Serum amyloid A triggers the mosodium urate -mediated mature interleukin-1β production from human synovial fibroblasts
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Monosodium urate (MSU) has been shown to promote inflammasome activation and interleukin-1β (IL-1β) secretion in monocyte/macrophages, but the cellular pathway and nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in synovial tissues, remain elusive. In this study, we investigated the effects of MSU on synovial fibroblasts to elucidate the process of MSU-mediated synovial inflammation.
Human synovial fibroblasts were stimulated with MSU in the presence or absence of serum amyloid A (SAA). The cellular supernatants were analyzed by immunoblotting using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or NLRP3 mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method.
Neither SAA nor MSU stimulation resulted in IL-1β or interleukin-1α (IL-1α) secretions and pro-IL-1β processing in synovial fibroblasts. However, in SAA-primed synovial fibroblasts, MSU stimulation resulted in the activation of caspase-1 and production of active IL-1β and IL-1α. The effect of SAA on IL-1β induction was impaired in cells by silencing NLRP3 using siRNA or treating with caspase-1 inhibitor. In addition, SAA induced the secretion of cathepsin B and NLRP3 mRNA expression in synovial fibroblasts.
Our data demonstrate that exposure of human synovial fibroblasts to SAA promotes MSU-mediated caspase-1 activation and IL-1β secretion in the absence of microbial stimulation. These findings provide insight into the molecular processes underlying the synovial inflammatory condition of gout.
KeywordsGout Synovial Fibroblast NLRP3 Inflammasome Inflammasome Activation NLRP3 Gene
apoptosis-associated speck-like protein containing CARD
Nod-like receptor family: pyrin domain containing 3
serum amyloid A
Gout is a paradigm for acute sterile inflammation that is triggered by interactions between monosodium urate (MSU) crystals and inflammatory cells in the joint connective tissues . Interleukin-1β (IL-1β) has been identified as a pivotal cytokine in gout and MSU crystal-induced inflammation . IL-1β is induced as an inactive pro-molecule by immune cells, such as macrophages and monocytes, and then cleaved into the active p17 form of IL-1 by caspase-1 [3, 4]. Tschopp et al. demonstrated that MSU is capable of activating the NLRP3 inflammasome to process and secrete active IL-1β . These findings suggest that macrophages can recognize MSU as danger-associated molecular patterns (DAMPs) in the damaged tissues and release proinflammatory IL-1β . Upon activation, NLRP3 binds to the ASC, which in turn recruits procaspase-1 for activation. Activated caspase-1 cleaves pro-IL-1β to form the mature IL-1β .
In vitro experiments have shown that triggering of the inflammasome to process IL-1β is a multistep process. Lipopolysaccharide, which belongs to pathogen-associated molecular patterns (PAMPs), had been shown to induce IL-1β from human synovial macrophages . In the absence of a first signal that induces pro-IL-1β, such as lipopolysaccharide, monocyte/macrophages do not spontaneously secrete mature IL-1β when stimulated with NLRP3 ligands [9, 10]. Ii proposed that the first signal modulates the threshold of NLRP3 and the second signal activates NLRP3 inflammasome and causes subsequent caspase-1 activation and IL-1β processing [11, 12]. Recent investigations demonstrated that IL-1β and IL-1 receptor are key players in MSU-mediated acute inflammation [13, 14]. However, the steps that associate cellular activity with MSU crystals that induce inflammasome activation in gouty arthritis are not completely understood.
Serum amyloid A (SAA) is an acute-phase protein present in serum. SAA is also known to possess proinflammatory properties and to mediate inflammatory disease pathogenesis [15, 16]. It has recently been demonstrated that β-amyloid fibrils in Alzheimer's disease signal through the NLRP3 inflammasome and drive caspase-1-dependent cleavage of IL-1β . Furthermore, SAA has been shown to induce the expression of IL-1β and activate the NLRP3 inflammasome via a cathepsin B- and P2X7-dependent manner . In this study, we investigated the MSU-mediated NLRP3 activation process using synovial fibroblasts isolated from human synovium and adjuvant activity induced by SAA.
