liu-bryan et al-2005-arthritis & rheumatism

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ARTHRITIS & RHEUMATISM

Vol. 52, No. 9, September 2005, pp 2936–2946

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 toMonosodium Urate Monohydrate Crystal–Induced Inflammation

Ru Liu-Bryan, Peter Scott, Anya Sydlaske, David M. Rose, and Robert Terkeltaub

Objective. In gout, incompletely defined molecu-

lar factors alter recognition of dormant articular and

bursal monosodium urate monohydrate (MSU) crystal

deposits, thereby inducing self-limiting bouts of charac-

teristically severe neutrophilic inflammation. To define

primary determinants of cellular recognition, uptake,

and inflammatory responses to MSU crystals, we con-

ducted 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 marrow–derived

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 condi-

tions. 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 crys-

tals in vitro. MSU crystal–induced production of

interleukin-1 (IL-1), tumor necrosis factor ,

keratinocyte-derived cytokine/growth-related oncogene

, and transforming growth factor 1 also were signif-

icantly suppressed in TLR-2 / and TLR-4 / BMDMs

and were blunted in MyD88 / BMDMs in vitro. Neu-

trophil influx and local induction of IL-1 in subcuta-

neous air pouches were suppressed 6 hours after injec-

tion 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 in-

flammation 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.

The deposition of monosodium urate monohy-drate (MSU) crystals in articular joints and bursaltissues can be asymptomatic in gout or can erupt viaincompletely defined factors into acute, episodic, self-limiting joint inflammation largely dependent onmarked neutrophil influx (1–3). Because neutrophilsare absent from normal joint fluid, the interaction of MSU crystals with resident cells in the joint is believedto be the primary factor stimulating neutrophil–endothelial adhesion in the synovial microvasculature(4), acute neutrophil ingress, and paroxysms of goutyinflammation (1,4,5).

Cells that encounter MSU crystals express a

broad array of inflammatory mediators that contributeto acute gouty inflammation, including cyclooxygenase2, tumor necrosis factor (TNF), interleukin-1 (IL-1),and IL-6 (5–9). Neutrophil chemotactic CXCR2-bindingchemokines, including keratinocyte-derived cytokine(KC)/growth-related oncogene (GRO)/CXCL1 andIL-8/CXCL4 (10), appear to be absolutely essential forthe neutrophil-dependent inflammation triggered by

Dr. Liu-Bryan’s work was supported by the NIH (grant AR-049416). Dr. Rose’s work was supported by a VA Merit ReviewEntry Program award, an Arthritis Foundation Arthritis Investigatoraward, and NIH grant P30-AR-43360. Dr. Terkeltaub’s work wassupported 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 MedicalCenter, University of California, San Diego.

Address correspondence and reprint requests to Ru Liu-Bryan, PhD, VA Medical Center, 111K, 3350 La Jolla Village Drive,San Diego, CA 92161. E-mail: [email protected].

Submitted for publication January 20, 2005; accepted inrevised form May 18, 2005.

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the MSU crystals, as demonstrated previously inCXCR2 / mice (11). Release of the neutrophil-expressed calgranulin heterodimer S100A8/A9, one of the most abundant protein constituents of the neutrophilcytoplasm (12), appears to substantially amplify neutro-

phil recruitment in gouty inflammation (13). Impor-tantly, the state of macrophage differentiation is a majorfactor in the uptake of MSU crystals and sequelae,including the capacity to express transforming growthfactor 1 (TGF1), a native suppressor of experimentalgouty inflammation (14–16).

