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The Journal of Immunology, 2006, 177: 6370-6378.
Copyright © 2006 by The American Association of Immunologists, Inc.

Engagement of CD14 Mediates the Inflammatory Potential of Monosodium Urate Crystals1

Peter Scott, Hong Ma, Suganya Viriyakosol, Robert Terkeltaub and Ru Liu-Bryan2

Veterans Affairs Medical Center, Department of Medicine, University of California San Diego, San Diego, CA 92161


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14–/– bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and ~90% less IL-1beta and CXCL1 release. Attenuated MSU crystal-induced IL-1beta release in CD14–/– BMDMs was mediated by decreased pro-IL-1beta protein expression and additionally by decreased caspase-1 activation and IL-1beta processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1beta and CXCL1 production in CD14–/– BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by~75%, and local induction of IL-1beta decreased by >80% in CD14–/– mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In gout, the deposition of monosodium urate (MSU)3 crystals in articular joints and bursal tissues can be asymptomatic or be associated with the pathogenesis of acute, episodic, self-limiting joint inflammation largely dependent on marked neutrophil influx (1, 2, 3). The interaction of MSU crystals with synovial lining cells, macrophages, and other resident cells in the joint appears to be the primary trigger for the acute neutrophil ingress that drives episodes of gouty arthritis (4). The induction by MSU crystals of phagocyte release of mediators, including arachidonate metabolites, the cytokines TNF-{alpha}, IL-1beta, CXCL1 (GRO{alpha}), CXCL8 (IL-8) (5, 6, 7, 8, 9), and the calgranulins S100A8 and S100A9 (10) drives and amplifies gouty inflammation.

The naked MSU crystal has a negatively charged, highly reactive surface that nonspecifically binds at least 25 different serum proteins (11) and also binds plasma membrane proteins, including certain integrins (12, 13). MSU crystal binding of C5 and C5 catalysis on the crystal surface (14) promotes C5b-C9 membrane attack complex assembly that contributes to both intraarticular CXCL8 expression and acute neutrophilic inflammation in experimental MSU crystal-induced knee arthritis (15). These collective findings implicated innate immunity in acute gouty inflammation, a notion recently reinforced by demonstration that the cytoplasmic NACHT-LRR-PYD-containing protein-3 (NALP)3 (cryopyrin) inflammasome complex, which is principally expressed in phagocytes, is pivotal for acute MSU crystal-induced inflammation (16). MSU crystals appear to be among the stimuli that trigger the pyrin domains of NALP3 and of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) to associate and thereby recruit and proteolytically cleave caspase-1 intracellularly in the inflammasome complex (17). In this context, MSU crystal-induced caspase-1 activation, and subsequent cleavage, maturation, and release of IL-1beta are markedly decreased in macrophages from mice deficient in NALP3, ASC, or caspase-1 in vitro (16). Moreover, MSU crystal-induced peritoneal neutrophil influx is blunted in ASC and caspase-1-deficient mice (16).

In this study, we investigated how pattern recognition by plasma membrane proteins could modulate the inflammatory potential of MSU crystals ingested by phagocytes. TLR2 and TLR4 are central players in innate immune recognition of components of numerous microbial pathogens (18). Significantly, MSU crystals also functionally engage the canonical signaling pathway from TLR2 to NF-{kappa}B activation mediated by the shared TLR and IL-1 receptor adaptor protein MyD88 (19). TLR2 and TLR4 each mediate macrophage uptake of the MSU crystal in vitro and MSU-crystal-induced induced inflammation in vivo (20). Moreover, MyD88 plays a major role in macrophage uptake of the MSU crystal and is essential for MSU crystal-induced inflammation in vivo (20).

The pattern recognition molecule CD14 (21) serves as a shared TLR2 and TLR4 adaptor molecule that increases TLR2- and TLR4-mediated functional responses to specific agonists, including peptidoglycan and LPS, respectively (22, 23). For example, CD14 directly binds to LPS and thereby efficiently presents LPS to the TLR4 complex with the adaptor protein MD-2 (24, 25). CD14 is highly expressed on the surface of myeloid linage cells as a GPI-anchored protein and also can be expressed at low copy numbers in nonmyeloid cells (26). Soluble (s)CD14 is released into serum and facilitates the capacity of certain agonists to activate cells lacking membrane-bound CD14 (27). CD14 lacks both transmembrane and intracellular domains. However, the association of CD14 with other plasma membrane molecules, including formation of cell surface complexes of CD14 with TLR2, TLR4 (22, 23), and certain integrins and FcR (28, 29) provide the means for CD14 to modulate signal transduction.

