In vivo imaging of NF-кB activity and correlation to pain in a model of inflammatory arthritis

Abstract

locomotion. Ccr2 null mice were protected from these locomotion decreases, despite similar joint damage at 8 weeks post DMM, and Ccr2 null DRG did not produce MCP-1. Therefore, we sought to further investigate the expression and regulation of MCP-1 in the DMM model. The current goals were to: 1) Test whether TNF-a induces MCP-1 production in DRG cells, since it has been shown that TNFa induces Mcp-1 mRNA in cultured DRG cell lines. 2) Analyze MCP-1 protein content in the knees of wild-type (WT) and Ccr2 null mice after DMM. Methods: Knee-innervating DRG, L3-L5, were collected from 10-week old naïve C57BL/6 WT or Ccr2 null mice. Cells were isolated and cultured for 2 days in basal medium before 48 h-stimulation with 0, 25, or 100 ng/mL TNF-a; supernatants were collected for MCP-1 ELISA. Hip cartilage explants were harvested from 5-week old naove C57BL/6 mice. Hip cartilage was used since it is not possible to culture mouse knee cartilage explants. Explants were rested overnight, stimulated with 100 ng/mL TNF-a for 48 h, and supernatants were collected for MCP-1 ELISA. DMM or sham surgery was performed in the right knees of 10-week old male C57BL/6 WT or Ccr2 null (Taconic #3736) mice. At 0, 4, and 8 weeks after surgery, whole knee joints were extracted for ELISA. At 4 or 8 weeks post surgery, DRG cells were cultured for 4 days and supernatants collected for ELISA. Results: In order to test whether TNF-a is able to stimulate primary DRG cells to produce MCP-1 protein, we treated naïve WT DRG cells with TNF-a. Cultures stimulated with 25 ng/mL TNF-a produced 37-fold increased amounts of MCP-1 compared to unstimulated cells (p<0.0001); a higher concentration of 100 ng/mL TNF-a did not further increase MCP-1. This confirms earlier reports of increased Mcp-1 mRNA following TNF-a stimulation. DRG cells from naïve Ccr2 null mice responded in the same way to TNF-a. We looked for the presence of TNF-a in cultures of DRG cells taken from WT or Ccr2 null naïve, sham, or DMM mice at 4 and 8 weeks post surgery. None of these cultures contained measurable amounts of TNF-a. Next, we determined MCP-1 levels in whole knee joint extracts after DMM in WT and Ccr2 null mice. In WT, knee MCP-1 protein levels were elevated 4 weeks post DMM, compared to sham and naïve age-matched controls (p<0.0001); by 8 weeks post DMM, levels had returned to baseline. MCP-1 was also increased in knee extracts from Ccr2 null mice, 4 weeks after DMM, but to a lesser extent than in WT. In order to determine the potential source of MCP-1 in the knee, we began by testing whether cartilage is able to produce MCP-1. Cartilage explants stimulated with 100 ng/mL TNF-a produced 86-fold increased levels of MCP-1 compared to unstimulated explants (p¼0.0008). When we looked for the presence of TNF-a in knee extracts, however, we found that DMM and naïve extracts contained similar low levels of TNF-a at 4 and 8 weeks post surgery. TNF-a levels in Ccr2 null knee extracts mirrored the WT results, with no difference at 4 or 8 weeks between DMM and naïve extracts. Conc...