Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 2, February 2006, pp 455–462 DOI 10.1002/art.21633 © 2006, American College of Rheumatology Induction of Triggering Receptor Expressed on Myeloid Cells 1 in Murine Resident Peritoneal Macrophages by Monosodium Urate Monohydrate Crystals Yousuke Murakami,1 Tohru Akahoshi,2 Izumi Hayashi,3 Hirahito Endo,2 Shinichi Kawai,4 Matsuhisa Inoue,2 Hirobumi Kondo,2 and Hidero Kitasato5 Objective. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface molecule that was recently identified on monocytes and neutrophils. TREM-1 has been implicated in the early inflammatory responses induced by microbes, but its pathophysiologic role in nonmicrobial inflammation remains unknown. In the present study, we investigated the role of TREM-1 in acute inflammation induced by monosodium urate monohydrate (MSU) crystals. Induction of TREM-1 expression by MSU crystal–stimulated murine resident peritoneal macrophages and infiltrating leukocytes in a murine air-pouch model of crystal-induced acute inflammation was determined. The biologic role of TREM-1 in crystal-induced cytokine production by resident peritoneal macrophages was also investigated. Methods. TREM-1 expression by resident peritoneal macrophages and infiltrating leukocytes in a mu- rine air-pouch model was determined by quantitative real-time polymerase chain reaction, Western blot analysis, and flow cytometry. Cytokine production by resident peritoneal macrophages after incubation with MSU crystals in the presence or absence of an anti– TREM-1 agonist antibody was determined by enzymelinked immunosorbent assay. Results. TREM-1 expression by resident peritoneal macrophages was significantly induced after stimulation with the crystals. Maximum expression of TREM-1 transcripts and protein occurred at 1 and 4 hours after exposure to the crystals, respectively. Costimulation of resident peritoneal macrophages with MSU crystals and an anti–TREM-1 agonist antibody synergistically increased the production of both interleukin-1␤ and monocyte chemotactic protein 1 compared with stimulation with the crystals alone. MSU crystals also induced TREM-1 expression in infiltrating leukocytes in a murine air-pouch model of crystalinduced acute inflammation. Conclusion. These findings suggest that rapid induction of TREM-1 expression on resident peritoneal macrophages and neutrophils by MSU crystals may contribute to the development of acute gout through enhancement of inflammatory responses. Supported in part by a research grant from the Ministry of Health, Labor, and Welfare of Japan, a project research grant from Kitasato University Graduate School of Medical Sciences, and a grant from Kitasato University School of Allied Health Sciences (grant-inaid for research project no. 2005-211). 1 Yousuke Murakami, PhD: Kitasato University School of Medicine, Kanagawa, Japan, and the Japan Health Sciences Foundation, Tokyo, Japan; 2Tohru Akahoshi, MD, Hirahito Endo, MD, Matsuhisa Inoue, PhD, Hirobumi Kondo, MD: Kitasato University School of Medicine and Graduate School of Medical Sciences, Kanagawa, Japan; 3Izumi Hayashi, PhD: Kitasato University School of Medicine, Kanagawa, Japan, and Nihon Pharmaceutical University, Saitama, Japan; 4Shinichi Kawai, MD: Toho University School of Medicine, Tokyo, Japan; 5Hidero Kitasato, PhD: Kitasato University School of Allied Health Sciences and Graduate School of Medical Sciences, Kanagawa, Japan. Address correspondence and reprint requests to Hidero Kitasato, PhD, Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. E-mail: email@example.com. Submitted for publication March 7, 2005; accepted in revised form November 10, 2005. Acute gouty arthritis is characterized by the deposition of monosodium urate monohydrate (MSU) crystals in articular and periarticular tissues (1). These crystals have been shown to cause massive infiltration of neutrophils into joints and to promote neutrophil activation, leading to tissue damage (2). MSU crystals have a remarkable capacity to induce the release of various inflammation mediators from synovial cells, macrophages, and infiltrating leukocytes (3,4). These mediators include interleukin-1␤ (IL-1␤), IL-8, and monocyte 455 456 chemotactic protein 1 (MCP-1), all of which have been shown to play an important role in promoting the infiltration and activation of inflammatory cells in acute gout (5,6). Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently identified cell surface molecule that is found on neutrophils and monocytes (7–9). TREM-1 is a member of the 30-kd immunoglobulin superfamily, and its expression is up-regulated by lipoteichoic acid or lipopolysaccharide (LPS) (7,8,10,11). TREM-1 activates monocytes through the transmembrane adaptor protein DAP12 (7–9). Targeting of TREM-1 expressed by neutrophils and monocytes with agonist monoclonal antibodies promotes the production of various inflammatory cytokines, such as IL-8, MCP-1, tumor necrosis factor ␣ (TNF ␣ ), granulocyte– macrophage colony-stimulating factor, and IL-1␤ (8,10,11). In addition, marked enhancement of TNF␣, IL-1␤, and MCP-1 production by monocytes incubated with agonist monoclonal antibodies occurs when LPS is added as a costimulant, indicating that TREM-1 can amplify inflammatory responses initiated by Toll-like receptors (TLRs) (8,10,11). Although natural ligands for TREM-1 remain to be identified, its pathophysiologic significance in acute inflammation has already been demonstrated in murine models of septic shock, in which competition for binding to TREM-1 by a recombinant TREM-1 fusion protein or synthetic soluble TREM-1 (sTREM-1) was shown to protect mice against lethal LPS challenge or bacterial sepsis (11,12). Several lines of evidence indicate that TREM-1 is strongly expressed in acute and chronic inflammatory lesions caused by bacterial or fungal infection, while it shows only weak expression in nonmicrobial inflammatory conditions, such as psoriasis, ulcerative colitis, and immune complex–mediated vasculitis (11,13). However, the mechanisms that regulate TREM-1 expression in various inflammatory diseases remain unknown. An essential role of TLR-mediated signaling in acute gout was recently demonstrated (14). Because TREM-1 may potentially amplify the inflammatory responses initiated by TLRs, we hypothesized that cooperation between TREM-1 and TLRs may occur during acute attacks of gout. Therefore, we investigated the expression of TREM-1 by MSU crystal–stimulated murine resident peritoneal macrophages, and also assessed the effect of a TREM-1 agonist antibody on cytokine production by MSU crystal–stimulated resident peritoneal macrophages. MURAKAMI ET AL MATERIALS AND METHODS Reagents. Uric acid, polymyxin B, and LPS were obtained from Sigma (St. Louis, MO), and IL-1␤ and TNF␣ were obtained from PeproTech (London, UK). Specific enzyme-linked immunosorbent assays (ELISAs) for murine IL-1␤ and MCP-1 were obtained from Biosource International (Camarillo, CA). Anti-mouse TREM-1 polyclonal antibody, phycoerythrin (PE)–conjugated anti-mouse TREM-1 monoclonal antibody (mAb), and the Mouse TREM-1 DuoSet were purchased from R&D Systems (Minneapolis, MN). Anti-actin polyclonal antibody and goat IgG1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and fluorescein isothiocyanate (FITC)–conjugated anti-mouse Gr-1 mAb and allophycocyanin (APC)–conjugated anti-mouse CD11b mAb were purchased from eBioscience (San Diego, CA). Horseradish peroxidase (HRP)–conjugated rabbit anti-goat IgG antibody was obtained from DakoCytomation (Kyoto, Japan). Preparation of MSU crystals. MSU crystals were prepared according to the method described by Seegmiller et al (15). Briefly, 8 gm of uric acid was dissolved in 1,600 ml of boiling distilled water containing 49 ml of 1N NaOH. After the pH of the solution was adjusted to 7.2 by addition of HCl, it was gradually cooled with stirring at room temperature and then stored overnight at 4°C. The crystals that formed were sterilized by heating at 180°C for 