Patients and controls
53 patients with RA (Table 1), 28 osteoarthritis (OA) patients, as well as 45 age- and sex-matched healthy control (HC) were enrolled in this study. All the patients met the 2010 American College of Rheumatology (ACR) revised criteria for RA [23] and 1986 ACR criteria for OA [24]. The study was approved by the Institutional Medical Ethics Review Board of Peking University People’s Hospital. Moreover, all participants provided informed consent.
Table 1
Demographic and clinical characteristics of RA patients
Characteristics | RA (n = 53) |
Age, mean (range), years | 53 (23–83) |
Sex, no, female/male | 40/13 |
Duration, mean (range), years | 14.4 (0.25–58) |
SJC, median (range) of 28 joints | 2 (0–28) |
TJC, median (range) of 28 joints | 5 (0–28) |
RF, mean (range), IU/ml | 297.5 (20–5660) |
Anti-CCP antibody, mean (range), IU/ml | 171 (2.72–311) |
ESR, mean (range), mm/h | 46.9 (6–115) |
CRP, mean (range), mg/l | 29.8 (0.27–124) |
DAS28-ESR, mean (range) | 6.47 (1.25–11.94) |
RA, rheumatoid arthritis; SJC, swollen joint count; TJC, tender joint count; RF, rheumatoid factor; Anti-CCP antibody, anti-cyclic citrullinated peptide antibody; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; DAS28, disease activity score 28. |
Clinical and laboratory indices of RA
The following data of patients with RA were recorded: gender, age, duration, swollen joint count (SJC), tender joint count (TJC) and laboratory parameters including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), platelets (PLT), immunoglobulin (Ig) A, IgG, IgM, anti-cyclic citrullinated peptide antibody (anti-CCP antibody), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP). Disease activity scores were calculated using the 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) in patients with RA. DAS28-ESR > 5.1 was considered a high disease activity according to the recommendations from the European League Against Rheumatism (EULAR).
Antibodies And Reagents
Recombinant human macrophage colony-stimulating factor (rhM-CSF) was obtained from PeoproTech GmbH (Rocky Hill, CT). Recombinant human RANKL (rhRANKL), recombinant human Gas6 (rhGas6), human anti-Tyro3TK antibody, human Tyro3TK PE-conjugated antibody, Mouse IgG2B PE-conjugated antibody were purchased from R&D Systems (Minneapolis, MN). Human TruStain FcX™ (Fc Receptor Blocking Solution) was purchased from BioLegend (San Diego, CA). Human CD14 FITC-conjugated antibody and human CD16 APC-conjugated antibody were purchased from eBioscience (San Diego, CA). Leukocyte Acid Phosphatase Kit was purchased from Sigma-Aldrich (St Louis, MO). α-minimum essential medium (α-MEM), 1% penicillin/streptomycin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA).
Flow cytometry analysis and sorting
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh EDTA blood samples using Ficoll density-gradient centrifugation. Before staining with antibodies, single-cell suspensions were incubated with human Fc Receptor Blocking Solution for 15 min at room temperature.
To detect the expression of Tyro3TK on CD14+CD16+ and CD14+CD16- monocytes, cells were stained with CD14 FITC-conjugated antibody, CD16 APC-conjugated antibody, and Tyro3TK PE-conjugated antibody. Corresponding negative isotype and fluorochrome-matched control (FMO) staining were also performed. The cells were then analyzed on FACS Aria II.
For CD14+CD16+ and CD14+CD16- monocytes sorting, cells were stained with CD14 FITC-conjugated antibody and CD16 APC-conjugated antibody. Then the stained cells were sorted with FACS Aria II. The purified CD14+CD16+ and CD14+CD16- monocytes were further analyzed after sorting; the purity of which used for experiments was about 95% - 99%.
In vitro osteoclast differentiation
CD14+CD16+ and CD14+CD16- monocytes from freshly isolated PBMCs were purified by FACS sorting. Then the cells were cultivated 17 days separately in 96-well plates (5×104 cells/200 μl per well) in α-MEM with 1% PenStrep, 10% heat-inactivated fetal bovine serum, 30 ng/ml rhM-CSF and 50 ng/ml rhRANKL. Different concentrations of rhGas6 and/or human anti-Tyro3TK antibody were added as indicated. The medium was changed with fresh medium every 6 days. Osteoclast differentiation was evaluated by staining cells for TRAP using a Leukocyte Acid Phosphatase kit (Sigma-Aldrich) according to the manufacturer’s instructions. TRAP-positive multinucleated cells were counted by an inverted fluorescence microscope (Olympus IX71-141, Tokyo, Japan).
Statistical analysis
All data were analyzed on the statistical software program SPSS 24.0 for windows (SPSS, Chicago, IL). Differences between groups were evaluated by Student’s t-test, non-parametric Mann-Whitney U test, one-way ANOVA test, and Spearman’s correlation test. P value less than 0.05 was considered statistically significant (*P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant).