Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

Neutrophil activation by inflammatory microcrystals of monosodium urate monohydrate utilizes pertussis toxin-insensitive and -sensitive pathways

The activation of leukocytes by particulates is a critical event in certain inflammatory syndromes, including acute gout associated with microcrystals of monosodium urate monohydrate. In this study we have evaluated mechanisms of human neutrophil activation by urate crystals. Both N-formyl-nor-leu-leu-phe-nor-leu-tyr-lys and uncoated urate crystals (0.5 to 5 mg/ml) but not urate crystals coated with human low density lipoprotein (which suppresses crystal-induced neutrophil responsiveness), stimulated pertussis toxin (PT)-sensitive GTPase activity in purified preparations of human neutrophil membranes. Hydroxyapatite crystals (up to 5 mg/ml) were inactive. Pretreatment of neutrophil membranes with cholera toxin also inhibited crystal-induced and formylated peptide-induced GTPase activity. Pretreatment of whole neutrophils with PT resulted in nearly complete inhibition of formylated peptide-induced cytosolic calcium mobilization, release of superoxide and release of the azurophil granule constituent alpha-mannosidase. In contrast, identical pretreatment of whole neutrophils with PT only partially suppressed urate crystal-induced alpha-mannosidase and superoxide release and failed to inhibit crystal phagocytosis and increases in cytosolic free calcium. Mechanisms of neutrophil activation by monosodium urate crystals appear to be heterogeneous in comparison with activation by formylated peptides and there is no absolute requirement for PT-sensitive membrane G proteins in neutrophil responsiveness to urate crystals.     

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil-hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.     

敲除 Sam68 基因导致白血病细胞系细胞生长迟缓和 G2/M 期延长

RNA 结合蛋白 Sam68 是细胞有丝分裂期 Src 酪氨酸磷酸化的靶蛋白 . 尽管确切机制尚不清楚,一些人还是认为 Sam68 可通过调控 RNA 的代谢参与细胞周期调控 . 利用基因打靶技术,在 DT40 细胞分离出 Sam68 基因缺失的细胞系 . 利用该细胞系,进行 Sam68 的功能解析 . 与野生型细胞系相比, Sam68 基因缺失细胞表现出明显的生长速度迟缓 . 通过细胞周期研究揭示 , 这些细胞生长速度延迟是由于细胞周期中的 G2/M 期延长 . 因为参与细胞周期 G2/M 期调控的周期因子 Cdc2 激酶的活性没有改变,所以提示 Sam68 不依赖于 Cdc2 激酶的活性参与细胞周期中 G2/M 期调控 .     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。     

Neutrophil CD16b crosslinking induces lipid raft-mediated activation of SHP-2 and affects cytokine expression and retarded neutrophil apoptosis

Two different types of FcRs for IgG are constitutively expressed on the surface of human neutrophils, namely, FcγRIIA (CD32a) and FcγRIIIB (CD16b). Unlike FcγRIIA, FcγRIIIb is GPI anchored to the cell membrane and its signal transduction is still ambiguous. To further understand the signal transduction of CD16b, we compared neutrophil cytokine expression and apoptosis by the cross-linking of CD32a and CD16b respectively. We found that both CD32a and CD16b crosslinking can activate neutrophils, but did not exactly share cytokine expression profiles. On the other hand, CD16b cross-linking retarded neutrophil apoptosis while CD32a promoted it. By interrupting the lipid raft with methyl-β-cyclodextrin (MβCD) and inhibiting the ITAM-SYK pathway with an SYK inhibitor (piceatannol), we found reduced apoptosis was at least partially mediated by lipid raft structure, but not the ITAM-SYK pathway. Additionally, CD16b but not CD32a cross-linking triggered SHP-2 phosphorylation and led to its translocation into lipid rafts. SHP-2 phosphorylation and translocation were inhibited by MβCD. Moreover, pre-inhibition of SHP-2 by a specific inhibitor (SHP099) converted IL-10 and SOCS3 expression level and promoted neutrophil apoptosis after CD16b crosslinking. In conclusion, these results, for the first time, collectively indicate that SHP-2 is activated by CD16b crosslinking in neutrophils and functions as a component of the raft-mediated signaling pathway.     

