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Uric acid nephropathy (UAN) is caused by purine metabolism disorders. UAN rat models were established in SD rats. The modeling rats received different doses of hispidulin (10, 20, 50 mg/mL). Febuxostat was applied as the positive drug. Serum creatinine, uric acid (UA), and cystatin-C (cys-C), neutrophil gelatinase-associated lipocalin (NGAL), IL-1β, IL-8, TNF-α, and IL-6 in rats were detected. HE staining was done to assess kidney injury. UAN rats possessed prominent levels of serum creatinine, UA, cys-C, and NGAL, which all reduced after hispidulin treatment in a dose-dependent manner. HE staining determined the improvement of kidney injury after treatment, which was comparable to the efficacy of febuxostat. Hispidulin inhibited the release of IL-1β, IL-8, TNF-α, and IL-6 in UAN rats. Hispidulin enhanced autophagy in UAN rats, presenting as ascending LC3II/I ratio and downregulated P62. The increasing trend of inflammasome-related proteins of NLRP3 and Caspase-1 was changeovered by hispidulin. The activation of NF-kB signaling was intercepted by hispidulin in UAN rats. Hispidulin can effectively improve renal function injury caused by UAN in rats. The mechanism may be related to the inhibition of inflammatory response induced by autophagy and activation of NF-κB pathway., (© 2023 John Wiley & Sons Ltd.)

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are involved in the production and reabsorption of uric acid, respectively. However, the currently available individual XOR- or URAT1-targeted drugs have limited efficacy. Thus, strategies for combining XOR inhibitors with uricosuric drugs have been developed. Previous virtual screening identified Compounds 1-5 as hits for the potential dual inhibition of XOR/URAT1. Nevertheless, in vitro experiments yielded unsatisfactory results. The first round of optimization work on those hits was performed, and two series of compounds were designed and synthesized. Compounds of the A series exerted moderate inhibitory effects on URAT1, but extremely weak inhibitory effects on XOR. Compounds of the B series exerted strong inhibitory effects on both XOR and URAT1. B 5 exhibited the greatest inhibitory activity, with similar inhibitory effects on XOR and URAT1. The half maximal inhibitory concentration (IC 50 ) of XOR was 0.012 ± 0.001 μM, equivalent to that of febuxostat (IC 50  = 0.010 ± 0.001 μM). The IC 50 of URAT1 was 30.24 ± 3.46 μM, equivalent to that of benzbromarone (IC 50  = 24.89 ± 7.53 μM). Through this optimization, the in vitro activity of most compounds of the A and B series against XOR and URAT1 was significantly improved versus that of the hits. Compound B 5 should be further investigated., (© 2023 John Wiley & Sons Ltd.)

Uric acid crystal is known to activate the NLRP3 inflammasome and to cause tissue damages, which can result in many diseases, such as gout, chronic renal injury and myocardial damage. Meanwhile, soluble uric acid (sUA), before forming crystals, is also related to these diseases. This study was carried out to investigate whether sUA could also activate NLRP3 inflammasome in cardiomyocytes and to analyse the mechanisms. The cardiomyocyte activity was monitored, along with the levels of mature IL-1β and caspase-1 from H9c2 cells following sUA stimulus. We found that sUA was able to activate NLRP3 inflammasome, which was responsible for H9c2 cell apoptosis induced by sUA. By elevating TLR6 levels and then activating NF-κB/p65 signal pathway, sUA promoted NLRP3, pro-caspase 1 and pro-IL-1β production and provided the first signal of NLRP3 inflammasome activation. Meanwhile, ROS production regulated by UCP2 levels also contributed to NLRP3 inflammasome assembly and subsequent caspase 1 activation and mature IL-1β secretion. In addition, the tlr6 knockdown rats suffering from hyperuricemia showed the lower level of IL-1β and an ameliorative cardiac function. These findings suggest that sUA activates NLRP3 inflammasome in cardiomyocytes and they may provide one therapeutic strategy for myocardial damage induced by sUA., (2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Toll-like receptors (TLRs) are expressed in the skin and airway epithelial tissues, which are the most important sites of host-pathogen interactions. TLRs recognize the 3-D structures of pathogen-associated molecules and are therefore useful markers of the innate immune response. Here, we investigated the role of lipopolysaccharides and monosodium urate (MSU) crystals in the activation of the TLR and NOD-like receptor (NLR) pathways in human keratinocytes. Analysis of the inflammasome compounds revealed that NOD-like receptor P3 and TLR4, both of which are components of inflammasome complexes involved in the activation of interleukin (IL)-1β, were not expressed in keratinocytes. Transcriptomic analysis showed that the combination of MSU and lipopolysaccharide priming did not elicit significant results compared to MSU treatment, which induced the expression of TLR2, IL-6 and IL-8/chemokine (C-X-C motif) ligand 8 CXCL8 in the keratinocyte cell line HaCaT. Furthermore, MSU promoted the phosphorylation of extracellular signal-regulated kinase 1/2 and MAPK14/p38α mitogen-activated protein kinases. We concluded that MSU stimulates a pro-inflammatory response in keratinocytes via mitogen-activated protein kinase pathway to induce production of IL-8/CXCL8 chemokine (C-X-C motif) ligand 8 and TLR2., (© 2017 John Wiley & Sons Australia, Ltd.)

