Nerve growth factor regulation and production by macrophages in osteoarthritic synovium

Abstract

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)-associated pain. Although recent studies suggest that tumour necrosis factor (TNF)-α and interleukin (IL)-1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF-α and IL-1β in freshly isolated CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL-1β and TNF-α on NGF mRNA expression in cultured CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF-α and IL-1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF-α and IL-1β mRNA levels in CD14-positive cells from the SYN of OA patients was significantly higher than that in CD14-negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14-positive and -negative cells with IL-1β and TNF-α enhanced NGF mRNA and protein levels. Expression of NGF, IL-1β and TNF-α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL-1β and TNF-α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

Keywords: macrophage; nerve growth factor; osteoarthritis; synovium.

Figure 1

Flow cytometric analysis of CD14‐negative and ‐positive synovial cells obtained from osteoarthritis patients. (a) Dot‐plot analysis of CD45⁺CD14⁺ cells in CD14‐positive fractions. x‐axis, CD45; y‐axis, CD14. (b–f) Dot‐plot analysis of CD14‐negative fractions. x‐axis, CD45; y‐axis, CD90 (b), CD31 (c), CD19 (d), CD3 (e), CD14 (f). Data represent mean ± standard error (s.e.) (n = 5). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Quantitative polymerase chain reaction (PCR) analysis of CD14, CD90, tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and nerve growth factor (NGF) mRNA levels in CD14‐negative and ‐positive synovial cells obtained from osteoarthritis patients. (a) CD14, (b) CD90, (c) TNF‐α, (d) IL‐1β and (e) NGF. Data represent mean ± standard error (s.e.) (n = 10). *Statistical difference between CD14‐negative‐ and ‐positive cells.

Figure 3

Flow cytometric analysis of CD14‐negative and ‐positive fractions in culture. (a and b) Dot‐plot analysis of CD45⁺CD14⁺ cells in the cultured CD14‐positive fraction (a) and CD14‐negative fraction (b). x‐axis, CD45; y‐axis, CD14. (c,d) CD45^–CD90⁺ cells in the cultured CD14‐positive fraction (c) and CD14‐negative fraction (d). x‐axis, CD45; y‐axis, CD90. (e) Percentage of CD45⁺CD14⁺ and CD45^–CD90⁺ cells in cultured CD14‐negative and ‐positive fractions. Data represent mean ± standard error (s.e.) (n = 5). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Effect of tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β on nerve growth factor (NGF) mRNA expression in synovial CD14‐negative and ‐positive fractions in culture. (a,b) Quantitative polymerase chain reaction (qPCR) analysis of CD14 (a) and CD90 (b) mRNA expression in cultured CD14‐negative and ‐positive fractions. (c) qPCR analysis of NGF mRNA expression in cultured synovial cells obtained from the knees of osteoarthritis (OA) patients following the treatment of CD14‐negative (white bars) and ‐positive cells (black bars) with α‐minimum essential culture medium (α‐MEM), 50 ng/ml human recombinant IL‐1β or 10 ng/ml human recombinant TNF‐α. Data represent mean ± standard error (s.e.) (n = 10). ^aStatistical difference between CD14‐negative and ‐positive cells under the same conditions; ^bstatistical difference between cytokine‐stimulated and non‐stimulated cells in each fraction.

Figure 5

Nerve growth factor (NGF) protein levels in the culture supernatant of synovial CD14‐negative and ‐positive fractions. NGF concentrations in the culture supernatants of CD14‐negative (white bars) and ‐positive cells (black bars) treated with or without human recombinant tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β were examined by enzyme‐linked immunosorbent assay (ELISA). NGF concentration was normalized to the number of cells in each well. Data represent mean ± standard error (s.e.) (n = 10). ^aStatistical difference between CD14‐negative (white bars) and ‐positive cells (black bars) under the same conditions; ^bstatistical difference between cytokine‐stimulated and non‐stimulated cells in each fraction.

Figure 6

Flow cytometric analysis of spleen‐derived CD11b⁺ F4/80⁺ macrophages following clodronate liposome treatment. Dot‐plot analysis of F4/80⁺ (x‐axis) and CD11b⁺ (y‐axis) cells from the spleens of STR/Ort mice injected with either phosphate‐buffered saline (PBS) (a) or clodronate liposomes (b). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7

Effect of macrophage depletion on synovial tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and nerve growth factor (NGF) expression in STR/Ort mice. Quantitative polymerase chain reaction (qPCR) analysis of TNF‐α (a), IL‐1β (b) and NGF (c) in the synovium of STR/Ort mice injected with phosphate‐buffered saline (PBS) or clodronate‐laden liposomes (Clo). *Significant difference between PBS and Clo groups. Data represent mean ± standard error (s.e.) (n = 10). (d) Western blot analysis of NGF in the synovium of STR/Ort mice injected with PBS or Clo.