Myelin Induces Inflammation Figure 7. Effect of myelin on FAK phosphorylation. Bone marrow-derived macrophages from WT and CR3 KO mice were treated with myelin for the indicated time and phosphorylation of FAK (pFAK) was assessed by western blot. Phosphorylation levels were normalized to non- phosphorylated total proteins. Similar results were obtained from three independent experiments. doi:10.1371/journal.pone.0009380.g007 entiation and intracellular trafficking [16]. As NF-kB can be activated through many different pathways, including phosphati- dylinositol 3-kinase (PI3K) and its major downstream kinase, Akt [18], we hypothesize that PI3K/Akt contributes to myelin-induced NF-kB activation. Figure 8A shows myelin treatment phosphor- ylated p85 regulatory subunit of PI3K and Akt in macrophages from WT mice but not in macrophages from CR3 KO mice. Pre- incubation with PI3K inhibitor, LY-294002 (50 mM) not only effectively inhibited myelin-induced phosphorylation of p85 in macrophages but also inhibited phosphorylation of IkB-a and its degradation induced by myelin (Figure 8B). The Akt inhibitor IV (1 mM), but not protein kinase C (PKC) inhibitor Go¨ 6983 (5 mM) inhibited IkB-a phosphorylation and degradation in macrophages (Figure 8C). Moreover, phosphorylation of Akt in macrophages can be abolished by both Akt inhibitor IV and PI3K inhibitor LY- 294002, but not by PKC inhibitor Go¨ 6983 and NF-kB inhibitor BAY 11-7082 (Figure 8D), suggesting that Akt is specifically activated by PI3K and Akt is the upsteam molecule of NF-kB. Accordingly, PI3K activating NF-kB was confirmed by immuno- fluorescent staining of p65 as the inhibitor of PI3K (LY-294002) suppressed p65 translocation from cytoplasm to the nuclei (Figure 8E, I, II and III). Moreover, the Akt inhibitor IV and NF-kB inhibitor BAY 11-7082 readily blocked nuclei translocation of p65 by myelin administration (Figure 8E, IV and V). In contrast, Go¨ 6983 had no inhibitory effect on p65 nuclei translocation (Figure 8E, VI). These results suggest that PI3K/ Akt is upstream kinases for NF-kB signaling and myelin activates the PI3K/Akt/NF-kB signaling pathway. We also showed that inhibitors of PI3K and Akt blocked the myelin induced cytokine production (Figure 9). Discussion Trauma and inflammation damage myelin, creating myelin debris. Myelin debris contains several inhibitors of axonal regeneration [19]. Although the inhibitory effect on axonal regeneration has been well documented, little direct evidence is available concerning myelin-induced changes in inflammation in spinal cord injury. Our findings presented in this study indicated that myelin is a strong inflammatory stimulus that CR3-myelin interaction enhances expression of pro-inflammatory mediators, down-regulates the production of anti-inflammatory cytokines in macrophages, and triggers undesirable inflammation in vivo. These data are crucial for our understanding of the role of myelin in CNS injury and inflammation. Myelin debris generated after CNS injury not only inhibits axonal regeneration and CNS remyelination [20] but also stimulates inflammatory responses. In our present study we showed that the myelin induced inflamma- tory response is myelin specific as sphingomyelin (the major lipid of myelin) and MBP (one of the major proteins of myelin) are not able to activate NF-kB signaling pathway. Other myelin specific lipids and proteins may have capacity to activate NF-kB. For example, sulfatide, one of major lipid component of myelin sheath, might be a target autoantigen in MS and EAE [21,22]. Recent in vitro results suggested that sulfatide can activate inflammatory response in microglia cells via activation of MAKPs and NF-kB [23]. It is not clear whether CR3 or other receptors is involved in sulfatide-induced NF-kB activation. Further study is needed to investigate whether NF-kB in macrophages can be activated by other myelin specific lipids and proteins. Myelin not only up-regulates expression of pro-inflammatory mediators such as TNF-a, IL-1b, IL-6, IL-12, MIP-1a and CXCL10 but also down-regulates production of anti-inflammato- ry cytokines such as IL-4 and TGF-b1. These cytokines play important roles in pathology of CNS injury and inflammation. For example, TNF-a and IL-1b are well-documented pathogenic mediators involved in CNS inflammation and axonal degenera- tion. Overexpression of IL-6 and IL-12 induced neurodegenera- tive and inflammatory pathology, respectively [24]. IL-6 and IL-12 deficient mice fail to respond to experimental autoimmune encephalomyelitis (EAE) [25,26]. IL-4, a well-known anti- inflammatory cytokine, has marked inhibitory effects on the expression and release of the pro-inflammatory cytokines [27,28]. TGF-b1 exerts potent anti-inflammatory and neuroprotective properties on a LPS-induced model of CNS inflammation and degeneration [29]. CXCL10 and its receptor CXCR3 play a critical role in multiple sclerosis, particularly in leukocyte recruitment into the CNS [30,31]. Taken together, myelin up- regulation of pro-inflammatory mediators in CNS injury and down-regulation of anti-inflammatory cytokines combine to amply the inflammatory responses. Kerschensteiner et al observed that axonal degeneration and myelin breakdown can happen as early as 30 minutes after injury [32]. In combination with our results, such early myelin debris provides rapid and strong stimuli that stimulate macrophages to produce cytokines including TNF-a and IL-1b and therefore the early increased cytokine expression can be observed after spinal cord injury by Pan et al [33] who showed that cytokines mRNA such as TNF-a, IL-6 and IL-1b were increased during the first 2 hours following spinal cord injury, prior to the appearance of circulating cells. Myelin debris is cleared rapidly and efficiently in peripheral nervous system (PNS) [34,35]. However, myelin debris can persist for years after degeneration of CNS axons as myelin debris can not be efficiently cleared in CNS [36]. Therefore, myelin debris not only provides an early the inflammatory PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9380