Interleukin (IL)-1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL-1β is dependent upon caspase-1-containing multiprotein complexes called inflammasomes and IL-1R1/MyD88/NF-κB pathway. In this study, we explored whether a potential anti-fibrotic agent fluorofenidone (FD) exerts its anti-inflammatory and anti-fibrotic effects through suppressing activation of NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and the IL-1β/IL-1R1/MyD88/NF-κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α-smooth muscle actin (α-SMA), fibronectin, collagen I, caspase-1, IL-1R1, MyD88 were measured by Western blot and/or RT-PCR. The human actue monocytic leukaemia cell line (THP-1) were incubated with monosodium urate (MSU), with or without FD pre-treatment. The expression of caspase-1, IL-1β, NALP3, apoptosis-associated speck-like protein containing (ASC) and pro-caspase-1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome-associated molecules were measured by Co-immunoprecipitation. RLE-6TN (rat lung epithelial-T-antigen negative) cells were incubated with IL-1β, with or without FD pre-treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), myeloperoxidase (MPO), α-SMA, fibronectin, collagen I, caspase-1, IL-1R1 and MyD88 in mice lung tissues. And FD inhibited MSU-induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome-associated molecules, decreased the level of caspase-1 and IL-1β in THP-1 cells. Besides, FD inhibited IL-1β-induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM-induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL-1β/IL-1R1/MyD88/ NF-κB pathway., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objective: Gout is a common cause of inflammatory arthritis and is provoked by the accumulation of monosodium urate (MSU) crystals. However, the underlying mechanisms of the pain associated with acute attacks of gout are poorly understood. The aim of this study was to evaluate the role of transient receptor potential ankyrin 1 (TRPA-1) and TRPA-1 stimulants, such as H2 O2 , in a rodent model of MSU-induced inflammation., Methods: MSU or H2 O2 was injected into the hind paws of rodents or applied in cultured sensory neurons, and the intracellular calcium response was measured in vitro. Inflammatory or nociceptive responses in vivo were evaluated using pharmacologic, genetic, or biochemical tools and methods., Results: TRPA-1 antagonism, TRPA-1 gene deletion, or pretreatment of peptidergic TRP-expressing primary sensory neurons with capsaicin markedly decreased MSU-induced nociception and edema. In addition to these neurogenic effects, MSU increased H2 O2 levels in the injected tissue, an effect that was abolished by the H2 O2 -detoxifying enzyme catalase. H2 O2 , but not MSU, directly stimulated sensory neurons through the activation of TRPA-1. The nociceptive responses evoked by MSU or H2 O2 injection were attenuated by the reducing agent dithiothreitol. In addition, MSU injection increased the expression of TRPA-1 and TRP vanilloid channel 1 (TRPV-1) and also enhanced cellular infiltration and interleukin-1β levels, and these effects were blocked by TRPA-1 antagonism., Conclusion: Our results suggest that MSU injection increases tissue H2 O2 , thereby stimulating TRPA-1 on sensory nerve endings to produce inflammation and nociception. TRPV-1, by a previously unknown mechanism, also contributes to these responses., (Copyright © 2013 by the American College of Rheumatology.)

Objective: Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout., Methods: Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively., Results: Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1β. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome., Conclusion: These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo., (Copyright © 2012 by the American College of Rheumatology.)

Problem: Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting that they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod-like receptor, Nalp3, leading to inflammasome activation and IL-1β processing. Because preeclampsia is associated with placental immune⁄ inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation., Method of Study: Isolated first- and third-trimester trophoblasts were assessed for expression of the inflammasome components, Nalp1, Nalp3, and ASC. First-trimester trophoblast cells were incubated with or without MSU, and after which, IL-1β secretion and processing and caspase-1 activation were determined., Results: Trophoblast cells expressed Nalp1, Nalp3, and ASC under basal conditions. Following incubation with MSU, first-trimester trophoblast IL-1β secretion was upregulated. This correlated with increased expression levels of active IL-1β and active caspase-1. ASC knockdown reduced MSU-induced IL-1β secretion., Conclusion: These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL-1β production. This may provide a novel mechanism for the induction of inflammation at the maternal–fetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia.

Objective: To profile monosodium urate monohydrate (MSU) crystal-recruited monocyte inflammatory function during the course of in vivo differentiation, in a murine model of peritoneal MSU crystal-induced inflammation., Methods: C57BL/6J mice were injected intraperitoneally with MSU crystals, and the peritoneal cells were harvested at different time points. The MSU crystal-recruited monocyte/macrophage population was analyzed for the expression of differentiation and activation markers, cytokine production following MSU crystal restimulation ex vivo and in vivo, expression of NLRP3-associated proteins (ASC, caspase 1) and pro-interleukin-1β (proIL-1β), and phagocytic capacity., Results: Monocytes recruited 8 hours after MSU crystal stimulation (F4/80(low) Gr-1(int) 7/4+) exhibited poor phagocytic capacity, expressed low levels of proIL-1β, and failed to produce proinflammatory cytokines in response to MSU crystal restimulation. In the absence of MSU crystal restimulation, differentiating monocytes produced low levels of transforming growth factor β1 ex vivo, and this was abrogated following MSU crystal restimulation. Over time these cells developed a proinflammatory phenotype in vivo, characterized by the production of IL-1β, tumor necrosis factor α, IL-6, CCL2 (monocyte chemotactic protein 1), and CXCL1 (cytokine-induced neutrophil chemoattractant) following ex vivo MSU crystal restimulation, and leading to IL-1β production and cell infiltration following MSU crystal rechallenge in vivo. Proinflammatory function was associated with differentiation toward a macrophage phenotype (F4/80(high) Gr-1-7/4-), an increase in phagocytic capacity, and an increase in the expression of proIL-1β., Conclusion: MSU crystal-recruited monocytes differentiate into proinflammatory M1-like macrophages in vivo. This proinflammatory macrophage phenotype is likely to play a key role in perpetuating inflammation in gouty arthritis in the presence of ongoing deposition of fresh MSU crystals., (Copyright © 2011 by the American College of Rheumatology.)

4-Hydroxyphenylacetic acid (HPA) and nitrite are present in human mixed whole saliva, and HPA can be nitrated by peroxidase/hydrogen peroxide (H(2)O(2))/nitrite systems in the oral cavity. Thus, the objectives of the present study were to estimate the concentrations of HPA, nitrated HPA [4-hydroxy-3-nitrophenylacetic acid (NO(2)HPA)], nitrite, and thiocyanate (SCN(-)) in saliva from 73 patients with periodontal diseases and to elucidate the conditions necessary to induce nitration of HPA. High concentrations of HPA, nitrite, and SCN(-) were found in the saliva of patients older than 50 yr of age. NO(2)HPA was detected in seven patients who were older than 60 yr of age. Nitrite-dependent formation of NO(2)HPA by a bacterial fraction prepared from mixed whole saliva was faster at pH 5.3 than at pH 7, and increased as the rate of H(2)O(2) formation increased. The formation of NO(2)HPA was inhibited by SCN(-) and by salivary antioxidants such as uric acid, ascorbic acid, and glutathione. These results suggest that nitration can proceed at an acidic site in the oral cavity where H(2)O(2) is produced under conditions of decreased concentrations of SCN(-) and of antioxidants.

Objective: To determine whether infiltrating monocytes, neutrophils, or resident macrophages contribute to the early inflammatory response to monosodium urate monohydrate (MSU) crystals in vivo., Methods: MSU crystal-induced inflammation was monitored using a peritoneal model of acute gout. The production of proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], IL-6) by resident macrophages, infiltrating monocytes, and neutrophils during the onset of gout was determined by flow cytometry. Infiltrating and resident peritoneal cells were cultured with MSU crystals ex vivo, and proinflammatory cytokine production was determined by multiplex cytokine array. Activated macrophages on the visceral epithelial lining of the peritoneum were identified by immunofluorescence histochemistry. The inflammatory immune response to MSU crystals was then compared with the inflammatory response in mice depleted of resident macrophages by pretreatment with clodronate liposomes., Results: The production of cytokines in vivo preceded the influx of Gr-1(intermediate)7/4+ monocytes. Monocytes and neutrophils recruited during the inflammatory phase of the response to MSU crystals failed to produce proinflammatory cytokines either in vivo, or ex vivo following restimulation with MSU crystals. Stimulation of the naive peritoneal resident cell population with MSU crystals ex vivo resulted in positive staining of resident macrophages for the proinflammatory cytokines IL-1beta, TNFalpha, and IL-6. Depletion of the resident macrophage population resulted in a significant decrease in both MSU crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo despite the presence of infiltrating monocytes., Conclusion: These data indicate that resident macrophages, rather than infiltrating monocytes or neutrophils, are important for initiating and driving the early proinflammatory phase of acute gout.

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 murine 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 enzyme-linked 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-1beta 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 crystal-induced 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.

