26,39)
in responsiveness to MSU crystals.
In conclusion, this study has established that host
expression of TLR-2, TLR-4, and their intracellular
adapter protein MyD88 is a major mediator of MSU
crystalinduced inflammation. Our results suggest that
acute gouty inflammation is triggered and regulated in
intensity at least in part by cellular recognition of the
naked MSU crystal as a function of TLR-dependent
innate immunity. Significantly, TLR-2 signaling, culminating in NF-B activation, critically transduces chondrocyte nitric oxide generation in response to MSU and
calcium pyrophosphate dihydrate (CPPD) crystals (25).
Since the acute inflammatory responses to CPPD crystals resemble those of MSU crystals, we speculate that
TLR-2, TLR-4, and MyD88 could also mediate CPPD
crystalinduced acute inflammation. Finally, TLR-2 and
TLR-4 sequence variants have been linked to altered
microbial carriage and phenotypic response of the host
to infection (54,55). Hence, it will be of interest to
determine if inherited or acquired alterations in the
structure and function of TLR-2 and TLR-4 contribute
to variability in the clinical phenotype of gout in humans
with hyperuricemia (1).
ACKNOWLEDGMENTS

2945

2.
3.
4.

5.
6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

We gratefully acknowledge Drs. Peter Tobias and


Richard Ulevitch (The Scripps Research Institute, La Jolla,
CA) for helpful comments relating to the design and execution
of these studies. We thank Monika Polewski (VA Medical
Center, San Diego) for expert technical assistance with the air
pouch model studies.

17.

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