Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of fibroblast

Abstract

Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2) (-) levels was observed by treatment of fibroblasts with SAA (r = 0·99 and P ≤ 0·001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0·001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2) (-) by 50%. Also, SAA raised fibroblast proliferation (P < 0·001) and this effect was completely abolished by the addition of anti-oxidants (P < 0·001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.

Fig. 1

(a) Representative kinetic of O₂^- production measured by lucigenin-enhanced chemiluminescence in 3T3 fibroblasts (2·5 10⁶ cells/ml) in the presence of serum amyloid A (SAA) (15 µg/ml). (b) Dose-dependent effect of SAA (5, 10 and 15 µg/ml) on O₂^- anion production measured by lucigenin-enhanced chemiluminescence in 3T3 fibroblasts (2·5 × 10⁶ cells/ml). (c) Effect of diphenyliodonium (DPI) (20 µM on the lucigenin-enhanced chemiluminescence of SAA (10 µg/ml)-treated 3T3 fibroblasts. ***P < 0·001 for comparison between control and SAA treatment. ##P < 0·01 for comparison between SAA and SAA plus DPI treatments.

Fig. 2

(a) Percentage of [³H]-thymidine incorporation into 3T3 cells treated with 1 and 10 µg/ml serum amyloid A (SAA). **P < 0·01 for comparison between control and SAA treatment. (b) Growth curves of 3T3 fibroblasts in the absence and presence of SAA (15 µg/ml). *Significant differences in relation to day 0; #between the control and SAA treatment curves. (c) Inhibitory effect of the anti-oxidants N-acetyl-l-cysteine (NAC) (10 mM) and α-tocoferol (400 µM) on [³H]-thymidine incorporation in 3T3 cells treated with SAA (1 ng/ml). The results are representative of three experiments performed in triplicate. ***P < 0·001 for comparison between control and SAA treatment; ###P < 0·001 for comparison between SAA and SAA plus anti-oxidant treatments.