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The Journal of Immunology, 1999, 163: 6132-6138.
Copyright © 1999 by The American Association of Immunologists

Inhibition of Oligodendrocyte Apoptosis by Sublytic C5b-9 Is Associated with Enhanced Synthesis of Bcl-2 and Mediated by Inhibition of Caspase-3 Activation1

Lucian Soane, Horea Rus, Florin Niculescu and Moon L. Shin2

Department of Pathology, University of Maryland, School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-{alpha}-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Demyelination in multiple sclerosis (MS)3 and its animal model experimental allergic encephalitis (EAE) is caused by damage to myelin and myelin-producing oligodendrocyte (OLG) by activated immune effectors. These effectors include macrophages, T cells, proinflammatory cytokines TNF-{alpha} and monocyte chemoattractant protein-1, and C5b-9 complexes generated during complement activation (1, 2, 3). Sequential interaction of C5b6, C7, C8, and C9 is associated with amphipathic conformational changes of C7, C8{alpha}, C8ß, and C9, resulting in assembly of membrane-inserted C5b-7, C5b-8, and C5b-9 complexes, collectively referred to as the terminal complement complexes (TCC) (4). Sublytic C5b-9 stimulates target cells and induces a variety of cellular activities in the absence of cell death (4, 5). One of the activities induced by C5b-9 is cell cycle induction (6, 7, 8, 9), which is mediated by Gi-dependent activation of Ras, Raf-1, and ERK1, and associated with expression of protooncogenes c-fos and c-jun, and increased DNA synthesis (8, 9, 10). Cell cycle activation by C5b-9 also occurs through release of fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) (6, 7). Increase in cytosolic Ca2+ and protein kinase C activation are responsible for some of the TCC activities, such as platelet activation and generation of arachidonic acid and metabolites, and are elicited by the pore-forming C5b-8 and C5b-9 complexes (4, 5, 11, 12, 13, 14). Additionally, membrane-inserted TCC, including C5b-7, are able to generate diacylglycerol and stimulate the Ras/Raf-1/ERK1 pathway via Gß{gamma} effectors of the G protein (10, 15, 16). However, C5b-9 is most effective in inducing DNA synthesis and cell cycle, in a Gi-ERK1-dependent manner (9, 10, 15, 16).

OLG that myelinate the central nerve axons differentiate from the O-2A progenitors, and this process requires axonal contact and soluble growth factors (17, 18, 19, 20). Survival of differentiated OLG also requires factors such as PDGF and basic FGF (17, 18, 19). In developing rat optic nerve, more than 50% of newly differentiated OLG undergo apoptotic death, which is an essential process for brain tissue modeling during development (17). In serum-free medium, O-2A cells differentiate into OLG concomitantly with apoptosis, as in vivo. Apoptosis in vitro is also inhibited by PDGF, insulin-like growth factor, ciliary neurotrophic factor, and leukemia-inhibitory factor (18, 19, 20, 21, 22).

A critical role of complement in EAE is supported by experiments in which abrogation of systemic complement activity by cobra venom factor or by soluble CR1 inhibited demyelination induced by encephalitogenic Ag or Ag-specific T cells (23, 24). Deposition of C5b-9 in MS and EAE brains and increased levels of soluble C5b-9 in MS spinal fluids indicated in situ activation and assembly of C5b-9 (25, 26, 27). The central nerve myelin, but not the peripheral nerve myelin, directly activates the classical pathway of complement (28, 29). Activation and assembly of C5b-9 on myelin cause hydrolysis of myelin basic protein (MBP) and extensive vesiculation with eventual loss of myelin membrane (30, 31). In addition, the complement-inhibitory proteins CD55 and CD46 are absent in myelin, causing the myelin membrane to be susceptible to C5b-9 (32). Therefore, C5b-9 can contribute to demyelination by directly damaging the myelin, even in the absence of myelin-specific Abs. In OLG, C5b-9 at a sublytic concentration induces cell cycle, as shown by activation of ERK1 and c-Jun N-terminal kinase 1, protooncogenes, and G1 progression to S phase (8, 33). Sublytic C5b-9 also induces phenotype changes in OLG by accelerating the decay of mRNA encoding myelin-specific genes (8, 34). While activating cell cycle, C5b-9 was also found to inhibit apoptosis of OLG associated with differentiation (8).