In this study, we demonstrate that CD14 directly binds MSU crystals and is central for the capacity of ingested MSU crystals to induce an inflammatory response that includes caspase-1 activation and IL-1 expression in vitro. Furthermore we demonstrate that CD14 is essential for a full-blown MSU crystal-induced neutrophilic inflammatory response in s.c. air pouches in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

All chemical reagents were obtained from Sigma-Aldrich unless otherwise indicated. Ultrapurified LPS (Escherichia coli O111:B4) was obtained from InvivoGen (mAb to human CD14 was from Alexis Biochemicals). Polyclonal Abs to caspase-1 and IL-1beta were from BioVision. Polyclonal Abs to phospho-specific p38 MAPK (Thr180/Tyr182) and total p38 MAPK were purchased from Cell Signaling Technology. SB202190 was from Calbiochem. The AlexaFluor 488 protein labeling kit was purchased from Invitrogen Life Technologies. Triclinic MSU crystals were prepared under pyrogen-free conditions, using uric acid pretreated for 2 h at 200°C before crystallization (20). MSU crystals were suspended at 25 mg/ml in sterile, endotoxin-free PBS, and verified to be free of detectable LPS contamination (<0.025 endotoxin U/ml) by the limulus amebocyte lysate assay (BioWhittaker). 14C-labeled MSU crystals were prepared as previously described (20), using as starting material trace 14C uric acid (Moravek Biochemicals) added to 1 g of cold uric acid. The specific activity of the 14C-labeled MSU crystals was 1.4 µCi/mg.

Chinese hamster ovary (CHO-K1) cell lines

TLR4, CD14, and TLR4/MD-2/CD14 stably transfected cells were generated from a CHO cell line bearing inducible plasma membrane CD25 under the transcriptional control of human E-selectin promoter NF-{kappa}B-binding sites, as described previously (30). TLR4 was engineered to express an N-terminal hemagglutinin tag (pDisplay; Invitrogen Life Technologies) and MD-2 was engineered to express a C-terminal His tag (pcDNA4/V5His; Invitrogen Life Technologies). CD14 was engineered into pRc/RSV (Invitrogen Life Technologies). Surface expression of these proteins was analyzed by flow cytometry. The CHO cell lines were cultured in DMEM/F12 (Invitrogen Life Technologies) supplemented with 10% FBS, and 50 µg/ml gentamicin and 1 mg/ml G418 were added for selection of TLR4 and CD14 expression and 500 µg/ml zeocin added for selection of MD-2 expression.

Purification of recombinant tagged mouse sCD14, sTLR2, and sTLR4 and nontagged human CD14

Using PCR, we cloned cDNAs encoding murine CD14 or the extracellular domains of TLR2 (aa 1–587) and TLR4 (aa 1–631) into pcDNA4/Myc-His that encodes a Myc tag and a 6x His tag at the C terminus (Invitrogen Life Technologies). After verification of retained normal sequences, the constructs were transfected in eukaryotic 293T cells using Lipofectamine (Invitrogen Life Technologies) following the manufacturer protocol. At 24 h after transfection, the cells were placed into serum-free medium, and the conditioned medium was harvested after an additional 48 h. Mouse sCD14, sTLR2, and sTLR4 were then purified from the conditioned medium using a column of nickel-nitrilotriacetic acid beads (Qiagen) following the manufacturer’s protocol. Detoxi-Gel endotoxin removing gel (Pierce) was used to remove any detectable endotoxin from the purified sCD14 and sTLR2, as confirmed by limulus amebocyte lysate assay. Recombinant human nontagged sCD14 was generated as described (31).

In vitro crystal protein binding assay

For assessment of binding of sCD14 to MSU crystals, we modified a previously described approach (11). In brief, MSU crystals (0.5 mg) or latex beads (0.5 mg) were blocked with 1% BSA (cell culture grade, endotoxin free) in sterile PBS for 2 h at room temperature, and then incubated with sCD14 in the same solution in the presence or absence of polymyxin B (10 µg/ml) at 4°C for 4 h. After washing four times with 0.02% Triton X-100 in PBS, the samples were separated by 10% SDS-PAGE and analyzed by Western blotting (19) using Abs to either Myc or CD14 (Santa Cruz Biotechnology). Where indicated, purified sCD14 was labeled with the fluoroprobe AlexaFluor 488 (Molecular Probes) following the manufacturer’s protocol, and then used to quantify binding to MSU crystals of the sCD14. The quantity of bound sCD14 was determined via fluorescence intensity of A488-sCD14 remaining on the crystals after washing (excitation 494 nm, emission 504 nm).

