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The Journal of Immunology, 2006, 176: 3173-3180.
Copyright © 2006 by The American Association of Immunologists

C5b-9 Terminal Complex Protects Oligodendrocytes from Apoptotic Cell Death by Inhibiting Caspase-8 Processing and Up-Regulating FLIP1

Cornelia Cudrici*, Florin Niculescu{dagger}, Timothy Jensen*, Ekaterina Zafranskaia*, Matthew Fosbrink*, Violeta Rus{dagger}, Moon L. Shin{ddagger} and Horea Rus2,*,§

* Department of Neurology, {dagger} Department of Medicine, Division of Rheumatology and Clinical Immunology, {ddagger} Department of Pathology, University of Maryland School of Medicine, and § Veterans Administration Maryland Health Care System Multiple Sclerosis Center of Excellence, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes (OLG) in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In the present study, we examined the possible involvement of the caspase-8 and Fas pathway in OLG apoptosis and the role of C5b-9 in this process. In a serum-free defined medium, OLG undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of cellular FLIP long isoform protein. The caspase-8 inhibitor Z-IETD-FMK inhibited cell death associated with differentiation in a dose-dependent manner. Exposure to C5b-9 induced an inhibition of caspase-8 activation, Bid cleavage, and a significant increase in expression of cellular FLIP long isoform. These C5b-9 effects were reversed by PI3K inhibitor LY294002. C5b-9 also down-regulated the expression of FasL and the Fas-induced apoptosis. These data suggest that C5b-9 through PI3K signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Oligodendrocytes (OLG)3 myelinate the axons of the CNS and undergo apoptosis during development (1, 2). In developing CNS, OLG are selectively rescued from apoptosis by survival signals provided by axonal contact and growth factors (3, 4). Apoptotic death of OLG is induced by a variety of factors, both in vitro and in vivo, including TNF-{alpha}, nerve growth factor, and the interaction of Fas (CD95) with FasL (CD178) (5, 6, 7). Apoptosis of OLG has been documented in lesions of multiple sclerosis (MS) (8, 9, 10), in which Fas expression on OLG is also increased (11, 12, 13). Activation of Fas results in recruitment of the death effector molecule Fas-associated death domain (FADD) to the cytoplasmic tail of Fas (14), which in turn binds procaspase-8 (15), leading to formation of a death-inducing signaling complex (DISC). DISC formation ultimately leads to caspase-8 activation and the full execution of apoptosis (16).

Bid, a BH3-only protein, plays a role in apoptosis initiated by the intrinsic pathway. Activation of caspase-8 leads to cleavage of Bid at Asp59, producing truncated Bid (tBid) (17). This is followed by mitochondrial translocation of tBid, which then induces a conformational change in Bax, causing oligomerization and insertion of Bax into the outer mitochondrial membrane and subsequent release of cytochrome c (18, 19, 20). Regulation of the FADD-caspase-8 proapoptotic signaling pathway is mediated through an intrinsic inhibitor, cellular FLIP (c-FLIP) (21, 22). It exists in long (c-FLIPL) and short (c-FLIPS) isoforms, both of which form heterodimers with procaspase-8 within the DISC and block its activation. c-FLIPS blocks caspase-8 processing, whereas c-FLIPL allows partial processing of both caspase-8. The c-FLIPL, which remain bound to the DISC as 43-kDa form, prevents the recruitment and processing of additional procaspase-8 (23). Recent evidence indicates that c-FLIPL not only functions to block caspase-8 activation but also positively signals the activation of the NF-{kappa}B and ERK pathways (24).

Complement activation with assembly of the terminal complement complex C5b-9, consisting of the C5b, C6, C7, C8, and C9 proteins, plays a significant role in the pathogenesis of a variety of CNS diseases, including MS (reviewed in Ref.25). By forming pores in the plasma membrane, C5b-9 can cause cell death and also induce apoptosis (26, 27, 28). However, OLG, like other nucleated cells, can survive limited C5b-9 complement attack through the protection provided by complement-inhibitory proteins and by elimination of membranes carrying C5b-9 complexes (29, 30). We have recently shown that C5b-9 at sublytic doses inhibits the mitochondrial pathway of apoptosis as well as caspase-3 activation induced in vitro by serum deprivation (31, 32). This C5b-9 effect effectively rescues OLG from apoptosis and this process is mediated by Gi protein-dependent and Gbeta{gamma}-mediated activation of the ERK1 and PI3K/Akt pathways (32, 33, 34). The PI3K/Akt signaling pathway appears to be responsible for phosphorylation of Bad at Ser112 and Ser136 and the dissociation of Bad from Bcl-xL (32). However, it is not clear at present whether caspase-8 activation and FLIP are involved in OLG apoptosis induced by serum deprivation, or how C5b-9 affects caspase-8 and the intrinsic pathway of apoptosis.