In this study, we have examined the differentiation-associated apoptosis of OLG in vitro by investigating involvements of caspase-3 and Bcl-2 as possible target sites regulated by C5b-9. We also tested the ability of C5b-9 to protect OLG from TNF-{alpha}-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Differentiation of OLG from O-2A progenitor cells in culture

Primary O-2A progenitor cells were prepared according to Saneto and de Vellis (35). Glial cells were isolated from neonatal Sprague Dawley rat brains, as described in detail (35, 34). Dispersed glia cells are grown for 10 days as stratified mixed glial cultures. O-2A progenitors growing on surface of the mixed culture were isolated by a series of differential shaking. Cells were placed in OLG defined medium consisting of serum-free DMEM/Ham’s F-12 containing 500 ng/ml transferrin (Sigma, St. Louis, MO), 75 ng/ml insulin (Sigma), 75 µg/ml basic FGF (Collaborative Research, Lexington, MA), and 1 mM sodium pyruvate. O-2A cells isolated by serial shaking at the time of plating showed 2–3% cell death, as determined by trypan blue dye exclusion. Differentiation was stepwise, as shown by the expression of MBP and proteolipid protein (PLP) mRNA before the expression of galactocerebroside (GC) (17, 35). After 56 h in OLG defined medium, more than 85% of cells expressed GC, MBP, and PLP. Less than 5% of the MBP-negative cells were astrocytes and microglia, and the remaining cells were O-2A cells in different stages of differentiation. O-2A cells grown in a defined medium for 3 days are designated as OLG.

Determination of cell viability

Viability of O-2A cells during differentiation and the effect of C5b-9 on cell viability were determined by using CellTiter 96 Aqueous cell proliferation assay, according to the instruction supplied by Promega (Madison, WI). Cells were seeded on poly(D-lysine)-coated 96-well plates at 105 cells/well in 200 µl of OLG defined medium and cultured at 37°C. At the indicated time points, 40 µl methyl tetrazolium salt (MTS) solution was added to each well. Plates were kept at 37°C for additional 2 h, followed by determination of OD at 540 nm under a condition in which absorbance was in linear range. The results are expressed as percentage of dead cells ± SD, relative to the initial cell number.

Analysis of apoptosis

DNA strand break was detected in cells by TdT-dependent incorporation of dUTP (Apoptag, Oncor, Gaithersburg, MD). O-2A cells were cultured on plastic slide chambers for the indicated time period. Cells were fixed in buffered Formalin at room temperature (RT), then treated with TdT in the presence of digoxigenin-dUTP for 1 h at 37°C. After washing, cells were treated with peroxidase-conjugated anti-digoxigenin IgG F(ab')2 fragments for 1 h; then color was developed using diaminobenzidine as a substrate. Approximately 600 cells with clearly defined nucleus were examined in each sample by TUNEL staining. The number of cells showing apoptosis was counted by identifying TUNEL-positive nuclei. The percentage of apoptotic cells was then calculated using the following formula: (number of cells with TUNEL-positive nuclei/total number of cells examined) x 100. Results are expressed as mean percentage of cells with TUNEL-positive nuclei ± SD.

Activation of serum complement and C5b-9 assembly

Normal human serum (NHS) pooled from several healthy donors was used as a source of serum complement. Rabbit antiserum to GC was used to sensitize rat OLG. The specific anti-GC activity was assayed by treating GC-expressing liposomes with trapped 86Rb aqueous marker with antiserum, then measuring the released marker (31). Because anti-GC Abs are mostly IgM isotype, IgM fraction of the antisera was used in most experiments. A sublytic dose of Ab was predetermined by titrating anti-GC Ab using an excess of NHS (8, 34). To evaluate the effect of serum C5b-9, OLG sensitized with a dose of anti-GC Ab for 30 min at RT were incubated with a 1/20 dilution of NHS depleted of C7 (C7D) reconstituted with C7 (10 µg/ml). Alternatively, sensitized cells were treated with NHS (1/10) and NHS treated with K76 (Otsuka Pharmaceutical, New York, NY) (NHS-K76) as a control (8, 34). K76 prevents C5b-9 assembly in serum by binding to C5 (36). Therefore, C7D and NHS-K76 allow complement activation to proceed up to C6 and C3, respectively. Purified human complement proteins C5-C9 were purchased from Quidel (San Diego, CA), and C5b6 complex was prepared from C5 and C6, as described (37). To assemble sublytic C5b-9 by using purified proteins, cells were incubated with C5b6 (30 µg) for 15 min, then with C7 (10 µg) for 5 min at RT, followed by addition of C8 (10 µg) and C9 (10 µg) in a final volume of 1 ml (8, 10). Cells were then incubated at 37°C for the indicated time periods.

