IL-1b and TNF-a were performed by Duoset ELISA Develop- ment kit from R&D Systems (Minneapolis, MN). ELISA for mouse MIF was done under the instruction of manual for human MIF ELISA kit from R&D Systems. Statistical Analysis Data in figures are presented as mean 6 SEM, with n representing the number of experiments. Statistical significance was evaluated using Student’s unpaired t-tests or one-way analysis of variance (ANOVA) followed by Dunnett’s post-test for multiple comparisons among groups. Statistical significance was set at P value ,0.05. Results Myelin Is a Direct Inflammatory Stimulus which Induces Inflammation in Animal Models Although it is well documented that degenerated myelin triggers undesirable inflammatory responses in MS and EAE, there has been very little study to show that myelin debris from injured spinal cord can trigger undesirable inflammation directly in vitro and in vivo. We first assessed whether myelin is an inflammatory stimulus in vivo. We used mouse peritonitis model, a well- documented experimental animal model for studying pathogenesis of inflammatory stimuli and evaluating in vivo efficacy of anti- inflammatory and anti-infectious treatments. We administrated myelin (25 mg/kg body weight) or PBS (as control) intraperito- neally (i.p.) to C57BL/6 WT and CR3 KO mice, respectively and then performed peritoneal lavage and collected cells at 0, 4, 24 and 72 hours after the myelin injection. The number of cells in peritoneal lavage rapidly increased after myelin injection in WT mice (Figure 1A) and few neutrophils were found in mice injected with PBS alone. The total number of cells in WT mice at all time points was significantly higher than that of CR3 KO mice (Figure 1B). Furthermore, neutrophil infiltration dramatically increased in WT mice by 4 h after myelin injection and then declined quickly at 24 h after injection (Figure 1C). In comparison, neutrophil infiltration in CR3 KO mice was significantly reduced at all time points (Figure 1C). Macrophage infiltration started at 72 h after myelin injection in WT mice (Figure 1C) and the CR3 KO mice showed the less macrophage infiltration compared to WT mice (Figure 1C). In addition, increased levels of IL-1b and TNF-a in peritoneal lavage fluid were detected at time 4 h and declined after 24 h (Figure 1D) and significantly higher levels of IL-1b and TNF-a were detected in WT mice compared to CR3 KO mice (Figure 1D). By contrast, peritoneal injection of PBS did not induce significant cell infiltration and cytokine production in both WT and CR3 KO mice (Figure 1). We further assessed whether myelin is able to generate inflammatory response in mouse spinal cord. Myelin at total volume of 0.5 mL (125 mg/kg body weight) or PBS was stereotaxically injected into the spinal cord at T8–T10 vertebrae by a T10 laminectomy in WT and CR3 KO mice. Mice were sacrificed at 1, 4 and 7 days after myelin injection. Myelin injection induced large amount of leukocyte infiltration (Figure 2). We assessed the infiltration of macrophages and neutrophils by using IBA-1 (ionized calcium-binding adaptor molecule 1) and Ly- 6G (lymphocyte antigen 6 complex, locus G, also known as Gr-1), respectively. The mouse Gr-1 antigen is a G-protein linked meyloid differentiation marker also known as Ly-6G [11]. Double immunostaining showed that infiltrated neutrophils were Ly-6G positive but negative for IBA-1 (Figure 2A). Kinetics of these cells infiltrated into the spinal cord is much earlier than IBA-1 positive macrophages (Figure 2C, D, E and F). Injection of PBS did not Myelin Induces Inflammation induce significant neutrophil infiltration in either WT or CR3 KO mice (Figure 2B and D). In myelin injection region in the spinal cord from CR3 KO mice, neutrophil infiltration was significantly reduced by more than 50% relative to that in WT mice at all time points after myelin injection (Figure 2C and D). Similar pattern can be observed in the marginal region of myelin injection (Figure 2C and D). After 7 days of myelin injection, few neutrophils can be observed in CR3 KO mice, while neutrophils in the spinal cord from WT mice still can be detected (Figure 2D). In addition, macrophages were not detected within the injection region in either WT or CR3 KO mice at 1 day after myelin injection. The number of macrophages significantly increased by 4 days at injection and marginal region in WT mice (Figure 2E and F). Macrophage infiltration was significantly suppressed in CR3 KO mice at 4 days after myelin injection (Figure 2E and F). However, there were no significant differences for macrophage infiltration between WT and CR3 KO mice at 7 days post- injection (Figure 2F). By contrast, injection of PBS did not induce significant macrophage infiltration in either WT or CR3 KO mice (Figure 2B and F). Effect of Myelin on Pro-Inflammatory Cytokine Expression In Vitro We then determined whether myelin treatment has effects on pro-inflammatory cytokine expression by inflammatory cells such as macrophages. We performed quantitative real-time (qRT)-PCR and ELISA on mouse bone marrow-derived macrophages to measure mRNA and protein levels of cytokines after myelin stimulation. As shown in Figure 3, mRNA levels of several of cytokines increased at 6 h and 12 h after myelin treatment. There was dramatic increase in expression of TNF-a, IL-1b, IL-6 and IL- 12. Macrophage migration inhibitory factor (MIF) and matrix metalloproteinase 9 (MMP9) were also increased by myelin stimulation (Figure 3A). Interestingly, the expression of anti- inflammatory cytokines such as IL-4 and TGFb1 was inhibited by myelin (Figure 3A). In addition to pro-inflammatory cytokines (e.g. TNF-a and IL-1b), expression of chemokines MIP-1a/CCL3 (chemokine (C-C motif) ligand 3) and CXCL10/IP-10 (interferon- inducible protein 10) significantly increased in macrophages stimulated by myelin (Figure 3A). However, myelin did not significantly enhance MCP-1/CCL2 production (Figure 3A). The protein levels of TNF-a, IL-1b and MIF secreted by macrophages treated with myelin were confirmed by ELISA (Figure 3B). Myelin exerts a time-dependent effect on expression of TNF-a, IL-1b and MIF. Myelin Induces NF-kB Activation We investigated signaling pathways triggered by myelin in macrophages. NF-kB is a key participant centrally in the inflammatory response to lipopolysaccharide (LPS) and other pro-inflammatory stimuli. We therefore examined the effects of NF-kB modulation on myelin-induced stimulation of macrophag- es. Myelin treatment increased the level of phosphorylated IkB-a, an inhibitory subunit of NF-kB, from 5 minutes up to 30 minutes (Figure 4A). Meanwhile, there was degradation of total IkB-a after myelin treatment (Figure 4A). Phosphorylation of IkB-a is considered to induce its degradation, which then releases NF-kB and confers it transcriptional activity. Pre-incubation with NF-kB inhibitor BAY 11-7082 (10 mM) for 30 minutes blocked IkB-a activation induced by myelin (Figure 4B). We also did immuno- fluorescence to detect subcellular distribution of p65, a major component of NF-kB. As shown in Figure 4C, p65 (stained as red) was located in the cytoplasm in the absence of myelin. After 90 minutes of exposure to myelin, p65 moved into the nuclei and this PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9380