Myelin Induces Inflammation Figure 4. Effect of myelin on NF-kB activation. (A) Bone marrow-derived macrophages were incubated with myelin for indicated time and phosphorylation/degradation of IkB-a were assessed by western blot. (B) Effect of NF-kB inhibitor BAY 11-7082 on myelin-induced IkB-a phosphorylation and degradation. (C) Myelin-induced p65 translocation in macrophages detected by immunofluorescent staining. p65 was stained red and nuclei were stained with DAPI (blue) (Scale bar = 10 mm). (D) Sphingomyelin (25 mg/mL) had no effect on IkB-a phosphorylation and degradation macrophages. (E) MBP (1 mg/mL) had no effect on IkB-a phosphorylation and degradation in bone marrow-derived macrophages. The inhibitor was used for 30-minute pre-incubation before myelin incubation. Phosphorylation levels were normalized to non-phosphorylated total proteins. Similar results were obtained from three independent experiments. doi:10.1371/journal.pone.0009380.g004 translocation can be abolished by pre-incubation of BAY 11-7082 (Figure 4C). The data indicate that myelin rapidly activates NF-kB in macrophages. By contrast, sphingomyelin (25 mg/mL), the major lipid component of myelin and MBP (1 mg/mL) are not able to activate NF-kB pathways (Figure 4D and E). Inhibition of NF-kB Activity Suppresses Myelin-Induced Cytokine Expression We further investigated whether inhibition of NF-kB activity could suppress the myelin-induced cytokine expression. Macro- phages were pre-incubated with BAY 11-7082 for 30 minutes and then incubated with myelin for 12 or 24 hours. BAY 11-7082 significantly inhibited myelin-induced expression of TNF-a, IL-1b, IL-6, IL-12, MIF and CXCL10 at mRNA level (Figure 5A) detected by qRT-PCR and the protein level by ELISA (Figure 5B). These data indicate that myelin is a strong pro-inflammatory stimulus which can enhance the expression of a variety of pro- inflammatory cytokines and chemokines in macrophages by activation of NF-kB. Inhibition of NF-kB activation abrogates the stimulatory effects of myelin on cytokines production by macrophages. Myelin-Induced NF-kB Activation and Cytokine Expression Is CR3-Dependent Binding and phagocytosis of myelin by macrophages is mediated by complement receptor 3 (CR3, CD11b/CD18), a member of integrin family [12,13]. CR3 interacts with many molecules in the extracellular matrix (ECM), such as iC3b and pathogens. Binding of ECM (e.g. fibrinogen, collagen, ICAM-1, and ICAM-2) with integrin, including CR3/MAC-1, can induce integrin activation and then regulate a variety of cell functions, including proliferation, apoptosis, migration and survival. We therefore ascertained whether myelin-induced NF-kB activation and cytokine expression were CR3-dependent. Myelin cannot phosphorylate and degrade IkB-a in bone marrow-derived macrophages from CR3 KO mice (Figure 6A). Furthermore, PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9380