Isolation, culture, and analyses of murine macrophages

All animal experiments were done humanely under institutionally peer-reviewed, approved protocols. Wild-type (WT; CD14+/+) and CD14-deficient (CD14–/–) mice on a C57BL/6 background (The Jackson Laboratory) were maintained under specific pathogen-free conditions. Bone marrow-derived macrophages (BMDMs) were prepared from 8- to 10-wk-old homozygous CD14–/– and congenic CD14+/+ mice as described previously (20). Macrophages were assessed by flow cytometry on a FACScan (BD Biosciences) via staining with allophycocyanin-conjugated Ab to F4/80 (Caltag Laboratories), a marker preferentially expressed by mature macrophages (32). To confirm macrophage CD14 expression, double staining used allophycocyanin-anti-F4/80 and PE-conjugated anti-CD14 (eBioscience).

BMDMs of individual genotypes were treated with MSU crystals (0.5 mg/ml) for 30 min and 2 h at 37°C. The proportion of the macrophages taking up MSU crystals was assessed by flow cytometry analysis based on increased side scatter (20). The amount of MSU crystals ingested by the macrophages was determined under the same conditions using 14C-labeled MSU crystals as described previously (20). The production of IL-1beta and CXCL1 in conditioned medium collected from mouse BMDMs (5 x 105/well) stimulated with MSU crystals (0.5 mg/ml) for 18 h was measured by DuoSet ELISA (R&D Systems). For SDS-PAGE/Western blot analyses, we studied aliquots of cell lysates (30 µg) or an equal amount of protein precipitated from conditioned medium using trichloroacetic acid (15% v/v). Quantitative RT-PCR of mouse IL-1beta and GAPDH was performed using the Light Cycler 2.0, and primers were designed using the LightCycler Probe Design software 2.0 (Roche Applied Science). The primers for mouse IL-1beta and GAPDH were: IL-1beta forward, 5'-TAACCTGCTGGTGTGTGAC-3'; IL-1beta reverse, 5'-TCATGGAGAATATCACTTGTTGG-3'; GAPDH forward, 5'-CATCCCAGAGCTGAACG-3'; and GAPDH reverse, 5'-CTGGTCCTCAGTGTAGCC-3'.

Crystal-induced inflammation in murine s.c. air pouches

The s.c. pouches were generated by the injection of sterile-filtered air to generate an accessible space that developed a synovium-like membrane lining within 7 days (20). MSU crystals (3 mg in 1 ml of sterile, endotoxin-free PBS) were injected into the pouch 7 days after the first injection of air. Air pouch fluids were harvested in euthanized mice via injection of 5 ml of PBS containing 5 mM EDTA, and cells infiltrating into the air pouch were counted manually using a hemocytometer (20). Smears of cells from the air pouches were generated and differential leukocyte subpopulation counts performed by Wright-Giemsa staining of cytospin slides (20). For histological analyses, frozen, sagittal sections of the air pouches were prepared and counterstained with eosin (20).

Statistical analyses

Data are presented as mean ± SD, except where otherwise indicated. Statistical analyses were performed using the two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Direct binding to MSU crystals of recombinant sCD14 in vitro

Because direct binding of CD14 to LPS modulates how LPS is presented to the TLR4-MD2 complex (24, 25), we first tested whether MSU crystals directly bind CD14. To do so, we performed in vitro binding assays using purified recombinant mouse sCD14 tagged with Myc and 6x His. MSU crystals (0.5 mg), but not latex beads, bound sCD14, as demonstrated by SDS-PAGE/Western blot analysis using anti-Myc Ab (Fig. 1A). Mouse sCD14 was detected as two bands consistent with alternative glycosylation (33). Further in vitro binding assays with purified recombinant nontagged human sCD14 (Fig. 1B) ruled out the possibility that binding of MSU crystals to tagged mouse sCD14 was dependent on binding of either the Myc or 6x His epitope tags (Fig. 1A). Binding of sCD14 to MSU crystals increased with increasing amounts of applied sCD14, as verified by fluorescence of bound AlexaFluor 488-labeled sCD14 (Fig. 1, A and B). Conversely, MSU crystal dose-dependent binding to purified sCD14 also was observed (data not shown). Binding of MSU crystals to AlexaFluor 488-labeled sCD14 was further confirmed by competition assay with unlabeled purified sCD14 (Fig. 1C). There was no significant difference in binding of sCD14 to MSU crystals in the presence or absence of the endotoxin inhibitor polymyxin B (10 µg/ml) (Fig. 1D).


