trigger undesirable inflammation in vitro and in vivo. Thus, study on understanding the mechanisms underlying inflammatory reaction induced by myelin is crucial to prevent further neuronal damage and develop the anti-inflammatory treatments of SCI. Our present results demonstrate that myelin debris contributes to inflammatory responses in animal models via stimulating cytokine production. We further show that myelin-increased cytokines expression is via activation of NF-kB through FAK/ PI3K/Akt signaling pathway and complement receptor 3 (CR3)- dependent. Inhibiting NF-kB activation abrogates myelin-induced cytokine production on macrophages. Our study provides the first direct evidence that myelin-CR3 interaction triggers undesirable inflammation in vitro and in vivo. Methods Reagents and Antibodies All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. The BAY 11-7082 was obtained from Biomol (Plymouth Meeting, PA). The antibodies against FAK, phospho-FAK (Tyr-576/577), p85, phospho-p85 (Tyr-458), Akt, phospho-Akt (Ser-473), IkB-a, phospho-IkB-a (Ser-32/36) and b- actin were purchased from Cell Signaling Technology (Danvers, MA). The rabbit-anti-p65 antibody, goat-anti-MIF antibody and rabbit-anti-MIF antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit-anti-IBA-1 antibody was from Wako USA (Richmond, VA). Rat-anti-Ly-6G (Gr-1) antibody was obtained from eBioscience (San Diego, CA). Alexa 546-conjugated goat-anti-rabbit IgG, FITC-conjugated rabbit- anti-rat IgG, HRP-conjugated goat-anti-rabbit IgG and HRP- conjugated rabbit-anti-mouse IgG antibodies were from Invitro- gen (Carlsbad, CA). Cells and Mice Bone marrows were harvested from C57BL/6 wild type (WT) and CR3 knockout (KO) mice (CD11b deleted) (The Jackson Laboratory, Bar Harbor, Maine) and cultured in DMEM containing 10% FBS and 15% L929 cells-conditioned medium as a source of M-CSF. Bone marrow-derived macrophages were used after 7–10 days of culture. All mice were maintained in pathogen-free animal facility in Rutgers University. Animal protocols were approved by Animal Care and Facilities Commit- tee in Rutgers University. Myelin Preparation Myelin was isolated from the brains of 3-month-old mice by sucrose density gradient centrifugation (0.32 M and 0.85 M) at 750006g, as described previously [10]. The protein content of myelin was determined by Bradford method with bovine serum albumin as the standard. The endotoxin concentration of myelin debris was below the detection limit by Limulus Amebocyte Lysate assay. Myelin was used to stimulate cells with concentration of 100 mg myelin protein per milliliter in all experiments. Myelin Stimulation in Peritoneal Cavity C57BL/6 WT and CR3 KO mice were injected i.p. with myelin (25 mg/kg body weight) or PBS alone for control. Mice were sacrificed at different time points and peritoneal lavage was performed. Cells in peritoneal lavage fluid were counted and stained with Diff-Quick staining kit (IMEB Inc., San Marcos, CA). Remaining lavage fluids were centrifuged at 4006g for 10 minutes and the cytokines in supernatants were detected by ELISA. Myelin Induces Inflammation Myelin Injection in Spinal Cord Myelin in PBS was adjusted to 125 mg/kg body weight and a total volume of 0.5 ml was stereotaxically injected into the spinal cord at T8–T10 vertebrae by a T10 laminectomy using a microliter syringe (Hamilton Company, NV) fixed in a stereotaxi frame. Injection of PBS alone was used for control. Mice were sacrificed and perfused at 1, 4 and 7 days after injection, and spinal cords were fixed and frozen sectioned for immunostaining. Immunofluorescent Staining and Quantitative Analysis Cells were seeded in 24-well plate at 16105 cells/well and incubated with culture medium containing myelin for different periods of time and then fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature (RT). For the frozen section, spinal cord tissues were fixed in 4% PFA at 4uC overnight. After being washed with PBS for three times, cells or tissues were blocked in PBS with 1% BSA, 0.4% Triton X-100 for 20 minutes, then incubated with primary antibody for at 4uC overnight. After the incubation with secondary antibody for 1 hour, all images were acquired at RT using Zeiss Axiovert 200 M Microscope (Carl Zeiss, Germany) with 206/0.4, equipped with AxioCam camera (Carl Zeiss, Germany) and software Axiovision 4.6 (Carl Zeiss, Germany). Injection regions and marginal regions were evaluated, six fields within each region were randomly selected. The numbers of Ly-6G and IBA1 positive cells were counted and divided by areas for each field (pixel6pixel). Numbers of cells per 100,000 pixel6pixel area were obtained as cell numbers per arbitrary unit for further analysis. Western Blot Cells were washed with PBS, and directly lysed in RIPA buffer containing phosphatase inhibitors and proteinase inhibitors. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the gel were transferred onto nitrocellu- lose membranes (GE Healthcare, UK) that were blocked with 5% milk in Tris-Buffered Saline containing 0.1% Tween 20 (TBST) for 1 hour at RT. Afterwards, the membranes were incubated with the indicated primary antibodies overnight at 4uC. After being washed with TBST, the membranes were incubated with the appropriate secondary antibodies. The immunoreactive bands were detected by using ECL Plus Western Blotting Detection System (GE Health- care, UK) and analyzed by Kodak Molecular Imaging Software (v4.0.4) for intensities. Phosphorylation levels were shown by normalizing intensities of phosphorylated proteins to those of non- phosphorylated total proteins. RNA Isolation and Quantitative Real-Time (qRT)-PCR Cells were incubated with myelin for 6 and 12 hours with or without the pre-incubation of inhibitors. Total RNA was isolated by TRIZOL method under the instruction of user manual and reverse-transcribed into cDNA by using oligo-dT primers and SuperScript II reverse transcriptase (Invitrogen). The primers used were shown in Supporting Information Table S1. The ABI 7900HT detection system (Applied Biosystems, UK) was used for qRT-PCR. SYBR Green dye (Applied Biosystems, UK) was used to monitor the replication of PCR products. Quantities of products were obtained by standard curve and then normalized to GAPDH quantity. The gene expression level was represented by the ratio of gene/GAPDH. Enzyme-Linked Immunosorbent Assay (ELISA) The supernatants of cells incubated with myelin were collected for detection of mouse TNF-a, IL-1b and MIF. ELISA for mouse PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9380