Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin
© Hadas et al.; licensee BioMed Central Ltd. 2012
Received: 21 March 2012
Accepted: 7 June 2012
Published: 9 July 2012
Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3) is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase) and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin) remodeling (i.e., disassembly and reassembly) by shifting between active unphosphorylated and inactive phosphorylated states.
Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin) decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia), which, as we also revealed, are instrumental in myelin phagocytosis.
CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive phosphorylated state of cofilin. Self-negative control of phagocytosis by the phagocytic receptor can be useful in protecting phagocytes from excessive phagocytosis (i.e., “overeating”) during extended exposure to particles that are destined for ingestion.
KeywordsMicroglia Macrophage Phagocytosis Myelin Complement receptor-3 Syk Cofilin
Intact myelin, which normally surrounds axons, breaks down during Wallerian degeneration following traumatic injury to axons [1–3] and during neurodegenerative diseases such as multiple sclerosis . Degenerated myelin thus formed impedes repair and exacerbates damage by arresting regeneration [5–7], inhibiting remyelination  and advancing production of membrane attack complexes [9, 10]. Therefore, rapid clearance of the degenerated myelin is critical for repair of the injured nervous system. Regarding this, it is important to elucidate the mechanisms that regulate degenerated myelin phagocytosis. We focused in this study on how Syk and cofilin regulate the myelin phagocytosis that CR3 (complement receptor-3; αMβ2 integrin) mediates in primary microglia and macrophages; the term "myelin" will replace "degenerated myelin" from here onward for simplicity.
The principal phagocytic receptors for myelin on macrophages and microglia are CR3, SRA (scavenger receptor-AI/II), and Fcγ [11–14]. CR3 functions both as a C3bi/opsonic and unopsonic receptor for C3bi-opsonized and unopsonized myelin, respectively. Unopsonic SRA primarily mediates phagocytosis of unopsonized myelin. However, Fcγ, but not CR3 and SRA, requires prior opsonization of myelin by anti-myelin Abs. We studied myelin phagocytosis by CR3 and SRA in the absence of anti-myelin Abs.
Syk is a non-receptor tyrosine kinase that phagocytic receptors Fcγ, CR3, and Dectin-1 recruit upon their activation [15–19]. However, Syk may or may not activate phagocytosis depending on the type of phagocyte, the identity of the phagocytic receptor, and the nature of the ingested particle [20–24]. It is important, therefore, to study each phagocyte, receptor, and phagocytosed particle combination on its own.
We document in this study that CR3 activated Syk, which in turn downregulated CR3-mediated phagocytosis of C3bi-opsonized and unopsonized myelin in CNS microglia and bone marrow-derived macrophages by advancing the inactive phosphorylated state of cofilin (Figure 1). Syk-dependent downregulation of phagocytosis and Syk-dependent control of cofilin activation have not been reported previously. These observations suggest a novel mechanism by which CR3 can both activate and downregulate myelin phagocytosis in parallel. This self-negative control of phagocytosis by CR3 can be useful in protecting phagocytes from excessive phagocytosis during extended exposure to degenerated myelin and possibly also to other particles that are destined for ingestion.
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.
Syk is activated during phagocytosis of degenerated myelin
Syk down-regulates CR3-mediated myelin phagocytosis
Filopodia like membrane protrusions are involved in myelin phagocytosis
Syk down-regulates cofilin activation by advancing the inactive phosphorylated state of cofilin during myelin phagocytosis
We predicted and then tested the following based on the inverse relationships between levels of phagocytosis and levels of p-cofilin. First, Syk-KD and control microglia will phagocytose about the same after 10 min since the two displayed similar levels of p-cofilin at that time. Second, Syk-KD microglia will phagocytose more than control microglia after 30 min since Syk-KD microglia displayed substantially lower levels of p-cofilin than control microglia at that time. Indeed, these predictions were confirmed as Syk-KD and control microglia phagocytosed about the same after 10 min, whereas Syk-KD microglia phagocytosed considerably more than control microglia after 30 min (Figure 6E and F).
We document in this study that CR3 downregulates the phagocytosis of C3bi-opsonized and unopsonized myelin in CNS microglia and bone marrow-derived macrophages through Syk, which advances the inactive phosphorylated state of cofilin. We previously documented that CR3 activates this phagocytosis through K-Ras and its downstream effector molecules in a galectin-3-dependent manner. Taken together, CR3 can both activate and downregulate myelin phagocytosis at the same time (Figure 1).
Syk down-regulates CR3-mediated phagocytosis of C3bi-opsonized and unopsonized myelin
Syk-dependent downregulation of CR3- but not SRA-mediated phagocytosis of C3bi-opsonized and unopsonized myelin is suggested by the following observations. First, Syk was continuously phosphorylated, thus continuously activated, during prolonged phagocytosis. Then, SYK inhibitor augmented phagocytosis in SRA−/− microglia where phagocytosis is CR3-dependent and SRA-independent, but not in CR3−/− microglia, in which phagocytosis is SRA-dependent and CR3-independent. In accord, SYK inhibitor augmented myelin phagocytosis, which is mediated by CR3 and SRA combined in wild-type BalbC and C57BL microglia and macrophages, and in Syk-KD microglia that express reduced Syk levels.