Objective: It has previously been shown that as monocytes differentiate into macrophages, they lose the ability to secrete proinflammatory cytokines in response to monosodium urate monohydrate (MSU) crystals. The purpose of this study was to investigate whether MSU crystals induce macrophages to secrete antiinflammatory factor instead., Methods: Human monocyte or macrophage isolates were prepared from samples obtained from healthy volunteer donors either by differentiation of blood monocytes in vitro or by collecting cells from skin blisters during the early or late phase of the dermal inflammatory response to cantharidin. Monocyte or macrophage isolates were then incubated with MSU crystals for 24 hours, and culture supernatants were assayed for candidate antiinflammatory mediators (by enzyme-linked immunosorbent assay) and for the capacity to activate or suppress endothelial cell E-selectin expression and secondary neutrophil recruitment under shear flow., Results: Analysis of supernatants from in vitro-differentiated macrophages revealed that transforming growth factor beta1 (TGFbeta1) was induced following MSU crystal stimulation (mean +/- SEM 1.50 +/- 0.24 ng/ml/10(6) cells), but there was no evidence of interleukin-10 (IL-10), IL-1 receptor antagonist, or tumor necrosis factor (TNF) receptor p55 release. Macrophage TGFbeta1 significantly suppressed endothelial cell E-selectin expression and secondary neutrophil capture on endothelial monolayers stimulated with supernatants from MSU-treated monocytes. Leukocytes isolated from resolving (40-hour) skin blisters similarly elaborated TGFbeta1 when challenged with MSU crystals (0.66 +/- 1.3 ng/ml/10(5) CD14+ cells). In contrast, cells isolated from acute (16-hour) skin blisters secreted TNFalpha (0.49 +/- 0.08 ng/ml/10(5) CD14+ cells) but no detectable TGFbeta1., Conclusion: These data provide further support for the concept that differentiated macrophages play a protective role in the pathophysiology of gout, and they identify macrophage TGFbeta1 as a mediator of paracrine suppression during the resolution phase of inflammation.

Nitric oxide (NO) is generated in central synapses on activation of N-methyl-D-aspartate (NMDA) receptors and exerts physiological effects by changing cGMP levels. NO has frequently also been claimed to engage a different mechanism, namely the covalent modification of thiol residues (S-nitrosation), and thereby exert a negative feedback on NMDA receptors. Tests of this hypothesis were conducted by recording NMDA receptor-mediated synaptic potentials in the CA1 area of rat hippocampal slices. Manipulations designed to increase or decrease endogenous NO levels had no effect. Addition of exogenous NO using a NONOate donor in concentrations up to 30-fold higher than those needed to evoke maximal cGMP accumulation also had no effect. Nevertheless, in agreement with previous findings, photolysis of a caged NO derivative with UV light led to an enduring block of synaptic NMDA receptors. To address these contradictory results, NMDA receptor-mediated currents were recorded from HEK-293 cells transfected with NR1 and NR2A subunits. As found in slices, photolysis of caged NO inhibited the currents whereas perfusion of NO (up to 5 microM) was ineffective. However, when NO was supplied at a concentration found to be effective when released photolytically (5 microM) and the cells simultaneously exposed to the UV light used for photolysis, NMDA receptor-mediated currents were inhibited. This effect was not observed at more physiological NO concentrations (10 nM range). The results indicate that neither endogenous NO nor exogenous NO in supra-physiological concentration inhibits synaptic NMDA receptors; the combination of high NO concentration and UV light can give an artifactual result.

Objective: To examine the role of chemokines, S100A8, and S100A9 in neutrophil accumulation induced by the causative agent of gout, monosodium urate monohydrate (MSU) crystals., Methods: MSU crystal-induced neutrophil migration was studied in the murine air-pouch model. Release of chemokines, S100A8, S100A9, and S100A8/A9 in response to MSU crystals was quantified by enzyme-linked immunosorbent assays. Recruited cells were counted following acetic blue staining, and the subpopulations were characterized by Wright-Giemsa staining of cytospins., Results: MSU crystals induced the accumulation of neutrophils following injection in the air pouch, which correlated with the release of the chemokines CXCL1, CXCL2, CCL2, and CCL3. However, none of these was found to play an important role in neutrophil migration induced by MSU crystals by passive immunization with antibodies directed against each chemokine. S100A8, S100A9, and S100A8/A9 were also found at high levels in the pouch exudates following injection of MSU crystals. In addition, injection of S100A8, S100A9, or S100A8/A9 led to the accumulation of neutrophils in the murine air pouch, demonstrating their proinflammatory activities in vivo. Passive immunization with anti-S100A8 and anti-S100A9 led to a total inhibition of the accumulation of neutrophils. Finally, S100A8/A9 was found at high concentrations in the synovial fluid of patients with gout., Conclusion: S100A8 and S100A8/A9 are essential to neutrophil migration induced by MSU crystals. These results suggest that they might be involved in the pathogenesis of gout.

