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. 2010 Nov;162(2):244-50.
doi: 10.1111/j.1365-2249.2010.04242.x. Epub 2010 Sep 14.

Induction of interleukin-23 p19 by serum amyloid A (SAA) in rheumatoid synoviocytes

K Migita  1 , T Koga, T Torigoshi, S Motokawa, Y Maeda, Y Jiuchi, Y Izumi, T Miyashita, M Nakamura, A Komori, H Ishibashi
Affiliations

Induction of interleukin-23 p19 by serum amyloid A (SAA) in rheumatoid synoviocytes

K Migita et al. Clin Exp Immunol. 2010 Nov.

Abstract

In this study, we investigated the roles of serum amyloid A (SAA) in T helper 17 (Th17)-related cytokine induction in rheumatoid arthritis (RA) synoviocytes. Synoviocytes isolated from rheumatoid arthritis (RA) patients were stimulated with recombinant SAA and IL-23 expression was investigated using reverse transcriptase-polymerase chain reaction and Western blot. The involvement of mitogen-activated protein kineases (MAPKs) and nuclear factor (NF)-κB in SAA-induced interleukin (IL)-23 p19 expression was investigated using pharmacological inhibitors. In RA synoviocytes, SAA induced the expression of IL-23 p19 and p40 mRNA expression. The SAA-stimulated expression of p19 was rapid (< 3 h), and insensitive to polymyxin B treatment. This SAA-stimulated expression of IL-23 p19 was inhibited completely by inhibitors of NF-κB, p38MAPK and dexamethasone. Interestingly, the SAA-induced IL-23, p19 and p40 production was accompanied by enhanced expression of IL-1β, but not transforming growth factor-β. These results indicate that SAA is a significant inducer of IL-23 and IL-1β in RA synoviocytes and potentially activates the IL-23/IL-17 pathway in the RA synovium. Our data present a novel interaction between inflammation and autoimmunity by an acute-phase protein.

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Figures

Fig. 1
Fig. 1
Semi-quantitative analysis of interleukin (IL)-23 p19 mRNA expression in rheumatoid arthritis (RA) synoviocytes. RA synoviocytes were stimulated by serum amyloid A (SAA) (5 µg/ml) for 6 h and total RNA was analysed by reverse transcription–polymerase chain reaction (RT–PCR) using primers specific for IL-23 p19 and β-actin, which was used as an internal control. Three experiments were performed using different RA synoviocytes and a representative finding is shown.
Fig. 2
Fig. 2
Serum amyloid A (SAA) stimulates interleukin (IL)-23 p19 and p40 mRNA expressions in rheumatoid arthritis (RA) synoviocytes. (a) RA synoviocytes were unstimulated or stimulated with SAA (0·5, 5 µg/ml) and lipopolysaccharide (LPS; 100 ng/ml) for 6 h in the presence or absence of polymyxin B (10 µg/ml). IL-23 p19 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was determined by real-time polymerase chain reaction (PCR). (b) RA synoviocytes were stimulated with SAA (0·5, 5 µg/ml) for 6 h. IL-23 p40 and GAPDH mRNA expression was determined by real-time PCR. (c) RA synoviocytes were stimulated with SAA (0·5, 5 µg/ml) for 6 h. IL-12 p35 and GAPDH mRNA expression was determined by real-time PCR. (d) RA synoviocytes were stimulated with SAA (5 µg/ml) for 0, 3, 6, 9 and 12 h. IL-23 p19 and p40 mRNA expressions were determined by real-time PCR. Data are means of three different RA–fibroblast-like synoviocytes run in triplicate ± standard deviation. *P < 0·0001 compared to untreated RA synoviocytes.
Fig. 3
Fig. 3
Dexamethasone suppressed the serum amyloid A (SAA)-induced p19 and p40 mRNA expressions. Rheumatoid arthritis (RA) synoviocytes were incubated with the indicated concentrations of dexamethasone for 1 h. Cells were then stimulated with 5 µg/ml of SAA for 6 h and IL-23 p19 (a), p40 (b) and glyceraldehyde-3-phosphate dehydrogenase mRNA expression was determined by real-time polymerase chain reaction (PCR). Data are the means of two different RA synoviocytes run in triplicate ± standard deviation. *P < 0·0001 compared to SAA-treated RA synoviocytes.
Fig. 4
Fig. 4
Nuclear factor (NF)-κB and p38 mitogen-activated protein kinease (MAPK) inhibition suppressed interleukin (IL)-23 p19 mRNA expression. Rheumatoid arthritis (RA) synoviocytes were incubated with vehicle (dimethylsulphoxide, media), BAY11-7082 (a, nuclear factor-κB inhibitor), PD98059 (b, extracellular regulated kinase pathway inhibitor), SB203580 (c, p38 inhibitor) and SP600125 (d, c-Jun N-terminal kinase inhibitor) for 1 h. Cells were then stimulated with 5 µg/ml of serum amyloid A (SAA) for 6 h and interleukin (IL)-23 p19 and glyceraldehyde-3-phosphate dehydrogenase mRNA expression was determined by real-time polymerase chain reaction. Data are the means of two different RA synoviocytes run in triplicate ± standard error of the mean. *P < 0·0001 compared to SAA-treated RA synoviocytes.
Fig. 5
Fig. 5
Serum amyloid A (SAA) stimulates interleukin (IL)-23 p19 protein expression in rheumatoid arthritis (RA) synoviocytes. RA synoviocytes were stimulated with SAA for 24 h. IL-23 p19 protein expression was determined by Western blotting using specific antibodies against p19. Three experiments were performed using different RA synoviocytes and a representative result is shown.
Fig. 6
Fig. 6
Serum amyloid A (SAA) stimulates interleukin (IL)-23 p40 protein synthesis in rheumatoid arthritis (RA) synoviocytes. RA synoviocytes were stimulated with various concentrations of recombinant SAA as indicated for 24 h. IL-23 p40 protein in the conditioned media was determined by enzyme-linked immunosorbent assay. Data represent the means of three independent experiments run in triplicate ± standard deviation. *P < 0·0001 compared to untreated RA synoviocytes.
Fig. 7
Fig. 7
Serum amyloid A (SAA) stimulates interleukin (IL)-1β mRNA expression in rheumatoid arthritis (RA) synoviocytes. RA synoviocytes were stimulated with SAA (0·5, 5 µg/ml) for 6 h. IL-1β and transforming growth factor-β1 mRNA expression was determined by real-time polymerase chain reaction. Data are means of three different RA–fibroblast-like synoviocytes run in triplicate ± standard deviation. P < 0·0001 compared to untreated RA synoviocytes.
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