Serum amyloid A triggers the mosodium urate -mediated mature interleukin-1β production from human synovial fibroblasts

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

SAA induces the transcription of pro-IL-1β in human synovial fibroblasts. A. Synovial fibroblasts were incubated with SAA (2 μg/ml) for the indicated periods. The cells were harvested and analyzed for pro-IL-1β mRNA levels by real-time PCR. B. Synovial fibroblasts were incubated with the indicated concentrations of SAA for 6 h. The cells were harvested and analyzed for IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of three independent experiments.*P <0.001 compared to SAA-untreated synovial fibroblasts.

Figure 2

MSU induces IL-1β synthesis from SAA-primed synovial fibroblasts. A. Synovial fibroblasts were pretreated or untreated with the indicated concentrations of SAA for 12 h. After pretreatment, the cells were stimulated with the indicated concentrations of MSU for 24 h and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of three independent experiments. B. Synovial fibroblasts were pretreated or untreated with the indicated concentrations of SAA for 12 h. After pretreatment, the cells were stimulated with the indicated concentrations of MSU for 24 h and supernatants were analyzed by immunoblot for the presence of mature IL-1β. Three experiments were performed using different synovial fibroblasts and a representative result is shown.

Figure 3

Endotoxin contaminution dose not contribute to IL-1β induction by SAA/MSU. A. Synovial fibroblasts were pre-treated with SAA (1 μg/ml) or LPS (500 pg/ml) for 12 h. Cells were stimulated with MSU (200 μg/ml) for 24 h. Supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments. P <0.01 compared to SAA-untreated synovial fibroblasts. B. Synovial fibroblasts were pre-treated with SAA (1 μg/ml) or LPS (500 pg/ml), which were exposed to 100°C (heat) in a water bath for 30 minutes, for 12 h. Cells were stimulated with MSU (200 μg/ml) for 24 h. Supernatants were analyzed for IL-1β production by ELISA. The endotoxin levels were also measured by limulus test using the same supernatants. Values represent the mean ± SD of two independent experiments.

Figure 4

SAA/MSU-induced IL-1β processing is dependent on caspase-1. A. Synovial fibroblasts were untreated or pretreated with SAA (2 μg/ml) for 12 h. Cells were stimulated with the indicated concentrations of MSU for 24 h. Supernatants were analyzed by immunoblot for the presence of procaspase-1 (p45) and cleaved caspase (p20). Three experiments were performed using different synovial fibroblasts and a representative result is shown. B. Synovial fibroblasts were untreated or pretreated with SAA (2 μg/ml) for 12 h. Cells were stimulated with MSU in the presence or absence of z-YVAD-fmk (10 μM) for 24 h. Supernatants were analyzed by immunoblot analysis for the presence of mature IL-1β. Two experiments were performed using different synovial fibroblasts and a representative result is shown.

Figure 5

SAA induces NLRP3 expression. A. Synovial fibroblasts were stimulated with SAA (2 μg/ml) for 12 h. The cells were analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different synovial fibroblasts and a representative result is shown. B. Synovial fibroblasts were stimulated with the indicated concentrations of SAA for 24 h. Cellular lysates were subjected to Western blotting using specific antibodies against NLRP3, pro-IL-1β and β-actin (internal control). Three experiments were performed and a representative result is shown. C. Synovial fibroblasts were transfected with non-targeting control siRNA or NLRP3 siRNA for 22 h and subsequently stimulated with SAA (2 μg/ml) for 12 h. The cells were analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different synovial fibroblasts and a representative result is shown. D. The transfected cells as described in C were pretreated with SAA (2 μg/ml) for 12 h and subsequently stimulated with MSU (200 μg/ml) for 24 h. Cell-free culture supernatants were analyzed for IL-1β by ELISA. Values represent the mean ± SD of three independent experiments. *P <0.01 compared to control siRNA transfected synovial fibroblasts.

Figure 6

SAA induces the secretion of cathepsin B from synovial fibroblasts. Synovial fibroblasts were untreated or pretreated with SAA (2 μg/ml) for 12 h and subsequently stimulated with of MSU (200 μg/ml) for 24 h. Supernatants were analyzed by immunoblot analysis for presence of cathepsin B. Three experiments were performed using different synovial fibroblasts and a representative result is shown.

Figure 7

MSU induces IL-1α synthesis from SAA-primed synovial fibroblasts. Synovial fibroblasts were pretreated or untreated with the indicated concentrations of SAA for 12 h. After pretreatment, the cells were stimulated with the indicated concentrations of MSU for 24 h and supernatants were analyzed for IL-1a production by ELISA. Values represent the mean ± SD of three independent experiments.