Objective: To evaluate the impact of monosodium urate monohydrate (MSUM) crystals on the synthesis of prostaglandin E(2) (PGE(2)) by human neutrophils, and to examine some of the mechanisms underlying these responses., Methods: The amount of PGE(2) released in the supernatants of stimulated human neutrophils was evaluated by enzyme immunoassay, and expression of cyclooxygenase 2 (COX-2) was monitored by immunoblot on cell lysates, as well as by cytofluorometry of buffy-coat cells., Results: We observed that MSUM crystals rapidly stimulated the synthesis of PGE(2), with levels peaking at 1 hour. This response was decreased by NS-398, a specific inhibitor of COX-2. We also detected a constitutive expression of COX-2 in unstimulated and unprimed neutrophils. This rapid COX-2-dependent PGE(2) accumulation was independent of translation and transcription. We also observed that piceatannol, but not colchicine, blocked the synthesis of PGE(2) stimulated by MSUM crystals., Conclusion: These results show that the interaction of MSUM crystals with human neutrophils stimulates a significant synthesis of PGE(2) mediated by constitutively expressed COX-2. The results of this study emphasize the potential importance of the neutrophil as a source of PGE(2), which may modulate, positively or negatively, the inflammatory response.

Objective: Peroxisome proliferator-activated receptor gamma (PPAR gamma) 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 PPAR gamma in self-limiting episodes of acute gouty arthritis. To do this, we investigated PPAR gamma expression by monosodium urate monohydrate (MSU) crystal-stimulated monocytes, and we studied the effects of PPAR gamma ligands on crystal-induced acute inflammation., Methods: PPAR gamma 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 PPAR gamma 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 PPAR gamma expression by monocytes. Gene expression was detected as early as 2 hours, and maximum expression was observed at 4 hours after stimulation. The induced PPAR gamma was functional, since a PPAR gamma ligand was able to up-regulate CD36 expression on monocytes. A natural ligand of PPAR gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15deoxy-PGJ(2)), 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-PGJ(2) significantly prevented cellular accumulation in a mouse air-pouch model of MSU crystal-induced acute inflammation., Conclusion: Rapid induction of PPAR gamma expression on monocytes by MSU crystals may contribute, at least in part, to the spontaneous resolution of acute attacks of gout.

Objective: To investigate the role of interleukin-10 (IL-10) in the inflammatory response, the antiinflammatory effect of retrovirally transfected IL-10 was evaluated both in vitro and in vivo., Methods: A recombinant retrovirus containing the murine IL-10 gene was constructed using the pLXSN vector and was designated as LXSN-IL-10. Murine IL-10 was introduced into embryonic C57BL/6J fibroblast cells using LXSN-IL-10 to create C57-IL-10 cells. The effect of IL-10 in the culture supernatant of these cells was then evaluated by determining changes in the production of tumor necrosis factor alpha (TNFalpha), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta by macrophages. The antiinflammatory effect of C57-IL-10 cells was also investigated using an in vivo model of monosodium urate monohydrate (MSU) crystal-induced acute inflammation., Results: The IL-10 gene transcript and its product were detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The level of IL-10 in the culture supernatant of C57-IL-10 cells was estimated to be 50 ng/ml. The culture supernatant of these cells exerted the biologic activity of IL-10, showing inhibition of TNFalpha, MIP-1alpha, and MIP-1beta production by macrophages. Injection of C57-IL-10 cells into murine air pouches significantly inhibited MSU crystal-induced cellular infiltration (P < 0.01) and production of the mouse CXC chemokine KC (P < 0.05). These findings were consistent with the results obtained by the injection of recombinant human IL-10 into air pouches., Conclusion: In this murine air pouch model of MSU crystal-induced inflammation, IL-10 seemed to inhibit the recruitment of neutrophils at least partly by suppressing KC production. These findings seem to suggest that IL-10 gene therapy may be useful for inflammatory diseases.

Objective: To quantify the inflammatory cell response in rat air pouch pseudosynovial membrane during monosodium urate monohydrate (MSU) crystal-induced inflammation., Methods: In the rat air-pouch model, we used a computer-assisted histomorphometric method to quantify cell distributions, based on cell linear densities, in histologic sections of membranes from pouches injected with MSU or saline. The volume, white blood cell (WBC) count, and histamine content of the pouch exudates were determined at several time points., Results: Injection of 10 mg of MSU crystals into the pouch produced an acute exudate. After peaking at 24 hours, the exudate volume and WBC count decreased spontaneously over the next 3 days, simulating the self-limited course of acute gout. Membrane thickness followed a parallel course. Membrane polymorphonuclear cell (PMN) linear densities were closely correlated with exudate WBC counts, suggesting PMN recruitment from the subintimal synovial membrane. Both monocyte/macrophage and mast cell linear densities increased in the subintimal layer 2 hours after crystal injection (P = 0.038 and P = 0.03, respectively, versus controls), whereas PMN linear densities showed 2 peaks, one at 4 hours and the other 24 hours. The exudate histamine content peaked 6 hours after crystal injection, when mast cell linear densities were minimal in the membranes, suggesting mast cell degranulation., Conclusion: An increase in monocyte/macrophage and mast cell densities in the membrane preceded the PMN influx in the pouch membrane and exudate, suggesting that mast cells may be involved in the early phase of MSU crystal-induced inflammation, at least in this rat model.

