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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

Peroxisome proliferator–activated receptor γ (PPARγ) is a member of the nuclear hormone receptor superfamily and functions as a key regulator of lipid and glucose metabolism, atherosclerosis, and inflammatory responses. This study was undertaken to evaluate the biologic role of PPARγ in self-limiting episodes of acute gouty arthritis. To do this, we investigated PPARγ expression by monosodium urate monohydrate (MSU) crystal–stimulated monocytes, and we studied the effects of PPARγ ligands on crystal-induced acute inflammation.

Methods

PPARγ expression by MSU crystal–stimulated human peripheral blood mononuclear cells was determined by reverse transcription–polymerase chain reaction and immunostaining. Expression of CD36 on monocytes was detected by flow cytometric analysis. The effects of PPARγ ligands on in vitro crystal-induced cytokine production and on in vivo cellular infiltration during crystal-induced acute inflammation were also investigated.

Results

MSU crystals rapidly and selectively induced PPARγ expression by monocytes. Gene expression was detected as early as 2 hours, and maximum expression was observed at 4 hours after stimulation. The induced PPARγ was functional, since a PPARγ ligand was able to up-regulate CD36 expression on monocytes. A natural ligand of PPARγ, 15-deoxy-Δ12,14-prostaglandin J2 (15deoxy-PGJ2), significantly reduced the crystal-induced production of cytokines by monocytes. Indomethacin inhibited cytokine production only at high concentrations, and an antidiabetic thiazolidinedione (troglitazone) failed to exert significant effects. Administration of troglitazone and 15deoxy-PGJ2 significantly prevented cellular accumulation in a mouse air-pouch model of MSU crystal–induced acute inflammation.

Conclusion

Rapid induction of PPARγ expression on monocytes by MSU crystals may contribute, at least in part, to the spontaneous resolution of acute attacks of gout.

Acute gouty arthritis is an inflammatory disease caused by the deposition of monosodium urate monohydrate (MSU) crystals in the articular and periarticular tissue (1). MSU crystals have a remarkable capacity to stimulate the generation of various inflammatory mediators from synovial cells, macrophages, and infiltrating leukocytes. Precipitation of these crystals has been shown to cause massive infiltration of neutrophils into the joints and to promote neutrophil activation, which in turn, brings about tissue damage (2). This is reflected in the clinical features of acute gouty arthritis, such as severe pain, edema, and periarticular erythema.

One of the characteristic features of acute gouty arthritis is its self-limiting course, since this arthritis generally subsides spontaneously after ∼1 week, even in the absence of any treatment (1, 3). A number of possible explanations for this spontaneous improvement have been postulated. Removal of MSU crystals by phagocytes, dissolution of the crystals, involvement of antiinflammatory factors, and alteration of the crystal surface by coating with serum factors have been suggested (1, 4–6). However, these explanations have not been generally accepted, and the precise mechanisms that actually cause spontaneous resolution of acute gouty arthritis remain unknown.

Peroxisome proliferator–activated receptor γ (PPARγ) is a member of the nuclear hormone receptor superfamily and acts as a transcriptional regulator of the related genes by forming heterodimers with the retinoid X receptor (RXR) (7). PPARγ is most highly expressed in white adipose tissue and has been implicated in the regulation of adipocyte differentiation, lipid storage, and glucose metabolism. PPARγ is activated by naturally occurring ligands, including 15-deoxy-Δ12,14-prostaglandin J2 (15deoxy-PGJ2) and oxidized low-density lipoprotein (ox-LDL), as well as by synthetic agents, such as thiazolidinedione antidiabetic drugs and nonsteroidal antiinflammatory drugs (NSAIDs). Accumulating evidence has indicated that PPARγ has diverse effects on a wide variety of cells and plays a crucial role in the regulation of monocyte differentiation, atherosclerosis, immune responses, apoptosis, and carcinogenesis (8). Of particular interest is the possibility that it may exert an antiinflammatory effect by inhibiting the gene expression of proinflammatory cytokines, inducible nitric oxide synthase (iNOS), proteinases, and cyclooxygenase (COX) (9, 10). In order to elucidate the regulatory mechanisms that cause acute gout to resolve, we conducted experiments investigating the expression of PPARγ by MSU crystal–stimulated monocytes and the therapeutic effect of PPARγ ligands on crystal-induced acute inflammation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Reagents.

Mouse monoclonal anti-human PPARγ antibody and fluorescein isothiocyanate (FITC)–conjugated anti-mouse IgG antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated anti-human CD36 antibody was purchased from BD PharMingen (San Diego, CA). Specific enzyme-linked immunosorbent assays (ELISAs) for human interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were obtained from R&D Systems (Minneapolis, MN), and a DIG-High Prime DNA Labeling and Detection Kit was obtained from Roche Diagnostics (Mannheim, Germany). We purchased 15deoxy-PGJ2 from Cayman Chemical (Ann Arbor, MI), and troglitazone was from Sankyo (Tokyo, Japan).

Preparation of MSU crystals.

MSU crystals were prepared according to the method described by Seegmiller et al (4). Briefly, 8 gm of uric acid (Sigma, St. Louis, MO) 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 the addition of HCl, the solution was cooled gradually with stirring at room temperature and then stored overnight at 4°C. The crystals were then 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 thus obtained were rod-shaped and uniform in size (5–25 μm in length). A Limulus amebocyte cell lysate assay verified the absence of endotoxin in the preparation.

Cell culture.

Heparinized peripheral blood was obtained from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation with Ficoll-Paque. After washing with PBS, the PBMCs were suspended in RPMI 1640 medium (Gibco, Detroit, MI) supplemented with 5% heat-inactivated fetal calf serum (Bioserum, Melbourne, Victoria, Australia), 10 mM HEPES, 2 mML-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. PBMCs were incubated in the presence or absence of varying concentrations of MSU crystals for the indicated periods at 37°C in a humidified incubator with an atmosphere of 5% CO2 and 95% air.

RNA analysis.

Total cellular RNA was extracted from cultured cells by the acid guanidinium thiocyanate–phenol–chloroform method, and reverse transcription (RT) was done using random hexamer primers and Rous-associated virus 2 reverse transcriptase (Takara, Kyoto, Japan). Gene expression was assessed by polymerase chain reaction (PCR) using the primers 5′-GCCTTGCAGTGGGGATGTCTCA-3′ and 5′-ATGCGGATGGCCACCTCTTTG-3′ for human PPARγ (11), 5′-AGGCGAGGGCGATCTTGACAG-3′ and 5′-AGGGCTTCCGCAGGCTTTTG-3′ for murine PPARγ (12), 5′-GGAATTCAAGTACCTGAGCTCGCCAGT-3′ and 5′-GGAATTCACAAAGGACATGGAGAACAC-3′ for human interleukin-1β (IL-1β), and 5′-CTTCTGCCTGCTGCACTTTGG-3′ and 5′-TCCCAAAGTAGACCTGCCCAGA-3′ for human tumor necrosis factor α (TNFα). The expected PCR products obtained for human PPARγ, murine PPARγ, human IL-1β, and human TNFα had sizes of 337 bp, 384 bp, 495 bp, and 549 bp, respectively.

Human β2-microglobulin (β2m) and murine GAPDH were used as the internal controls. The primers for β2m were 5′-TTCTGGCCTGGAGGGCATCC-3′ and 5′-ATCTTCAAACCTCCATGATG-3′, while the primers for murine GAPDH were 5′-TTGGATCCGCAGTGCCGGCCTCGTCTCATAG-3′ and 5′-TTCTCGAGGACCCTTTTGGCACCACCCTTCAG-3′. The expected sizes of the PCR products for β2m and GAPDH were 340 bp and 380 bp, respectively.

PCR products were electrophoresed on 2% agarose gel, transferred to a nylon membrane, and hybridized with digoxigenin end-labeled oligonucleotide probes. The probes used in this study were 5′-CCCTCGCCTTTGCTTTGGTCA-3′ for human PPARγ, 5′-AGGGAGGCCAGCATCGTGTAGA-3′ for murine PPARγ, 5′-GGTGCATCGTGCACATAAGC-3′ for human IL-1β, 5′-GAAGACCCCTCCCAGATAGAT-3′ for human TNFα, 5′-ACACGGCAGGCATACTCATC-3′ for human β2m, and 5′-ACGGCAAGTTCAATGGCACAG-3′ for murine GAPDH. We performed 3′ end–labeling of each probe and its detection after hybridization, using a DIG-High Prime DNA Labeling and Detection Kit, after which the membrane was exposed to RX-U film (Fuji Photo Film Company, Tokyo, Japan).

Immunocytochemistry.

PBMCs (4 × 106) were cultured in a chamber slide (Nalge Nunc International, Roskilde, Denmark) at 37°C for 2 hours. After the slide was washed, adherent monocytes were incubated with MSU crystals (100 μg/ml) for 12 hours. The cells were then fixed in 2% paraformaldehyde in PBS (pH 7.4), washed with 0.01M Tris, 0.5M NaCl (pH 7.4), and permeabilized in cold methanol/acetone (1/1 volume/volume). After incubation in 0.01M Tris, 0.5M NaCl, 0.5% ovalbumin to block nonspecific staining, the cells were stained with a monoclonal anti-human PPARγ antibody and a FITC-conjugated anti-mouse IgG antibody. Fluorescence microscopy was used to visualize PPARγ staining in the monocytes.

Assay of cytokine production.

PBMCs (1 × 106) were incubated with MSU crystals in the presence or absence of varying concentrations of PPARγ ligands for 12 hours, after which the culture supernatant was obtained by centrifugation and stored at −20°C until use. The levels of IL-1β and TNFα in the supernatant were then determined using specific ELISAs.

Detection of CD36 expression on monocytes.

Peripheral blood monocytes were incubated with MSU crystals (100 μg/ml) in the presence or absence of troglitazone (10 μM) for the indicated periods at 37°C. The cells were subsequently incubated with a FITC-conjugated anti-human CD36 antibody, and the expression of CD36 was detected by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA).

Air-pouch model of MSU crystal–induced inflammation.

Subcutaneous air pouches were created in mice according to the method previously described (13). Briefly, anesthetized male C57BL/6 mice (age 8–12 weeks) were injected in the subcutaneous tissue of the back with 3 ml of air, followed by injection with another 5 ml of air after 3 days. On day 7 after the first injection, the air pouches thus created were used for experiments. MSU crystals (3 mg in a volume of 1 ml of sterile PBS) were coinjected into the air pouches with or without PPARγ ligand. After the indicated periods, the pouch fluid was harvested by injecting 3 ml of PBS. The infiltrating cells were then counted using a hemocytometer and stained with Wright-Giemsa solution to determine the differential leukocyte count.

Troglitazone emulsion was made by mixing troglitazone with 0.15M NaCl, 5 mM phosphate buffer, 1% Tween 80, and 1% benzyl alcohol. Indomethacin was dissolved in ethanol and diluted with PBS. 15deoxy-PGJ2 was diluted with PBS. Troglitazone and indomethacin were intraperitoneally administered to mice every day from day 4 to day 7 after the first injection of air into the subcutaneous tissue. 15deoxy-PGJ2 was coinjected with the crystals into the air pouches.

Statistical analysis.

Results are expressed as the mean and SD. Statistical analysis was performed using Student's paired t-test, and P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Induction of PPARγ expression by MSU crystals.

In order to investigate the effects of MSU crystals on PPARγ expression, PBMCs were incubated with the crystals, and PPARγ gene expression was evaluated by RT-PCR. As shown in Figure 1A, MSU crystals (100 μg/ml) significantly induced PPARγ gene expression by PBMCs in a time-dependent manner. Induction of gene expression occurred quite rapidly and was seen as early as 2 hours after stimulation. Maximum induction occurred at 4 hours after stimulation, and gene expression subsequently declined gradually up to 24 hours. When PBMCs were incubated with varying concentrations of MSU crystals for 6 hours and gene expression was evaluated, the crystals enhanced PPARγ messenger RNA (mRNA) expression in a dose-dependent manner, and maximum expression occurred after stimulation at 10 μg/ml or 100 μg/ml (Figure 1B).

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Figure 1. Induction of peroxisome proliferator–activated receptor γ (PPARγ) gene expression by monosodium urate monohydrate (MSU) crystals. A, Peripheral blood mononuclear cells (PBMCs) were incubated with MSU crystals (100 μg/ml) for the indicated periods. B, PBMCs were incubated with various concentrations of MSU crystals for 6 hours. Gene expression of PPARγ was determined by reverse transcription–polymerase chain reaction followed by Southern blot hybridization. Results are representative of 3 separate experiments using PBMCs obtained from 3 different donors.

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PPARγ gene expression by adherent and nonadherent PBMCs was investigated to determine which cells expressed this gene. MSU crystals enhanced PPARγ expression by adherent cells (monocytes), but not by nonadherent cells (data not shown).

Immunohistochemical analysis using a specific anti-PPARγ antibody was also performed to identify PPAR-γ-expressing cells. Adherent monocytes were incubated in the presence or absence of MSU crystals (100 μg/ml) for 12 hours, and PPARγ expression was determined. PPARγ was faintly detected after monocytes were incubated with medium alone (Figure 2A), whereas increased expression of PPARγ was clearly found when monocytes were incubated with the crystals for 12 hours (Figure 2B). These findings indicated that MSU crystals up-regulated PPARγ expression by monocytes.

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Figure 2. Immunostaining of PPARγ in monocytes. PBMCs (4 × 106) were cultured in a chamber slide at 37°C for 2 hours. After the slide was washed, adherent monocytes were incubated in the absence (A) or presence (B) of MSU crystals (100 μg/ml) for 12 hours, and PPARγ expression was determined by immunohistochemical analysis. Results are representative of 3 separate experiments using monocytes isolated from 3 different donors. (Original magnification × 100.) See Figure 1 for definitions.

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In order to determine whether MSU crystals could promote PPARγ expression in vivo, gene expression was investigated in murine subcutaneous air pouches after injection of the crystals. No PPARγ expression was detected in control pouches, while injection of 3 mg of MSU crystals into air pouches caused the rapid induction of tissue PPARγ mRNA expression in a time-dependent manner (Figure 3). Expression was evident after only 2 hours and was elevated for up to 12 hours after stimulation. Injection of the crystals also induced PPARγ expression in infiltrating leukocytes, but the level of gene expression was significantly lower than that in the inflamed soft tissues around the air pouches (data not shown). In the present study, we could not determine whether the crystals caused an increase in PPARγ expression by resident cells localized in the soft tissue, or whether they induced the recruitment of PPARγ-expressing cells into the air pouch. However, it was clearly demonstrated that MSU crystals caused a rapid increase of PPARγ expression both in vitro and in vivo.

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Figure 3. Gene expression of PPARγ in mouse subcutaneous air pouches. MSU crystals (3 mg) were injected into the air pouches created in the subcutaneous tissue of mice. The subcutaneous tissue of the air pouches was harvested after the indicated periods, and gene expression of PPARγ was determined by reverse transcription–polymerase chain reaction followed by Southern blot hybridization. Results are representative of 3 separate experiments. See Figure 1 for definitions.

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Induction of CD36 expression on MSU crystal–stimulated monocytes by PPARγ ligand.

Expression of CD36 has been shown to depend on PPARγ (14). Therefore, we investigated the effect of troglitazone on the induction of CD36 expression in crystal-stimulated monocytes in order to evaluate the biologic role of PPARγ. PBMCs were incubated with MSU crystals (100 μg/ml) in the presence or absence of troglitazone (10 μM) for up to 72 hours, and CD36 expression on monocytes was determined by flow cytometric analysis. As shown in Figure 4, troglitazone significantly increased the expression of CD36 by crystal-stimulated monocytes, indicating that functioning PPARγ was induced in the monocytes.

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Figure 4. Effect of a PPARγ ligand on CD36 expression by MSU crystal–stimulated monocytes. PBMCs were incubated with MSU crystals (100 μg/ml) in the presence (hatched bars) or absence (open bars) of troglitazone (10 μM) for the indicated periods. CD36 expression on monocytes was determined by flow cytometric analysis. Values are the mean and SD of triplicate experiments and are representative of 3 separate experiments using PBMCs obtained from 3 different donors. MFI = mean fluorescence intensity. See Figure 1 for other definitions.

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Effect of PPARγ ligands on MSU crystal–induced cytokine production.

PPARγ has been shown to down-regulate the inflammatory response of monocytes and macrophages. Accordingly, we conducted experiments to investigate the effects of several PPARγ ligands, including 15deoxy-PGJ2, indomethacin, and troglitazone, on MSU crystal–induced production of inflammatory cytokines. PBMCs were incubated with 100 μg/ml of MSU crystals in the presence or absence of PPARγ ligands for 6 hours, and the expression of IL-1β and TNFα genes was determined by RT-PCR. As shown in Figure 5, MSU crystals significantly induced expression of the genes for both cytokines. The 3 PPARγ ligands exerted different effects on cytokine expression. 15deoxy-PGJ2 significantly decreased the expression of genes for both cytokines, and even a concentration as low as 10 μM caused a reduction of gene expression. The NSAID indomethacin also reduced expression of these cytokine genes, but inhibition occurred only at a high concentration of 10−4M. On the other hand, the thiazolidinedione antidiabetic agent troglitazone was able to slightly reduce TNFα expression at a high concentration of 30 μM, but it failed to have any effect on IL-1β expression.

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Figure 5. Effects of PPARγ ligands on MSU crystal–induced expression of cytokines. PBMCs were incubated with MSU crystals (100 μg/ml) in the presence or absence of the PPARγ ligands 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), indomethacin, and troglitazone for 6 hours. Gene expression of interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was determined by reverse transcription–polymerase chain reaction followed by Southern blot hybridization. Results were confirmed in 3 separate experiments performed with PBMCs isolated from different donors. See Figure 1 for other definitions.

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To further investigate the effects of the PPARγ ligands on cytokine production, PBMCs were incubated with MSU crystals (100 μg/ml) in the presence or absence of various concentrations of the ligands for 12 hours, and cytokine production was determined using specific ELISAs. The results are shown in Figure 6. Incubation with 15deoxy-PGJ2 significantly reduced the production of both cytokines in a concentration-dependent manner, and it inhibited cytokine production at concentrations as low as 1–3 μM (Figures 6A and B). The effect of 15deoxy-PGJ2 on cytokine production was quite marked, with a concentration of 3 μM yielding 61.1% and 25.4% inhibition of IL-1β and TNFα production, respectively. These findings were consistent with the results obtained by RT-PCR analysis. Indomethacin was only able to decrease the production of the two cytokines at a high concentration of 10−4M (Figures 6C and D). At a high concentration of 30 μM, troglitazone slightly decreased IL-1β production but failed to modulate TNFα production (Figures 6E and F).

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Figure 6. Effects of PPARγ ligands on cytokine production from MSU crystal–stimulated PBMCs. PBMCs were incubated with MSU crystals (100 μg/ml) in the presence or absence of various concentrations of 15d-PGJ2 (A and B), indomethacin (C and D), and troglitazone (E and F) for 12 hours. Production of IL-1β (A, C, and E) and TNFα (B, D, and F) was determined using specific enzyme-linked immunosorbent assays. Results are representative of 3 separate experiments using PBMCs isolated from independent donors. See Figures 1 and 5 for definitions.

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In vivo effect of PPARγ ligands on MSU crystal–induced acute inflammation.

Injection of 3 mg of MSU crystals into subcutaneous air pouches caused acute inflammation resembling that seen in acute gouty arthritis (13). The crystals induced infiltration of leukocytes into the air pouch, which was maximal at 8 hours after crystal injection and declined thereafter (Figure 7). Neutrophils were the predominant cell type among the infiltrating cells. Since MSU crystals caused the rapid induction of PPARγ expression in air pouch soft tissues, the biologic activity of PPARγ ligands during MSU crystal–induced cellular accumulation was also investigated using this model.

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Figure 7. Effect of PPARγ ligands on a mouse air-pouch model of MSU crystal–induced acute inflammation. MSU crystals (3 mg) were coinjected with phosphate buffered saline (open bars) or 1 mg/kg weight of 15-deoxy-Δ12,14-prostaglandin J2 (dotted bars) into the subcutaneous air pouches. MSU crystals were also injected into the air pouches of mice that had been pretreated with 0.5 mg/kg weight of indomethacin (solid bars) or 100 mg/kg weight of troglitazone (hatched bars). After the indicated periods, the pouch fluid was harvested and the infiltrating cells were counted. Values are the mean and SD accumulated cells (12 replicates/group). ∗ = P < 0.05 versus control mice. See Figure 1 for definitions.

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Indomethacin and troglitazone were injected intraperitoneally, and cellular infiltration was determined. Pretreatment with indomethacin (0.5 mg/kg weight) slightly reduced cellular accumulation at 8 hours after stimulation, but the change was not significant. Troglitazone at a dose of 100 mg/kg of body weight significantly prevented cellular infiltration at 4 hours after stimulation, and inhibition of leukocyte accumulation was estimated to be 41.9%. Troglitazone did not inhibit cellular infiltration at 8 hours and 12 hours after injection. 15deoxy-PGJ2 was coinjected into air pouches with MSU crystals, and cellular infiltration was determined. Coinjection of 15deoxy-PGJ2 at a dose of 1 mg/kg of body weight caused a significant decrease in leukocyte infiltration. Inhibitory effects were evident at 4 hours and 8 hours after treatment, and inhibition of leukocyte accumulation at these time points was estimated to be 61.3% and 54.8%, respectively. At 12 hours after injection, 15deoxy-PGJ2 did not exert any inhibitory effect. The effect of 15deoxy-PGJ2 was dose dependent, and the coinjection of 15deoxy-PGJ2 at a dose of 100 μg/kg of body weight failed to prevent cellular accumulation (data not shown). These findings indicated that MSU crystals could up-regulate PPARγ expression, and PPARγ ligands seemed to attenuate in vivo cellular infiltration in a model of MSU crystal–induced acute inflammation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The present study provided evidence that MSU crystals can potentially up-regulate PPARγ expression by monocytes. In addition, MSU crystals induced functioning PPARγ, since the PPARγ ligand was able to enhance CD36 expression by crystal-stimulated monocytes. 15deoxy-PGJ2 caused significant inhibition of cytokine production by MSU crystal–stimulated monocytes in vitro. Both 15deoxy-PGJ2 and troglitazone prevented cellular infiltration in this mouse air-pouch model of crystal-induced acute inflammation.

Deposition of MSU crystals in the articular and periarticular tissues is an essential part of the pathogenesis of acute and chronic gouty arthritis (1, 2). MSU crystals have the potential to stimulate various types of cells, including monocytes, macrophages, synovial lining cells, and neutrophils. MSU crystals have been shown to stimulate monocytes and macrophages, resulting in the production of proinflammatory cytokines (IL-1, IL-6, and TNFα (15–17), chemokines (IL-8 and monocyte chemoattractant protein 1) (18), arachidonic acid metabolites, oxygen radicals, and proteinases. Several lines of evidence indicate that the release of these inflammatory mediators plays an important role in the infiltration and activation of inflammatory cells in patients with acute gout (1, 2). Therefore, activation of monocytes and macrophages by MSU crystals seems to be critical for the initiation of crystal-induced inflammation.

Self-limiting episodes are a characteristic feature of acute gouty arthritis. Although the mechanisms related to the initiation of acute attacks of gout have been well characterized, the mechanisms involved in the termination of these attacks are poorly understood. Removal of MSU crystals by phagocytes and dissolution of the crystals by increased solubility modulated by an increase of temperature, a decrease of pH, and a decrease of sodium and urate levels have been proposed (4–6, 19). However, these explanations have not been widely accepted because crystals may remain in the inflamed joints for a long time after the acute attack has subsided (4). Antiinflammatory factors (local or systemic) have also been demonstrated to exist, even though little is known about their properties. Matsukawa et al recently reported that a locally produced IL-1 receptor antagonist might have an antiinflammatory effect on MSU crystal–induced acute inflammation (18). Alteration of the crystal surface by the deposition of immunoglobulins and apolipoproteins has been shown to make these crystals less inflammatory (2, 20). However, these mechanisms may not be sufficient to explain the spontaneous resolution of acute gout attacks.