Northern blot analysis

RNA was isolated from cells lysed with buffer containing guanidine isothiocyanate and 2-ME, and total RNA was purified by ultracentrifugation on 5.7 M CsCl (as described in Ref. 38). Poly(A)+ RNA was prepared from total RNA using Dynabead mRNA purification system (Dynal, Great Neck, NY). Poly(A)+ RNA was denatured and electrophoresed on 0.8% agarose-formaldehyde gels, then transferred to a nitrocellulose membrane. After baking for 2 h at 80°C, the membrane was hybridized with 32P-labeled cDNA probes. The probe binding was quantitated by measuring band densities of autoradiogram using Computing Densitometer (Molecular Dynamics, Sunnyvalle, CA). Integrated volume of each band was calculated using the ImageQuant software (Molecular Dynamics), and the results are expressed by density ratio to actin. Caspase-3 cDNA probe was obtained by RT-PCR cloning of rat caspase-3 cDNA with the forward (5'-GCGAAGCTTAAGTGACCATGGACAACCAAC) and reverse (5'-GCGTCTAGACCCAGTCATTCCTTTAGTGA) primers designed according to rat CPP32 cDNA (39). The rat bcl-2 and bax cDNA were gifts from Dr. E. Podack (University of Miami) and Dr. S. Korsmeyer (Washington University, St. Louis, MO), respectively. The cDNA was labeled with [{alpha}-32P]dCTP (New England Nuclear, Boston, MA) using reagents for DNA labeling from Pharmacia (Piscataway, NJ).

Western blot analysis of caspase-3, PARP protein, and BCL-2

The levels of caspase-3, PARP, and their cleavage products were determined by Western and immunoblot. Cells were lysed with RIPA buffer (30 mM Tris-HCl, pH 7.4, 0.15 M NaCl (NaCl), 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 2 mM MgCl2, 1 mM NaVO4, 0.5 mM PMSF, 100 µg/ml aprotinin, and leupeptin), as described (16). An equal amount of protein from each cell lysate was used directly for SDS-PAGE and Western blot, a method sufficient to detect caspase-3 proenzyme and high levels of the cleavage fragment. To detect the cleavage fragments, cell lysates (100 µg protein) were immunoprecipitated with rabbit anti-caspase-3 IgG (Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of protein A/G agarose at 4°C overnight. Cell lysates or immunoprecipitates were analyzed on 10% SDS-PAGE, then by Western blotting using the same rabbit anti-caspase-3 IgG. For PARP, immunoprecipitates using polyclonal anti-PARP IgG (Boehringer Mannheim, Indianapolis, IN) were analyzed by 7% SDS-PAGE, and monoclonal anti-PARP IgG1 (Zymed, San Francisco, CA) was used for immunoblotting. This was followed by reaction with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology), then by enhanced chemiluminescence (ECL; Pierce, Rockford, IL). BCL-2 protein was determined similarly by immunoprecipitation of cell lysates. The BCL-2 Western blot reagents were from Oncogene (Cambridge, MA).

Effects of caspase-3 inhibitor on OLG viability

To test whether caspase-3 activity is required for differentiation-induced apoptosis, the cell-permeable caspase-3 inhibitor DEVD-CHO (Calbiochem, San Diego, CA) was used. O-2A cells were seeded in 96-well plates at 105 cells/well in 200 µl of OLG defined medium and cultured for 24 h. Cells were further incubated for 48 h in the presence of 10–100 µM of DEVD-CHO. Cell viability was then determined, as described earlier.

Effect of C5b-9 on OLG apoptosis induced by TNF-{alpha}.

To test whether sublytic C5b-9 also protects OLG from apoptotic cell death induced by TNF-{alpha} (21, 22, 40), O-2A cells were differentiated in 96-well plates, then cells were exposed to sublytic NHS or NHS-K76 for 1 h. After addition of 100 ng/ml of human rTNF-{alpha} (R&D Systems, Minneapolis, MN), cells were incubated for 18 h at 37°C, and viability was determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apoptotic cell death of O-2A progenitor cells during in vitro differentiation

Differentiation of OLG is associated with cell death in developing brains and during in vitro differentiation (17, 18, 19). As shown in Fig. 1GoA, cell death reached 36% at 48 h, and was increased further to 70% at 96 h. Many of these cells showed the characteristic features of apoptosis, including cell process retraction, chromatin condensation, and DNA cleavage with ladder formation (data not shown). By TUNEL stain, 31.3 ± 6.5% of cells were apoptotic after 48 h in OLG defined medium (Fig. 1GoB).



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  		FIGURE 1. O-2A progenitor cells undergo cell death by apoptosis in serum-free defined medium. A, O-2A cells were placed in 96-well plates at 105 cells/well in 200 µl of OLG defined medium to allow differentiation. Cell viability was assessed by a method using MTS, as described in Materials and Methods. Background cell death at the time of plating was 2–3%. The percentage of cell death was expressed relative to the initial viable cell number, and the results are shown as mean ± SD of three experiments performed in triplicate. B, O-2A cells were differentiated in chamber slides as above. Apoptosis was determined by TUNEL method, as described in Materials and Methods. Approximately 600 cells with clearly defined nucleus were examined in each sample, and apoptotic cells were identified by the presence of TUNEL-positive nuclei. Results are expressed as mean percentage of cells with TUNEL-positive nuclei relative to the total number of cells examined ± SD. Data were derived from two separate experiments performed in triplicate.

 
Bcl-2, Bax, and caspase-3 expression during OLG differentiation

The expression of caspase-3, bcl-2, and bax during OLG differentiation was examined. Northern blot analysis of poly(A)+ RNA showed a gradual increase in caspase-3 mRNA expression and a rapid decline of bcl-2 mRNA as early as 2 h (Fig. 2GoA–C). The changes in bcl-2 and caspase-3 mRNA level are associated with differentiation, as shown by the robust expression of PLP mRNA (Fig. 2GoA). The bax mRNA expression was reduced by 30% at 6 h, as shown by density ratios to actin, obtained by quantitative densitometry (Fig. 2GoC). The bax mRNA level after 3 days in differentiation medium, determined in a separate experiment, was similar to the initial level (data not shown). Western blot analysis of cell lysates showed an increase in 32-kDa caspase-3 proenzyme after 24 h (1 day) (Fig. 3Go, A and B), which correlated with increased expression of caspase-3 mRNA. The caspase-3 proenzyme began to decrease on day 3, as shown by the density ratio to ß-actin on the same blot. Because caspase-3 activity requires the proenzyme cleavage to active subunits (41), the appearance of 17- and 12-kDa subunits was evaluated by anti-caspase-3 immunoprecipitation, followed by Western blot. The appearance of anti-caspase-3-reactive 17- and 12-kDa bands was identified on day 3 (Fig. 3GoC). Decreased caspase-3 proenzyme with increased cleavage products on day 3 (Fig. 3GoC) is clearly evident in experiments when cell lysates were immunoprecipitated, then examined by Western blotting.



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  		FIGURE 2. Expression of caspase-3 and bcl-2 and bax mRNA during OLG differentiation. O-2A cells cultured in OLG defined medium for the indicated time periods were examined by Northern blot for mRNA encoding caspase-3 and PLP (A), and for bcl-2 and bax (B) using 1 µg poly(A)+ RNA/lane. The data shown represent one of two identical experiments. The results are also expressed by density ratios to ß-actin (C).

 


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  		FIGURE 3. Caspase-3 proenzyme and its activation during OLG differentiation. A, Cell lysates (50 µg protein) of O-2A cells cultured for 1, 3, and 4 days were examined for caspase-3 proenzyme by 10% SDS-PAGE and Western blot. The blot was immunostained with anti-caspase-3 IgG, then with anti-ß-actin, as loading control. B, Densitometric analysis of the radiographic bands is shown as density ratios to ß-actin. Representative data from three experiments are shown. C, Caspase-3 activation during OLG differentiation was determined by detection of the 17- and 12-kDa caspase-3 fragments. O-2A cells in culture for 1, 2, and 3 days were lysed, and the lysates, 100 µg protein, were immunoprecipitated with rabbit anti-caspase-3 IgG. The immunoprecipitates were then analyzed for the cleavage products, as in A.

 
To determine whether caspase-3 activation is required for cell death, O-2A cells in medium for 24 h were cultured for additional 48 h with the cell-permeable caspase-3 inhibitor DEVD-CHO. Cell death was inhibited in a dose-dependent manner, with 50% protection at 25 µM and 100% at 100 µM of DEVD-CHO (Fig. 4Go). DMSO at concentrations used to resuspend DEVD-CHO was not toxic to the cell.



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  		FIGURE 4. Inhibition of caspase-3 activity prevented cell death associated with differentiation. O-2A cells were cultured for 24 h in 96-well plates at 105 cells/well in OLG defined medium. Cells were incubated for additional 48 h with 10–100 µM of DEVD-CHO. 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 in which cells were incubated with or without the inhibitor for the last 48 h. Results are expressed as mean percentage of cell death ± SD of three experiments performed in triplicate.

 
Inhibition of caspase-3 activation by C5b-9

To evaluate the antiapoptotic activity of C5b-9 previously shown (8), OLG exposed to serum C5b-9 were examined for caspase-3 cleavage. As shown in Fig. 5GoA, a prominent 17-kDa cleavage product was seen in control cells treated with C7D for 18 h, which was inhibited by addition of C7 to C7D. Because the data were obtained by direct analysis of the cell lysates by SDS-PAGE/Western blotting, presence of the cleavage fragment in unstimulated cells was not detected. When cell lysates were immunoprecipitated first, then examined by Western/immunoblot (Fig. 5GoB), the caspase-3 cleavage fragment increased with time in cells exposed to NHS-K76. This increase was inhibited when C5b-9 assembly was allowed in NHS, in contrast to NHS-K76. To exclude a possibility that C5b-9 may have enhanced the serum effect on caspase-3, identical experiments were performed by treating cells with C5b-9 assembled using purified proteins (Fig. 5GoC). A cleavage fragment of caspase-3 was detected in unstimulated OLG. This 17-kDa band increased with time in control cells exposed to C5b6, C8, and C9 without C7. However, the cleavage product was barely detected in cells exposed to C5b-9. We have also examined the effect of C5b-9 on cleavage of PARP, a specific substrate for caspase-3 (41, 42). On Western blotting, the 89-kDa fragment of PARP protein was detected in unstimulated OLG. The PARP cleavage was significantly reduced in cells treated with serum C5b-9, compared with the level of NHS-K76 (Fig. 6Go).



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  		FIGURE 5. Inhibition of caspase-3 activation in OLG by C5b-9. A, Differentiated OLG, 5 x 106 cells/flask, were sensitized with anti-GC Ab, then exposed to C7D ± C7, as described in Materials and Methods. At the indicated time points, cell lysates were examined by 10% SDS-PAGE and Western blot. B, Cells were exposed to TCC as above, except that they were treated with Ab and NHS or NHS-K76 (K76). Cell lysates, 100 µg protein, were immunoprecipitated with rabbit anti-caspase-3 IgG in the presence of a mixture of protein A- and G-coated agarose. The Ag-Ab complexes were eluted from the beads; then the eluates were examined by 10% SDS-PAGE and Western blotting. Generation of a 17-kDa fragment of caspase-3 was detected as in A using the same anti-caspase-3 IgG. Densitometric scan of the band is also shown. C, Similar experiment as in B was conducted by treating cells with purified proteins to assemble C5b-9. Control cells were treated with C5b6, C8, and C9 without C7.

 


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  		FIGURE 6. Inhibition of PARP cleavage by C5b-9. OLG treated with Ab and NHS or NHS-K76 for 3 and 6 h were lysed. Cell lysates, 100 µg protein, were immunoprecipitated with anti-PARP IgG, as in caspase-3; then the precipitates were analyzed by 7.5% SDS-PAGE and Western blot. A representative data of three experiments is shown.

 
Expression of BCL-2 in OLG exposed to C5b-9

In view of the ability of C5b-9 to inhibit caspase-3 activation, the steps upstream to caspase-3 activation that may be affected by C5b-9 were explored. Caspase-3 can be activated by caspase-9 through mitochondrial pathway or by caspase-8 in death receptor-dependent pathway (43). We have analyzed the effect of C5b-9 on Bcl-2, a potent antiapoptotic factor, which inhibits caspase-3 activation by regulation of mitochondrial pathway (44). The expression of bcl-2 mRNA was not affected by C5b-9, as shown in Fig. 7GoB. However, C5b-9 significantly increased the level of BCL-2 protein within 4 h, and to the maximum level at 8 h (Fig. 7GoA). BCL-2 protein was not detected in unstimulated OLG and in OLG treated with control C5b6.



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  		FIGURE 7. Effects of C5b-9 on expression of bcl-2 mRNA and protein in OLG. A, O-2A cells differentiated in vitro for 3 days (OLG) were exposed to C5b-9 or C5b6 assembled with purified components. After 4, 8, and 18 h, cells were lysed. Cell lysates were immunoprecipitated with anti-BCL-2 IgG, and the protein was analyzed by 10% SDS-PAGE and Western blot, using anti-BCL-2 IgG (Oncogene, Cambridge, MA). Purified BCL-2 protein (Pierce, Rockford, IL) was used as a positive control. B, OLG were exposed to C5b-9 assembled with purified components as in A. Northern blot was performed at 3 and 6 h, using 1 µg of poly(A)+ RNA/lane, for bcl-2 and ß-actin mRNA expression.

 
Protection of TNF-{alpha}-induced cell death by C5b-9

We have examined whether C5b-9 also protects OLG from apoptosis induced by other factors. TNF-{alpha} was tested, since TNF-{alpha} induces apoptotic cell death in OLG both in vivo and in vitro (21, 22, 45). In our system, 100 ng/ml of TNF-{alpha} induced 50% cell death after 18 h (Fig. 8GoA). Pretreatment with NHS, but not with NHS-K76, protected OLG from cell death (Fig. 8GoA). The cleavage product of caspase-3 proenzyme, which was increased by TNF-{alpha}, was abolished in OLG treated with NHS (Fig. 8GoB).



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  		FIGURE 8. Effect of C5b-9 on cell death induced by TNF-{alpha}. A, Anti-GC-sensitized OLG were incubated with NHS or NHS-K76 for 1 h at 37°C. Then 100 ng/ml of human rTNF-{alpha} was added, and cells were incubated for 18 h before determining the cell viability. B, An identical experiment as in A was performed, except that cell lysates were prepared and the protein (100 µg) was immunoprecipitated with anti-caspase-3 IgG, then examined for the 17-kDa cleavage fragment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The C5b-9 complex is a pleiotropic effector generated during inflammation and immune response. When inserted into the target cell membrane, C5b-9, depending on doses, causes cell death or cell activation (4, 5). We have previously shown that at a sublytic concentration, C5b-9 enhances OLG survival in vitro through inhibition of apoptosis (8). C5a and C5b-9 have been implicated in cell injury associated with apoptosis seen in a rat ischemia/reperfusion model (46). However, regulation of apoptosis by sublytic C5b-9 has not been reported. In addition, C5a receptor is not expressed by OLG (47). Therefore, the antiapoptotic activity of C5b-9 may be a new biological function with a potential significance.

Although apoptosis is induced by a variety of stimuli, execution of the apoptotic program involves a common mechanism, which relies on the activation of caspases, cysteine proteases belonging to the IL-1-converting enzyme/CED-3 family. The role of individual caspases and their relative importance in apoptosis have been recently clarified (43). Caspase-8 and caspase-10 are activated early in apoptotic process and are considered initiators, while caspase-3 and caspase-7 activated at a later phase of apoptosis are effectors acting on a large number of substrates. Death receptor-induced pathways of apoptosis require activation of the caspase-8 and caspase-3, whereas apoptosis following growth factor deprivation and stress-induced cell injury appears to be through mitochondrial dysfunction by releasing cytochrome c, which triggers activation of caspase-9, then caspase-3 (43, 44, 45, 48, 49, 50, 51). In addition, apoptosis may also be mediated by a poorly understood caspase-independent pathway (52). Disruption of caspase-8 gene produces fetal death without anomalies of the nervous system. In contrast, disruption of caspase-3 or caspase-9 genes results in abnormal neural development, in addition to fetal death (48, 49, 50). Despite the finding that caspase-1 and caspase-3 are expressed in OLG and involved in TNF-{alpha}-induced apoptosis (53), the specific caspases responsible for differentiation-induced OLG apoptosis have not been clearly defined.

