Effects of CD14 Macrophages and Proinflammatory Cytokines on Chondrogenesis in Osteoarthritic Synovium-Derived Stem Cells

We investigated the effects of CD14 macrophages and proinflammatory cytokines on chondrogenic differentiation of osteoarthritic synovium-derived stem cells (SDSCs). Osteoarthritic synovial fluid was analyzed for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and IL-6. Levels of stem cell surface markers in osteoarthritic SDSCs were evaluated using flow cytometry. CD14-negative cells were obtained using magnetically activated cell sorting. We compared chondrogenic potentials between whole cells and CD14-negative cells in CD14(low) cells and CD14(high) cells, respectively. To assess whether nuclear factor-kappa B (NF-kappa B) and CCAAT/enhancer-binding protein beta (C/EBP beta) modulate IL-1 beta-induced alterations in chondrogenic potential, we performed small interfering RNA transfection. We observed a significant correlation between the CD14 ratio in osteoarthritic SDSCs and IL-1 beta and TNF-alpha in osteoarthritic synovial fluid. Phenotypic characterization of whole cells and CD14-negative cells showed no significant differences in levels of stem cell markers. mRNA expression of type II collagen was higher in CD14-negative cell pellets than in whole cell pellets. Immunohistochemical staining indicated higher levels of type II collagen in the CD14-negative cell pellets of CD14(high) cells than in whole cell pellets of CD14(high) cells. As expected, IL-1 beta and TNF-alpha significantly inhibited the expression of chondrogenic-related genes in SDSCs, an effect which was antagonized by knockdown of NF-kappa B and C/EBP beta. Our results suggest that depletion of CD14(+) synovial macrophages leads to improved chondrogenic potential in CD14(high) cell populations in osteoarthritic SDSCs, and that NF-kappa B (RelA) and C/EBP beta are critical factors mediating IL-1 beta-induced suppression of the chondrogenic potential of human SDSCs.