Wild-type BalbC and C57BL6 mice (Harlan, Israel), and BalbC mice lacking SRAI/II (SRAI/II−/−) or C57BL6 mice lacking αM/CD11b subunit of CR3 (CR3−/−) were used and handled in accordance with the National Research Council’s guide for the care and use of laboratory animals and the approval of the institutional committee.
Primary microglia were isolated from neonate mice brains . In brief, brains were stripped of their meninges, enzymatically dissociated, cells plated on poly-L-lysine-coated flasks for 1 week, replated for 1 to 2 h on bacteriological plates, and non-adherent cells washed away. The vast majority of adherent cells (over 95 %) are microglia judged by morphology and positive immunoreactivity to the murine-specific monocyte, macrophage, and microglia marker F4/80, and the activation markers galectin-3/MAC-2 and CR3. Microglia were propagated by incubation in medium containing 10 % conditioned medium by the L-cell line, which produces M-CSF.
Primary peritoneal macrophages were harvested in cold DMEM/F12 3 to 4 days after intraperitoneal injection of 1.5 ml of 3 % thioglycollate (Difco, USA), plated in the presence of 10 % heat-inactivated FCS in 96-well culture plates (Nunc International, USA) for 2 h, and non-adherent cells washed away. The remaining adhered cells are macrophages since they express F4/80, galectin-3/MAC-2, and CR3 .
Myelin was isolated from mice brains as previously described .
C3bi-opsonization was carried out by pre-incubating myelin in 50 % fresh mouse or rat serum in DMEM/F12 for 40 min at 37° C, followed by washing in serum-free media as previously described [34, 36]. Levels of phagocytosis were the same whether mouse or rat sera were used, indicating similar opsonization efficiency by the two. Additionally, experiments that were performed in the presence of 10 % HI (heat inactivated)-rat serum, 10 % HI-mouse serum, 10 % HI-FCS, or 0.1 % delipidated BSA did not differ, indicating that heat inactivation of the complement system was effective in all sera. We used, therefore, mostly rat serum, which is easier to obtain in the quantities required to opsonize myelin, and HI-FCS as supplement in the phagocytosis assays. The efficiency of C3bi-opsonization was tested in each individual experiment by validating that levels of phagocytosis of C3bi-opsonized myelin are about two fold higher than those of unopsonized myelin.
Microglia or macrophages were plated in 96-well tissue culture plates at a density that minimizes cell-cell contact (2.5 × 104/well) in the presence of DMEM/F12 supplemented with 10 % heat inactivated (HI) rat serum, 10 % HI mouse serum, 10 % HI-FCS, or 0.1 % delipidated BSA (Sigma-Aldrich, Israel), thus in the absence of externally provided complement. Phagocytes were left to rest overnight, washed, and myelin was added for the indicated times. Then unphagocytosed myelin was washed out and levels of phagocytosis quantified. At this time all remaining myelin has already been internalized/phagocytosed . When phagocytosis was assayed in the presence of Syk inhibitors, phagocytes were pre-incubated in the presence of either SYK-inhibitor or piceatannol (Calbiochem, USA), or control/vehicle for 15 min, and phagocytosis assayed thereafter in the continued presence of inhibitor/vehicle.
ELISA assay to quantify myelin phagocytosis is based on the detection of the myelin-specific MBP (myelin basic protein) in lysates of phagocytes . Since MBP is unique to PNS and CNS myelin, and is not produced by phagocytes, MBP levels detected in cytoplasm are proportional to levels of phagocytosed myelin. In brief, after non-phagocytosed myelin is washed away, phagocytes are lysed, lysates transferred to high-protein absorbance plates (Nalge Nunc International, USA), and levels of MBP determined by ELISA using rat anti-MBP (Serotec, Oxford, UK). We determined previously that more than 95 % of the detected MBP arises from phagocytosed/internalized myelin . We further verified the validity of this phagocytosis assay by testing the ability to detect inhibition of myelin phagocytosis by cytochalasin-D .