Figure 1
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  		FIGURE 1. Direct binding of MSU crystals to recombinant sCD14 in vitro. Increasing amounts of purified recombinant Myc-tagged mouse sCD14 (A) or a single dose of 20 µg of purified recombinant nontagged human sCD14 (B) were added in the in vitro binding assays with MSU crystals (0.5 mg) described in Materials and Methods. Latex beads (0.5 mg) were used as a negative control. SDS-PAGE/Western blot analyses with anti-Myc (A) or anti-CD14 Ab (B) were conducted to qualitatively detect and compare sCD14 binding to MSU crystals and latex beads (A and B). Increasing amounts of fluorescent AlexaFluor 488-labeled sCD14 were added to MSU crystals (0.5 mg) in the in vitro binding assays performed as above (C). Here, and in D and E, sCD14 binding to MSU crystals was determined by measuring fluorescence intensity as described in Materials and Methods. MSU crystals (0.5 mg), AlexaFluor 488-labeled sCD14 (10 µg), and differing amounts of unlabeled sCD14 were studied by the same in vitro binding assay approach (D). MSU crystals (0.5 mg) and AlexaFluor 488-labeled sCD14 (30 µg) were studied in the in vitro binding assay in the presence or absence of polymyxin B (10 µg/ml) (E). Data shown in each panel are representative of three different experiments. *, p < 0.05.

 
Mediation of macrophage responsiveness to MSU crystals by CD14 in vitro

We studied the effects of endogenous CD14 expression on macrophage responsiveness to MSU crystals by isolating BMDMs from congenic CD14–/– and CD14+/+ mice. The mature macrophage differentiation state of isolated BMDMs was confirmed by flow cytometry using the marker F4/80 (32). In all experiments, ≥85% of the isolated BMDMs were F4/80+, and ≥90% of CD14+/+F4/80+ BMDMs expressed CD14, as assessed by flow cytometry (not shown). All in vitro experiments were done under serum-free conditions to avoid potential masking effects of free serum proteins (including sCD14), of serum proteins bound to the MSU crystals before exposure to the cells, and of crystal-induced complement activation (14, 15). Under these conditions, CD14–/– and CD14+/+ BMDMs were treated with endotoxin-free MSU crystals (0.5 mg/ml) and the proportion of macrophages containing MSU crystals measured by flow cytometry, based on increase in side scatter (20). Unlike the case described previously for TLR2-, TLR4-, and MyD88-deficient macrophages (20), there was no impairment in the fraction of CD14–/– BMDMs taking up MSU crystals or the amounts of 14C-labeled MSU crystals ingested by CD14–/– BMDMs (Fig. 2B). However, release of IL-1beta and CXCL1 in response to stimulation with MSU crystals was suppressed in CD14–/– BMDMs (Fig. 2, C and D). To rule out possible artifacts of trace endotoxin contamination of MSU crystals, we studied cells treated in the presence of polymyxin B. We observed that polymyxin B did not significantly alter MSU crystal-induced IL-1beta release in CD14+/+ macrophages (Fig. 3A), in contrast with marked inhibition by polymyxin B of LPS-induced IL-1beta release (Fig. 3B).


Figure 2
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  		FIGURE 2. Effects of CD14 deficiency on BMDM phagocytosis and cytokine production in response to MSU crystals in vitro. BMDMs prepared from CD14+/+ and CD14–/– mice were incubated with MSU crystals (0.5 mg/ml) for 30 min and 2 h (A and B) or 18 h (C and D) under serum-free conditions, as described in Materials and Methods. The percentages of BMDMs taking up the unlabeled MSU crystals were estimated by flow cytometry based on increase in the side-scatter profile (A). In parallel experiments, amounts of 14C-labeled MSU crystals ingested by BMDMs were determined by measuring the 14C associated with washed cells after coincubation of crystals with the cells for 30 min and 2 h (B), as described in Materials and Methods. Conditioned medium was assayed for cytokines IL-1beta (C) and CXCL1 (D) by ELISA, as described in Materials and Methods. Data shown in each panel are representative of three different experiments, using cells from more than three different mice of each genotype. *, p < 0.05 relative to CD14+/+ cells.

 

Figure 3
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  		FIGURE 3. Lack of effect of polymyxin B (PMB) on MSU crystal-induced IL-1beta release. BMDMs prepared from WT mice were incubated with either MSU crystals (0.5 mg/ml) or ultrapurified LPS (1 µg/ml) in the presence or absence of polymyxin B (10 µg/ml) for 18 h (A and B). Conditioned medium was assayed for IL-1beta by ELISA. Data shown are representative of three different experiments on cells from three separate CD14+/+ mice. *, p < 0.05.