2 hours and were suspended in phosphate buffered saline (PBS) at a concentration of 10 mg/ml. The crystals obtained by this method were rod-shaped and fairly uniform in size (5–25 m long). A Limulus amebocyte cell lysate assay verified the absence of endotoxin in the crystal preparation. Resident peritoneal macrophages. Resident peritoneal macrophages were isolated from male ICR mice (ages 6–8 weeks), as reported elsewhere (16). After washing with PBS, the cells were suspended in RPMI 1640 medium (Sigma) supplemented with 5% heat-inactivated fetal calf serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA). Next, the resident peritoneal macrophages were incubated in the presence or absence of various concentrations of MSU crystals for the indicated periods at 37°C in a humidified incubator with an atmosphere of 5% CO2 and 95% air. The cells were also incubated with or without polymyxin B (5 g/ml) for 1 hour and then were incubated for 1 hour in the presence or absence of various inflammatory agents, such as IL-1␤, TNF␣, LPS, and MSU crystals. Expression of TREM-1 was determined by quantitative real-time PCR and Western blot analysis. Production of sTREM-1 in a culture supernatant was determined using the TREM-1 DuoSet (R&D Systems). Quantitative real-time PCR and reverse transcription–PCR. Total RNA was extracted from resident peritoneal macrophages, infiltrating cells, and soft tissues of murine air pouches by using an RNeasy Mini Kit (Qiagen, Tokyo, Japan) and was treated with DNase I (Qiagen). Complementary DNA (cDNA) was synthesized from 2 g of random-primed total RNA in a total volume of 20 l using Omniscript reverse transcriptase (Qiagen). Murine TREM-1 expression was assessed by quantitative real-time PCR using the oligonucleotide primers 5⬘-CCAGAAGGCTTGGCAGAGACT-3⬘ and 5⬘-ACTTCCCCATGTGGACTTCACT-3⬘. Murine GAPDH was used as the internal control, being RAPID INDUCTION OF TREM-1 IN ACUTE GOUT amplified with the primers 5⬘-TGCAGTGGCAAAGTGGAGATT-3⬘ and 5⬘-CCATCAACGACCCCTTCATTGACCTC-3⬘. The expected sizes of the PCR products for TREM-1 and GAPDH were 101 bp and 97 bp, respectively. PCR was performed in duplicate with a 25-l reaction mixture containing 1 l of cDNA, 12.5 l of QuantiTect SYBR Green PCR Kit (Qiagen), and 300 nM of the forward and reverse primers. The PCR mixture was incubated for 15 minutes at 95°C to activate HotStarTaq DNA polymerase (Qiagen). Subsequently, amplification was performed for 40 cycles of denaturation at 94°C for 15 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds. During the extension step, the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Tokyo, Japan) monitored real-time amplification by quantitative analysis of the emitted fluorescence. The amount of sample messenger RNA (mRNA) was estimated relative to the control sample, which was assigned a value of 1 arbitrary unit. Western blot analysis. Resident peritoneal macrophages (1.0 ⫻ 106 cells) were solubilized in 200 l of sample buffer (350 mM Tris [pH 6.8], 10% sodium dodecyl sulfate [SDS], 30% glycerol, 600 mM dithiothreitol [DTT], and 0.05% bromophenol blue), loaded onto 10% SDS–polyacrylamide gel electrophoresis gel, and run at 20 milliampere for 1.5 hours. Next, cellular proteins were transferred to a polyvinylidene difluoride membrane (Roche Diagnostics, Mannheim, Germany) for 1.5 hours at 200 mA by the semi-dry blot method. The membrane was blocked with 5% skim milk in Tris buffered saline (TBS) containing 0.05% Tween 20 for 1 hour at 37°C, washed with TBS containing 0.1% Tween 20, and incubated with anti-mouse TREM-1 polyclonal antibody or anti-actin polyclonal antibody overnight at 4°C. The blots were washed 4 times with TBS and incubated for 30 minutes with HRP-conjugated rabbit anti-goat IgG antibody. Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus; Amersham Biosciences, Piscataway, NJ). Cytokine production. Flat-bottomed plates were precoated overnight at 4°C with 5 g/ml anti–TREM-1 polyclonal antibody or an isotype-matched control (goat IgG1). After washing with PBS, resident peritoneal macrophages (1.0 ⫻ 105 cells) were added to the wells, and then incubated in the presence or absence of MSU crystals (100 g/ml) for 24 hours. Culture supernatant was obtained by centrifugation and was stored at ⫺20°C until the IL-1␤ and MCP-1 levels were determined using specific ELISAs. Murine air-pouch model. Subcutaneous air pouches were created by the injection of sterile filtered air, as described previously (17). Briefly, C57/BL6 mice (6–8 weeks old) were anesthetized, and 5 ml of air was injected into the subcutaneous tissue of the back, followed by reinjection of an additional 3 ml of air after 3 days. On day 7 after the first injection, the air pouches thus created were used for these experiments. First, MSU crystals (3 mg in 1 ml of sterile PBS) were injected into the air pouches. After the indicated time periods, pouch fluid was harvested by injecting 3 ml of cold PBS. Next, the cells that had accumulated inside the air pouches were counted using a hemocytometer and were stained with Wright Giemsa solution to determine the differential leukocyte count. Expression of TREM-1 by the infiltrating cells and soft tissues of the 457 air pouches was investigated by quantitative real-time PCR. Soluble TREM-1 in the pouch fluid was determined using the TREM-1 DuoSet (R&D Systems). Flow cytometric analysis. Cells infiltrating into the air pouches were harvested 8 hours after stimulation. Thioglycolate-elicited peritoneal neutrophils were harvested 20 hours after peritoneal injection of 2 ml of 3% thioglycolate, and cells were used as a negative control. Cells were incubated with an Fc blocking agent for 5 minutes at room temperature to block Fc receptors. Subsequently, cells were incubated with PE-labeled anti–TREM-1 mAb, FITC-labeled anti–Gr-1 mAb, or APC-labeled anti-CD11b mAb for 20 minutes on ice. Gating on neutrophils was based on characteristic forward and side scatter parameters as well as the binding of anti–Gr-1 mAb. In order to ensure specific staining, appropriate isotype control mAb were used. Samples were analyzed by flow cytometry. RESULTS MSU crystal–induced TREM-1 expression by resident peritoneal macrophages. In order to investigate the role of MSU crystals in TREM-1 expression, resident peritoneal macrophages were stimulated with crystals (100 g/ml), and TREM-1 expression was evaluated by quantitative real-time PCR and Western blot analysis. As shown in Figure 1A, exposure to MSU crystals rapidly induced TREM-1 mRNA expression by resident peritoneal macrophages in a time-dependent manner. The maximum induction of TREM-1 mRNA occurred 1 hour after stimulation, with a return to the basal level at 4 hours. Expression of TREM-1 protein was also induced 2–8 hours after stimulation with the crystals (Figure 1B). Maximum induction occurred at 4 hours, and expression returned to the basal level by 12 hours after stimulation. Actin (used as a control) was also detected in all of the samples. Resident peritoneal macrophages were also incubated with various concentrations of MSU crystals for the indicated periods of time, and TREM-1 expression was evaluated. The crystals enhanced TREM-1 mRNA expression at 1 hour and TREM-1 protein expression at 4 hours, in a concentration-dependent manner; maximum expression occurred after stimulation with 100 g/ml of the crystals (Figures 1C and D). It has been demonstrated that LPS could enhance the production of sTREM-1 and expression of TREM-1 on the cell surface. Therefore, we investigated the production of sTREM-1 from MSU crystal– stimulated resident peritoneal macrophages. We could not detect sTREM-1 in a culture supernatant of MSU crystal–stimulated resident peritoneal macrophages. 