The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites

We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.     

Specificity in the recognition of crystals by antibodies

We show that IgG molecules isolated from the serum of rabbits injected with crystals of monosodium urate monohydrate, magnesium urate octahydrate and allopurinol, can each catalyze the nucleation of the same type of crystal to which they were exposed. These results generalize previous findings related to monosodium urate monohydrate and assess the idea that the invasion of a foreign crystal into an organism may amplify a population of antibodies which bear in their binding sites an imprint of the crystal surface structure. Such antibodies further act as nucleating templates which accelerate crystal formation in vitro. Antibodies from rabbitscan injected with sodium urate crystals do not cross-react or cross-react only to a low extent with antibodies isolated from rabbits injected with crystals of either magnesium urate or allopurinol. These results indicate a high specificity of the elicited antibodies, as these can distinguish between nuclei of crystals having similar molecular and structural characteristics.     

Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes

Introduction  

The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane.     

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.     

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.     

Ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) regulates neutrophil chemotaxis by hydrolyzing released ATP to adenosine

Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.     

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway.

Accumulating evidence suggests that enhanced peroxynitrite (ONOO-) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO- action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO- (0.1 to 200 5M) to isolated neutrophils resulted in a concentration-dependent down-regulation of L-selectin expression, and up-regulation of CD11b/CD18 expression. ONOO- stimulation of Erk activity was accompanied by activation of Ras, Raf-1 and MEK (mitogen-activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO- also evoked activation of neutrophil p38 MAPK. Neither ONOO--induced up-regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO- by itself had little effect on expression of ICAM-1 and E-selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide-activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO- were blocked by an anti-CD18 monoclonal antibody. These data suggest that ONOO- activates Erk in neutrophils via the Ras/Raf-1/MEK signal transduction pathway, leading to up-regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.     

How to regulate neutrophils in gout

Most research in gout has concentrated on the proinflammatory mechanisms to explain the inflammation that is generated when leucocytes are in contact with monosodium urate crystals. However, the episodic nature of gout and the absence of inflammation even when crystals are present suggest that there are natural counter-regulatory mechanisms to limit the inflammatory response. Gagné and colleagues showed that myeloid inhibitory C-type lectin, a C-type lectin inhibitory receptor expressed on neutrophils, modulates monosodium urate-induced neutrophil responses in vitro.Neutrophil recruitment and activation play a key role in the acute inflammatory response to monosodium urate (MSU) crystals. In acute gout, our current treatments such as nonsteroidal anti-inflammatory drugs, colchicine or corticosteroids all act on different steps of neutrophil activation. These drugs form part of the first treatment objective in gout – to relieve the painful symptoms of the acute attack – but do not address the second objective, which is to treat the underlying metabolic disorder hyperuricemia. Can neutrophil activation be manipulated or regulated? Are there signals that can be modulated and can this be of clinical relevance?The article by Gagné and colleagues provides evidence for an inhibitory pathway of neutrophil activation that acts through a recently described C-type lectin receptor called the myeloid inhibitory C-type lectin (MICL) [1]. This membrane receptor, also known as CLEC12A, inhibits neutrophil activation when it is engaged. C-type lectin receptors form a large family of proteins that have a common type of carbohydrate-binding domain that mediate cell adhesion and ligand binding in a calcium-dependent manner. Members of the C-type lectin receptors are known to participate in immune regulation, with well-known examples including Dectin-1 (CLE7A), DC-sign (CD209 or CLEC4L) and natural killer cell receptors (Ly49 or KLRA1). The MICL protein is encoded on chromosome 12p13, closely linked to the natural killer gene complex. MICL contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif and is expressed mainly on neutrophils and monocytes. Previous work has shown that the receptor could inhibit cellular activation [2]. The ligands that lead to MICL activation are currently unknown, as there is only a small body of data to show that the receptor interacts with ligands expressed in the bone marrow, thymus and kidney [3].In their studies, Gagné and colleagues showed that MSU crystals as well as a MICL-specific antibody downmodulated MICL expression on neutrophils. Reducing the expression of MICL by transfecting small interfering RNA or by antibody modulation of the receptor led to enhanced production of IL-8 when MSU was added to neutrophils, but no changes in IL-1β secretion were observed. The mechanisms of MICL signaling probably involve tyrosine phosphorylation as well as calcium flux, differing from previous results that showed MICL associated with the phosphatases SHP-1 and SHP-2 [2]. Finally, the addition of colchicine to neutrophils abrogated the negative effect of MSU on MICL expression.These results showed that reduced MICL expression is associated with augmented inflammatory responses from neutrophils, and a higher level of neutrophil MICL expression is associated with a reduced IL-8 production in vitro. As IL-8 is a major neutrophil chemoattractant, this can have important effects on neutrophil recruitment to an inflammatory site in gout. By extrapolation, if MICL expression or signaling could be enhanced or maintained during inflammation, the inhibitory signal may be reinforced and thereby downregulate inflammation. The effect of colchicine in this system is to elevate the expression of MICL, thereby increasing the inhibitory signaling mechanisms that counteract the inflammatory process.A number of caveats need to be mentioned in the interpretation of these results. The data presented were based on in vitro models of inflammation using MSU, and we need to see how this works in vivo before coming to any conclusions, as we have had examples where the in vivo results did not recapitulate the in vitro findings. They convincingly showed that reducing MICL expression on the surface of neutrophils enhanced the proinflammatory signature, but they did not show the converse – that enhanced MICL signaling can further downmodulate inflammation. Furthermore, the ligands that bind and activate MICL are unknown, so we have no idea what is the signal or how to reinforce or manipulate this signaling system. The results presented show that MSU had dual effects on neutrophils – the first is to downregulate MICL expression, and the second is to activate IL-8 production. How are these two mechanisms linked? If MSU acts mainly on the cell membrane internalization of MICL, what is the trigger for the IL-8 secretion? Notwithstanding these uncertainties, the finding that MICL modulates neutrophil activation in gout suggests that there are a number of counter-regulatory mechanisms in operation during an inflammatory process. Identifying these mechanisms may help us to understand the nature of gout as well as open up new therapeutic perspectives.     