In the present report, we demonstrate for the first time that caspase-8 is activated and that FLIP is down-regulated in OLG in response to growth factor withdrawal and this was followed by Bid cleavage. Sublytic C5b-9 inhibited caspase-8 processing and Bid cleavage and increased the level of FLIPL in a PI3K-dependent manner. These findings suggest that sublytic C5b-9, through inhibition of the Fas pathway of apoptosis, may play a significant role in OLG survival in inflammatory CNS disorders.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Primary cultures of OLG progenitor cells and OLG

Neonatal OLG were purified from brains of 1-day-old Sprague-Dawley rats, as described previously (32). After removal of the meninges, the brain were minced and sequentially passed through nylon meshes. The dissociated cell suspension was plated on 75-cm2 plates in DMEM/Ham’s F-12 medium containing 10% FBS. OLG progenitor cells (OPC) were separated from the astrocyte monolayer by shaking overnight at 200 rpm on a rotary shaker. The OPC cell suspension was collected and resuspended in defined medium containing serum-free DMEM/Ham’s F-12, transferrin (500 ng/ml; Sigma-Aldrich), insulin (75 ng/ml; Sigma-Aldrich), basic fibroblast growth factor (75 µg/ml; PeproTech), and 1 mM sodium pyruvate (Sigma-Aldrich). The cells were differentiated at 37°C for 52 h. Over 85% of the cells of the OLG expressed myelin basic protein, proteolipid protein, and galactocerebroside. Fewer than 3% of the cells were negative for myelin basic protein and were astrocytes or OPC in varying stages of differentiation.

Membrane assembly of sublytic C5b-9 using terminal complement proteins

Purified human complement protein C5b6 and C7, C8, C9 were obtained from Quidel and Advanced Research Technologies. In brief, OLG were incubated at 37°C with 18 U of C5b6 and 10 µg/ml each of C7-C9 in a final volume of 2 ml for the indicated periods of time. Inactive C5b-9 was prepared by assembling the complexes from purified proteins in the fluid phase at room temperature. In some experiments, normal human serum (NHS) pooled from several healthy donors was used as a source of serum complement. Rabbit antiserum to galactocerebroside was used to sensitize rat OLG; the cells were then incubated with NHS or K76-NHS (at a final dilution of 1/10) for various time periods. As previously described, K76 COONa (K76; Otsuka Pharmaceuticals) prevents C5b-9 assembly in serum by binding to C5 (33, 35). The concentrations of complement proteins used in this study were sublytic for OLG, as determined by staining cells with the vital dye trypan blue and by measuring release of cytoplasmic lactate dehydrogenase as an indicator of cell death (35).

Functional caspase-8 assay

Activation of caspase-8 was evaluated using a Caspase-8/FLICE Colorimetric Assay kit (BioSource International) according to the manufacturer’s instructions: equal amounts of cell lysate (150 µg) were incubated with 200 µM IETD-pNA substrate for 2 h at 37°C in buffer containing 10 mM DTT. OD values at 405 nm were determined for each sample and corrected for spontaneous release of pNA. The increase in caspase activity is expressed as the fold increases relative to untreated controls. Measurements at all time points were made in triplicate, and the results were expressed as mean ± SEM.

Cell death as assessed by MTS assay

Cell viability was determined using a CellTiter 96 Aqueous cell proliferation assay (Promega). OLG were seeded onto poly-D-lysine-coated 96-well plates at 1 x 105 cells/well in 200 µl of defined medium and cultured at 37°C. After exposure of the cells to the caspase-8 inhibitor Z-IETD-FMK (25–100 µM; EMB Biosciences), methyltetrazolium salt (40 µl) was added at the indicated time points. The plates were incubated for 2 h at 37°C, and the OD was measured at 540 nm. Results were expressed as mean percentage of surviving cells ± SEM using the initial cell number at the beginning of the experiment as 100%. Each sample was assayed in triplicate. The Student t test was used to determine the statistical significance of the results.

