Chondrocyte isolation and primary culture
The study was approved by the institutional review board (approval #0004-03). OA articular cartilage was obtained during total knee replacement surgery. Cartilage slices from normal appearing portions of the tibia plateau were removed and washed in Dulbecco's modified Eagle's medium (DMEM). Chondrocytes were isolated from cartilage as previously described [15
]. Briefly, small pieces of cartilage were minced with a scalpel and digested with pronase (2 mg/ml; Boehringer Roche, Indianapolis, IN, USA;) in Hank's balanced salt solution for 30 minutes at 37°C subjected to shaking. After digestion solution was removed, tissue pieces were washed once with DMEM and digested with crude bacterial collagenase (type IA, C 2674; 1 mg/ml; Sigma, Saint Louis, MO USA.) for 6–8 hours at 37°C subjected to shaking. The enzyme reaction was stopped by adding DMEM containing 10% fetal bovine serum. Residual multicellular aggregates were removed by filtration and the cells were washed three times in DMEM.
Chondrocytes were incubated in DMEM containing 10% fetal calf serum, l-glutamine and antibiotics, and were allowed to attach to the surface of the culture dishes. For use in the experiments, cells were trypsinized, washed once, and plated either in eight-well chamber (Nalge Nunc International Corp., Naperville, IL, USA) at 1 × 105 cells/well or in 100 mm diameter culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 1 × 106 cells/plate. At 90% confluence, cells were cultured under serum-free conditions overnight before treatment with a mAb anti-Fas CH 11 (100 ng/ml; Panvera, Madison, WI, USA) in serum-free medium for 17 hours, or with SB203580 (10 μmol/l) for 2 hours before anti-Fas treatment. Control cells were treated with either dimethyl sulphoxide or a mouse isotype control antibody IgM (M 5909; Sigma), as indicated.
Measurement of cell death
After chondrocytes were stimulated as indicated, supernatants containing floating cells were harvested and adherent cells were scratched off the plate with a disposable cell lifter. Cells were combined, spun down, and washed with phosphate-buffered saline (PBS). Cell viability was analyzed by trypan blue dye exclusion assays. Apoptotic cells were detected by in situ cell death fluorescein detection kit (terminal dUTP nick-end labeling [TUNEL]; Boehringer Mannheim) and quantified by flow cytometry. Briefly, collected cells were washed twice with PBS containing 1% bovine serum albumin at 4°C. Cell suspensions were fixed with 100 μl freshly prepared paraformaldehyde solution (4% in PBS; pH 7.4) for one hour at room temperature, followed by centrifugation to remove fixative. Cells were washed once with 200 μl PBS, resuspended in 100 μl permeabilization solution (0.1% triton X-100 in 0.1% sodium citrate) for two minutes on ice. After cells were washed two times with 200 μl PBS, they were resuspended in 50 μl TUNEL reaction mixture or in 50 μl label solution as a negative control. Cells were incubated for 30 minutes at 37°C in a humidified atmosphere in the dark, before apoptotic cells were quantified by fluorescence-activated cell sorter (FACS) analysis. Positive control of apoptosis was generated by incubating cells with DNase I (grade I; 0.5 mg/ml) in 50 mmol/l Tris-HCl (pH 7.5), 1 mmol/l MgCl2, and 1 mg/ml bovine serum albumin for 10 min at room temperature before the TUNEL labeling reactions.
Apoptotic and necrotic cells were also analyzed with an Annexin-V-Fluos Staining Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA). Cells at early stages of apoptosis were labeled by annexin V, whereas necrotic cells were labeled by propidium iodide, which permeated them and stained their nuclei. About 1 × 106 cells were incubated with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide simultaneously, before quantification by FACS analysis.
Immunocytochemical analyses of chondrocyte phenotype were performed as described previously [16
]. using anti-type I and anti-type II collagen mAb (Chemicon International, Temecula, CA, USA). A secondary antibody, rhodamine-conjugated donkey anti-mouse IgG (H+L; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA), was diluted 1:500 in PBS containing 1% bovine serum albumin. Slides were mounted in FluorSave™ reagent (Calbiochem-Novabiochem Corporation, La Jolla, CA, USA) and viewed under a fluorescent microscope (Nikon microscope E 800).