PPARγ is a member of the nuclear hormone receptor superfamily and acts as a ligand-dependent transcription factor by forming heterodimers with the RXR (7). PPARγ is primarily found in adipose tissue and is well characterized as a regulator of various genes related to lipid and glucose metabolism. It has recently been demonstrated that this receptor is also expressed in a wide variety of cells, including monocytes and macrophages. Activation by various stimuli, including phorbol myristate acetate, lipopolysaccharide, advanced glycation end products, and phagocytosis of ox-LDL, could enhance PPARγ expression by monocytes and macrophages (10, 21, 22). Increased expression of PPARγ has also been documented at sites of inflammation in arthritis and colitis and in foam cells from atherosclerotic plaques (23–25). PPARγ has recently been suggested to function as a negative regulator of inflammatory responses. PPARγ ligands are capable of reducing the expression of genes for cytokines (e.g., TNFα, IL-6, and IL-1β), iNOS, gelatinase B, scavenger receptor A, and COX-2 in activated macrophages (9, 10, 26).

The present study clearly demonstrated that MSU crystals could potently stimulate PPARγ expression in monocytes and in a mouse air-pouch model of MSU crystal–induced inflammation. Immunohistochemical analysis indicated that MSU crystals caused a selective stimulation of PPARγ expression by the monocytes among PBMCs. Although the PPARγ-expressing cells in the soft tissues of mouse air pouches were not identified, we suspect that these were resident or infiltrating macrophages. We could not detect expression of the PPARγ gene in either peripheral blood neutrophils or synovial fibroblasts following stimulation with MSU crystals (data not shown); therefore, MSU crystals may selectively up-regulate PPARγ expression by monocytes and macrophages.

The PPARγ expressed by crystal-stimulated monocytes was shown to be functional, since exposure to troglitazone significantly enhanced the expression of CD36 by these monocytes. In addition, PPARγ ligands inhibited the production of IL-1β and TNFα by MSU crystal–stimulated monocytes. We found that 15deoxy-PGJ2, a naturally occurring derivative of PGD2, reduced the MSU crystal–induced production of proinflammatory cytokines, and its inhibitory effect was greater than that of synthetic PPARγ ligands, such as indomethacin and troglitazone. In fact, indomethacin could only prevent cytokine production at a high concentration (10−4M), and troglitazone had only a slight effect on cytokine production.

Previous studies have demonstrated that 15deoxy-PGJ2 was more effective than synthetic PPARγ ligands in reducing the expression of iNOS and cytokines by monocytes (27). It has been shown that high concentrations of thiazolidinedione antidiabetic drugs are required to obtain antiinflammatory activities. These pharmacologic discrepancies suggested a PPARγ-independent pathway for 15deoxy-PGJ2. Chawla et al evaluated the biologic effects of PPARγ ligands on macrophages from receptor-deficient mice and concluded that the induction of CD36 expression by macrophages was PPARγ dependent, while the inhibition of cytokine production might be PPARγ independent (28). Rossi et al reported the direct inhibition of inhibitor of nuclear factor κB (NF-κB) kinase activity by 15deoxy-PGJ2 (29). However, Straus et al demonstrated that 15deoxy-PGJ2 may reduce NF-κB binding by alkylating p50/p65 dimers through PPARγ-dependent pathways (30). Thus, 15deoxy-PGJ2 appears to inhibit NF-κB activation at different levels and may exert its activity in a PPARγ-dependent and -independent manner.

