Myelin Induces Inflammation Figure 5. Effect of inactivation of NF-kB on myelin-induced cytokine expression in macrophages. (A) Macrophages were pre-treated with BAY 11-7082 for 30 minutes and then treated with myelin for 12 hours. Cytokines at transcriptional level were detected by qRT-PCR. (B) Macrophages were pre-treated with BAY 11-7082 for 30 minutes and then treated with myelin for 24 hours. Cytokines at protein level were detected by ELISA. Data were expressed as mean 6 SEM and similar results were obtained from three independent experiments. (n = 3, *P,0.05 vs. control; #P,0.05 vs. BAY treatment; t-test). doi:10.1371/journal.pone.0009380.g005 macrophages from CR3 KO mice did not respond to myelin by increasing mRNA (Figure 6B) and protein expression (Figure 6C) of cytokines. Myelin Induces FAK Phosphorylation Next we studied to reveal the upstream molecules involved in myelin-induced NF-kB activation. Myelin interacts with CD11b subunit of CR3 and the ligand-CR3 interaction regulates a variety of biological functions through activation of its downstream effector, focal adhesion kinase (FAK) [14,15]. FAK is a protein tyrosine kinase that is important in serving integrin signaling pathways [16]. Activated FAK interacts with Src family kinases or PI3K to initiate downstream signaling pathways [17]. We therefore determined whether NF-kB activation by myelin is via FAK activation. Figure 7 shows that FAK underwent transient FAKTyr-576/577 phosphorylation at 5 minutes after myelin treatment in macrophages. The phosphorylation of FAK (pFAK) by myelin was also CR3-dependent as myelin failed to phosphorylate FAK in macrophages from CR3 KO mice (Figure 7). These results suggest that myelin induces rapid FAK phosphorylation in CR3-dependent manner. PI3K/Akt Serves as Upstream Kinases for NF-kB Activation by Myelin FAK can activate CrK/CAS, PI3K and Grb2/Ras cell signaling pathways to regulate cell proliferation, motility, differ- PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9380