 
MSU crystal-induced caspase-1 activation, assessed by release of cleaved caspase-1, was diminished in CD14–/– BMDMs, as was pro-IL-1beta (p35) protein expression (Fig. 4). MSU crystal-induced IL-1beta mRNA expression also was suppressed in CD14–/– BMDMs (data not shown). The mature form of IL-1beta (p17) was observed in the cell medium of CD14+/+, but not CD14–/–, BMDMs as assessed by SDS-PAGE/Western blotting (Fig. 4B).


Figure 4
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  		FIGURE 4. CD14 deficiency limits caspase-1 activation, pro-IL-1beta protein expression, and IL-1beta processing in cultured BMDMs in response to MSU crystals. WT (CD14+/+) and CD14–/– BMDMs were treated with MSU crystals (0.5 mg/ml) for 18 h under serum-free conditions as above. Cell lysates and trichloroacetic acid-precipitated protein from conditioned medium (supernatants) were studied by SDS-PAGE/Western blotting using Abs to pro- and processed forms of caspase-1 and IL-1beta, respectively (A and B).

 
Next, we tested whether CD14 on the surface of MSU crystals could restore defective cytokine production by MSU crystals in CD14–/– macrophages. To do so, aliquots of naked MSU crystals (0.1 mg) were coated with murine recombinant sCD14 alone, or alternatively, murine recombinant sTLR2 or sTLR4 (10 µg) alone in a volume of 0.1 ml for 4 h at 4°C, followed by repeated washing of the crystals as for the in vitro crystal protein binding assays above. Under these conditions, the concentration of each of the soluble recombinant proteins remaining on the crystals after washing was ~0.05–0.1 µg/25 µg MSU crystals, and qualitative binding to the MSU crystals of sCD14, sTLR2, and sTLR4 was verified by SDS-PAGE/Western blotting (data not shown). CD14–/– BMDMs were then treated with the MSU crystals (0.5 mg/ml) that had been coated with sCD14, sTLR2, or TLR4. We observed that the impaired production of IL-1beta and CXCL1 in response to MSU crystals in CD14–/– BMDMs was restored to levels ~85% of those for CD14+/+ BMDMs when MSU crystals were coated with sCD14, but not with sTLR2 or sTLR4 (Fig. 5).


Figure 5
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  		FIGURE 5. Restoration of defective MSU crystal-induced IL-1beta and CXCL1 release in CD14–/– BMDMs by coating MSU crystals with sCD14. Aliquots of naked MSU crystals (0.1 mg) were preincubated with murine recombinant sCD14, sTLR2, or sTLR4 (10 µg) in a volume of 0.1 ml for 4 h at 4°C, followed by repeated washing as above for in vitro binding assays. Aliquots of the coated MSU crystals (0.5 mg/ml) were then added to CD14+/+ and CD14–/– BMDMs for 18 h, as above. Conditioned medium was assayed for IL-1beta and CXCL1 by ELISA (A and B). Data shown are representative of two experiments using different sets of donor cells. *, p < 0.05.

 
CD14 in MSU crystal-induced p38 MAPK pathway and TLR4-mediated signaling

MSU crystal-induced p38 MAPK pathway signaling is a central dynamic in MSU crystal-induced CXCL8 expression in human monocytes (34). In this study, we first pretreated CD14+/+ macrophages with the p38 MAPK selective inhibitor SB202190 (25 µM), as previously validated in monocytes (34) (Fig. 6, A and B). MSU crystal-induced release of both IL-1beta and CXCL1 was suppressed by SB202190 in normal mouse BMDMs. Activation of the p38 MAPK pathway by MSU crystals in macrophages was CD14 dependent, as impaired MSU crystal-induced phosphorylation of p38 was observed in CD14–/– BMDMs (Fig. 6C). CD14 has no intrinsic signaling capacity but operates in conjunction with TLR2 or TLR4. Here, comparative studies demonstrated diminished phosphorylation of p38 in response to MSU crystals in TLR2–/–, TLR4–/–, and MyD88–/– BMDMs (Fig. 6D).


Figure 6
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  		FIGURE 6. CD14 deficiency suppresses p38 MAPK activation required for induction of IL-1beta and CXCL1 in response to MSU crystals in BMDMs. CD14+/+ BMDMs were treated with MSU crystals (0.5 mg/ml) for 18 h, with and without 1 h pretreatment using the selective p38 MAPK pathway inhibitor SB202190 (25 µM) (A and B). Conditioned medium was assayed for IL-1beta (A) and CXCL1 (B) by ELISA. BMDMs of CD14+/+ and CD14–/– mice (C) or BMDMs of WT, TLR2–/–, TLR4–/–, and MyD88–/– mice (D) were stimulated with MSU crystals (0.5 mg/ml) for the times indicated, and p38 phosphorylation determined by SDS-PAGE/Western blotting, as described in Materials and Methods. Data shown are representative of two experiments using different sets of donor cells.