Generation of microglia with stable reduced Syk and cofilin expression
Reduction of Syk expression was achieved through lentiviral infection of wild-type BalbC microglia with short hairpin RNAs directed against mouse Syk mRNA using pLKO.1 puro plasmids (Sigma-Aldrich, Israel). The shRNA sequence selected in the Syk cDNA coding sequence was 5'CGGCGAAGGGAAAGTATTGCACTACTCGAGTAGTGCAATACTTTCCCTTCGTTT3'. The plasmid was transfected into a 293 T-based packaging cell line, and the resulting culture supernatant was used for lentiviral infection of microglia. Infected microglia were selected on the basis of their resistance to puromycin (Sigma-Aldrich, Israel) brought by the pLKO.1 plasmid, and their level of Syk protein expression was monitored by immunoblotting. As control, microglia were infected in a similar way with the shRNA sequence 5'CTTACGCTGAGTACTTCGA-3' against the non-target firefly Luciferase gene (a gift from Dr. I. Ben-Porath).
Phagocytes were washed in PBS and lysed by incubation in ice-cold lysis buffer (Tris-HCL 1 M, pH 7.5, MgCl2 1 M, NaCl 4 M, 0.5 % NP-40, 0.1 % DTT, and 0.1 % NaVa) supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, Israel), cellular debris was removed by centrifugation, and total protein determined using Bradford reagent (Sigma-Aldrich, Israel). Equal protein content lysates were separated on 10 % SDS-PAGE to detect Syk and 12 % SDS-PAGE to detect cofilin. Proteins were blotted to nitrocellulose membranes, blocked with 10 % non-fat milk or 5 % BSA in TBS (Tris-buffered saline) for 1 h at RT, incubated over night at 4° C in the presence of primary Abs rabbit anti-cofilin, rabbit anti-p-cofilin-1 and rabbit anti-Syk (Santa Cruz Biotechnology, USA), rabbit anti-p-Syk (Cell Signaling, USA), and mouse anti-actin mAb (MP Biomedicals, USA). Blots were washed with TBST, and incubated with respective secondary Abs goat anti-rabbit and goat anti-mouse conjugated to HRP (Jackson ImmunoReserach, USA) for 40 min at RT. Proteins were visualized with EZ-ECL kit for HRP detection (Beit Haemek, Israel). The intensities of immunoblot bands were determined by TINA software, and quantification was carried out as indicated in the figure legends.
Confocal fluorescence microscopy and time-lapse cinematography
Myelin was pre-labeled by DiIc18 (Molecular Probes). Then 1 μl of DiIc18 stock solution (5 mM in DMSO) was added to 1 ml of myelin for 15 min at 37° C; myelin washed (× 3 in culture medium) and then added to macrophages. Macrophage plasma membrane was labeled by dye RH237 (N-(4-sulfutyl)-4-(6-(p-dibutylamynophenyl) hexatrenyl) pyridinium, inner salt (Molecular Probes). A stock solution of 10-mM RH237 in ethanol was diluted before use in culture medium to a final concentration of 0.5 μM. Images were taken in the continuous presence of the dye. Therefore, some of the myelin, which was pre-labeled by DiIc18, acquired some of the RH237. Ca+2 sensor Fluo-4 (Molecular Probes) was used to image the cytoplasm of macrophages by imaging intracellular Ca+2. Macrophages were incubated in Fluo-4/AM solution (50 μg Fluo-4/AM dissolved in 10 μl DMSO to which 500 μl culture medium and 10 μl of 1 μg/μl pluronic acid were added) for 40 min and then washed (× 3 in culture medium).
The system used for confocal imaging consisted of an Olympus microscope IX70 and a Bio-Rad Radiance 2000/AGR-3 confocal imaging system. RH237 was excited at 514 nm (argon laser) and emitted fluorescence collected by a 660-nm low-pass filter. DiIc18 was excited at 543 nm (green HeNe laser) and emitted fluorescence collected at 555–625 nm. Fluo-4 was excited at 488 nm (argon laser) and emitted fluorescence collected at 500-530 nm. The argon laser excitation intensity was usually lowered to 5-10 %. The pinhole was set to 1.6 to 2.5 mm. A single optical slice was continuously but sequentially scanned for DiIc18, RH237, and Fluo-4 (each lasting 9 s). Images were collected and processed using LaserSharp, LaserPix, and LaserVox (BioRad software).
Media products DMEM, DMEM/F12, FCS, HI-FCS, gentamycin sulfate, and L-glutamine were obtained from Biological Industries (Beit-Haemek, Israel).
Mann-Whitney and one- and two-way ANOVA analyses were carried out as indicated in the figure legends.