The naked MSU crystal has a highly negativelycharged, reactive surface that nonspecifically binds morethan 25 serum proteins (17) and also binds plasmamembrane proteins, including integrins (18,19). Signifi-cantly, MSU crystals activate both the classic and alter-native complement pathways in vitro (20–23). In studies

of C6-deficient rabbits, we recently discovered that localassembly of the C5b–9 membrane attack complex playeda major role in both intraarticular IL-8 expression andacute neutrophilic inflammation in experimental MSUcrystal–induced arthritis of the knee joint (24). Thesefindings implicated the complement arm of innate im-munity as one of the factors that controls acute goutyinflammation. Furthermore, we observed that MSUcrystals activate the chondrocyte, a cell of mesenchymalorigin, in a manner that requires canonical signal trans-duction via Toll-like receptor 2 (TLR-2) (25). TLRs playa vital role in host defense by initiating the innateimmune response against pathogens (26). Here, we

tested the hypothesis that innate immune–mediatedcellular recognition of the naked MSU crystal throughcertain TLRs is critical in the capacity of MSU crystalsto launch acute gouty inflammation.

There are more than 10 defined members in theTLR group (26). TLRs are type I transmembrane re-ceptors characterized by the presence of extracellularleucine-rich repeat motifs that recognize pathogen-associated molecular patterns (26). Most TLRs have acytoplasmic Toll/IL-1 receptor (IL-1R) domain, which isrequired for the activation of downstream signalingpathways that lead to the activation of NF-B (26), a

transcription factor activated rapidly by MSU crystalsand centrally involved in MSU crystal–induced cellactivation (27,28). In response to pathogen-associatedmolecular patterns, the canonical TLR signaling path-

way recruits the cytosolic TLR adapter protein my-eloid differentiation factor 88 (MyD88), IL-1R–activated kinase, and TNF receptor–associated factor6 to activate IKKs, and the process culminates in

NF-B–mediated expression of proinflammatory mes-senger RNA (26).

Ligation of certain TLRs also activates signaltransduction pathways, leading to phagocytosis, killing,and clearance of pathogens by leukocytes (29). Recently,

we obser ved that TLR-2–mediated and MyD88-dependent signaling in chondrocytes played critical rolesin NF-B activation and nitric oxide generation inresponse to MSU crystals (25). However, the directexposure of MSU crystals to chondrocytes is generallylimited in the joint, unlike the case with fibroblast-likeand macrophage-like synovium lining cells (1). Hence, inthis study, we sought to determine the roles of phagocyteexpression of TLR-2, MyD88, and TLR-4 (30) in in-flammatory responses to MSU crystals by macrophagesin vitro, and in the synovium-like mouse subcutaneousair pouch (11,31) in vivo.

MATERIALS AND METHODS

Reagents. All chemical reagents were obtained fromSigma-Aldrich (St. Louis, MO), unless indicated otherwise.Triclinic MSU crystals were prepared under pyrogen-freeconditions, using uric acid pretreated for 2 hours at 200°Cprior to crystallization (10). The crystals were suspendedat 25 mg/ml in sterile, endotoxin-free phosphate bufferedsaline (PBS), and verified to be free of detectable lipopoly-saccharide contamination (0.025 endotoxin units/ml) bythe Limulus amebocyte cell lysate assay (BioWhittaker,Walkersville, MD). MSU crystals (14C labeled) were pre-pared as described above, using as a starting material trace14

C uric acid (Perkin Elmer, Boston, MA) added to 1 gm of cold uric acid. The specific activity of the 14C-labeled MSUcrystals was 1.4 Ci/mg.

Isolation and culture of murine macrophages. Allanimal experiments were performed humanely under institu-tionally approved protocols. TLR-2–knockout (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 pathogen–free condi-tions and genotyped by polymerase chain reaction, as previ-ously described (32–34). Macrophages were prepared frombone marrow obtained from 8–10-week-old homozygous TLR-2 / , TLR-4 / , MyD88 / , and congenic wild-type (WT)control mice on the C57BL/6 background.