Immunoprecipitation and Western blot analysis

Immunoprecipitation and Western blotting were performed as previously described (33). OLG were washed with PBS before lysis in a buffer consisting of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton X-100, 0.1% SDS, 100 mM NaCl, 10% glycerol, 0.5% sodium deoxycholate, and 1 mMNa3VO4. One tablet of complete mini protease inhibitor mixture (Roche Applied Science) was added just before use. After scraping, whole cell lysates were placed in ice for 30 min, and protein concentrations were determined using a BCA protein assay kit (Pierce). Immunoprecipitation was performed with rabbit polyclonal anti-FLIPL/S (Santa Cruz Biotechnology), anti-Bid (Cell Signaling Technology), or anti-caspase-8 (Santa Cruz Biotechnology), and protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4°C. Samples were centrifuged at 10,000 rpm for 3 min; the supernatant was discarded, and the precipitate was resuspended in Laemmli sample buffer. Immunoprecipitates were fractionated on a 4–20% gradient SDS-polyacrylamide gel (ISC BioExpress Gel) and transferred to a nitrocellulose membrane (Millipore). The membrane was blocked with 0.1% Tween 20 TBS containing 1% BSA for 1 h and incubated with primary Ab overnight at 4°C. Goat anti-rabbit or anti-mouse IgG-HRP conjugated Abs (Santa Cruz Biotechnology) were applied for 1 h at room temperature. After washing, the immune complexes were detected using ECL (Pierce). The primary Abs used for Western blotting were: rabbit IgG anti-Bid (Cell Signaling Technology), rabbit IgG anti-tBid (BioSource International), rabbit IgG anti-caspase-8 (StressGen Biotechnology), rabbit IgG anti-FLIPL (Upstate Biotechnology), and rabbit IgG anti-FLIPS (Santa Cruz Biotechnology). Membranes were stripped using Restore Western Blot Stripping Buffer (Pierce) and reprobed for the expression of beta-actin (Santa Cruz Biotechnology). The radiographic band density was measured using UN-SCAN-IT software (Silk Scientific), and the results were expressed as ratios of the density to the density of beta-actin.

DISC analysis

The DISC proteins were immunoprecipitated overnight at 4°C using anti-Fas Ab (Santa Cruz Biotechnology) and anti-protein G/A agarose (Santa Cruz Biotechnology). After immunoprecipitation, the beads were washed with lysis buffer, resuspended in Laemmli sample buffer, and boiled. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with Abs specific for Fas, FADD, FLIP, or procaspase-8, and resolved by ECL.

Flow cytometry

Cultured cells (1 x 106/sample) were collected and washed in PBS with 2% BSA and 0.02% sodium azide and then incubated with affinity-purified Ab specific for biotin anti-FasL (BD Biosciences/BD Pharmingen) or anti-Fas (StressGen Biotechnology) for 30 min at 4°C. Cells were then washed twice and incubated for another 30 min with streptavidin-PE (BD Pharmingen) or goat anti-rabbit FITC-conjugated Ab (Stratagene). Cells were then washed, resuspended, and subsequently analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences). The percentage of cells that was positive for FasL or Fas was determined using the CellQuest software (BD Biosciences) gated for 10,000 events. Caspase-3 cleavage was tested by FACS using a kit and following manufacturer’s recommendation (Stratagene).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OLG apoptosis is associated with DISC formation and caspase-8 activation

Because OLG undergoes apoptosis in the absence of serum growth factors, we have investigated the formation of the DISC during this process. To examine DISC formation during differentiation, we immunoprecipitated Fas from cell lysates and looked for the presence of caspase-8, Fas, and FADD by Western blotting (Fig. 1A). We found that procaspase-8 and FADD were present in the Fas immunoprecipitates from cell lysates at time 0 as well as after 48 h of differentiation in serum-free medium (Fig. 1A). The decrease seen in procaspase-8 levels suggests that DISC formation might lead to caspase-8 activation and cleavage.


