Bregina Pool

Cellular characterization of the gouty tophus: A quantitative analysis

OBJECTIVE To characterize the cellular architecture of the tophus and to determine the presence of cytokines implicated in the initiation and resolution of gouty inflammation. METHODS Sixteen fixed, paraffin-embedded, uninfected tophus samples were surgically obtained from 12 patients with microscopically proven gout and were analyzed by quantitative immunohistochemistry. The number of cells present in the corona and fibrovascular zones of the tophus was analyzed by Genmod mixed models analysis. RESULTS Numerous CD68+ mononucleated and multinucleated cells were present within the corona zone. Mast cells were identified in all tophus samples and at similar densities throughout the corona and fibrovascular zones. In contrast, neutrophils were rarely observed. Plasma cells were present in very high numbers within the corona zone. The overall number of CD20+ B cells was much lower. However, in 6 of 12 patients (50%), at least 1 B cell aggregate was present in the fibrovascular zone. Large numbers of cells expressing interleukin-1beta (IL-1beta) were observed in the corona zone. Transforming growth factor beta1 (TGFbeta1)-expressing mononucleated cells were also identified. The number of CD68+ cells correlated with the number of cells expressing IL-1beta (r = 0.691, P = 0.009) and the number expressing TGFbeta1 (r = 0.518, P = 0.04). CONCLUSION The tophus represents a complex and organized chronic inflammatory tissue response to monosodium urate monohydrate crystals involving both innate and adaptive immune cells. The coexpression of IL-1beta and TGFbeta1 suggests that both proinflammatory and antiinflammatory factors present within the tophus contribute to a cycle of chronic inflammation, attempted resolution, and tissue remodeling.

Circulating mediators of bone remodeling in psoriatic arthritis: implications for disordered osteoclastogenesis and bone erosion

IntroductionDiverse bone pathologies are observed in patients with psoriatic arthritis (PsA). Uncoupling of bone remodeling with disordered osteoclastogenesis has been implicated in the pathogenesis of PsA. The aim of this study was to examine the role of soluble mediators of bone remodeling within the circulation of patients with PsA.MethodsPatients with PsA (n = 38), with psoriasis (n = 10), and healthy controls (n = 12) were studied. Serum was obtained for testing of Dikkopf-1 (Dkk-1), macrophage-colony stimulating factor (M-CSF), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) with ELISA. Patients with PsA also had bone densitometry, plain radiographs of the hands and feet, and assessment of peripheral blood osteoclast precursors. Radiographs were scored for erosion, joint-space narrowing, osteolysis, and new bone formation.ResultsCompared with those with psoriasis and healthy controls, patients with PsA had higher circulating concentrations of Dkk-1 and M-CSF. In patients with PsA, M-CSF and RANKL, but not Dkk-1, concentrations positively correlated with radiographic erosion, joint-space narrowing, and osteolysis scores. Mediators of bone remodeling did not correlate with the number of joints with new bone formation or with total hip-bone mineral density. Peripheral blood CD14+/CD11b+ cells, and the number of osteoclast-like cells and resorptive pits after culture with RANKL and M-CSF also correlated with radiographic damage scores. Circulating M-CSF concentrations correlated with the percentage of peripheral blood CD14+/CD11b+ cells.ConclusionsSystemic expression of soluble factors that promote osteoclastogenesis is disordered in patients with PsA and may contribute to periarticular bone loss in this disease.

Acute effect of milk on serum urate concentrations: a randomised controlled crossover trial

Objectives Recent observational studies have highlighted the beneficial role of dairy ingestion in gout prevention. The aims of this study were to determine the acute effects of milk ingestion on serum urate concentrations and examine the mechanisms of these effects. Methods This was a short-term randomised controlled crossover trial of milk in 16 healthy male volunteers. The following products were tested (each 80 g protein): soy control, early season skim milk, late season skim milk (containing high concentrations of orotic acid, a naturally occurring uricosuric agent) and ultrafiltrated MPC 85 skim milk. Each participant received a single dose of each product in random order. Serum and urine were obtained immediately before and then hourly over a 3 h period after ingestion of each study product. Results Ingestion of the soy control led to an increase in serum urate concentrations by approximately 10%. In contrast, ingestion of all milks led to a decrease in serum urate concentrations by approximately 10% (p<0.0001). All products (including soy) rapidly increased the fractional excretion of uric acid (FEUA). Late season milk led to a greater increase in FEUA than MPC 85 (p=0.02) and early season milk (p=0.052). There were no differences over time in serum oxypurines or purine-containing nucleosides. However, all products increased the fractional excretion of xanthine. Conclusions Intact milk has an acute urate-lowering effect. These data provide further rationale for long-term intervention studies to determine whether such dietary interventions have an adjunctive role in the management of individuals with hyperuricaemia and gout.

Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout

Background Bone erosion is a common manifestation of chronic tophaceous gout. Objectives To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. Methods The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. Results MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. Conclusions MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus–bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.