Briefly, bone marrow cells were isolated from thefemurs and tibias of the mice by flushing the medullarycavity with PBS containing 2% fetal calf serum (FCS). After 1 wash in the same solution, cells were seeded in tissue culturedishes in low-glucose Dulbecco’s modified Eagle’s mediumsupplemented with 10% FCS, 100 g/ml of streptomycin, 100IU/ml of penicillin, and 40 ng/ml of recombinant granulocyte–macrophage colony-stimulating factor (BioSource Interna-tional, Camarillo, CA) (35) at 37°C for 7–9 days. Macrophages were then assessed by flow cytometry using a FACScan(Becton Dickinson Biosciences, San Jose, CA) by staining with

TLRs AND GOUTY INFLAMMATION 2937



allophycocyanin (APC)–conjugated anti-F4/80 (Caltag, Burlin-game, CA), a marker preferentially expressed by maturemacrophages (36). Double staining was performed using APC-conjugated anti-F4/80 and phycoerythrin-conjugated anti–TLR-2 or anti–TLR-4 (eBioscience, San Diego, CA) forTLR-2 or TLR-4 expression, or APC-anti-F4/80 and MyD88polyclonal primary antibodies (eBioscience, San Diego, CA)and fluorescein isothiocyanate–conjugated goat anti-rabbitIgG (Jackson ImmunoResearch, West Grove, PA) as second-ary antibodies for MyD88 expression. For double-stainingstudies, cells were permeabilized in Cytofix/Cytoperm (BectonDickinson), following the manufacturer’s protocol.

Assays of phagocytosis and cytokine production.

BMDMs of individual genotypes were treated with MSUcrystals (0.5 mg/ml) for 2 hours at 4°C or 37°C and then washed3 times with cold PBS containing 5 m M EDTA and harvestedin the same buffer. The proportion of macrophages taking upMSU crystals was assessed by flow cytometry based on increasedside scatter properties (36). The amount of MSU crystals ingestedby the macrophages was determined under the same condi-

tions using 14

C-labeled MSU crystals by measuring 14

C radio-activity in each sample of cells after they were washed 3 timesin cold PBS containing 5 m M EDTA.

The generation of IL-1, TNF, KC/GRO, andTGF1 was evaluated by DuoSet enzyme-linked immuno-sorbent assay (ELISA; R&D Systems, Minneapolis, MN),following the manufacturer’s protocol, by testing conditionedmedia 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 developeda synovium-like membrane within 7 days, as previously de-scribed (10). Briefly, anesthetized 10–12-week-old WT, TLR-2 / , TLR-4 / , and MyD88 / mice were injected with 5 ml

of sterile air into the subcutaneous tissue of the back, followedby a second injection of 3 ml of sterile air into the pouch 3days later. MSU crystals (3 mg), in 1 ml of sterile, endotoxin-free PBS, were injected into the pouch 7 days after the firstinjection of air. Mice were killed and pouch fluids wereharvested at specific time points by injecting 5 ml of PBScontaining 5 m M EDTA, and cells infiltrating the air pouch were counted manually using a hemocytometer. Smears of cells from the air pouches were prepared by centrifugationof either 50 l of the pouch exudates or 105 of cells incytofunnels (ThermoShandon, Pittsburgh, PA) in a Cytospin4 centrifuge (ThermoShandon) at 110 g for 2 minutes. Differ-ential leukocyte subpopulation counts were measured byWright-Giemsa staining of cytospin slides. IL-1 in superna-tants 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% ethanoland then stained with hematoxylin for 2–5 minutes. Sections were washed 3 times with water and incubated in ammoniumhydroxide 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 3–5minutes.

Statistical analysis. Data are expressed as the mean SD. Statistical analyses were performed using Student’s2-tailed t-test. P values less than 0.05 were considered signifi-cant.

RESULTS

Roles of TLR-2 and MyD88 in macrophage re-

sponsiveness to MSU crystals in vitro. We first testedthe hypothesis that both TLR-2 and MyD88 mediatedthe capacity of MSU crystals to induce macrophageexpression of selected cytokines previously impli-cated in regulating gouty inflammation and known tobe directly and rapidly induced in cells via simpleexposure to MSU crystals (IL-1, TNF, KC/GRO,and TGF1, as cited above). To do so, we generatedBMDMs from TLR-2 / , MyD88 / , and congenic WTmice. The differentiation state of isolated BMDMs

was confirmed by flow cytometry using F4/80 as amature, macrophage-specific marker (36). In all experi-ments and for all genotypes studied, 85% of theisolated BMDMs were F4/80 positive. In F4/80-positiveWT BMDMs, 95% expressed TLR-2, 85% expressedTLR-4, and 90% expressed MyD88, as assessed byflow cytometry of permeabilized cells.