Materials and methods
Recombinant human SAA was purchased from Peprotech (Rocky Hills, NJ, USA). According to the manufacturer, the endotoxin level of the product is 0.1 ng/mg protein. MSU crystals were purchased from Alexis (Lausen, Switzerland). Polyclonal anti-IL-1β, pro-IL-1β and anti-cleaved caspase-1 (D57A2) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-caspase-1 polyclonal antibodies (sc-622) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NLRP3 antibodies were obtained from Abcam (Cambridge, UK). Anti-cathepsin B antibodies and caspase-1 inhibitor (z-YVAD-FMK) were obtained from Calbiochem (San Diego, CA, USA)
Preparation of synovial fibroblasts
Synovial tissues were obtained from patients with rheumatoid arthritis at the time of total joint replacement. Synovial fibroblasts were isolated from the synovial tissues by enzymatic digestion. The study was approved by the Ethics Committees Nagasaki Medical Center and informed consent was obtained from each of the individuals. Synovial fibroblasts were used from passages 4 through 6 during which time they are a homogeneous population of cells (<1% CD 45 positive).
Measurement of cytokine secretion and immunoblot analysis
Synovial fibrablasts (5 × 104) were seeded in 24-well plates containing RPMI1640 supplemented with 10% heat-inactivated FBS and stimulated with MSU for 24 hours. In some experiments, synovial fibrablasts were pre-treated with SAA for 12 hours before stimulation. Cell-free supernatants were collected by centrifugation at 400 g for five minutes and assayed for IL-1β or IL-1α with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) without the steps for concentrations or precipitations. The same supernatants were also subjected to 12% SDS-PAGE, followed by immunoblotting with Abs for human IL-1β (dilution 1:400), caspase-1 (dilution 1:500), and cathepsin B (dilution 1:500) with an ECL Western blotting kit (Amersham, Little Chalfont, UK). Endotoxin was measured by chromogenic limulus test (Toxicolor LS-50M Kit, SEIKAGAKU CORPORATION, Tokyo, Japan).
Small interfering RNA experiments
Synovial fibroblasts were transfected with 100 nM non-targeting control small interfering RNA (siRNA; AllStars Negative Control siRNA; Qiagen, Hilden, Germany) or with 50 nM two NLRP3 siRNAs (CIAS1_6 and CIAS1_9; Qiagen), combined with the HiPerFect Transfection Reagent (Qiagen) under serum-free condition, as instructed by the manufacturer. The medium was subsequently replaced, pretreated with SAA for 12 h and stimulated with another 24 h with MSU with medium containing 10% FBS. The cell-culture medium was collected for IL-1β ELISA analysis. In some experiments cells were harvested for total RNA purification after SAA pretreatment and analyzed by semi-quantitative RT-PCR (NLRP3), as described below.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from synovial fibroblasts using the RNeasy total RNA isolation protocol (Qiagen, Crauley, UK) according to the manufacturer's protocol. First-strand cDNA was synthesized from 1 μg of total cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. Thereafter, cDNA was amplified using specific primers respectively. The specific primers used were as follows:
NLRP3: forward primer 5'- AAAGAGATGAGCCGAAGTGGG -3' reverse primer 5'- TCAATGCTGTCTTCCTGGCA -3' β-actin; forward primer 5'-GTGGGGCGCCCCAGGCACCA-3' reverse primer 5'-CTCCTTAATGTCACGCACGATTTC-3'.
The product sizes were 79 bp for NLRP3 and 234 bp for β-actin. The thermocycling conditions (35 cycles) 94°C for 60 s and 62°C for 60 s, and 72°C for 60 s.
The amplification of the IL-1-β transcripts was also accomplished on a Light Cycler (Roche Diagnostics, Mannheim, Germany) using specific primers. The housekeeping gene fragment of glyceraldehydes-3-phosphates dehydrogenase (GAPDH) was used for verification of equal loading.
Cell lysis and immunoblot
Synovial fibroblasts were stimulated with SAA with the indicated concentrations of SAA for 24 h. Cells were washed by ice-cold PBS and lysed with a lysis buffer (1% Nonidet P 40, 50 mM Tris, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA , 20 mM β-glycerophosphate, 1.0 mM sodium orthovanadate, 10 μg/mL aprotinin and 10 μg/mL leupeptin) for 20 minutes at 4°C. Insoluble material was removed by centrifugation at 15,000 × g for 15 minutes at 4°C. The supernatant was saved and the protein concentration was determined using the Bio-Rad protein assay kit (Bio Rad, Hercules, CA, USA). An identical amount of protein (50 μg) for each lysate was subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. Immunoblot analysis using anti-NLRP3, pro-IL-β and β-acitin antibodies was performed with an ECL Western blotting kit (GE Healthcare, BUCKS, UK). In brief, the membrane was probed with primary antibodies and washed and incubated with donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:10,000 diluation; GE Healthcare). After being washed, the membrane was reacted with an ECL advance Western blot detection kit (GE Healthcare). Protein bands were visualized using a lumino-image analyzer (LAS3000; Fujifilm, Toyo, Japan).
Differences between groups were examined for statistical significance using Wilcoxon-Mann-Whitney U test. P-values less than 0.05 were considered statistically significance.
SAA priming induces mature IL-1β secretion from MSU-treated synoval fibroblasts
Endotoxin contamination dose not contribute to the IL-1β induction by SAA/MSA
SAA/MSU-induced IL-1β processing is dependent on caspase-1
SAA induces NLRP3 expression and cathepsin B release in synovial fibroblasts
SAA priming induces IL-1a secretion from MSU-treated synoval fibroblasts
Gout is a form of inflammatory arthritis caused by formation of MSU crystals in the synovial tissues of joints . IL-1β has been identified as a pivotal cytokine in gout and MSU crystal-induced inflammation . Recent studies suggested that MSU-mediated NLRP3 inflammasome activation and subsequent IL-1β production in macrophages as key events in initiation of gout . The aim of this study was to determine whether MSU-mediated inflammasome activation could be induced in non-myeloid synovial fibroblasts. A variety of structurally diverse molecules, including ATP, bacterial toxins, crystals, and amyloid proteins, are known to activate the NLRP3 inflammasome leading to IL-1β secretion . Here we found that SAA, which is endogenously induced as an acute phase reactant, sensed the MSU-mediated caspase-1 activation and pro-IL-1β processing. The NLRP3 inflammasome pathway should be pivotal in this SAA/MSU-mediated IL-1β induction, since silencing NLRP3 using siRNA resulted in the abortive IL-1β induction. These data implicate a casual role of SAA in the pathogenesis of MSU-mediated inflammasome activation as well as acute inflammation seen in gouty arthritis.
IL-1β requires cleavage via caspase-1 for proper secretion, which is facilitated as a consequence of inflammasome assembly and activation . The NLRP3 inflammasome has emerged as a critical sensor for a number of endogenous mediators, including MSU, that are capable of promoting IL-1β secretion . However, our study demonstrated that MSU alone did not induce caspase-1 activation or IL-1β secretion in human synovial fibroblasts. Because of its pro-IL-1β inducing effect, SAA-priming of synovial fibroblasts could be essential for MSU-induced IL-1β secretion. Our data also suggest that SAA-induced NLRP3 mRNA expression and cathepsin B secretion may contribute to MSU-mediated NLRP3 activation.
Several lines of evidence indicate that toll-like receptor (TLR) ligands can elicit inflammasome activation . Our findings suggest that SAA, a non-bacterial endogenous product, is sufficient to trigger caspase-1 activation and IL-1β processing in response to MSU, providing a mechanism for activation of the NLRP3 inflammasome in human synovial tissues. Endogenous molecules may be the first signal to prime the activation of the NLRP3 inflammasome, resulting in cooperative signaling . The second signal is provided by stimuli that specifically activate NLRP3 and leads to caspase 1 activation and IL-1β processing . Our results suggest that an endogenous proinflammatory molecule, SAA, could be the first signal to prime the activation of the NLRP3 inflammasome.
Our data indicate that SAA induced MSU-mediated NLRP3 inflammasome activation and post-translational processing of IL-1β in human synovial fibroblasts. These findings highlight the potential role of SAA, a highly sensitive acute phase reactant, in the triggering of MSU-mediated acute synovial inflammation. The innate immune systems, including TLRs, are thought to be essentially involved in inflammasome-mediated inflammation . However, our data show that interaction of an endogenous and non-bacterial acute phase protein, SAA, and MSU crystals synergistically enhance the inflammatory response by activating the inflammasome pathway. These findings provide a new insight into the mechanisms underlying acute gout.
This work was supported by a Grant-in-Aid for Research on intractable diseases from Ministry of Health, Labour and Welfare of Japan, "Study group of national-wide survey for FMF in Japan"
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