Previous studies documented that Syk either activated or did not regulate phagocytosis at all depending on the type of phagocyte, the identity of the phagocytic receptor, and the nature of the ingested particle. Syk activated FcγR-mediated phagocytosis of IgG-opsonized erythrocytes in macrophages [20, 21], and Syk activated CR3-mediated phagocytosis of C3bi-opsonized zymosan in transformed HL60 cells , but not in primary macrophages ; finally, Syk activated Dectin-1-mediated phagocytosis of unopsonized zymosan in NIH 3T3 fibroblasts transfected with Dectin-1 , but neither in primary macrophages nor the macrophage cell line RAW164.7 [23, 24]. We presently add the novel category of Syk-dependent downregulation phagocytosis, namely the phagocytosis of C3bi-opsonized and unopsonized myelin by CR3 in primary microglia and macrophages. Our present findings suggest further that the occurrence of Syk-dependent downregulation of myelin phagocytosis is also determined by the identity of the phagocytic receptor since SYK-inhibitor augmented myelin phagocytosis by CR3 and not by SRA.
The two pharmacological inhibitors of Syk that we used produced contrasting effects, SYK-inhibitor augmented whereas piceatannol inhibited myelin phagocytosis. Of the two, SYK inhibitor (IC50 value of 15nM)  is Syk-specific in our assay system since knocking down Syk expression augmented myelin phagocytosis, as did SYK inhibitor. Inhibition by piceatannol can be explained on two grounds. First, our previous observations showed that PKA (protein kinase-A), PKC (protein kinase-C), and MLCK (myosin light chain kinase) activated myelin phagocytosis (Figure 1 and [30–33]). Second, piceatannol inhibits Syk, PKA, PKC, and MLCK with extremely close and also overlapping IC50 values (10, 3, 8, and 12 μM, respectively; Calbiochem). Therefore, it is very likely that piceatannol reduced myelin phagocytosis by inhibiting PKA, PKC, and MLCK alone or combined. Of note, also others have questioned the specificity of piceatannol towards Syk in some cell types [38–40].
Cofilin regulates myelin phagocytosis
Cofilin-dependent regulation of myelin phagocytosis is suggested by the findings that levels of phagocytosis correlated positively with shifting the balance from inactive p-cofilin to active cofilin (Figure 1). First, control microglia displayed transient reduction in p-cofilin levels during prolonged phagocytosis. Second, microglia in which Syk was either inhibited or its expression reduced displayed augmentation of prolonged phagocytosis that was associated with a continuous decrease in p-cofilin. Third, control and Syk-KD microglia phagocytosed about the same at times when both displayed similar levels of p-cofilin.
Cofilin could regulate myelin phagocytosis through its ability to control remodeling of F-actin and thereby production of membrane protrusions [26–28]. This proposition is supported by our present and previous observations. Live imaging in this study revealed the involvement of filopodia-like membrane protrusions in myelin phagocytosis. Additionally, we demonstrated previously that remodeling of F-actin localized preferentially to sites of myelin phagocytosis, and, furthermore, that RhoA and its effector ROCK downregulated CR3-mediated myelin phagocytosis by stabilizing F-actin  (see also Figure 1).
Syk down-regulates CR3-mediated myelin phagocytosis by advancing the inactive phosphorylated state of cofilin
Syk-dependent inactivation of cofilin is suggested by current observations that p-cofilin levels decreased when Syk activity and Syk expression were reduced. It is unclear, and yet to be determined, how Syk advanced the inactive phosphorylated state of cofilin as it could promote phosphorylation and/or impede dephosphorylation (Figure 1). To the best of our knowledge, Syk-dependent regulation of cofilin activation has not been previously reported.
Finally, the concomitant occurrences of Syk-dependent downregulation of CR3-mediated myelin phagocytosis, Syk-dependent increase in p-cofilin, and the negative correlation between phagocytosis and p-cofilin levels all together suggest that Syk downregulated phagocytosis by advancing the inactive phosphorylated state of cofilin.
CR3 both activates and down-regulates myelin phagocytosis
All together, our present and previous [30–33] observations reveal that CR3 both activated and downregulated myelin phagocytosis, indicating that phagocytosis is controlled by the balance between the two (see also Figure 1). The dominance of activation over downregulation was accomplished, at least in part, by a transient decrease in p-cofilin levels. This transient decrease in p-cofilin was Syk-dependent since reducing Syk activity and expression resulted in a sustained decrease in p-cofilin levels that was further associated with an increase in phagocytosis.
Self-negative regulation of phagocytosis by the phagocytic receptor can be useful in protecting phagocytes from excessive phagocytosis (i.e., “overeating”) during extended exposure to particles that are destined for ingestion. In our study, the phagocytic receptor was CR3 and the ingested particles the tissue debris of degenerated myelin.
actin depolymerizing factor
central nervous system
myelin basic protein
myosin light chain kinase
protein kinase A
protein kinase C
peripheral nervous system
short hairpin RNA
scavenger receptor AI/II
spleen tyrosine kinase
This study was supported by a grant from the Niedersachsen-Israeli Research Cooperation fund.
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