CD14+ monocytes and soluble CD14 of synovial fluid are associated with osteoarthritis progression

Abstract

Objectives: This study aims to investigate the role of cluster of differentiation 14 (CD14) expressed monocytes and soluble CD14-mediated pathway in the synovial inflammation of knee osteoarthritis (OA).

Patients and methods: Between May 2012 and July 2013, a total of 35 patients with knee OA (9 males, 26 females; mean age: 66.3±8.8 years; range, 52 to 79 years) were included in this cross-sectional study. Synovial fluid was obtained from knee joints of 35 OA patients. The CD14⁺ monocytes from synovial fluid mononuclear cells (SFMCs) were isolated using the MACS. The fibroblast-like synoviocytes (FLSs) isolated from knee joint tissue were incubated with recombinant CD14 and lipopolysaccharide (LPS) for 24 h. Cytokine profiling was performed with the Luminex® Performance Assay or magnetic bead panel kit. The expression of CD14 and CD16 was analyzed by immunohistochemistry and flow cytometry.

Results: The concentration of sCD14 in synovial fluid was correlated with the interleukin-6 (IL-6) level (n=35) (ρ=0.654, p<0.001). The culture supernatants of CD14⁺ monocytes isolated from SFMC (n=15) showed a correlation between sCD14 and IL-6 (ρ=0.784, p=0.001), along with complement component 3 (ρ=0.756, p=0.010), IL-1b (ρ=0.652, p=0.012), and tumor necrosis factor-alpha (ρ=0.806, p=0.001). Following recombinant CD14 and LPS treatment, OA FLS synergistically enhanced the secretion of IL-6, IL-8, and matrix metalloproteinase 3 (n=3, p<0.05). In five paired-samples from identical patients, the proportions of CD14⁺ monocytes were significantly elevated in recurred synovial fluid compared to those in initial synovial fluid (p=0.043). When monocyte subsets were analyzed in SFMC (n=26), CD14⁺CD16⁺monocytes were abundant (p=0.019) and had higher toll-like receptor 4 expression than CD14⁺CD16^- (p<0.001).

Conclusion: Our study results suggest that CD14⁺ monocytes and the sCD14-mediated pathway play an important role in OA aggravation through inflammatory cytokine secretion.

Keywords: Cytokines; monocytes; osteoarthritis; synovial fluid..

Figure 1. Positive correlation between sCD14 and IL-6 on OA patients. (a) Expression of CD14, CD16, and CD56 in the synovium from OA patients was analyzed using immunohistochemistry. Representative pictures are shown. Magnification is x100 or x400. Black scale bar is 300 µm and gray scale bar is 60 µm. (b) The levels of inflammatory cytokines and sCD14 were examined in OA synovial fluids according to the manufacturer’s instructions. Scatterplot shows the correlation between sCD14 and IL-6 concentration of synovial fluid from OA patients (n=35). (c) CD14+ cells were isolated from OA SFMC (n=15) and were incubated in FBS containing RPMI at 37°C in 5% CO2. After 24 h, the supernatants were analyzed according to the manufacturer’s instructions. Each symbol represents an individual donor. The resulting Spearman’s correlation coefficient (p) and corresponding p value are reported. CD: Cluster of differentiation; SF: Synovial fluid; OA: Osteoarthritis; RPMI: Roswell Park Memorial Institute; SFMC: Synovial fluid mononuclear cells; FBS: Fetal bovine serum; IL-6: Interleukin-6; C3: Complement component 3.

Figure 2. Synergistic increase of inflammatory mediators with sCD14/LPS treatment in FLS. Isolated FLS from patients (n=3) with OA were stimulated with recombinant CD14 (0.5 µg/mL) and LPS (100 ng/mL) in RPMI containing 10% FBS at 37°C in 5% CO2. After 24 h, levels of secreted IL-6, IL-8, and MMP-3 in stimulated and unstimulated OA FLS were measured according to the manufacturer’s instructions. The bar represents the mean. Statistical analysis was performed by one-way ANOVA, followed by Duncan’s post hoc test. Different letters (a-c) show values having significant difference at p<0.05. The same letter represents groups that are not statistically significant. IL: Interleukin; MMP-3: Matrix metalloproteinase-3; rCD14: Recombinant CD14; LPS: Lipopolysaccharide; RPMI: Roswell Park Memorial Institute; FBS: Fetal bovine serum; FLS: Fibroblast-like synoviocytes.

Figure 3. Percentage repartition of CD14+ monocytes on SFMC from OA patients. (a) SFMCs were isolated from OA patients and stained with specific markers. The single cells were gated in the isolation SFMC using FSC-A and FSC-H, and monocytes were identified by CD14 and CD16 expressions. Representative data are shown. (b) The population of CD14+ was examined on SFMC from five OA patients who underwent arthrocentesis at both baseline (first) and follow-up (recurrence) times. Each symbol corresponds to one pair from the same individual donor. (c) To study monocyte population, SFMC were isolated and analyzed with a specific marker as schemed. The single cells were gated in the isolation SFMC using FSC-A and FSC-H, and T cells were identified by CD3 and CD4 expressions. To detect monocytes, CD14 and CD16 expressions were gated in singlets. Representative data are shown. (d) SFMC from OA patients (n=26) were divided by CD14 and CD16 expressions and analyzed by flow cytometry. Each symbol represents an individual donor, and the bar represents the mean. p=0.019 (e) The surface expression of TLR4 was detected in a monocyte subset of OA SFMC. Isotype control is represented by the grey line, CD14+CD16- expressed TLR4 is indicated by the black line, and CD14+CD16+ expressed TLR4 is represented by the bold line. Data represent results of one experiment, which was performed in triplicate with similar results. CD: Cluster of differentiation; SFMC: Synovial fluid mononuclear cells; TLR4: Toll-like receptor 4; OA: Osteoarthritis; FSC-H: Forward scatter height; FSC-A: Forward scatter area.