To avoid potential masking effects of both serumprotein opsonization of the crystals (1) and crystal-induced complement activation (24), we treated BMDMs

with endotoxin-free MSU crystals at a concentration of 0.5 mg/ml under entirely serum-free conditions, basedon our previous studies (27). At 24 hours, MSU crystalsinduced the production of several cytokines, includingIL-1, TNF, KC/GRO, and TGF1 in WT BMDMs(Figure 1A), as determined by ELISA of conditionedmedia. 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 inresponse 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 MSUcrystals promotes cell activation in phagocytes

(12,37,38), and TLR-2 signaling is known to mediate aphagocytic program (29) and to physically cooperate

with other receptors in promoting phagocytosis of specific microbial pathogens (39). Therefore, we hypoth-esized that there was impaired uptake of MSU crystalsin TLR-2 / cells. First, we validated that our primaryassay system discerned crystal uptake rather than non-specific crystal–cell association by treating murine

2938 LIU-BRYAN ET AL



BMDMs with MSU crystals for 2 hours at 4°C versus37°C, again under serum-free conditions. The propor-tion of macrophages containing MSU crystals was mea-sured by flow cytometry, based on an increase in sidescatter profile, as previously described (36). As seen inFigure 2A, there was an absence of detectable uptake of MSU crystals by either WT or TLR-2 / BMDMs at4°C. At 37°C, the proportion of TLR-2 / BMDMs thattook up MSU crystals was reduced by 50% relative toWT BMDMs.

To ascertain the effects of TLR-2 deficiency onthe amount of crystals taken up by the BMDM popula-tion as a whole, we treated cells with 14C-labeled MSUcrystals. The amount of 14C-labeled MSU crystals in-gested by BMDMs of TLR-2 / mice was decreased by70% compared with WT BMDMs (Figure 2B). Simi-larly, the proportion of MyD88 / BMDMs that took upMSU crystals was reduced by 50% compared with WT

BMDMs, with inhibitory effects of MyD88 deficiencyobserved as early as 15 minutes after stimulation withMSU crystals (Figure 2C). Moreover, the amount of 14C-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

crystal–induced inflammation in the mouse synovium-

like subcutaneous air pouch. Next, we tested the rolesof TLR-2 and MyD88 in MSU crystal–induced inflam-mation in vivo. To do so, we injected endotoxin-freeMSU crystals into subcutaneous air pouches of TLR-2 / , MyD88 / , and WT mice, a model system char-acterized by generation of a synovium-like lining celllayer containing fibroblastic and phagocytic cells (31). Inorder to screen for inhibitory effects on a submaximalinflammatory response, we chose a dose of MSU crystals(3 mg in 1 ml of PBS) that was 70% lower than thatused in our previous studies of MSU crystal–induced airpouch inflammation (11). At 0, 6, and 24 hours post–MSU crystal injection, mice were killed, and we mea-sured both IL-1 production and leukocyte influx in theair pouch exudates. IL-1 expression (Figure 3C) andleukocyte ingress (Figure 3A) were robust in WT mice 6hours after MSU crystals were injected into the airpouch, with neutrophils accounting for the majority of infiltrated leukocytes (Figure 3B). Both cytokine induc-tion and neutrophilic inflammation were self-limiting by24 hours postinjection (Figure 3). Six hours after MSUcrystal injection, IL-1 expression and the total number

of infiltrated leukocytes and neutrophils were partially,but significantly, suppressed in TLR-2 / mice relativeto WT mice (Figure 3). Furthermore, there was a virtualabsence of IL- induction as well as leukocyte ingress inthe air pouch of MyD88 / mice in response to MSUcrystals (Figure 3).

