Clinical specimens
Synovial tissues were obtained from OA and RA patients (all Chinese) upon joint replacement or synovectomy at the First Affiliated Hospital of USTC and the First Affiliated Hospital of Anhui Medical University, Hefei, China. This study was approved by the Ethics Committee of the First Affiliated Hospital of USTC and the First Affiliated Hospital of Anhui Medical University. The informed consent was signed by each of the patients and their guardians. All RA patients (n = 23) include in this study were diagnosed with RA according to the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria [8]. The diagnosis of primary knee OA (n = 23) conformed to the criteria from the American College of Rheumatology (ACR) [9], radiographic changes were grade III or IV according to the Kellgren and Lawrence classification method [10]. RA and OA patients induced by trauma, inflammation, tuberculosis, or sepsis were excluded.
Isolation and in vitro culture of cells from OA and RA synovia
The method used for the isolation of FLS from synovial tissues was modified from a method previously described [11]. The synovium was removed in joints of patients with OA and RA. Removed hyperplastic synovial tissues were washed and minced in DMEM (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% FBS (Gibco, Grand Island, USA), penicillin and streptomycin (Gibco, Grand Island, USA). Dissected synovial tissues were digested in culture media containing 1 mg/ml collagenase IV (Sigma, St. Louis, MO, USA) and 0.1 mg/ml deoxyribonuclease I (Sigma, St. Louis, MO, USA), and then it was incubated for 2 h at 37 °C. Tissues were then vortexed and resuspended in fresh media. The media containing the digested tissues was centrifuged, and the cell pellet was resuspended in fresh media for cell sorting. The sorted OA FLS and RA FLS were cultured at 37 °C in 5% CO2. Culture media were replenished every 2 days, and cells were subcultured when they reached 90% confluence.
Flow cytometry
OA and RA FLS were incubated with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- or allophycocyanin (APC)-conjugated antibodies at 37 °C for 1 h: CD45-FITC (5 μg/test), CD31-FITC (5 μg/test), CD146-FITC (5 μg/test), CD235a-FITC (5 μg/test), PDPN-PE (2 μg/test), CD90-APC (2 μg/test), CD106 (VCAM1)-FITC (5 μg/test), CD54 (ICAM1)-PE (2 μg/test). All flow antibodies were from Miltenyi Biotechnology Company (Bergisch Gladbach, Germany). After 3 times of washing by pre-cooled PBS, the cells were analysed by flow cytometer (BD FACSAria II) and BD FACSDiva software. For each test, 2 × 108 total synovial cells from RA and OA synovium were used. It’s around (1.3 ± 0.4) × 108 and (1.0 ± 0.3) × 108 total count (events) in gate P4 for RA samples and OA samples. Isotype controls were included in the FACS detection for those proteins with relatively low expression (IgG1-FTIC for CD146, and REA control-FITC for CD31 and CD235a, Additional file 1: Figure S1). Unlabelled cells were used in other FACS assays as negative controls.
H&E staining, Masson staining and immunohistochemistry (IHC)
OA and RA synovial tissues were fixed with acetone for 15 min, washed twice with PBS, and then incubated for 1 h in a humid chamber with primary antibodies (PCNA, Santa Cruz Biotec, Santa Cruz, CA, USA; VCAM-1, Santa Cruz Biotec; ICAM-1, Santa Cruz Biotec). The synovial tissues were then washed 3 times with PBS and incubated for an additional hour with an isotype-matched horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rat IgG, Santa Cruz Biotec). After 3 additional washes, the HRP reaction was developed with diaminobenzidine (DAB) per the manufacturer’s instructions.
Paraffin-embedded OA and RA synovial tissues were sliced and then blocked with 5% goat serum for 30 min. The tissues were incubated in primary anti-PCNA (Santa Cruz Biotec), anti-CD90 (Santa Cruz Biotec), anti-ICAM1 (Abcam, Cambridge, UK) or anti-VCAM1 (Abcam, Cambridge, UK) antibodies overnight at 4 °C. IHC staining was generally performed as previously reported. The primary antibody was omitted in negative controls. H&E staining and Masson staining were performed according to standard protocols.
Proliferation
The proliferation abilities of OA and RA FLS were evaluated by CCK-8 assay (Bestbio, Nanjing, China) and Ki-67 staining (Miltenyi Biotec). All procedures were performed according to standard protocols.
Migration
Transwell (Costar) cell culture plates were used for migration and invasion assays. A total of 5 × 104 FLS were seeded into the upper chamber in serum-free medium, and serum-containing medium was added into the lower wells. After 24 h or 48 h, the upper chamber was washed with PBS twice and fixed with 4% paraformaldehyde for 20 min, and then 1% crystal violet was used to stain the invasive cells.
Apoptosis
Annexin V/propidium iodide (PI) staining was performed for the detection of apoptotic cells. After the desired treatment, 1 × 106 cells were collected and washed twice with ice-cold PBS. The cells were then stained using the Alexa Fluor®488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 annexin V and PI (Miltenyi Biotec) for flow cytometry according to the manufacturer’s guidelines. The untreated cells served as a negative control for double staining.
Western blot
OA and RA FLS were lysed in SDS buffer. The protein concentrations were determined via a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of cell lysates were run on a gel, transferred onto a PVDF membrane (Millipore, Billerica, MA, USA), and probed with the following primary antibodies: interleukin-1 beta (IL-1β) (1:200, Santa Cruz Biotec), interleukin-6 (IL-6) (1:500, Santa Cruz Biotec) and GAPDH (1:4000, Abcam). GAPDH served as a loading control. After incubation with secondary antibodies, signals were visualized by enhanced chemiluminescence (GE system).
Elisa
A total of 2 × 105 cells were seeded in a 12-well plate for 48 h. Supernatants were collected to measure the amounts of secreted IL-6 and TNF-α. IL-6 and TNF-α were detected via a human IL-6 ELISA kit and a human TNF-α ELISA kit (Miltenyi Biotec), respectively.
qRT-PCR
Cultures of OA and RA FLS were grown to confluence in 6-well culture plates. Cells were treated for 24 h with TNF-α (20 ng/ml) or Methotrexate (MTX, 100 μM). Twelve hours after stimulation, whole-cell RNA was collected by TRIzol (Life Technologies), and it was analysed by qRT-PCR for gene expression. GAPDH was used as an endogenous control. A reverse transcription kit and qRT-PCR dye were obtained from Takara Biomedical Technology Company (Shiga, Japan).
Statistics
The error bars mean ± Standard Error of Mean (SEM) of six independent experiments. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.0001, respectively (Student’s t-test).