1. Human osteoarthritic chondrocytes are impaired in matrix metalloproteinase-13 inhibition by IFN-γ due to reduced IFN-γ receptor levels 
R. Ahmad, M. El Mabrouk, J. Sylvester, M. Zafarullah 
Osteoarthritis and Cartilage 
Volume 17, Issue 8, Pages (August 2009)
DOI: /j.joca
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Impaired suppression of IL-1-induced MMP-13 gene expression by IFN-γ in human OA chondrocytes. Human chondrocytes from different healthy control subjects (C) or OA patients (P) were treated with vehicle (0.1% BSA), IL-1β (10ng/ml), IFN-γ (300units/ml) alone or in combination for 24h. Cells were harvested for MMP-13 (A, upper panel) and GAPDH (A, lower panel) mRNA analysis by RT-PCR. (B) The supernatants were used for MMP-13 protein (48kDa, active enzyme) analysis by Western blotting. (C) Band densities of MMP-13 RT-PCR products were quantified in arbitrary units and normalized with GAPDH. The values are the mean and SE. (D) The densities of MMP-13 protein bands were quantified and shown as mean values with SE. The mean values of two groups differed significantly (P<0.05). (E) Human chondrocytes were treated with vehicle control (BSA), IL-1, IFN-γ or both together for 24h and MMP-13 mRNA and protein analyzed as above. IFN-γ alone does not affect MMP-13 expression. (F) Dose-dependent induction of MMP-13 mRNA and protein by IL-1β providing justification for the use of 10ng/ml dose. (G) Dose-dependent inhibition of IL-1-induced MMP-13 mRNA and protein by IFN-γ in human OA chondrocytes justifying the 300Units/ml as the effective dose.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Analysis of IFN-γR1 expression on the chondrocytes derived from patients with OA (P) and control (C) subjects. (A) Chondrocyte surface expression of IFN-γR1 was evaluated by flow cytometry using Jurkat cells (upper panel) as positive controls. The histograms represent the percentage of cells expressing IFN-γR1 (right panels, white histogram or background of mouse IgG1 negative control gray histogram). The histogram is representative of three independent experiments for each patient. (B) The mean percentages of chondrocytes expressing IFN-γR1. A star shows statistically significant (P<0.05) difference between the two groups. (C) The band densities of OA patient and control IFN-γR1 protein were presented as mean values and SE, which differed significantly. (D) Total cell lysates were immunoprecipitated with anti-IFN-γR1 antibody or control non-immune mouse IgG, resolved by SDS-PAGE and analyzed by Western immunoblotting with anti-IFN-γR1 antibody (upper panel). Jurkat cells and PBMCs extracts were used as positive controls. Lysates (20μg) were probed for beta-actin analysis (lower panel). (E) For monitoring chondrocyte metabolism and phenotype, collagen II (upper panel) and beta-actin (lower panel) protein levels in both groups of chondrocytes were determined by Western blotting using purified type-II collagen as positive control (lane 1). (F) Detection of IFN-γR1 in chondrocytes by using fluorescein-labeled secondary antibody. Cells were grown in 96-well flat bottom microplates for 48h. Cells were washed with PBS containing 0.2% BSA and incubated with a mouse anti-human IFN-γR1 monoclonal antibody (2μg/ml; Chemicon International Cat# MAB1154) for 45min. Cells were washed twice and then probed with FITC-goat anti-mouse secondary antibody (BD Biosciences Cat# 58859) (2μg/ml). Cells washed and then fluorescent images were recorded through fluorescence microscopy. C=Control; P=OA patients. Light microscope images of the corresponding regions are also shown.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Differential IFN-γ-stimulated STAT1 phosphorylation in control and OA chondrocytes. Chondrocytes were incubated for 30min with IFN-γ or 0.1% BSA and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-phospho-STAT1 (A) and respective total (lower panels) antibodies. (B) The normalized densities of STAT1 bands shown in the form of mean values with SE. (C) Neutralization of IFN-γR1 blocks IFN-γ-stimulated STAT1 phosphorylation. Human OA chondrocytes from two different individuals were grown to confluence and treated with control IgG or neutralizing IFN-γR1 antibody (Nab) for 1h and then stimulated with IFN-γ for 20min followed by Western blotting with anti-phospho- and total STAT1. The resulting bands are shown. (D) IFN-γR1 Nab alone did not affect STAT1 phosphorylation.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Transfection of IFN-γR1 vector restores the ability of IFN-γ to repress IL-1-induced MMP-13 expression in OA chondrocytes. Cells from two different OA patients were transfected with 1μg of pCMV-XL5 (empty vector) or CMV-IFN-γR1 expression vector and then treated with IL-1 and IFN-γ for 24h as indicated. Results of MMP-13 and GAPDH control mRNA RT-PCR as well as MMP-13 protein Western blot are depicted.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions