, 2010). To address whether Raf/MEK/ERK signaling in Schwann cells is involved in the recruitment of inflammatory cells, we compared P0-RafTR nerves to control nerve sections using a panel of inflammatory
cell markers (Figures 5 and S5). Remarkably, in nerves from injected P0-RafTR mice, we observed a large increase in the number of macrophages, GSK 3 inhibitor mast cells, neutrophils, and T cells, all of which have been shown to be recruited into nerves following injury (Figures 5A–5E and S5A–S5C). These results were confirmed by analysis of EM sections, where large numbers of macrophages and mast cells could be seen (Figures 5A and S5C). However, one cell type, fibroblasts, which are found in large numbers in damaged nerves were not recruited into P0-RafTR nerves following tamoxifen injection (Figure S5D), suggesting that Schwann cells may not be responsible for recruiting fibroblasts to damaged nerves. Interestingly, the influx of inflammatory cells mirrored the response seen following an injury, with neutrophils entering the nerve at day 3, followed by macrophages, mast cells and T cells at around day 5 with the numbers increasing over time (Hall, 2005 and Mueller et al., 2003). Importantly, at day 5, there was no observable myelin breakdown—suggesting that signals from dedifferentiated
Schwann cells, rather than www.selleckchem.com/products/AZD8055.html axons or myelin debris, are responsible for recruiting inflammatory cells. The simplest explanation for our findings is that substances secreted by Raf-activated Schwann cells were directly responsible for the inflammatory response. We therefore tested whether conditioned medium (CM) from cultures of tamoxifen-treated rat Schwann cells expressing and the RafTR (NSRafER) (Lloyd et al., 1997) was able to attract inflammatory cells in a similar fashion to that seen in vivo. We collected blood from adult rats, purified the white blood cell fraction and found that CM from tamoxifen-treated cells attracted significantly more monocytes, T cells, and granulocytes compared to vehicle-treated controls (Figures 6A and S6A). Moreover, this response was dependent
on signaling through the ERK pathway confirming the specificity of the response. In contrast, CM from tamoxifen-treated cells was unable to attract more fibroblasts suggesting that cytokines produced by dedifferentiated Schwann cells do not promote fibroblast attraction which is consistent with the lack of a fibroblast response in the RafTR nerves (Figure 6A). To determine the Schwann cell-derived molecules which may be involved in mediating the inflammatory response, we reexamined a microarray analysis performed on NSRafER cells (Parrinello et al., 2008) and found that a number of mRNAs encoding secreted factors were upregulated following Raf activation in dedifferentiated Schwann cells (Table 1). These included cytokines, some of which had been previously implicated in attracting inflammatory cells following nerve trauma such as the c-kit ligand and MCP-1 (Perrin et al.