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.
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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.