 
For CD14 gain of function studies, we stably transfected CD14 alone, TLR4 and MD-2, or the combination of CD14, TLR4, and MD-2 into CHO cells expressing human CD25 reporter gene under the control of the NF-{kappa}B-dependent ELAM-1 promoter. CHO cells with high levels of surface expression of the transfectants were isolated using flow cytometry, cell sorting, and limiting dilution cloning. Under conditions where CD14 enhanced TLR4-mediated signaling in response to MSU crystals, CD14 was validated to enhance TLR4-mediated signaling in response to ultrapurified LPS (Fig. 7). No significant induction of CD25 expression by MSU crystals was observed in the reporter CHO cell line expressing CD14 alone, and <2-fold CD25 induction was seen in the reporter cell line expressing TLR4/MD-2, but >4-fold induction of CD25 expression was observed in the reporter cell line expressing CD14/TLR4/MD-2 (Fig. 7).


Figure 7
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  		FIGURE 7. Enhancement of MSU crystal-induced TLR4-mediated signaling by CD14 in gain of function transfection studies in CHO cells. The CHO reporter cell line (EL1) was generated by stable transfection with a reporter plasmid comprised of the human CD25 as a reporter under the control of the NF-{kappa}B-dependent ELAM-1 promoter. The CHO cell lines designated CD14-EL1, TLR4/MD-2-EL1, and CD14/TLR4/MD-2-EL1 were derived from the parental EL1 cell line stably transfected with the cDNA(s) indicated, as described in Materials and Methods. The CHO cell lines were stimulated with either MSU crystals (0.5 mg/ml) or ultrapurified LPS (100 ng/ml) for 18 h, and flow cytometry analyses of surface CD25 expression was conducted to assess NF-{kappa}B activation. Data are shown as mean ± SEM pooled from three different experiments, with results displayed as fold-induction over baseline CD25 expression in the parental EL1 cell line. *, p < 0.05.

 
CD14 mediates MSU crystal-induced inflammation in vivo

Leukocyte ingress (Fig. 8A) and expression of IL-1beta and CXCL1 (Fig. 8) were robust at 6 h postinjection of MSU crystals into s.c. air pouches in CD14+/+ mice, with neutrophils accounting for the majority of infiltrated leukocytes (Fig. 8B). Both neutrophilic inflammation and cytokine release were confirmed (20) to be self-limiting by 24 h postinjection of crystals (Fig. 8, A, B, E, and F). Under the same conditions, MSU crystal-induced total leukocyte and neutrophil infiltration (Fig. 8, A and B), as well as levels of IL-1beta and CXCL1 in the air pouch exudates (Fig. 8, E and F) were significantly suppressed in CD14–/– mice. To verify that MSU crystal-induced leukocyte ingress was not due to trace endotoxin contamination, we injected polymyxin B together with either MSU crystals or LPS into the air pouches of CD14+/+ mice in control studies. At 6 h postinjection, LPS-induced total leukocyte ingress were markedly inhibited by polymyxin B, whereas MSU crystal-induced total leukocyte ingress were not significantly affected by polymyxin B (Fig. 8C). MSU crystal-induced leukocyte aggregation in air pouch fluid exudates was demonstrable in CD14+/+ mice, but only minimally in CD14–/– mice (Fig. 8D).