Figure 1
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  		FIGURE 1. DISC formation and activation of caspase-8 in apoptotic OLG. A, OPC were cultured in defined medium for the indicated times, and DISC formation was determined in anti-Fas immunoprecipitates (IP) by Western blotting. IP were analyzed for the presence of procaspase-8, FADD, and Fas. Procaspase-8 was present in the complex in the OPC cells (0 h), and the levels decreased during the next 48 h of differentiation. B and C, The effect of differentiation on caspase-8 was tested by evaluation of cleavage products (A) and analysis of caspase-8 activity (B). Both caspase-8 cleavage and the functional activity increased significantly (p < 0.003) during OLG differentiation.

 
To determine whether DISC had the capacity to convert procaspase-8 into active caspase-8 subunits, we tested for the presence of caspase-8 cleavage. We found that caspase-8 cleavage products were increased in OLG cultured in defined medium. The 25-kDa cleavage fragment was present at only low levels in OPC and showed a 2-fold increase after 48 h of differentiation in defined medium (Fig. 1B). Caspase-8 activity was increased during differentiation and reached its highest level at 48 h (p < 0.003) (Fig. 1C). These data suggest that caspase-8 might be involved in the induction of apoptosis in OLG under conditions of serum deprivation. To further test this hypothesis, we used a cell-permeable caspase-8 inhibitor, z-IETD-FMK, to examine the effect on serum deprivation-induced OLG cell death. Cell death was almost completely inhibited at a concentration of 100 µM (Fig. 2). These data clearly indicate that caspase-8 activation plays a major role in OLG apoptosis induced by serum deprivation.


Figure 2
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  		FIGURE 2. Effect of caspase-8 inhibition on OLG apoptosis. OPC were cultured for 24 h in 96-well plates at 105 cells/well in OLG-defined medium. Cells were incubated for an additional 48 h with the caspase-8 cell-permeable inhibitor, z-IETD-FMK FMK (25 –100 µM). Cell viability was determined by a method using MTS, as described in Materials and Methods, and the percentage of cell death was assessed at 24 h in culture and also at 72 h, with cells being incubated with or without the inhibitor for the last 48 h. Results are expressed as mean percentage of cell death ± SEM. Data are derived from two separate experiments performed in triplicate.

 
Apoptosis of OLG is associated with Bid cleavage and down-regulation of c-FLIP

Next, we tested whether Bid, a caspase-8 substrate involved in mitochondrial apoptosis, is cleaved during OLG differentiation. Cleavage of Bid by caspase-8 has been shown to directly trigger the release of cytochrome c from mitochondria (18). We monitored the cleavage of Bid by measuring the loss of intact Bid and the increase in tBid. Bid was present as a 22-kDa protein in OPC (Fig. 3A); during differentiation, cleavage of Bid leads to the increased formation of the15-kDa tBid fragment (Fig. 3C). We found that tBid levels were increased during OLG differentiation with a parallel decrease by 70% in Bid levels (Fig. 3). These data suggest that activation of caspase-8 results in Bid cleavage. Because regulation of caspase-8 activation is mediated by c-FLIP, an endogenous inhibitor (21), we tested the effect of differentiation on c-FLIPL and FLIPS levels. Our data showed that c-FLIPL levels were high in OPC and decrease significantly during differentiation (Fig. 4). After 48 h of differentiation, c-FLIPL protein had almost completely disappeared. c-FLIPS was expressed in OPC and its level was reduced by 40% after 48 h of differentiation (Fig. 4). Because a reduction in c-FLIP levels is associated with an increased susceptibility to apoptosis (21), we can speculate that c-FLIP down-regulation is promoting OLG apoptosis by allowing caspase-8 activation during differentiation.


Figure 3
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  		FIGURE 3. Activation of Bid in apoptotic OLG. A and B, OPC cultured in defined medium for 0, 6, 24, and 48 h were lysed, immunoprecipitated with the anti-Bid Ab, and examined by Western blotting (A). Results are shown as density ratios to IgG in the lower panel (B). C and D, The same cell lysates as in A were examined by Western blotting using a rabbit IgG anti-tBid. The same blot was probed for beta-actin. Results are shown as density ratios to beta-actin in D. Bid levels decreased during OLG apoptosis, with a concomitant increase in tBid.

 

Figure 4
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  		FIGURE 4. c-FLIPL and c-FLIPS levels during OLG apoptosis. OPC cultured in defined medium for 0, 6, 24, and 48 h were lysed and c- FLIPL (A) and c-FLIPS (C) expression was examined by Western blotting. Results are shown as density ratios to beta-actin in the lower panel (B and D). c- FLIPL levels were significantly lower at 6 h and almost absent at 48 h. The decrease in c-FLIPS was less dramatic.