In comparisons of TLR-2 / and WT mice, weanalyzed the histologic features of the air pouch follow-ing MSU crystal injection. The resting air pouchespossessed a thin synovium-like lining and a thickersubcutaneous layer of vascularized fibrous and adiposetissue beneath the synovium-like lining (Figure 4). Six hours after the injection of MSU crystals, the architec-

ture of the WT synovium-like lining layer became dis-rupted, and there was marked swelling of the tissuesbeneath the air pouch lining layer and massive infiltra-tion of leukocytes throughout the air pouch lining andtissue immediately surrounding it (Figure 4). By com-parison, at the same time point following MSU crystalinjection in the TLR-2 / air pouch, there was markedlyless swelling of the synovium-like layer (Figure 4). There

Figure 1. Effects of Toll-like receptor 2 (TLR-2) and myeloid differ-

entiation factor 88 (MyD88) deficiency on cytokine production in bonemarrow–derived 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 serum-free conditions, as described in Materials and Methods. Levels of

interleukin-1 (IL-1), tumor necrosis factor (TNF), keratinocyte-

derived cytokine (KC)/growth-related oncogene (GRO), and trans-forming growth factor 1 (TGF1) in conditioned media were mea-

sured 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.




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-labeledMSU 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 4°C or 37°C, or with 14C-labeled MSU crystals for 2 hours at 37°C. In C and D, MyD88 /

BMDMs were incubated with unlabeled or 14C-labeled MSU crystals for 15 minutes and 2 hours at37°C. 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).




also was less intense infiltration of leukocytes in theTLR-2 / air pouch, a result that correlated well withgross analyses of Wright-Giemsa–stained smears of cells in the air pouch exudates (Figure 4) and with theaforementioned counts of infiltrated leukocytes (Figures3A and B).

Mediation of MSU crystal–induced macrophage

activation and inflammation by TLR-4. Results to this

point indicated that TLR-2 deficiency partially impairedMSU crystal–induced inflammatory responses, as op-posed to the nearly complete attenuation of these sameresponses in macrophages and air pouches of micedeficient in MyD88. Hence, we hypothesized that 1 ormore TLRs other than TLR-2 also contributed signifi-cantly to the triggering of MSU crystal–induced inflam-mation. We elected to assess TLR-4, which, like TLR-2,is constitutively expressed in macrophages (30) and canmodulate recognition and phagocytosis of microbial

pathogens (29,40). TLR-4 / BMDMs showed markedimpairment of production of TNF, KC/GRO, andTGF1 in response to MSU crystals (Figure 5A) as wellas impaired uptake of MSU crystals (Figures 5B and C).Finally, the capacity of MSU crystals to induce IL-1expression, leukocyte ingress, and neutrophilic inflam-mation in the air pouch was markedly suppressed inTLR-4 / mice (Figures 6A and B).

DISCUSSION

In this study, we demonstrated that host expres-sion of TLR-2, TLR-4, and their shared adapter proteinMyD88 was a major determinant of the capacity of endotoxin-free MSU crystals to turn on the macrophagein vitro and to trigger acute neutrophilic inflammation in

vivo. To avoid confounding effects of serum opsonins onthe in vitro results, we limited this study to an evaluation

Figure 3. Suppressed infiltration of leukocytes and induction of IL-1 in response to MSU crystalsin 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 m M 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 WTmice, 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 forother definitions.




of the effects of uncoated MSU crystals under serum-free conditions, including an analysis of cultured macro-phage uptake of MSU crystals and cytokine release. But

it was notable that the decreases in inflammation in-duced by injected MSU crystals in vivo in the airpouches of mice deficient in TLR-2, TLR-4, and MyD88paralleled the decreases in cytokine production ob-served in MSU crystal–stimulated macrophages underserum-free conditions in vitro.

The inert MSU crystal has the capacity to avidlybind at least 20 different plasma proteins (1,14,15).