Figure 8
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  		FIGURE 8. Suppression of MSU crystal-induced infiltration of leukocytes and inflammatory cytokine expression in the s.c. air pouch in CD14–/– mice. s.c. air pouches with a synovium-like lining were created in mice via injections of sterile air, as described in Materials and Methods. At 7 days after the first injection of air, a 1-ml suspension of 3-mg MSU crystals in PBS was injected into the air pouches. Mice were euthanized at the times indicated, and the air pouch exudates were harvested by washing with 5 ml of PBS containing 5 mM EDTA. The leukocytes in the pouch exudates were counted using a hemocytometer, and the fraction of neutrophils determined using Wright-Giemsa staining, as described in Materials and Methods (A and B). The leukocyte counts were measured at 6 h in the exudates of air pouches of CD14+/+ mice after injection with polymyxin B (10 µg), where indicated, in conjunction with either MSU crystals (3 mg) or ultrapurified LPS (10 µg) (C). Smears of cells from air pouch exudates after injection of MSU crystals were stained using Wright-Giemsa method (D). IL-1beta and CXCL1 production were measured by ELISA from the supernatants of air pouch exudates after cells were removed by sedimentation (E and F). Data shown in A and B are mean ± SEM of nine CD14+/+ and nine CD14–/– mice. *, p < 0.05 relative to CD14+/+ mice. Data shown in C–F are representative of three different experiments on three individual mice of each genotype for each condition and time point shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Pattern recognition receptors and certain of their adaptor molecules directly engage exogenous and endogenous danger signals and trigger primitive innate immune responses fundamental to host defense and inflammation (35). MSU crystals provide extracellular and intracellular danger signals (36) whose direct recognition at the plasma membrane by TLR2 and TLR4 stimulates crystal uptake and canonical signal transduction (19, 20). Delivery of MSU crystals to the NALP3 inflammasome by fully functional phagocytes promotes apparent recognition of MSU crystals in the cytosol by one or more inflammasome components, thereby stimulating caspase-1 activation and IL-1beta processing (16). In this study, we demonstrated that MSU crystals, but not latex beads, directly engage the recombinant soluble form of the pattern recognition molecule CD14, an adaptor molecule for TLR2 and TLR4. MSU crystal–CD14 interaction did not significantly modulate the capacity of MSU crystals to be taken up by macrophages but did drive the capacity of MSU crystals to induce IL-1beta and CXCL1 in macrophages, suggestive of altered intracellular recognition of MSU crystals.

Ingestion of MSU crystals by phagocytes is central to MSU-crystal induced phagocyte inflammatory responses (1, 37, 38). However, macrophages take up a variety of infectious and noninfectious particulates (e.g., latex beads and certain other microspheres) without an acute inflammatory response but with potential effects on Ag presentation and adaptive immunity (39). Indeed, noninflammatory uptake of MSU crystals is a disposal mechanism for the crystals exerted by subsets of mature macrophages and certain macrophage cell lines in vitro (40). Our results suggest that CD14 appears to be a central determinant of inflammation-inducing MSU crystal uptake response as opposed to noninflammatory removal of MSU crystals by macrophages in vitro.

Naked MSU crystals nonspecifically bind multiple serum and cell membrane proteins (11), and the binding of sCD14 to MSU crystals was nonsaturable at the concentrations used in this study. Moreover, MSU crystals are unlikely to have a selective binding site for CD14, as we have noted the binding of sCD14 to MSU crystals, assessed via in vitro binding assays, to be competitively inhibited by heterogeneous purified proteins, including membrane protein-derived sTLR2, and serum proteins, including haptoglobin (R. Liu-Bryan, unpublished observations). Though nonspecific, the binding interaction between MSU crystals and sCD14 alone was nevertheless observed to be sufficient to selectively restore defective IL-1beta and CXCL1 in response to MSU crystals in CD14–/– BMDMs. The dominant effect of coating the naked synthetic MSU crystal with whole serum is to inhibit the capacity of MSU crystals to bind and activate cells, via LDL apoB binding to the crystal surface (41). Hence, free sCD14 and macrophage membrane-bound CD14 may have to compete with other proteins for access to the surface of MSU crystals in the joint. Naked MSU crystals, either newly formed or liberated from the protein-walled confines of remodeling tophi, would be predicted to have a higher likelihood of engaging CD14 and thereby activating macrophages.

MSU crystal-induced pro-IL-1beta expression was suppressed in CD14–/– macrophages, and the linked findings of diminished caspase-1 activation and IL-1beta processing in CD14–/– macrophages stimulated with MSU crystals suggested a role for CD14 in MSU crystal-induced NALP3 inflammasome activation. The molecular mechanisms by which NALP3 inflammasome is activated, a process stimulated by a small number of diverse agents (16, 17, 42, 43), are not completely defined. However, both CD14 and NALP3 contain leucine-rich repeat (LRR) sequences that mediate protein-protein as well as protein-danger signal interactions (17). Indeed, LRR sequences were defined in glycoproteins of the extracellular matrix before being characterized in TLRs (44). Our results suggest the possibility that MSU crystal surface-bound CD14 may bridge crystal uptake with subsequent, direct engagement of the NALP3 inflammasome by the internalized crystals presented as a danger signal via CD14 LRR sequences.

Results of this study partly reflected a fundamental role of macrophage expression of CD14 as a mediator of MSU crystal-induced p38 MAPK pathway signaling, which drives several inflammatory responses in phagocytes (6, 34). In this study, CD14 directly enhanced TLR4/MD-2-mediated signaling for NF-{kappa}B activation in response to MSU crystals in gain of function studies. Hence, it is possible that CD14 binding to MSU crystals more efficiently presents the crystals to TLR4/MD-2 or vice versa.

