Interleukin-29 Enhances Synovial Inflammation and Cartilage Degradation in Osteoarthritis

Abstract

We have recently shown that IL-29 was an important proinflammatory cytokine in pathogenesis of rheumatoid arthritis (RA). Inflammation also contributes to the pathogenesis of osteoarthritis (OA). The aim of this study was to investigate the effect and mechanism of IL-29 on cytokine production and cartilage degradation in OA. The mRNA levels of IL-29 and its specific receptor IL-28Ra in peripheral blood mononuclear cells (PBMCs) were significantly increased in OA patients when compared to healthy controls (HC). In the serum, IL-29 protein levels were higher in OA patients than those in HC. Immunohistochemistry revealed that both IL-29 and IL-28Ra were dramatically elevated in OA synovium compared to HC; synovial fibroblasts (FLS) and macrophages were the main IL-29-producing cells in OA synovium. Furthermore, recombinant IL-29 augmented the mRNA expression of IL-1β, IL-6, IL-8, and matrix-metalloproteinase-3 (MMP-3) in OA FLS and increased cartilage degradation when ex vivo OA cartilage explant was coincubated with OA FLS. Finally, in OA FLS, IL-29 dominantly activated MAPK and nuclear factor-κB (NF-κB), but not Jak-STAT and AKT signaling pathway as examined by western blot. In conclusion, IL-29 stimulates inflammation and cartilage degradation by OA FLS, indicating that this cytokine is likely involved in the pathogenesis of OA.

Figure 1

The expression of IL-29 and its receptor IL-28Rα in the peripheral blood mononuclear cells (PBMCs) and serum of OA patients. The mRNA levels of IL-29 (a) and IL-28Rα (b) in the OA PBMC (n = 30), when compared with that in HC PBMC (n = 30), were detected by real-time PCR. (c) The levels of IL-29 in the serum from OA patients (n = 30) and HC (n = 30) were assessed by ELISA. Data in graphs are the mean ± SEM.

Figure 2

The expression and cellular distribution of IL-29 and IL-28Rα in the synovial tissues. IL-29 and IL-28Rα in synovial tissues of OA patients (n = 5) and HC (n = 3) were detected by immunostaining (a) and semiquantification (b). Values in (b) are the mean ± SEM. ^∗∗ P < 0.01 versus medium control. (c) Colocalization of IL-29 with CD68 or FGF-2 in OA synovium was detected by double immunofluorescence staining, and nuclei were counterstained with DAPI. All of the magnification in this figure was ×400.

Figure 3

Immunostaining for IL-28Rα in OA fibroblasts and viability of OA FLS in response to IL-29. The expression of IL-28Rα in OA FLS, detected by immunofluorescent staining (a) and flow cytometric analysis (b). The magnification in (a) was ×400. (c) OA FLS viability after being incubated with IL-29 at 1, 10, and 100 ng/mL for 72 h was measured by MTT assay. The data present the mean ± SEM of three independent experiments.

Figure 4

IL-29-induced cytokine expression by OA synovial fibroblasts. mRNA levels of IL-1β (a), IL-6 (b), IL-8 (c), and MMP-3 (d) in OA FLS after exposure to IL-29 (1, 10, and 100 ng/mL) with or without its blocking antibody for 48 h were determined by real-time PCR. The results shown are representative of one of three independent experiments. The error bars represent mean ± SEM for triplicate wells. ^∗ P < 0.05 versus medium control. ^# P < 0.05 versus IL-29 (100 ng/mL) treatment group.

Figure 5

Effects of IL-29 on cartilage degradation by OA FLS ex vivo. (a) The OA cartilage depletion after being cocultured with OA FLS for 72 h was visualized in Safranin O/Fast Green-stained sections. Representative images from one experiment are reported (a), and the reduced intensity of red stain denotes proteoglycan loss (original magnification ×100). (b) The depth of GAG depletion (μm) in cartilage was measured from the articular surface to the red/orange tidemark (marked by arrow). (c) The ratio of MM-1, MM-2, MM-3, and MM-13 to TIMP-1 was determined by real-time PCR in OA FLS. (d) MMP-3 in culture supernatants harvested from each well after 72 h was measured by ELISA. The error bars represent mean ± SEM for triplicate experiments. ^∗ P < 0.05 versus medium control. ^∗∗ P < 0.01 versus medium control.

Figure 6

Effect of IL-29 on the phosphorylation of Jak-STAT, AKT, MAPK, and NF-κB signaling pathways. OA FLS were treated with IL-29 at 100 ng/mL for 0 , 20, 40, and 60 min, and the activation of STAT 1/2/3/4/5/6, AKT, JNK, ERK, P38, p65, and NF-κB was evaluated by western blot. The relative quantification of target proteins was calculated by comparison of the bands density levels between samples. The results were expressed as mean ± SEM from three independent experiments. ^∗ P < 0.05 versus nontreatment group.