Effects of CD14 macrophages and proinflammatory cytokines on chondrogenesis in osteoarthritic synovium-derived stem cells

Abstract

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-1β (IL-1β), tumor necrosis factor-α (TNF-α), 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-κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ) modulate IL-1β-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-1β and TNF-α 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-1β and TNF-α significantly inhibited the expression of chondrogenic-related genes in SDSCs, an effect which was antagonized by knockdown of NF-κB and C/EBPβ. 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-κB (RelA) and C/EBPβ are critical factors mediating IL-1β-induced suppression of the chondrogenic potential of human SDSCs.

FIG. 1.

Levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and CD14 in synovial fluid and correlation of IL-1β and TNF-α levels with CD14 levels of synovium-derived stem cells (SDSCs) in osteoarthritis patients. (A) IL-1β, TNF-α, and IL-6 levels in the synovial fluid of osteoarthritis patients were measured by enzyme-linked immunosorbent assay (ELISA). (B) Phenotype analysis of the expression of CD14 marker was conducted by flow cytometry in osteoarthritic CD14^high and CD14^low SDSCs. CD14^low indicates the CD14^low cell group and CD14^high indicates the CD14^high cell group. (C) Synovial fluid levels of IL-1β and TNF-α correlate with the CD14 ratio of CD14^low SDSCs in osteoarthritis patients. (D) Synovial fluid levels of IL-1β and TNF-α correlate with the CD14 ratio of CD14^high SDSCs in osteoarthritis patients. r=Spearman's correlation coefficient. p<0.05.

FIG. 2.

Flow cytometric analysis of whole cells and CD14-negative SDSCs. Cell surface marker expression of whole cells of CD14^high SDSCs (A), CD14-negative cells of CD14^high SDSCs (B), whole cells of CD14^low SDSCs (C), and CD14-negative cells of CD14^low SDSCs (D), showing negativity for CD14, CD31, and CD34 and positivity for CD44, CD90, and CD105.

FIG. 3.

Effects of CD14-negative SDSCs on chondrogenesis. (A) Analysis of the expression of chondrogenesis-related genes by real-time polymerase chain reaction (PCR) after chondrogenic differentiation in whole cells and CD14-negative cells of SDSCs. The expression of marker genes was calculated as the percentage of the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CD14^low indicates the CD14^low cell group and CD14^high indicates the CD14^high cell group. Col I, type I collagen; Col II, type II collagen; Col X, type X collagen. (B) Glycosaminoglycan (GAG) synthesis in pellets increased production levels in CD14-negative cells of CD14^high cells. (C) Safranin-O staining and immunohistochemical analysis. Extracellular matrix formation was evident by Safranin-O staining, and type I and II collagen levels were elevated in CD14-negative cell pellets of CD14^high cells. Magnification,×100. Color images available online at www.liebertpub.com/tea

FIG. 4.

Effects of IL-1β or TNF-α on chondrogenesis. (A) Effects of CD14-positive cells with 1 μg/mL lipopolysaccharide (LPS) on IL-1β or TNF-α determined by ELISA. (B) Effects of IL-1β or TNF-α on expression of chondrogenesis-related genes determined by real-time PCR (*p<0.05 vs. control). (C) Effects of IL-1β on p300, cAMP-response element-binding protein-binding protein (CREBBP), CCAAT/enhancer-binding protein β (C/EBPβ), and nuclear factor-κB (NF-κB) gene expression determined by real-time PCR in CD14-negative cells of SDSCs (*p<0.05 vs. control). (D) Effect of IL-1β treatment on collagen type II as shown by fluorescent immunohistochemical analysis in CD14-negative cells of SDSCs (green, Col II; blue, 4′,6-diamidino-2-phenylindole [DAPI]) (magnification,×100). (E) Effect of IL-1β treatment on proteoglycan levels determined by safranin-O staining (Saf-O, safranin-O) (magnification,×100). Color images available online at www.liebertpub.com/tea

FIG. 5.

Effects of NF-κB and C/EBPβ small interfering RNAs (siRNAs) on chondrogenesis. Real-time PCR (A) and western blot (B) for NF-κB and C/EBPβ knockdown. (C) NF-κB and C/EBPβ knockdown with 1 ng/mL IL-1β enhances chondrogenic gene expression in CD14-negative cells of SDSCs. (siCon, control siRNA; siNF-κB, NF-κB siRNA; siC/EBPβ, C/EBPβ siRNA; siN+siC, NF-κB siRNA, and C/EBPβ siRNA) (*p<0.05 vs. control). (D) The effects of NF-κB and C/EBPβ knockdown with 1 ng/mL IL-1β on Col II levels assessed by fluorescent immunohistochemistry analysis in CD14-negative cells of SDSCs (green, Col II; blue, DAPI) (magnification,×100). Color images available online at www.liebertpub.com/tea