 
FasL expression is induced in OLG during apoptosis

To identify the trigger of caspase-8 activation in serum-deprived OLG, we tested the involvement of death receptors by investigating the first expression of FasL and Fas. As shown in Fig. 5A, the number of FasL-positive cells increased during OLG differentiation from 14% at time 0 h to 60% at 48 h. Furthermore, a 1.4-fold increase in Fas expression was detected in differentiated OLG when whole lysates were analyzed by Western blotting (Fig. 5B). By FACS analysis, the Fas receptor was found to be expressed on the surface of 90% of OLG after 48 h of differentiation (data not shown). These results indicate that Fas is expressed on OLG under conditions of serum deprivation and by interacting with FasL will trigger DISC assembly and caspase-8 activation. We further tested whether FasL can trigger apoptosis of OLG using serial dilutions of membrane-bound FasL (Upstate Biotechnology), which was provided as membranous vesicles. Exposure to FasL induced a dose-dependent reduction in OLG survival (Fig. 6, A and B), an increase in caspase-8 activity (Fig. 6C), and caspase-3 cleavage as measured by FACS analysis (data not shown).


Figure 5
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  		FIGURE 5. FasL and Fas expression during OLG apoptosis. A, OPC were cultured in defined medium, and FasL expression was measured by FACS analysis. FasL is expressed in OPC (0 h), and the number of cells expressing FasL significantly increased after 48 h of culture in defined medium. CTR, Isotype control. B, Fas and actin expression was measured by Western blotting. As compared with OPC, a 1.4-fold increase was detected in differentiated OLG after 48 h.

 

Figure 6
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  		FIGURE 6. Effect of FasL on OLG survival. A, To determine the effect of FasL on OLG survival, various concentrations of membrane-bound FasL (Upstate Biotechnology) were used to treat OLG differentiated for 48 h in serum-defined medium. The number of surviving cells was determined by the MTS method. Exposure to FasL significantly reduced the number of surviving OLG at all three concentrations used. All experiments were performed in triplicate. B, Effect of C5b-9 and FasL on OLG cell death. OPC plated in 96-well plates at 105 cells/well were cultured in defined medium for 56 h. Cells were exposed to C5b-9 for 1 h and then exposed to FasL (2 ng/ml). After 18 h, cell survival was assessed by MTS assay. The number of live cells at the beginning of the experiment (0 h) was considered to be 100%. Results of three separate experiments performed in triplicate are expressed as percent surviving cells ± SEM, relative to the value at 0 h. C, Effect of FasL on caspase-8 activity. The effect of FasL on caspase-8 was tested by analysis of caspase-8 activity. Caspase-8 activity was increased significantly by exposure for 18 h to FasL (p < 0.003). C5b-9 was able to reverse the effect of FasL. Results are expressed as the percentage of control untreated OLG.

 
These data indicate that FasL induced apoptosis of OLG in vitro. Exposure to sublytic C5b-9 before treatment with FasL prevented OLG apoptosis (Fig. 6B), inhibited caspase-8 activation (Fig. 6C), and caspase-3 cleavage (data not show).

Sublytic C5b-9 does not affect formation of DISC but suppresses processing of caspase-8

To examine at which level in the Fas-signaling pathway C5b-9 activation is suppressing apoptosis, we first investigated the effect on the DISC formation. The presence of procaspase-8 in Fas immunoprecipitates, from unstimulated OLG, and in C5b6- and C5b-9-stimulated cells, indicated that caspase-8 is recruited to the DISC (Fig. 7A). However, there were no significant differences in recruitment of procaspase-8, FADD, or Fas to the DISC in C5b-9-treated OLG and C5b6-treated cells (Fig. 7A). Hence, the DISC seemed to assemble in OPC, and this assembly was maintained after differentiation and exposure to sublytic C5b-9 exposure. It is clear from these results that C5b-9 did not affect DISC formation. We then asked whether C5b-9 has the ability to inhibit DISC-induced processing of procaspase-8 into active caspase-8 subunits. OLG were cultured in defined medium for 18 h in the presence of C5b-9 or C5b6. We found that sublytic concentrations of C5b-9 inhibited caspase-8 cleavage (Fig. 7B) and reduced the activity by 65% (p < 0.0007) (Fig. 7C). Treatment of OLG with inactive C5b-9 had no effect on caspase-8 activity (Fig. 7C).


