CD11c(+) macrophages and levels of TNF-α and MMP-3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia

Abstract

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage-regulated matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c(+) F4/80(+) CD11b(+) macrophage populations in the ST and AT of 9-month-old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real-time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)-α, MMP-3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced-macrophage depletion were also evaluated by real-time PCR. Expression of TNF-α in CD11c(+) cells, which were isolated by magnetic beads, was compared to CD11c(-) cells. In addition, the effect of TNF-α on cultured synovial fibroblasts and adipocytes was investigated. CD11c(+) F4/80(+) CD11b(+) macrophages were increased in ST and AT of STR/Ort mice. The CD11c(+) cell fraction highly expressed TNF-α. Expression of TNF-α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF-α treatment of cultured synovial fibroblasts and adipocytes markedly up-regulated MMP-3. CD11c(+) F4/80(+) CD11b(+) macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF-α. Our findings suggest that increased numbers of CD11c(+) macrophages and elevated levels of TNF-α and MMP-3 in AT and ST may explain the relationship between hyperlipidaemia and OA.

Keywords: hyperlipidaemia; macrophage; matrix metalloprotease-3; osteoarthritis; tumour necrosis factor-α.

Fig 1

Flow cytometric analysis of CD11c⁺F4/80+CD11b macrophage cells in the synovial tissue (ST) and adipose tissue (AT) of STR/Ort (STR) and C57BL/6J (C57) mice. (a-1,-2, b-1,-2) Dot-plot analysis of F4/80⁺CD11b⁺ cells in ST of C57 (a-1) and STR (a-2) and AT of C57 (b-1) and STR (b-2); x-axis, CD11; y-axis, F4/80. (a-3,-4, b-3,-4) Histogram analysis of CD11c⁺ in cells in gated regions in the dot-plot in ST of C57 (a-3) and STR (a-4) and AT of C57 (b-3) and STR (b-4). Percentage of F4/80- and CD11b-positive cells in ST of C57 and STR (a-5) and AT of C57 and STR (b-5) in gated regions of the dot plot (n = 5). Percentage of CD11c⁺ cells in F4/80- and CD11b-positive gated regions is shown in a-6 (AT) and b-6 (AT) (n = 5).

Fig 2

Real-time polymerase chain reaction (PCR) analysis for the expression of the CD11c, tumour necrosis factor (TNF)-α, matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in synovial (ST; a–d) and adipose tissues (AT; e–h) of C57BL/6J (C57) and STR/Ort (STR) mice. *Statistically significant difference between C57 and STR mice. All data are presented as the mean ± standard error (s.e.) (n = 10).

Fig 3

Flow cytometric analysis of CD11c⁺F4/80⁺CD11b macrophage cells in synovial tissue (ST) and adipose tissue (AT) of phosphate-buffered saline (PBS)-injected STR/Ort mice [phosphate-buffered saline (PBS)] and clodronate-injected STR/Ort mice (Clo). (a-1,-2, b-1,-2) Dot-plot analysis of F4/80⁺CD11b⁺ cells in ST of PBS (a-1) and Clo (a-2) and AT of PBS (b-1) and Clo (b-2); x-axis, CD11; y-axis, F4/80. (a-3,-4, b-3,-4) Histogram analysis of CD11c⁺ in cells in gated regions in the dot-plot in ST of PBS (a-3) and Clo (a-4) and AT of PBS (b-3) and Clo (b-4). Percentage of F4/80- and CD11b-positive cells in ST of PBS and Clo (a-5) and AT of PBS and Clo (b-5) in gated regions of the dot-plot (n = 5). Percentage of CD11c⁺ cells in F480- and CD11b-positive gated regions is shown in a-6 (ST) and b-6 (AT) (n = 5).

Fig 4

Real-time polymerase chain reaction (PCR) analysis for the expression of the CD11c, tumour necrosis factor (TNF)-α, matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in synovial (ST; a–d) and adipose tissue (AT; e–h) of phosphate-buffered saline (PBS)-injected STR/Ort (PBS) and clodronate-injected STR/Ort (Clo) mice. *Statistically significant difference between PBS and Clo mice. All data are presented as the mean ± standard error (s.e.) (n = 10).

Fig 5

Expression of CD11c, tumour necrosis factor (TNF)-α, matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in CD11c-negative and -positive cell fractions of synovial (ST) and adipose tissues (AT) of STR/Ort mice. All data are presented as the mean standard error (s.e.) of three experiments (n = 3).

Fig 6

Effect of TNF-α on matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) expression in cultured synovial fibroblasts and adipocytes. Expression of MMP-3 in synovial fibroblasts (a) and adipocytes (c); expression of ADAMST4 in synovial fibroblasts (b) and adipocytes (d). All data are presented as the mean ± standard error (s.e.) of four experiments (n = 4).