Objective: The objective of this study was to determine if nitric oxide (NO)-mediated cutaneous vasodilation depends on urate concentrations., Methods: Capillary blood cell velocities (BCV) in the tail capillary vessels of anesthetized, heat-stressed Sprague-Dawley rats were measured by the videomicroscopy method, and the femoral mean arterial pressure (MAP) was recorded on a Grass polygraph. The effects of urate or urate and NO production on the above parameters were determined by using specific inhibitors of these compounds, for example, allopurinol (All) or All or N-nitro-L-arginine (NNA), respectively. Urate concentrations were determined by an enzymatic, colorimetric method, and total nitrite levels were measured fluorometrically., Results: During heat stress, All or All and NNA significantly decreased plasma urate or urate and total nitrite concentrations, respectively, and the response of vascular conductance (estimated as a ratio of BCV to MAP). Urate administration, after All and NNA pretreatment, restored plasma urate and total nitrite concentrations and increased BCV/MAP., Conclusions: Urate at physiological concentration helps to maintain vascular conductance of cutaneous capillaries in the rat tail during body heating. The effect of urate on vascular conductance may be mediated by NO.

Objective: Monosodium urate monohydrate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals cause acute gout and pseudogout, respectively. Because acute gout and pseudogout appear to be dependent on interleukin-8 (IL-8)-induced neutrophil ingress, this study was undertaken to define and compare how MSU and CPPD crystals stimulate IL-8 messenger RNA (mRNA) expression in mononuclear phagocytes., Methods: MSU and CPPD crystal-induced mitogen-activated protein kinase (MAPK) signal transduction and IL-8 transcriptional activation were studied in human monocytic cells, using the THP-1 cell line., Results: MSU and CPPD crystals (0.5 mg/ml) induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase 1 (ERK-1)/ERK-2, and p38 MAPK pathways in THP-1 cells. Activation of the ERK-1/ERK-2 pathway was essential for MSU and CPPD crystal-induced IL-8 mRNA expression, whereas the p38 pathway played a greater role in IL-8 mRNA expression in response to CPPD crystals in comparison with MSU crystals. Both crystals induced the binding of nuclear factor kappaB (NF-kappaB), including the NF-kappaB complex c-Rel/RelA, and activator protein 1 (AP-1, including N-terminal phosphorylated c-Jun) to the IL-8 promoter. Both crystals induced transcriptional activation of the IL-8 promoter, which was dependent on activation of c-Rel/RelA and AP-1. Activation of the NF-IL-6 transcription factor played a lesser role. Finally, crystal-induced IL-8 promoter activation was mediated by activation of the ERK-1/ERK-2 pathway, as demonstrated by transfection of dominant-negative raf-1., Conclusion: These results indicate that ERK-1/ ERK-2 signaling and transcriptional activation through AP-1 and NF-kappaB are essential for the induction of IL-8 expression in mononuclear phagocytes in response to CPPD and MSU crystals.

1. Dideoxyinosine (ddI) has recently been approved for the treatment of patients with HIV infection. As increasing numbers of such patients are pregnant, we wished to define the rate and mechanism(s) of ddI transfer by the placenta to the foetus. Using isolated single perfused human term placental cotyledons, the drug was shown to cross the placenta from mother to foetus at a rate of 25% that of a freely diffusible marker, antipyrine, and at about half the rate of zidovudine (AZT). The transfer of ddI was similar in both directions (maternal to foetal and the reverse), equal to that of L-glucose, a passively transported sugar, and was not inhibited by excess inosine or uric acid (structural analogues of ddI). ddI did not cross to the foetus against a concentration gradient. The transport process appeared to be passive and it was not altered by AZT. 2. ddI was not metabolized in the Krebs Ringer buffer/albumin perfusate, and placental homogenates converted only 4% of ddI to hypoxanthine over the 4 h incubation. However, when maternal term or cord blood was incubated with ddI for 3 h, 50% of the drug was converted to hypoxanthine in maternal blood and to hypoxanthine and uric acid in cord blood. 3. Thus, ddI metabolism in maternal blood should decrease its net transfer to the foetus in vivo. In the foetal circulation, ddI will be further metabolized by erythrocytes to hypoxanthine and possibly to uric acid. Hence, the fraction of administered ddI delivered to foetal tissues should be much lower than that of AZT.

Objective: To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD)., Methods: Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl-phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1-0-3H-alkyl-2-acetyl-sn-glycero-3-phosphocholine. The cells were stimulated with microcrystals of triclinic morphology., Results: Both MSU and CPPD crystals induced a time- and concentration-dependent accumulation of phosphatidic acid mass and elevation in levels of alkyl-phosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal-stimulated production of superoxide anions., Conclusion: These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil-microcrystal interactions may be dependent on this biochemical pathway.