Figure 7
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  		FIGURE 7. Effect of C5b-9 on DISC formation and activation of caspase-8. A, OLG were cultured in defined medium for 18 h in the presence of C5b-9 or C5b6, and DISC formation was determined in anti-Fas IP by Western blotting. Procaspase-8 was present in the DISC in the differentiated OLG (CTR) and remained detectable in the complex after C5b-9 or C5b6 treatment. Procaspase-8, FADD and Fas levels were similar in OLG exposed to C5b-9 and C5b6. B and C, The effect of differentiation on caspase-8 expression was tested by evaluation of cleavage products (B) and analysis of caspase-8 activity (C). Both caspase-8 cleavage and its functional activity were significantly inhibited by exposure to C5b-9. Exposure to inactive C5b-9, assembled in the fluid phase, had no effect on caspase-8 activity (C).

 
Sublytic C5b-9 induces up-regulation of FLIP expression and inhibition of Bid cleavage

To further investigate the mechanisms of C5b-9-induced OLG survival, we examined the effect of C5b-9 on c-FLIPL and c-FLIPS expression. c-FLIP, FADD, and procaspase-8 contain death effector domains (DEDs), protein modules that facilitate binding via homophilic protein interactions. c-FLIP heterodimerizes with FADD or procaspase-8 by binding to their DEDs, thus acting as a molecular inhibitor of DISC formation by blocking procaspase-8 recruitment to FADD. Exposure of OLG to sublytic C5b-9 for 18 h induced a 2-fold increase in the levels of the c-FLIPL and a 1.5-fold increase in c-FLIPS (Fig. 8). In contrast, exposure to C5b6 had no effect on FLIPL and FLIPS levels. Both FLIP splice forms are recruited to the DISC in unstimulated OLG (Fig. 9). A 43-kDa c-FLIPL cleavage fragment was also observed. This finding is not unusual, because it has previously been shown that the p43 c-FLIPL fragment remains bound to DISC and prevents the recruitment and processing of additional procaspase-8 (23). DISC in C5b-9-stimulated cells also contains full-length c-FLIPL, the p43 c-FLIPL cleavage fragment and c-FLIPS (Fig. 9). Interestingly, DISC c-FLIPS levels were lower in C5b-9-treated cells than in control or C5b6-stimulated cells. These results suggest that C5b-9 prevents further caspase-8 processing by the DISC, possibly through a c-FLIPL-dependent mechanism.


Figure 8
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  		FIGURE 8. Effect of C5b-9 on c-FLIPL and c-FLIPS. OLG cultured for 18 h in the presence of C5b-9 or C5b6 were lysed and tested for the expression of c-FLIPL (A) and c-FLIPS (C) by Western blotting. Results are shown as density ratios to beta-actin in the lower panels (B and D). c-FLIPL levels increased 2-fold after C5b-9 treatment while c-FLIPS increase was only 1.5-fold. Three experiments were performed, all of which gave similar results.

 

Figure 9
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  		FIGURE 9. Effect of C5b-9 on DISC FLIP levels. OLG were cultured in defined medium for 18 h in the presence of C5b-9 or C5b6, and presence of FLIPL and FLIPS was examined in the DISC by Western blotting. c-FLIPL, p43 c-FLIPL, and c-FLIPS were present in the DISC in the differentiated OLG (CTR), and remained detectable in the complex after C5b-9 or C5b6 treatment. Although c-FLIPS decreased after C5b-9 treatment, there was a slight increase in the p43 c-FLIPL cleavage fragment, which is known to bind to partially processed caspase-8 and inhibit further caspase-8 processing.

 
To evaluate the downstream effects of C5b-9 on caspase-8 targets, we tested the effect of C5b-9 on the cleavage of Bid, a substrate of caspase-8 and a link to the mitochondrial pathway of apoptosis. C5b-9 increased the level of total Bid and reduced tBid by 65% when compared with the C5b6 control (Fig. 10). These findings indicate that C5b-9 inhibits caspase-8 activation and the subsequent Bid cleavage, which plays an essential role in apoptosis induced through the mitochondrial pathway.


