Fig 1.
Expression of IL-20 and its receptors in patients with OA and in OA rats.
(A) Interleukin (IL)-20 expression in osteoarthritis (OA) synovial tissue and cartilage was detected using immunohistochemical (IHC) staining with anti-IL-20 monoclonal antibody (7E). Staining with mouse immunoglobin G1 (mIgG1) isotype as the primary antibody was the negative control for IL-20. (B) IL-20, IL-20R1, IL-20R2, and IL-22R1 expression in OA synovial fibroblasts (OASFs) and OA chondrocytes (OACCs) isolated from OA rats were detected using anti-IL-20, anti-IL-20R1, anti-IL-20R2, and anti-IL-22R1 monoclonal antibodies. Staining with mIgG1 isotype as the primary antibody was the negative control for IL-20, IL-20R1, IL-20R2, and IL-22R1. The reaction was detected using AEC chromogen stain (red), and the nuclei were counterstained with hematoxylin (blue). Magnification was 200×. (C) The knees and patellae from OA rats at the indicated time periods were collected, mRNA was isolated, and the IL-20 transcript was measured using real-time quantitative polymerase chain reaction (RTQ-PCR). β-actin was an internal control. Data are the mean ± standard deviation (SD) of 5 mice. Data are representative of three independent experiments. *P < 0.05, **P < 0.01 compared with week 0.
Fig 2.
(A-D) OASFs were treated with IL-20 (200 ng/ml) for 4 and 8 hours. mRNA was isolated and the transcripts of tumor necrosis factor (TNF)-α, IL-1β, matrix metalloproteinase (MMP)-1, and MMP-13 were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. *P < 0.05, **P < 0.01 versus untreated controls. Data are representative of three independent experiments, each done in triplicate. (E) MMP activity in the conditioned media of OASFs treated with IL-20 (200 ng/ml) for 24 and 48 hours. Results are shown as a percentage of the untreated control. *P < 0.05, **P < 0.01 versus untreated controls. Data are representative of three independent experiments, each done in triplicate. (F) OASFs were incubated with IL-20 (200 ng/ml) for the indicated time periods. Total cell lysates were analyzed using Western blotting with specific antibodies against phosphor-ERK-1/2, JNK, and β-actin. Data are representative of three independent experiments.
Fig 3.
(A) OACCs were treated with IL-20 (200 ng/ml) for 8 hours. mRNA was isolated and the transcripts of monocyte chemoattractant protein (MCP)-1, IL-6, MMP-1, and MMP-13 were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. *P < 0.05, **P < 0.01 versus untreated controls. Data are representative of three independent experiments, each done in triplicate. (B) MMP activity in the conditioned media of OACCs treated with IL-20 (200 ng/ml) for 24 and 48 hours. Results are shown as a percentage of the untreated control. **P < 0.01 versus untreated controls. Data are representative of three independent experiments, each done in triplicate. (C) OACCs were treated with IL-20 (200 ng/ml) for 6 hours. mRNA was isolated and the transcripts of TGF-β1, BMP-2, aggrecan, and type 2 collagen (Col 2) were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. **P < 0.01 versus untreated controls. Data are representative of three independent experiments, each done in triplicate.
Fig 4.
Correlation of IL-20 and chondrogenic differentiation.
(A) Mouse mesenchymal stem cells (MSCs) were incubated with IL-20 (800 ng/ml) and cultured in chondrogenic medium. The cell pellets formed free-floating aggregates within the first 24 hrs. Aggregates cultured for 21 days were fixed and paraffin-embedded, and then sections were stained with Safranin O. (B-C) Mouse MSCs were incubated with IL-20 (800ng/ml), 7E (5 μg/ml), or mIgG (5 μg/ml) and cultured in chondrogenic medium for 7 days. Total RNAs were isolated from aggregates and analyzed using RTQ-PCR with primers specific for Sox9 and type 1 collagen (Col 1). The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. **P < 0.01 versus untreated controls. #P < 0.05 versus mIgG controls. Data are representative of three independent experiments, each done in triplicate. (D) Aggregates were cultured with IL-20 (800 ng/ml), 7E (5 μg/ml), or mIgG (5 μg/ml) for 7 days. Total cell lysates were analyzed using Western blotting with specific antibodies against phosphor-Sox9 and with β-actin. β-actin was used as an internal control. Data are representative of three independent experiments. (E) Bands were quantified using ImageJ software. *P < 0.05 versus untreated controls. Data are the means ± SD of three independent experiments.
Fig 5.
Arthritis was surgically induced in 3 groups of rats (n = 5/group) on day 0. A fourth group of sham-operated rats were negative controls (n = 5). (A) Rats with OA were injected subcutaneously with 7E or mIgG (3 mg/kg) 3 times a week throughout the study. OA rats treated with phosphate buffered saline (PBS) were positive controls. Knee thickness as an indictor of disease activity was measured on the indicated days. *P < 0.05 versus mIgG-treated controls. Data are representative of three independent experiments. (B) On day 56, the rats were anesthetized, and radiographs were taken. Arrows indicate subchondral cysts. Representative radiographs are shown. (C) Histological sections of the knee joints of OA rats were stained using toluidine blue. Representative photos are shown. (D) On day 57, synovial tissue from rats was collected, mRNA was isolated and the IL-20, IL-1β, and MMP-13 transcripts were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. *P < 0.05 versus mIgG-treated controls. Data are representative of three independent experiments.
Fig 6.
The effects of 7E and MSCs on OA mice.
(A) Arthritis was surgically induced in 4 groups of mice (n = 5/group) on day 0. A fifth group of sham-operated mice were negative controls (n = 5). OA mice were given intra-articular injections of PBS, MSCs (5x105/mouse/single injection), mIgG (2μg/mouse 3 times a week), or 7E (2μg/mouse 3 times a week) 14 days after surgery. Knee joint sections of the mice were stained with Safrainin O to analyze the effects of 7E and MSCs on destabilization of the medial meniscus (DMM) surgery-induced OA 42 days after treatment. Representative Safranin O stained images of cartilage sections are shown. (B) The severity of cartilage destruction was evaluated based on the scoring system using Safranin O images. Data are mean ± standard error of the mean (SEM) (n = 5 mice per group). *P < 0.05 versus mIgG-treated controls. (C-G) On day 56 after surgery, the knee joints from mice were collected, mRNA was isolated, and the IL-20, IL-1β, TNF-α, MMP-9, and MMP-13 transcripts were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; β-actin was the housekeeping gene. #P < 0.05 versus OA controls. *P < 0.05 versus mIgG-treated controls. Data are representative of three independent experiments.