In this study, CD14 expression was required for full expression of an IL-1beta and chemokine-mediated inflammatory response to MSU crystals in vivo. It will be of interest to define the respective roles of CD14 on resident cells and infiltrating phagocytes in MSU crystal-induced inflammation in vivo. Macrophages and macrophage-like synovial lining cells express membrane-bound CD14 (45). In addition, neutrophil membrane-bound CD14 (46) would be predicted to mediate activation of infiltrating neutrophils in the joint, thereby amplifying synovitis once the gouty inflammatory process has been initiated (1). In contrast, CD14 expression is generally sparse on nonmyeloid lineage cells (26). Cells first to respond to MSU crystals in the normal joint space are likely to include resident cells of mesenchymal origin such as synovial fibroblasts, with little or no CD14 expression, rather than phagocytes that enter the joint as gouty inflammation erupts and evolves (1). Recognition of naked MSU crystals by constitutively expressed TLR2 modulates the capacity of inert MSU crystals to turn on chondrocytes (19). However, it is not yet known whether the binding of free sCD14 to MSU crystals can modulate MSU crystal-induced activation of synovial fibroblasts.

Limitations of this study include issues related to the in vivo model system used. In humans, acute gouty arthritis has been suggested to commonly supervene in joints already manifesting low-grade, subclinical inflammation associated with established tophaceous deposits of MSU crystals (47). In contrast, the mouse air pouch model system does not study a synovial joint and is triggered by injection of relatively large amounts of MSU crystals into a previously noninflamed cavity of animals naive to MSU crystals. Unlike humans, mice express uricase and are able to rapidly degrade exogenous MSU crystals and thereby to potentially alter crystal morphology and to additionally generate hydrogen peroxide as a byproduct of urate oxidation. To avoid confounding effects on our in vitro results of serum (which contains sCD14), of serum MSU crystal binding opsonins such as IgG and C3b (48), and of MSU crystal binding anti-opsonins such as apoB (41), we limited studies of cultured cells to evaluation of effects of naked MSU crystals under serum-free conditions. It was notable that the significant decrease in inflammation induced by injected MSU crystals in vivo in the air pouches of mice deficient in CD14 paralleled the marked decreases in cytokine production observed in crystal-stimulated CD14-deficient macrophages under serum-free conditions in vitro. However, we have not yet determined whether experimental addition of sCD14, or chimerism devised by delivery of congenic CD14-expressing phagocytes, can modulate MSU crystal-induced inflammation of air pouches in the CD14-deficient mouse. Last, MSU crystals engage the integrin CD11b/CD18, and engagement of CD11b/CD18 and phagocyte activation by certain bacterial products are mediated by complex formation of CD14, TLR2, and CD11b/CD18 (28). Hence, it is conceivable that the role of CD14 in MSU crystal-induced inflammation is primarily via macromolecular complex formation with TLRs and integrins that are directly responsible for modulating cellular responsiveness to MSU crystals.

In conclusion, this study establishes that direct engagement of CD14 is a major determinant of the inflammatory potential of the MSU crystal. Our results suggest that acute gouty inflammation is triggered by cellular recognition of the naked MSU crystal as a function of CD14 in innate immunity. CD14 binding to MSU appears to be a central switch in stimulating an inflammatory rather than noninflammatory response to ingested MSU crystals. CD14 may bridge TLR-mediated recognition events for MSU crystals with intracellular engagement of the MSU crystal by the NALP3 inflammasome to drive caspase-1 activation, IL-1beta processing and release, and inflammation. The CD14/-260C>T promoter gene polymorphism has been found to affect the risk of inflammatory diseases (49), and it will be of interest to determine whether inherited or acquired alterations in CD14 function contribute to clinical variability in gouty inflammation. Our results identify CD14 as a potential novel therapeutic target for gouty inflammation. Last, activated leukocytes can alter membrane-associated CD14 expression and shed sCD14 polypeptides (50). Therefore, shedding of sCD14 by activated phagocytes that have entered the joint space may contribute to the spontaneous self-limitation of acute gouty arthritis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Veterans Affairs Research Service and National Institutes of Health Grants AR049416 and HL077360. Back

2 Address correspondence and reprint requests to Dr. Ru Liu-Bryan, Veterans Affairs Medical Center, 111K, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail address: rliu{at}vapop.ucsd.edu Back

3 Abbreviations used in this paper: MSU, monosodium urate; ASC, adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain; BMDM, bone marrow-derived macrophage; CHO, Chinese hamster ovary; s, soluble CD14; LRR, leucine-rich repeat; WT, wild type; NALP, NACHT-LRR-PYD-containing protein. Back

Received for publication May 10, 2006. Accepted for publication August 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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