Figure 10
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  		FIGURE 10. Inhibition of Bid cleavage by C5b-9. A and B, OLG were exposed to either C5b-9 or C5b6, lysed and immunoprecipitated with the anti-Bid Ab then examined by Western blotting (A). Results are shown as density ratios to IgG in the lower panel (B). C and D, The same cell lysates as in A were examined by Western blotting for the expression of tBid. The same blot was stripped and then probed with beta-actin. Results are shown as density ratios to beta-actin in D. Bid levels increased after C5b-9 exposure, but C5b6 had no effect. tBid levels were significantly decreased in the presence of C5b-9 when compared with cells treated with C5b6.

 
PI3K inhibition reverses the effects of C5b-9 on caspase-8, c-FLIPL, and Bid in OLG

We examined the role of PI3K in mediating the C5b-9 effects on caspase-8, c-FLIPL, and Bid activation. OLG were pretreated with PI3K inhibitor LY294002 for 30 min, and then exposed to C5b-9 for 18 h. As shown in Fig. 11, A and B, the effect of C5b-9 on procaspase-8 was reversed by pretreatment with LY294002. A similar effect on Bid cleavage (Fig. 11, C and D) and c-FLIPL down-regulation (Fig. 12) was also noted. These data suggest that the PI3K pathway is the primary signaling pathway activated by C5b-9 and is required to rescue OLG from apoptosis.


Figure 11
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  		FIGURE 11. Effect of C5b-9 on caspase-8 and Bid is reversed by PI3K inhibition. OLG were pretreated with LY294002 (10 µM) for 1 h, then exposed to C5b-9 for 18 h. Lysates were analyzed by Western blotting for procaspase-8 and Bid cleavage as described above. Exposure to LY294002 was able to reverse the effect of C5b-9 on caspase-8 (A and B) and tBid expression (C and D).

 

Figure 12
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  		FIGURE 12. Effect of PI3K inhibition on C5b-9-induced c-FLIPL expression. OLG were pretreated with LY294002 (10 µM) for 30 min, and then exposed to C5b-9 for 18 h. Cell lysates were analyzed by Western blotting for c-FLIPL expression. Inhibition of PI3K was able to reverse the effect of C5b-9 on c-FLIPL (A). Lower panel results are expressed as c-FLIPL to beta-actin ratio (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Assembly of lytic C5b-9 induces cell death by necrosis or apoptosis (27, 36). DNA fragmentation (28) and caspase-3 activation (36) were detected in apoptotic cell death mediated by a lytic dose of complement. In contrast, sublytic levels of C5b-9 promote cell survival (31, 32, 37). In this study, we examined the role of sublytic C5b-9 in rescuing OLG from serum deprivation-induced, Fas-mediated apoptosis. Apoptosis of OLG through Fas-mediated mechanisms plays a significant role in myelin loss in MS (5, 6, 7). Although previous studies have linked sublytic C5b-9 with cell survival, a direct role for C5b-9 in preventing Fas-mediated apoptosis in OLG has not to our knowledge been demonstrated. Fas-induced apoptosis can be associated with two alternate biochemical pathways (38). In type I cells, ligation of Fas primarily leads to the recruitment and activation of caspase-8 at the DISC, resulting in caspase-3 activation and cell death. In type II cells, the level of procaspase-8 recruited to the DISC is very low. Procaspase-8, in type II cells, is cleaved with slower kinetics, and this condition results in amplification of death signals through the cleavage of Bid by caspase-8 and triggering of the mitochondria-mediated apoptosis. Cells may also acquire Fas sensitivity by down-regulation of the Fas-signaling inhibitor c-FLIP, which inhibits caspase-8 processing (39).

Our results demonstrate that OLG resemble type II cells, because these cells undergoing apoptosis in serum-free medium showed DISC formation, caspase-8 processing, and then Bid cleavage. Together with increased expression of FasL in these cells in serum-free medium, Fas-FasL-mediated cell death appeared to be a component of apoptosis in OLG. These findings are also consistent with our previous data, in which serum deprivation of OLG was associated with cytochrome c release, at a time when caspase-3 processing was not yet present (31, 32). In this study, we showed the involvement of PI3K/Akt-mediated phosphorylation of Bad and dissociation of the Bad-Bcl-xL complex, leading to cytochrome c release (32). It is significant to note that the exposure of OLG to a sublytic dose of C5b-9 did not suppress the functional DISC formation but inhibited the DISC-mediated processing of caspase-8. Under these conditions, C5b-9 may have inhibited both the activation of caspase-8 and the mitochondrial amplification loop of apoptosis through Bid. Therefore, the regulation of caspase-8 processing constitutes a newly recognized upstream target for C5b-9 in rescuing cells from Fas-mediated apoptosis.