The effects of monosodium urate and calcium pyrophosphate dihydrate crystals on the levels of cytoplasmic free calcium and on the oxidative burst in normal human blood neutrophils were examined. The pattern of sensitivity to granulocyte-macrophage colony-stimulating factor, colchicine, cytochalasin B, pertussis toxin, diglyceride kinase, and protein kinase C inhibitors differentiated the mechanism(s) of neutrophil activation by the crystals from that involved in the responses to soluble chemotactic factors and indicated that individual crystals can use several activation pathways.

Monosodium urate crystals (MSU) stimulate suspensions of washed platelets or neutrophils. When MSU crystals are coated with IgG, as occurs in plasma, stimulation is markedly enhanced. These studies which use MSU-induced human platelet serotonin secretion as a model examine the nature of cellular recognition mechanisms for MSU crystals and IgG-coated MSU crystals. F(ab')2 fragments of specific anti-Fc antibody blocked and the lipopolysaccharide of Salmonella minnesota R595 enhanced human platelet secretion induced by IgG-coated urate crystals. These agents had little effect on stimulation by uncoated crystals. This indicated that urate crystals stimulate platelets independently of fluid phase IgG. Urate crystals directly stimulated suspensions of washed rabbit platelets which lack Fc receptors. In contrast to human cells, stimulation was blocked by IgG. This again demonstrated IgG-independent cell stimulation by urate crystals. Calcium pyrophosphate dihyrate crystals could trigger human platelet secretion only when coated with IgG. This suggests that when crystals are coated with IgG, the surface-bound IgG alone may be the stimulus to the cell. This was supported by the finding that polyvinylpyridine-N-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated, but not IgG-coated, urate crystals. These data indicate that urate crystals (and potentially other surface or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of adsorbed IgG or, when coated with IgG, by triggering the cell via immunoglobulin receptors.

We studied the capacities of naked and protein-coated monosodium urate (MSU) crystals to stimulate superoxide anion (O(2)) release by human polymorphonuclear leukocytes (PMN). Uncoated MSU estimated significant O(2) production by cytochalasin B-treated PMN. Precoating MSU with IgG caused an increase in mean O(2) production, whereas precoating heated MSU with serum or plasma inhibited O(2) release. Unheated MSU crystals, which activate complement to a greater extent than heated crystals, also provoked O(2) generation, an effect again abrogated by precoating with serum but not with plasma. Coincubation of unheated MSU and plasma resulted in an enhancement of O(2) generation. The results of these experiments support the hypothesis that adsorbed proteins modulate the phlogistic potential of MSU and that the surface activation of humoral mediators contributes to the local inflammatory response.

Monosodium urate monohydrate (MSUM) is a potent activator of the complement system as measured by electrophoretic conversion of beta1C to beta1A. Activation of C3 in human serum by MSUM is both time- and dose-dependent. The sensitivity of the assay allows detection of C3 activation by as little as 0.2 mg/ml of MSUM. It was observed that C3 activation is calcium dependent and elimination by both EDTA and EGTA, a finding that demonstrated the major role of the classic pathway of complement activation. Excess calcium or magnesium alone inhibited C3 activation by MSUM in accord with the inhibitory effect of these cations on sensitized sheep cell hemolysis by complement. Heating of MSUM at 200 degrees C for 2 hours removes the water of crystallization such that heated crystals may no longer be considered MSUM. Such treatment has a variable effect on C3 activation. Of the crystals and other material studied, only zymosan was more potent than MSUM in activating C3. Calcium prophosphate dihydrate and hydroxyapatite activated significant amounts of C3. Asbestos, glass wool, or a variety of microcrystalline steroids activated little or no C3.

We found previously that crystals of sodium urate and silicon dioxide (silica) can stimulate the production of endogenous pyrogen (EP), now called interleukin-1 (IL-1), the polypeptide mediator of fever and other aspects of inflammation. We have confirmed and extended the work with urate crystals and have examined 2 other crystals associated with joint problems, hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD). The crystals were added to suspensions of human blood leukocytes (2.5 X 10(6) monocytes/dose, with 10% fresh autologous plasma); after 18 hours of incubation, the EP content of the supernatants was assayed in the rabbit pyrogen test. HA and CPPD crystals neither induced EP production nor reduced the amount of staphylococci-induced EP. Presized (10 - 40 micron) urate crystals were pyrogenic, but less so than the unsized and aggregated urate crystals investigated previously and reexamined here. On ultrasonication, the aggregated urate crystals became first more pyrogenic and then less so as the crystals were dispersed and broken down. Ultrasound did not impart pyrogenicity to HA or CPPD crystals: their failure to stimulate EP/IL-1 production from leukocytes in vitro indicates a difference in their phlogistic properties, compared with crystals of urate or silica. The results with urate crystals have pathogenetic implications in a number of areas of gouty inflammation: initiation of the acute attack, other aspects of the acute-phase response, polyarticular involvement, and the inflammatory consequences of chronic stimulation by tophaceous material.

