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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
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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
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
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.
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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).
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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.
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LIU-BRYAN ET AL
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-
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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).
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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.
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