A number of studies have also documented PPARγ-dependent antiinflammatory functions of PPARγ ligands. Inoue et al demonstrated that 15deoxy-PGJ2 could reduce PGE2 production by monocytes in a PPARγ-dependent manner (31). Moreover, 15deoxy-PGJ2 and synthetic PPARγ ligands are capable of inducing cellular apoptosis of monocytes and endothelial cells and of inhibiting IL-1β–induced production of NO and matrix metalloproteinase in human chondrocytes through PPARγ-dependent pathways (32–34). Overall, both naturally occurring and synthetic ligands for PPARγ exert considerable influences on inflammatory responses through receptor-dependent and -independent pathways.

CD36 is a scavenger receptor for ox-LDL and cells undergoing apoptosis (14, 35), and the expression of CD36 on monocytes and macrophages is up-regulated by PPARγ ligands. Since ox-LDL is a ligand for PPARγ, CD36-mediated phagocytosis of ox-LDL can stimulate CD36 expression. Fadok et al recently demonstrated that CD36-mediated phagocytosis of apoptotic cells by macrophages was able to prevent cytokine production through the endogenous release of transforming growth factor β, PGE2, and platelet-activating factor (36). This suggests that MSU crystal–induced expression of PPARγ in monocytes may enhance CD36 expression, and CD36-mediated phagocytosis of apoptotic cells may inhibit inflammatory responses.

Because of the increased expression of PPARγ in inflamed foci in various inflammatory diseases, therapeutic advantages of PPARγ ligands have been investigated. Su et al demonstrated that thiazolidinedione drugs markedly reduced colonic inflammation in a mouse model of inflammatory bowel disease (24). Kawahito et al also reported that intraperitoneal administration of 15deoxy-PGJ2 and troglitazone could ameliorate adjuvant-induced arthritis in rats through the suppression of pannus formation and cellular infiltration in the inflamed joints (23). The present study demonstrated that administration of 15deoxy-PGJ2 and troglitazone could inhibit cellular infiltration in an air-pouch model of MSU crystal–induced acute inflammation. Inhibitory effects of PPARγ ligands were evident at 4 hours and 8 hours after stimulation. However, PPARγ ligands failed to prevent cellular accumulation at 12 hours after stimulation. Although precise mechanisms are not known, the short half-life of 15deoxy-PGJ2 and the partial inhibitory effects of PPARγ ligands on cellular accumulation may be implicated in the progression of inflammatory responses at 12 hours after stimulation.

It was recently demonstrated that PGD2 and its metabolite, 15deoxy-PGJ2, are present in vivo during the late phase of inflammation. Gilroy et al recently showed that NSAIDs could prevent early cellular infiltration by the inhibition of PGE2 production and could exacerbate the late inflammatory response through the inhibition of PGD2 production in rats with carrageenan-induced pleurisy (37). These findings suggest that 15deoxy-PGJ2 may act as a feedback regulator of inflammatory responses. A previous study demonstrated that injection of MSU crystals into mouse peritoneum rapidly generated increased amounts of various eicosanoid species, including PGD2 (38), indicating a possible role of PGD2 and its metabolite on the resolution of crystal-induced acute inflammation.

Based on the results of this study, we hypothesize that rapid induction of PPARγ may contribute to the spontaneous resolution of MSU crystal–induced acute inflammation through interaction with endogenous PPARγ ligands, such as 15deoxy-PGJ2 and ox-LDL. Further investigation should be directed toward the in vivo effects of PPARγ inhibition on the spontaneous remission of MSU crystal–induced acute inflammation by using antisense oligonucleotides for PPARγ mRNA or decoy oligonucleotides for PPARγ response elements. Such studies may elucidate the precise role of PPARγ in acute gouty arthritis and could possibly provide a new strategy for the treatment of acute gout.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Schumacher HR Jr. Crystal-induced arthritis: an overview. Am J Med 1996; 100: 46S52S.
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    Seegmiller JE, Howell RR, Malawista SE. The inflammatory reaction to sodium urate. JAMA 1962; 180: 46975.
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    Pascual E, Jovani V. A quantitative study of the phagocytosis of urate crystals in the synovial fluid of asymptomatic joints of patients with gout. Br J Rheumatol 1995; 34: 7246.
  • 6
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