458 MURAKAMI ET AL Figure 1. Rapid induction of triggering receptor expressed on myeloid cells 1 (TREM-1) expression in monosodium urate monohydrate (MSU) crystal– stimulated resident peritoneal macrophages. A, TREM-1 mRNA level as determined by quantitative real-time polymerase chain reaction (PCR). Resident peritoneal macrophages were incubated with MSU crystals (100 g/ml) for the indicated time periods. Murine GAPDH was used as the internal control. The relative amount of TREM-1 mRNA was evaluated by comparison with the level in vehicle-treated resident peritoneal macrophages, which was defined as 1 arbitrary unit. B, Western blot analysis of the TREM-1 protein level. Murine actin was used as the loading control. C, TREM-1 mRNA level as determined by quantitative real-time PCR in resident peritoneal macrophages incubated with or without various concentrations of MSU crystals for 1 hour. D, Western blot analysis of TREM-1 protein level 8 hours after stimulation. Data are expressed as the mean and SD results of triplicate determinations. Expression of TREM-1 in a murine air-pouch model of MSU crystal–induced acute inflammation. In order to determine whether MSU crystals could promote TREM-1 expression in vivo, a murine air-pouch model of MSU crystal–induced acute inflammation was used. Injection of MSU crystals (3 mg) caused infiltration of cells into the air pouches (Figure 2), and expression of TREM-1 mRNA by the infiltrating cells increased in a time-dependent manner (Figure 2). Maximum induction occurred at 4 hours, and expression subsequently declined 8 hours after stimulation. We previously demonstrated increased expression of IL-1␤, macrophage inflammatory protein 2, and KC in infiltrating cells in this air-pouch model (18,19). The time course of TREM-1 mRNA expression closely resembles gene expression of inflammatory cytokines and chemokines. Gene expression of TREM-1 in soft tissue around air pouches was also evaluated, and it increased ⬃4-fold relative to the control pouch (data not shown). However, the TREM-1 expression level in soft tissues was lower than that in infiltrating cells. Neutrophils are a predominant cell type among the accumulated cells in MSU crystal–induced acute inflammation. In order to evaluate TREM-1 expression on neutrophils, flow cytometric analysis was performed on the infiltrating cells. As shown in Figure 2B, 8 hours after stimulation, infiltrating neutrophils expressed TREM-1 at an increased level in comparison with thioglycolate-elicited neutrophils. We failed to determine TREM-1 expression in infiltrating neutrophils at different time points because of the limited number of cells. This result indicated that MSU crystals could also up-regulate TREM-1 expression in infiltrating neutrophils. Furthermore, sTREM-1 was also detected in pouch fluid 8 hours (but not 4 hours) after stimulation (Figure 2C). Overall, these findings clearly demon- RAPID INDUCTION OF TREM-1 IN ACUTE GOUT 459 Figure 2. TREM-1 expression in a murine air-pouch model of MSU crystal–induced acute inflammation. A, MSU crystals (3 mg in 1 ml of sterile phosphate buffered saline) were injected into subcutaneous air pouches created in mice. Infiltrating cells in the air pouches were harvested after the indicated periods of time. Expression of TREM-1 by infiltrating cells was determined by quantitative real-time PCR. Murine GAPDH was used as the internal control. Results are the mean and SD values from 3–6 mice. (Similar results were obtained in 2 independent experiments.) B, Infiltrating cells into the air pouches were harvested 8 hours after stimulation. Thioglycolateelicited neutrophils were used as a negative control. Cellular expression of TREM-1 was determined by flow cytometry. Representative histogram illustrates TREM-1 expression by infiltrating neutrophils (bold solid line) and control neutrophils (solid line). Broken line indicates background staining of infiltrating cells with an isotypematched control monoclonal antibody. C, Pouch fluid was harvested 8 hours after stimulation. Soluble TREM-1 levels were determined by enzyme-linked immunosorbent assay. Results are the mean and SD. See Figure 1 for definitions. strated that MSU crystals are capable of promoting TREM-1 expression in soft tissue as well as