Exposure to monosodium urate crystals (MSU) stimulated the respiratory burst of human neutrophils as measured by increased O2 consumption and the generation of superoxide radicals (O(2)). From the comparison of data derived from nitroblue tetrazolium and cytochrome C reduction (two methods of detecting O(2) release), it appears tht O(2) production in response to MSU may be compartmentalized, i.e., occur predominantly in the intracellular space. After exposure to MSU, neutrophils from patients with chronic granulomatous disease lost viability at the normal rate; thus products of the respiratory burst are not likely to be responsible for cell death.

Synthetic hydroxyapatite (HA) crystals in 1% serum stimulated 3H thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures, as did 10% serum. The onset of stimulation and peak uptake of thymidine after crystal addition were delayed by 2-3 hours as compared with the effects produced by 10% serum. Stimulation of 3H thymidine uptake was proportional to the serum concentration used. HA crystals (50 micrograms/ml) stimulated nuclide uptake at each serum concentration used. 3H thymidine uptake was also proportional to the dose of HA or calcium pyrophosphate dihydrate crystals, although larger doses of the latter crystal were required to produce equivalent effects. Not all particulates were effective mitogenic agents. Latex beads and diamond crystals had no effect. Monosodium urate crystals modestly stimulated and calcium urate crystals markedly stimulated nuclide uptake. The more complex crystals found in a naturally occurring condition (calcinosis) were as mitogenic as the pure synthetic HA. The synovial cell hyperplasia sometimes associated with crystals might be explained in part by their mitogenic activity.

The generation of polymorphonuclear cell (PMN) superoxide ion (O2-) by monosodium urate (MSU) crystals may be important in the pathogenesis of acute gout. Coating MSU crystals with IgG prior to exposure to PMN markedly augmented O2- generation. This augmentation was inhibited by supernates, termed cell lysate, derived from sonicated PMN or PMN exposed to MSU crystals for 5 hours at 37 degrees C. Lysate was effective in inhibiting O2- production when incubated with MSU crystals prior to, during, or after MSU crystals were exposed to IgG. No IgG could be eluted from crystals exposed to both lysate and IgG. Immunoelectron microscopy showed virtually no IgG on crystal surfaces after incubation of crystals with lysate and IgG. These data suggest that products of PMN injury can modulate further PMN responses to MSU crystals. This phenomenon provides a negative feedback loop and is one possible mechanism for the self-limitation of acute gouty attacks.

Crystal-related joint diseases are often associated with systemic inflammatory manifestations, including increased levels of acute-phase proteins, leukocytosis, and fever. Recently, interleukin-6 (IL-6) has been identified as a pluripotent mediator of inflammatory and immunologic responses and the major hepatocyte-stimulating factor. In this study, we demonstrated that monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, and to a lesser extent, hydroxyapatite crystals, increased IL-6 production by synoviocytes and monocytes in vitro. Immunoprecipitation experiments showed that MSU and CPPD crystals, but not hydroxyapatite crystals, were able to increase the release of newly synthesized IL-6. Crystal-induced IL-6 stimulated acute-phase protein synthesis, immunoglobulin production, and hybridoma cell proliferation, which was neutralized by a specific antibody to IL-6. High levels of IL-6 were found in synovial fluid from patients with gout and pseudogout. These results demonstrate that MSU and CPPD crystals can induce IL-6 production in synoviocytes and monocytes, and that synovial fluid from patients with gout and pseudogout contains high levels of IL-6. Crystal-induced IL-6 is likely to be an important mediator of inflammatory responses in acute gout and pseudogout.

To test whether urate crystal-membrane protein interactions mediate platelet stimulation, platelet membrane proteins were radiolabeled by lactoperoxidase, extracted with 1% Triton X-100, and incubated with urate crystals. The crystal-associated and supernate fractions were isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by 2-dimensional isoelectric focusing followed by SDS-PAGE. Four of the lactoperoxidase-radiolabeled polypeptides that associated with urate crystals had reduced molecular weights and pIs of Mr = 105,000, pI 4.9; Mr = 123,000, pI = 4.9; Mr = 123,000, pI = 5.3; and Mr = 132,000, pI = 4.8-6.3, respectively. These proteins were characterized with regard to their labeling intensities, apparent isoelectric points, apparent molecular weights (reduced and nonreduced), lectin-binding properties, carbohydrate- and protein-staining characteristic, and presence or absence in Glanzmann's thrombasthenia. They have been identified as derived from glycoproteins Ib, IIb, and III (Phillips-Agin nomenclature) and an unidentified membrane protein. To test whether removal of these proteins would result in a diminution of platelet response to urate, intact platelets were digested with chymotrypsin, resulting in cleavage of more than 80% of these proteins and a 5-fold reduction in secretory responsiveness to urate crystals. Response to a second platelet stimulus, collagen, was unaffected, indicating an intact secretory mechanism. In addition, when platelets were preincubated with F(ab')2 fragments of an antibody directed against these proteins, platelet secretory response to urate was inhibited by 50%, whereas the responses to collagen and thrombin were unaffected. Thus, membrane proteins appear to mediate platelet stimulation by urate crystals.