Pieces of cartilage from normal appearing areas of the OA-affected tibial plateaus were collected and fixed in 4% paraformaldehyde before they were embedded in tissue freezing medium and processed for cryostat section. Sections (5 μm thick) were cut perpendicular to the cartilage surface. Distribution of Fas antigen in cartilage was examined by immunohistochemistry with a Histostain SP kit (Zymed, San Francisco, CA, USA) using anti-Fas mAb Ch-11 (Panvera) as primary antibody. Sections were fixed at -20°C with 70% ethanol and 50 mmol/l glycine (pH 2.0) for 20 minutes, treated with hyaluronidase (2 mg/ml; Sigma Chemical Co., St Louis, MO) for 30 minutes at 37°C, and incubated in 0.2% Triton X-100/PBS for 5 minutes at room temperature. Slides were washed with PBS and treated with peroxidase quenching solution to eliminate endogenous peroxidase activity. Sections were then incubated with primary antibodies for 1 hour at 37°C followed by biotinylated secondary antibodies for 10 minutes at room temperature. After washing with PBS, sections were incubated with a streptavidin–peroxidase conjugate for ten minutes at room temperature followed by a solution containing diamino-benzidine (chromogen) and 0.03% hydrogen peroxide for 5 minutes at room temperature. Sections were counterstained with hematoxylin, dehydrated, and mounted. Photography was performed using a Nikon microscope.
Total protein was extracted from cells and quantified as described with BAC Protein Assay Reagent Kit (Pierce, Rockford. IL). For each sample, 10 μg total protein was electrophoresed in 10% SDS PAGE under reducing conditions before blotting and probing with polyclonal antibodies against p38 MAPK (SC535; Santa Cruz, CA, USA), phospho-p38 MAPK (pTGPY, Santa Cruz) and activating transcription factor (ATF)-2 (SC187, Santa Cruz), and a mAb against phospho-ATF-2 (SC8398, Santa Cruz). All of the antibodies were diluted 1:1,000 in PBS-Tween containing 1% bovine serum albumin. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (H+L; Bio-Rad Laboratories, Richmond, CA, USA) were diluted 1:3,000 in PBS-Tween, and used as secondary antibodies. Visualization of immunoreactive proteins was achieved using ECL Western blotting detection reagents (Amersham, Arlington Heights, IL, USA) and by subsequently exposing the membrane to Kodak X-Omat AR film.
Caspase-3 activity assay
A caspase-3 cellular activity assay kit (BIOMOL® Research Laboratories, Inc., Plymouth Meeting, PA, USA) was used to measure caspase-3 activity, in accordance with the manufacturer's instructions. Briefly, chondrocytes treated with anti-CD95 antibody, SB203580 and anti-CD95 antibody, or SB203580 alone were harvested, washed in PBS, and resuspended in cell lysis buffer. Cytosolic extract was collected from supernatant after centrifugation at 10,000 g for ten minutes at 4°C, before it was incubated in microtiter plate with assay buffer. After the reaction was started by the addition of 10 μl Ac-DEVD-pNA substrate (final substrate concentration 200 μmol/l), plate absorbance at 405 nm was read by a microtiter plate reader. Caspase-3 activity was calculated as pmol/min per 2 × 106 cells.
Cell proliferation assay
Proliferation of chondrocytes was determined using BrdU Kit I (Roche, Indianapolis, IN, USA), in accordance with the manufacturer's instruction. Briefly, after cells were treated by anti-Fas antibody, SB, or SB-positive anti-Fas antibody, they were incubated with BrdU labeling medium in eight-well chambers for 60 minutes at 37°C in 5% carbon dioxide. Cells were fixed with ethanol fixative for 20 minutes at -20°C, washed, and incubated with anti-BrdU working solution for 30 minutes at 37°C. After incubation with anti-mouse-immunoglobulin-fluorescein working solution for 30 minutes at 37°C in the presence of Hoechest solution (1:1,000), cells were washed and examined in a fluorescence microscope.
Real-time RT-PCR was performed as previously described [11
]. Briefly, total RNA was isolated from chondrocytes with RNeasy isolation kit (Qiagen). One microgram of RNA was reverse transcribed using Superscript™ II Rnase H Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). Of the resulting cDNA, 30 ng/μl was used as the template to quantify the relative content of mRNA by real-time PCR (ABI PRISM 7700 sequence detection system, Applied Biosystems, Foster City, CA USA) using respective primers and SYBR Green. The primers of Fas ligand were designed using Primers Express software (BioTools Incorporated, Edmonton, AB, T5J 3H1, Canada): forward primer sense ACA CCT ATG GAA TTG TCC TGC, and antisense AGT TTC ATT GAT CAC AAG GC. PCR reactions were performed with TaqMan PCR master mix kit (Applied Biosystems, Foster City, CA, USA). The 18S RNA was amplified at the same time and used as an internal control. The cycle threshold values for 18S RNA and that of CD95L were measured and calculated using computer software. Relative transcript levels were calculated as x = 2-ΔΔCt
, in which ΔΔCt = ΔE - ΔC, and ΔE = CtEXP
, and ΔC = CtCTL
Statistical analysis was performed by analysis of variance followed by a Tukey's test for multiple comparisons at a rejection level of 5%. Data are expressed as mean ± standard deviation.