p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K.

The 68 kDa Src substrate associated during mitosis is an RNA binding protein with Src homology 2 and 3 domain binding sites. A role for Src associated in mitosis 68 as an adaptor protein in signaling transduction has been proposed in different systems such as T-cell receptors. In the present work, we have sought to assess the possible role of Src associated in mitosis 68 in insulin receptor signaling. We performed in vivo studies in HTC-IR cells and in vitro studies using recombinant Src associated in mitosis 68, purified insulin receptor and fusion proteins containing either the N-terminal or the C-terminal Src homology 2 domain of p85 phosphatidylinositol-3-kinase. We have found that Src associated in mitosis 68 is a substrate of the insulin receptor both in vivo and in vitro. Moreover, tyrosine-phosphorylated Src associated in mitosis 68 was found to associate with p85 phosphatidylinositol-3-kinase in response to insulin, as assessed by co-immunoprecipitation studies. Therefore, Src associated in mitosis 68 may be part of the signaling complexes of insulin receptor along with p85. In vitro studies demonstrate that Src associated in mitosis 68 associates with the Src homology 2 domains of p85 after tyrosine phosphorylation by the activated insulin receptor. Moreover, tyr-phosphorylated Src associated in mitosis 68 binds with a higher affinity to the N-terminal Src homology 2 domain of p85 compared to the C-terminal Src homology 2 domain of p85, suggesting a preferential association of Src associated in mitosis 68 with the N-terminal Src homology 2 domain of p85. This association may be important for the link of the signaling with RNA metabolism.     

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.     

Characterization of Itk tyrosine kinase: contribution of noncatalytic domains to enzymatic activity.

Itk is a Tec family tyrosine kinase found in T cells that is activated upon ligation of the T cell receptor (TCR/CD3), CD2, or CD28. Itk contains five domains in addition to the catalytic domain: pleckstrin homology, Tec homology which contains a proline-rich region, Src homology 3, and Src homology 2. To provide a basis for understanding the contribution of these various domains to catalysis, recombinant Itk was purified and its substrate specificity determined by steady-state kinetic methods. Measurements of the rates of phosphorylation of various protein substrates, including Src associated in mitosis 68K protein (SAM68), CD28, linker for activation of T cells, and CD3 zeta, at a fixed concentration indicated that SAM68 was phosphorylated most rapidly. Wild-type Itk and three Itk mutants were characterized by comparing their activity (k(cat)) using the SAM68 substrate. A deletion mutant removing the pleckstrin homology domain and part of the Tec homology domain (Itk(Delta152)) had approximately 10-fold less activity than wild type, a mutant with an altered proline-rich domain (P158A,P159A) had a more dramatic 100-fold loss of activity, and the catalytic domain alone was essentially inactive. Itk(Delta152) had K(m) values for ATP and SAM68 nearly identical to those of the wild-type enzyme, while Itk(P158A,P159A) had approximately 3-fold higher K(m) values for each substrate. SAM68 phosphorylation by the wild-type and mutant enzymes in the presence of several tyrosine kinase inhibitors were compared using a homogeneous time-resolved fluorescence assay. Both the Itk(Delta152) deletion mutant and the Itk(P158A,P159A) mutant had IC(50) values similar to those of the wild-type enzyme for staurosporine, PP1, and damnacanthal. These comparisons, taken together with the similar K(m) values for ATP and SAM68 substrate between the wild-type and the mutant enzymes, indicate that the amino acids in the N-terminal 152 residues and proline-rich domains enhance catalysis by affecting turnover rate rather than substrate binding.     

Trikatu,a herbal compound that suppresses monosodium urate crystal‐induced inflammation in rats,an experimental model for acute gouty arthritis

Gout is an inflammatory joint disorder characterized by hyperuricaemia and precipitation of monosodium urate crystals in the joints. In the present study, we aimed to investigate the anti‐inflammatory effect of trikatu, a herbal compound in monosodium urate crystal‐induced inflammation in rats, an experimental model for acute gouty arthritis. Paw volume and levels/activities of lysosomal enzymes, lipid peroxidation, anti‐oxidant status and histopathological examination of ankle joints were determined in control and monosodium urate crystal‐induced rats. In addition, analgesic (acetic acid‐induced writhing response), anti‐pyretic (yeast‐induced pyrexia) and gastric ulceration effects were tested. The levels of lysosomal enzymes, lipid peroxidation and paw volume were significantly increased, and anti‐oxidant status was found to be reduced in monosodium urate crystal‐induced rats, whereas the biochemical changes were reverted to near normal levels upon trikatu (1000 mg/kg b.wt) administration. The trikatu has also been found to exhibit significant analgesic and anti‐pyretic effects with the absence of gastric damage. In conclusion, the present results clearly indicated that trikatu exert a potent anti‐inflammatory effect against monosodium urate crystal‐induced inflammation in rats in association with analgesic and anti‐pyretic effects in the absence of gastrointestinal damage. Copyright © 2013 John Wiley & Sons, Ltd.     

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Gout occurs in individuals with hyperuricemia when monosodium urate (MSU) crystals precipitate in tissues and induce acute inflammation via phagocytic cells such as monocytes. MSU crystals have been demonstrated in skin diseases such as tophaceous gout or psoriasis; however, the importance of MSU crystals in the skin is totally unknown. In this study, we found that MSU crystals, through P2Y(6) receptors, stimulated normal human keratinocytes (NHK) to produce IL-1α, IL-8/CXCL8, and IL-6. P2Y(6) receptor expression increased in MSU-stimulated NHK. Both P2Y(6)-specific antagonist and P2Y(6) antisense oligonucleotides significantly inhibited the production of IL-1α, IL-8/CXCL8, and IL-6 by NHK. Similarly, the P2Y(6)-specific antagonist completely inhibited the MSU-induced production of IL-1β by THP-1 cells, a human monocytic cell line. Remarkably, the P2Y(6)-specific antagonist significantly reduced neutrophil influx in both mouse air pouch and peritonitis models. Thus, these results indicate that the P2Y(6) receptor signaling pathway may be a potential therapeutic target for MSU-associated inflammatory diseases, such as tophaceous gout.