neutrophils in vivo. Regulation of MSU crystal–induced TREM-1 expression. Because MSU crystals significantly induced TREM-1 expression by resident peritoneal macrophages, we investigated the mechanism regulating this change in TREM-1 expression. Because MSU crystals are well known to promote cytokine production by macrophages, the effects of inflammatory cytokines such as IL-1␤ and TNF␣ on TREM-1 expression by resident peritoneal macrophages were investigated. After resident peritoneal macrophages were incubated with IL-1␤ (20 ng/ml) or TNF␣ (20 ng/ml) for 1 hour, gene expression was determined by quantitative real-time PCR. As shown in Figures 3A and B, these cytokines enhanced the expression of both TREM-1 mRNA and protein, but the potency of their effect on TREM-1 appeared to be lower than that of LPS. Enhancement of TREM-1 expression by MSU crystals at a concentration of 100 g/ml appeared to be equivalent to that caused by LPS at a concentration of 20 ng/ml. A Limulus amebocyte cell lysate assay indicated the absence of LPS in the crystal preparation. However, we also used polymyxin B to eliminate possible contamination of the crystals by LPS. Polymyxin B is a cationic, cyclic peptide antibiotic that can inhibit the activity of LPS (20). Treatment with polymyxin B at a concentration of 5 g/ml for 1 hour completely abolished LPSinduced TREM-1 expression by resident peritoneal macrophages but failed to block crystal-induced TREM-1 expression (Figure 3A). These findings indicated that MSU crystal–induced up-regulation of TREM-1 expression was caused by direct interaction of 460 resident peritoneal macrophages with the crystals rather than by induction of proinflammatory cytokines or LPS. Synergistic effect of agonistic TREM-1 antibodies on MSU crystal–induced cytokine production. It was previously shown that expression of TREM-1 on macrophages is induced by LPS, and that treatment of macrophages with LPS and an agonist antibody for TREM-1 synergistically enhances the production of proinflammatory cytokines. In order to evaluate whether antibody binding to TREM-1 also had a synergistic effect on cytokine production by crystal-stimulated macrophages, resident peritoneal macrophages were incubated with MSU crystals in the presence or absence of anti– TREM-1 agonist polyclonal antibodies for 24 hours, and cytokine production was determined by ELISA. As shown in Figure 4, IL-1␤ production by resident peritoneal macrophages incubated with both MSU crystals and anti–TREM-1 agonist polyclonal antibodies was MURAKAMI ET AL Figure 4. Synergistically enhanced production of proinflammatory cytokines by macrophages, by binding of TREM-1 and MSU crystals. Resident peritoneal macrophages were incubated with an isotype control antibody (goat IgG1) or polyclonal anti–TREM-1 agonist monoclonal antibodies in the presence of MSU crystals for 24 hours. Production of IL-␤ (A) and macrophage chemotactic protein 1 (MCP-1) (B) was determined using specific enzyme-linked immunosorbent assays. Values are the mean and SD of triplicate determinations. PBS ⫽ phosphate buffered saline (see Figure 3 for other definitions). significantly increased, with production being 18-fold greater than that by cells stimulated with crystals alone (Figure 4A). Costimulation of resident peritoneal macrophages with crystals and anti–TREM-1 polyclonal antibodies also promoted an 8-fold increase of MCP-1 production in comparison with that by crystal-stimulated cells (Figure 4B). These results clearly indicated that antibody binding of TREM-1 and exposure to MSU crystals synergistically up-regulated the production of proinflammatory cytokines by resident peritoneal macrophages. DISCUSSION Figure 3. Induction of triggering receptor expressed on myeloid cells 1 (TREM-1) expression in resident peritoneal macrophages by various inflammatory agents. Resident peritoneal macrophages were incubated with or without polymyxin B (polyB; 5 g/ml) for 1 hour, and were subsequently stimulated with interleukin-1␤ (IL-1␤), tumor necrosis factor ␣ (TNF␣), monosodium urate monohydrate (MSU) crystals, or lipopolysaccharide (LPS) for 1 hour. A, TREM-1 mRNA levels were determined by quantitative real-time polymerase chain reaction. Murine GAPDH was used as the internal control. Values are the mean and SD of triplicate determinations. B, TREM-1 protein levels were determined by Western blot analysis. The results of this study provide the first evidence that a nonmicrobial agent, MSU crystals, can induce TREM-1 expression by macrophages in vitro. In addition, TREM-1 expression was significantly up-regulated in an in vivo (murine air-pouch) model of MSU crystal– induced acute inflammation. Activation of TREM-1 in combination with exposure to MSU crystals synergistically increased the production of IL-1␤ and MCP-1 by macrophages. Deposition of MSU crystals in articular and periarticular tissues is an essential pathologic finding in acute and chronic gouty arthritis (1,2). MSU crystals stimulate various types of cells, including monocytes, macrophages, neutrophils, and synovial cells, resulting in a rapid increase in the production of proinflammatory RAPID INDUCTION OF TREM-1 IN ACUTE GOUT cytokines and chemokines. Several lines of evidence indicate that release of these inflammatory mediators plays an important role in the infiltration and activation of inflammatory cells in acute gout (5,6,21). Therefore, activation of inflammatory cells seems to be essential for the initiation of MSU crystal–induced acute inflammation. It has previously been shown that direct contact with or phagocytosis of these crystals by phagocytes may promote cellular activation in acute gout, but the precise mechanisms of cellular activation by the crystals remain unknown. In this study, we demonstrated that rapid induction of TREM-1 by MSU crystals may be involved in the crystal-induced inflammatory response that occurs during acute gout attacks. TREM-1 is a recently discovered cellular receptor that belongs to the immunoglobulin superfamily and is expressed on monocytes and neutrophils. It promotes cellular activation mediated by an associated signal transduction molecule, DAP12, and a role of TREM-1 in both the innate and adaptive immune responses has recently been documented (10). A pathophysiologic role of TREM-1 as an amplifier of inflammation has also been confirmed by in vivo studies (11,12). In animal models of severe bacterial infection, blockade of signaling via TREM-1 with an sTREM-1 immunoglobulin fusion protein or synthetic sTREM-1 was able to protect mice against septic shock and death (11,12). It was recently reported that TREM-1 expression is induced by stimulation with microbial products, but not by nonmicrobial agents (11,22). We observed that proinflammatory cytokines (e.g., IL-1␤ and TNF␣) produced by crystal-stimulated macrophages caused only slight induction of TREM-1 expression, whereas exposure to MSU crystals led to a marked increase of TREM-1 expression even in the presence of polymyxin B. This may be the first report of TREM-1 expression being caused by a nonmicrobial agent, i.e., MSU crystals. MSU crystals induced TREM-1 expression of infiltrating cells and sTREM-1 production in a murine air-pouch model of crystal-induced acute inflammation. Rapid induction of TREM-1 mRNA expression in infiltrating cells was observed, and maximal gene expression of TREM-1 was found 4 hours after stimulation. Flow cytometric analysis clearly demonstrated increased expression of TREM-1 in infiltrating neutrophils. In a murine air-pouch model, expression of TREM-1 in monocytes and macrophages among infiltrating cells could not be identified because of the limited number of cells. However, TREM-1 mRNA expression was detected in the soft tissue. These findings indicated that MSU crystals could induce TREM-1 expression in both 461 macrophages and neutrophils in the setting of crystalinduced acute inflammation. Furthermore, the addition of agonistic TREM-1 antibodies significantly enhanced cytokine