The role of the caspase-8 inhibitor c-FLIP in serum deprivation-induced OLG apoptosis has not been examined. Our data showed a marked down-regulation of c-FLIPL that reached a maximum at 48 h of serum deprivation, in parallel with increased apoptosis. Thus, a reduction in c-FLIPL is likely involved in enhancing the sensitivity of these cells to apoptosis. To our surprise, we found that expression of c-FLIPL was significantly increased by C5b-9. We also found c-FLIPL and its cleavage p43 fragment in the DISC, both of which are able to prevent caspase-8 processing (23). c-FLIPL also acts in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (40, 41). It is possible that c-FLIPL expression induced by C5b-9 plays a similar role in promoting OLG survival through stimulation of the signaling pathways of cell growth. In contrast, c-FLIPS was less significantly changed during differentiation and was only slightly increased by C5b-9. Moreover, lower levels of c-FLIPS were detected in the DISC after C5b-9 stimulation. Because c-FLIPS prevents both FLIPL and caspase-8 cleavage at the DISC (23), we can speculate from our data that c-FLIPS does not play a major role in protecting OLG from apoptosis.

We also observed that the C5b-9 effect on caspase-8 processing was mediated through the activation of the PI3K pathway. Because PI3K is also involved in up-regulation of c-FLIPL and inhibition of Bid cleavage, the inhibitory effect of C5b-9 on caspase-8 may be exerted entirely through the regulation of c-FLIPL. Alternatively, PI3K might work at multiple levels, up-regulating c-FLIPL and inhibiting procaspase-8 processing. Our data are in agreement with the reported involvement of PI3K in the restriction of caspase-8 processing in the presence of DISC assembly (42). PI3K has also been shown to inhibit lateral diffusion and aggregation of Fas in Th2 cells without affecting DISC assembly or the procaspase-8 recruitment to DISC (43). In fact, caspase-8 has been implicated as a target for several other cell survival kinases, such as Akt and protein kinase C. Akt may directly inhibit the recruitment of procaspase-8 to DISC as well as caspase-8 processing in T cells, and these effects of Akt are implicated in rescuing T cells from Fas-induced apoptosis (44). Apoptosis induced by Fas has also been found to be inhibited by activation of protein kinase C (45) or MAPK (46). The presence of these redundant pathways that promote cell survival by regulating apical steps of the cell death cascade appears to be beneficial for the cell, as only in this way can cells effectively escape the harmful effects of a partially activated apoptotic machinery. This type of regulation would also be useful in situations in which the initiator caspase activation is required for signaling processes, such as those involved in cell proliferation or differentiation (47, 48, 49).

In conclusion, our experimental results indicate that C5b-9 contributes to OLG survival by inhibiting caspase-8 processing. The C5b-9 effect results in inhibition of downstream caspase activation and of the mitochondrial amplification loop through Bid. This function of C5b-9 may have an additional significance in situations in which the cell cycle and proliferation of OLG progenitors are induced by C5b-9 in experimental autoimmune encephalomyelitis and MS, in which complement activation and C5b-9 assembly occurs ubiquitously (9, 50).


   Acknowledgments
 
We thank Deborah McClellan for editing this manuscript and Adrian German for help with the illustrations.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported, in part, by U. S. Public Health Grants RO-1 NS42011 (to H.R.) and the Veterans Administration Maryland Health Care System, Multiple Sclerosis Center of Excellence, Baltimore, MD (to H.R.). Back

2 Address correspondence and reprint requests to Dr. Horea Rus, Department of Neurology, University of Maryland School of Medicine, 655 West Baltimore Street, Bressler Research Building 12-016, Baltimore, MD 21201. E-mail address: hrus{at}umaryland.edu Back

3 Abbreviations used in this paper: OLG, oligodendrocyte; MS, multiple sclerosis; FADD, Fas-associated death domain; DISC, death-inducing signaling complex; tBid, truncated Bid; c-FLIP, cellular FLIP; c-FLIPL, c-FLIP long isoform; c-FLIPS, c-FLIP short isoform; OPC, OLG progenitor cell; NHS, normal human serum; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt. Back

Received for publication June 2, 2005. Accepted for publication December 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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