Population-specific influence of SLC2A9 genotype on the acute hyperuricaemic response to a fructose load

Background SLC2A9 is a strong genetic risk factor for hyperuricaemia and gout. SLC2A9 (GLUT9) is a high capacity urate transporter and reportedly transports glucose and fructose. Intake of fructose-containing beverages is associated with development of hyperuricaemia and gout. Objective To determine whether genetic variation in SLC2A9 influences the acute serum urate response to a fructose load. Methods Following an overnight fast, 76 healthy volunteers (25 Māori, 26 Pacific, 25 European Caucasian) drank a solution containing 64 g fructose. Serum and urine were obtained immediately before and then 30, 60, 120 and 180 min after ingestion. The SLC2A9 single nucleotide polymorphism (SNP) rs11942223 was genotyped and data were analysed based on the presence or absence of the gout protective minor allele (C). Results The rs11942223 C allele was present in 17 participants (22%). In the entire group, fructose intake led to an increase in serum urate, which peaked 60 min following fructose ingestion (analysis of variance p=0.006). The presence of the C allele was associated with an attenuated hyperuricaemic response (p(SNP)<0.0001) and increased fractional excretion of uric acid (FEUA) (p(SNP)<0.0001) following the fructose load. The effects of rs11942223 variants on serum urate and FEUA in response to fructose were present only in Caucasian ancestral subgroups but not in the Māori and Pacific ancestral subgroup. Conclusions Variation in SLC2A9 influences acute serum urate and FEUA responses to a fructose load. SLC2A9 genotype may influence the development of gout on exposure to fructose-containing beverages, particularly in European Caucasian populations.

Myeloid-Related Proteins 8 and 14 Contribute to Monosodium Urate Monohydrate Crystal-Induced Inflammation in Gout

Monosodium urate monohydrate (MSU) crystal–induced interleukin‐1β (IL‐1β) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL‐1β–inducing factor, MSU crystals alone are insufficient to induce IL‐1β secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid‐related protein 8 (MRP‐8) and MRP‐14 (endogenous Toll‐like receptor 4 [TLR‐4] agonists) in MSU crystal–induced IL‐1β secretion and their relevance in gout.

Identification of dairy fractions with anti-inflammatory properties in models of acute gout

Aims Large epidemiological studies have shown that low-fat dairy intake reduces the risk of developing gout. It was hypothesised that factors within dairy fractions inhibit the inflammatory response to monosodium urate monohydrate (MSU) crystals. Methods Dairy fractions were tested in MSU crystal-stimulated THP-1 cell assays. Fractions with inhibitory effects were then tested in the murine urate peritonitis model. Results Two dairy fractions were found to have consistent inhibitory effects. Glycomacropeptide (GMP) and G600 milk fat extract both inhibited interleukin-1β (IL1β) gene and protein expression in the THP-1 cell assay. Conversely, standard milk fat increased IL8 protein expression in the THP-1 cell assay. Oral administration of GMP and G600 milk fat extract inhibited cellular influx in the urate peritonitis model. Conclusions Both protein and lipid fractions within dairy products are capable of modulating the inflammatory response to MSU crystals.

Role of miR-146a in regulation of the acute inflammatory response to monosodium urate crystals

Objectives MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional repressors of gene expression. We hypothesised that miRNA regulate gene expression of proinflammatory cytokines in response to monosodium urate (MSU) crystals. Methods We stimulated human monocytic THP-1 cells with MSU crystals and examined miRNA and proinflammatory cytokine gene expression. The effects of miR-146a overexpression were examined by transfecting THP-1 cells with miR-146a precursor. miR-146a expression was examined in the urate peritonitis model, in peripheral blood mononuclear cells from people with gout and control participants, and in gouty tophus samples. Results MSU crystals increased miR-146a expression in THP-1 cells, but not other miRNA implicated in interleukin (IL)-1β regulation. Overexpression of miR-146a expression reduced MSU crystal-induced IL-1β, tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and IL-8 gene expression. In the urate peritonitis model, reduced miR-146a expression was observed during the acute inflammatory response to MSU crystal injection. In people with intercritical gout, peripheral blood mononuclear cells expressed significantly higher levels of miR-146a, compared with normouricaemic and hyperuricaemic control participants and those with acute gout flares. Expression of miR-146a was also observed in all tophus samples. Conclusions Collectively, these data suggest that miR-146a is a transcriptional brake that is lost during the acute inflammatory response to MSU crystals.

Interactions between tenocytes and monosodium urate monohydrate crystals: implications for tendon involvement in gout

Objectives Advanced imaging studies have demonstrated that urate deposition in periarticular structures, such as tendons, is common in gout. The aim of this study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on tenocyte viability and function. Methods The histological appearance of tendons in joints affected by advanced gout was examined using light microscopy. In vitro, colorimetric assays and flow cytometry were used to assess cell viability in primary rat and primary human tenocytes cultured with MSU crystals. Real-time PCR was used to determine changes in the relative mRNA expression levels of tendon-related genes, and Sirius red staining was used to measure changes in collagen deposition in primary rat tenocytes. Results In joint samples from patients with gout, MSU crystals were identified within the tendon, adjacent to and invading into tendon, and at the enthesis. MSU crystals reduced tenocyte viability in a dose-dependent manner. MSU crystals decreased the mRNA expression of tendon collagens, matrix proteins and degradative enzymes and reduced collagen protein deposition by tenocytes. Conclusions These data indicate that MSU crystals directly interact with tenocytes to reduce cell viability and function. These interactions may contribute to tendon damage in people with advanced gout.

The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

Objective. Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. Methods. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. Results. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. Conclusion. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.