production by crystal-stimulated macrophages, indicating that the TREM-1 molecules induced by crystal stimulation were functional. Although natural ligand(s) for TREM-1 have not yet been identified, our findings indicate that endogenous MSU crystals may promote an inflammatory response, at least partly via TREM-1–mediated signaling. Thus, it is possible to hypothesize that natural ligands are capable of amplifying the acute inflammatory response in patients with acute gout after being recognized by TREM-1 expressed on crystal-stimulated phagocytes. TREM-1 and TLRs are both recently identified cellular receptors that regulate innate immune responses, and an interaction between these 2 kinds of receptors has been demonstrated. For example, TREM-1 is significantly up-regulated by various ligands for TLRs, including lipoteichoic acid (TLR-2), polyinosinic–polycytidylic acid (TLR-3), and LPS (TLR-4) (10). In addition, binding of agonistic mAb to TREM-1 on monocytes in combination with the ligands for TLR-2, TLR-3, or TLR-4 synergistically amplified the cellular production of proinflammatory cytokines (8,10,11). Liu-Bryan et al recently demonstrated an essential role of TLR-2 in MSU crystal–induced acute inflammation (14). This indicates that MSU crystals are potent ligands for TLR-2, and recognition of the crystals by TLR-2–expressing inflammatory cells promotes rapid induction of various inflammation mediators. However, because it is not known whether MSU crystal–induced up-regulation of TREM-1 expression is mediated by TLR-2, further investigations should be conducted to elucidate the biologic interactions between TLRs and TREM-1 in MSU crystal–induced acute inflammation. Mechanisms for the synergistic effect of TREM-1 and TLRs have been postulated. For example, NF-B is activated by the TLR signaling pathway. TREM-1– mediated tyrosine phosphorylation, activation of MAPK, and mobilization of Ca2⫹ might also lead to the activation of transcription complexes, which could have a synergistic effect with NF-B in promoting the expression of proinflammatory genes. It has also been shown that MSU crystals potentially activate the NF-B signaling pathway (23). Overall, these findings suggest that TREM-1 may act synergistically with MSU crystals in promoting the production of proinflammatory cytokines through activation of NF-B and MAPK, tyrosine phosphorylation, and mobilization of Ca2⫹. 462 MURAKAMI ET AL Crosslinking of TREM-1 not only induces cytokine production but also up-regulates the expression of various cell surface molecules, including CD40 and intercellular adhesion molecule (ICAM) (8). Activation of CD40 by binding to CD40 ligand amplifies the production of proinflammatory cytokines by macrophages (24), while ICAM is an adhesion molecule that facilitates recruitment of neutrophils and macrophages to inflammation foci. Thus, induction of these molecules by TREM-1 may lead to initiation of the inflammatory response in acute gout (25). Based on the present findings, we hypothesize that rapid induction of TREM-1 may contribute to the enhancement of MSU crystal–induced acute inflammation through recognition of a natural ligand for TREM-1. Further investigation is needed to evaluate the pathologic role of TREM-1 in increasing MSU crystal–induced acute inflammation in vivo by using an sTREM-1–immunoglobulin fusion protein. Such studies may define the precise role of TREM-1 in acute gouty arthritis and could also provide a novel therapeutic target for the management of acute gout. ACKNOWLEDGMENTS 9. 10. 11. 12. 13. 14. 15. 16. 17. We thank Mrs. Rie Hasegawa and Terumi Mizuno for excellent technical support. 18. REFERENCES 1. Schumacher HR. Crystal-induced arthritis: an overview. Am J Med 1996;100:46S–52S. 2. Terkeltaub RA. Pathogenesis and treatment of crystal-induced inflammation. In: Koopman WJ, editor. Arthritis and allied conditions. 14th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 2329–47. 3. 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