Although such MSU crystal binding interactions arenonspecific via hydrogen and electrostatic bonds,MSU does associate preferentially with selected pro-teins in complex mixtures, as illustrated by apoB andcomplement pathway protein binding in crystals ex-

posed to human plasma (41,42). Clearly, naked MSUcrystals can directly promote inflammation by activat-ing complement and the contact coagulation system(1,24). But our results support a model for triggeringgouty inflammation in which the capacity of the MSUcrystal to directly activate resident cells in the joint ispivotal (1).

MSU crystals bind IgG, which can promote rec-

ognition by phagocytes and enhanced cellular responsesto the crystals (1,43). However, the predominant effectof coating MSU crystals with plasma or serum is marked

inhibition of cell activation via apoB binding to thecrystal surface, which physically suppresses MSUcrystal–cell binding (44). Synovial MSU crystals depos-ited in microscopic tophi have been observed to betightly packed in a core contained by a protein-rich wallthat includes fibrinogen (45). As such, our results here,and the known association of acute gouty attacks withrapid rises and falls in the levels of serum urate, suggestthat gouty inflammation is triggered either by the denovo formation of uncoated MSU crystals in the joint orby the release from synovial tophi of MSU crystalsliberated from coating proteins by factors includingpartial crystal dissolution.

MSU crystals preferentially bind to certain cellmembrane proteins, such as integrins and the Fc recep-tor CD16 (18,19). In this context, both macrophages andsynovium lining cells express TLR-2 and TLR-4(30,46,47). Although expression of both TLR-2 andTLR-4 is relatively low in normal synovium, TLR-2 andTLR-4 expression is subject to regulation, as illustratedby cytokine-inductive effects in vitro and up-regulated

Figure 4. Comparison of histologic features of MSU crystal–induced 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-Giemsa–stained smears of cells from the same air pouches,following centrifugation of 50 l of air pouch exudates at 110 g 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.




expression in RA synovium in vivo (46,47). We speculatethat TLR-2 and TLR-4 or closely interacting proteinsare among the plasma membrane proteins directly en-gaged by MSU crystals. Articular chondrocytes havebeen particularly advantageous in testing this notion,because we observed that normal articular chondrocytesconstitutively expressed TLR-2 but not several otherTLRs, including TLR-4, in vitro (25). Moreover, directup-regulation and down-regulation of MSU crystal–induced chondrocyte nitric oxide production was induc-ible by specific “gain-of-function” of TLR-2 expression

versus “loss-of-function” of TLR-2–dependent signaling

(25). It will be interesting to determine if TLRs otherthan TLR-2 and TLR-4 expressed by macrophages andsynovium lining cells (48) also mediate MSU crystal–induced inflammation. We speculate that MSU crystalsalso mediate the activation of infiltrating neutrophils viaTLRs to amplify synovitis once the acute gouty inflam-matory process has been initiated.

The markedly reduced capacity of both TLR-2–

deficient and TLR-4–deficient macrophages to ingestand respond to MSU crystals is compelling, becausedeficiencies of TLR-2 and TLR-4 are associated withselective rather than generalized phagocytosis defects(29), mediated partly by divergent modes of TLR-dependent and TLR-independent phagosome matura-tion (40). For example, in TLR-2 / macrophages,phagocytosis of inert latex beads is intact (30,40), as isingestion of zymosan particles, although TLR-2 / mac-rophages demonstrate decreased activation by zymosan(49,50). Furthermore, macrophages from TLR-2/TLR-4double-knockout mice and MyD88 / mice demon-

strate no differences in phagocytosis of apoptotic cellsrelative to WT cells under conditions in which phagocy-tosis of bacteria is impaired (40). Although our resultssuggest that TLR-2 and TLR-4 recognize the inert MSUcrystal surface as a pathogen-associated molecular pat-tern to directly promote phagocytosis, the current studydid not unequivocally prove this. One alternative sce-nario is that TLR-2 ligands promote phagocytosis

Figure 5. Effects of TLR-4 deficiency on MSU crystal uptake and MSU crystal–induced 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 byflow 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 arerepresentative 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).




through induction of a function-specific gene expressionprogram that includes up-regulation of scavenger recep-tor A expression, an activity shared by ligands of certainother TLRs (29). Given this, it is possible that deficientTLR-2– and/or TLR-4–mediated induction of otherplasma membrane proteins that recognize the inertMSU crystal may have contributed to our findings.

We limited our analysis of cultured cells tounfractionated bone marrow macrophage preparations.The state of differentiation of macrophages clearlymediates the ability of these cells to not only take upMSU crystals (14), but to also respond in a proinflam-

matory or antiinflammatory manner to the crystals(14,15). Hence, the observed effects of host TLR-2 andTLR-4 expression on the capacity of macrophages toingest and react to MSU crystals were possibly at leastpartially mediated by indirect effects on macrophagedifferentiation. Previously described effects of expres-sion of certain TLRs on bacterial phagocytosis by macro-phages mediated by regulation of signal transduction

and gene expression (29) might provide an analogy tothe situation for MSU crystal–macrophage interactions.However, further elucidation of direct and indirecteffects of TLR-2 and TLR-4 on true “recognition” of theMSU crystal by the macrophage will clearly requirespecific TLR “loss-of-function” and “gain-of-function”studies in mature macrophages.

Striking suppression of MSU crystal uptake inTLR-2 / and TLR-4 / macrophages was mirrored inacross-the-board suppression of the capacity of MSUcrystals to induce expression by macrophages of proin-flammatory cytokines and of TGF1 in the same cells in

this study. Importantly, fully differentiated macrophagesthat clear MSU crystals express TGF1, thereby pro-moting resolution of acute MSU crystal–induced inflam-mation (4,15). Therefore, our results suggest that undersome conditions, TLR-2 and TLR-4 expression mightnot only promote the triggering of acute gout, but alsocontribute to the spontaneous self-limitation so charac-teristic of gouty inflammation (1,4).

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 crystal–induced 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.




Limitations of this study include the fact that that we did not assess whether MSU crystals regulate inflam-mation indirectly through TLR and MyD88 signaling viainduced cellular release of endogenous ligands of TLR-2and TLR-4, such as Hsp70 and HMGB-1 (51,52).

MyD88 is one of several cytosolic adapter proteins forTLR-2 and TLR-4 (26), and MyD88 also transducesIL-1R–mediated responses (53). These are points to beconsidered because we did not test to determine whetherthe blunting effects of MyD88 deficiency on MSUcrystal–induced macrophage activation and inflamma-tion were mediated partly by impaired IL-1 signaling.Moreover, we have not yet evaluated the potential rolesof the extracellular TLR-2 and/or TLR-4 adapter mole-cules 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 intracellularadapter protein MyD88 is a major mediator of MSUcrystal–induced inflammation. Our results suggest thatacute gouty inflammation is triggered and regulated inintensity at least in part by cellular recognition of thenaked MSU crystal as a function of TLR-dependentinnate immunity. Significantly, TLR-2 signaling, culmi-nating in NF-B activation, critically transduces chon-drocyte nitric oxide generation in response to MSU andcalcium pyrophosphate dihydrate (CPPD) crystals (25).Since the acute inflammatory responses to CPPD crys-tals resemble those of MSU crystals, we speculate thatTLR-2, TLR-4, and MyD88 could also mediate CPPD

crystal–induced acute inflammation. Finally, TLR-2 andTLR-4 sequence variants have been linked to alteredmicrobial carriage and phenotypic response of the hostto infection (54,55). Hence, it will be of interest todetermine if inherited or acquired alterations in thestructure and function of TLR-2 and TLR-4 contributeto variability in the clinical phenotype of gout in humans

with hyperuricemia (1).

ACKNOWLEDGMENTS

We gratefully acknowledge Drs. Peter Tobias andRichard Ulevitch (The Scripps Research Institute, La Jolla,

CA) for helpful comments relating to the design and executionof these studies. We thank Monika Polewski (VA MedicalCenter, San Diego) for expert technical assistance with the airpouch model studies.

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