In this study, we have investigated caspase-3 and Bcl-2 in OLG apoptosis and the effect of sublytic C5b-9 in this process. In view of the potent antiapoptotic activity of BCL-2 (54) and the key role of caspase-3 as an apoptosis effector (43, 48), rapid loss of bcl-2 mRNA expression concomitant with increasing caspase-3 mRNA and protein at the onset of cell differentiation suggested a role for these two proteins in differentiation-induced apoptosis. Proteolytic activation of caspase-3, as indicated by the generation of 17- and 12-kDa subunits and the 89-kDa cleavage fragment of PARP, was detected during OLG differentiation. Furthermore, caspase-3 inhibitor DEVD-CHO effectively protected OLG from cell death. Together, these findings indicated that caspase-3 activation is essential for differentiation-induced apoptosis. Caspase-3 activation was abrogated by C5b-9, as shown by inhibition of caspase-3 proenzyme cleavage into its active subunits. This finding was consistent with the inhibition of PARP cleavage, a substrate for activated caspase-3. Regulation of BCL-2 expression was examined as a possible step upstream to the caspase-3 affected by C5b-9. In OLG, bcl-2 mRNA was expressed at a very low level without detectable protein, as examined by sensitive methods, such as the use of poly(A)+ RNA for Northern blot and analysis of cell lysates by immunoprecipitation and Western immunoblot. Interestingly, C5b-9 was able to increase BCL-2 protein without significantly affecting the mRNA level, suggesting a possible role of C5b-9 in posttranscriptional regulation of Bcl-2. Detection of bcl-2 mRNA in the absence of BCL-2 protein in germinal center B cells (55) and in a trophoblastic tumor cell line when induced to differentiate (56) also suggested a step of translational regulation of Bcl-2. A specific cis element within the promoter has been identified as a regulatory site involved in the translational control of bcl-2 gene (57). How BCL-2 synthesis is regulated by C5b-9 remains unclear. We have shown that sublytic C5b-9 induces ERK1 pathway, and this is through activation of phosphatidylinositol-3 (PI-3) kinase (9, 10, 33). In OLG, ERK1 activated by C5b-9 are responsible for enhanced DNA synthesis (33), and C5b-9 increased the p70 S6 kinase activity (33), a ribosomal kinase responsible for protein synthesis (58). PI-3 kinase has been shown to inhibit apoptosis, and this is thought to be through activation of Akt kinase and by increasing BCL-2 (59, 60). In postmitotic cells such as OLG, C5b-9, instead of inducing proliferation, may enhance cell survival. The putative antiapoptotic signaling generated by C5b-9 may include PI-3 kinase. C5b-9, by increasing BCL-2, may stabilize mitochondrial inner membrane permeability, or inhibit the interaction of BAX with outer membrane proteins (61). BCL-2 prevents cytochrome c release and inhibits activation of caspase-9 and caspase-3 (54). Therefore, up-regulation of BCL-2 protein by C5b-9 in OLG may precede the inhibition of caspase-3 activation. C5b-9 also inhibited cell death and caspase-3 activation induced by TNF-{alpha}. TNF-{alpha} induces apoptosis via caspase-8 through the recruitment of TRADD/FADD (TNFR-associated death domain/Fas-associated death domain) proteins to the TNFR1 (62). However, TNF-{alpha} also generates ceramide, which induces caspase-9 activation and apoptosis, in a caspase-8-independent manner (49, 63). We can speculate that C5b-9 inhibits OLG apoptosis induced by differentiation and by TNF-{alpha}, and this is mediated through up-regulation of BCL-2 protein and inhibition of caspase-9 and caspase-3.

Our finding that C5b-9 rescues OLG from differentiation-induced apoptosis and apoptosis caused by TNF-{alpha} may have a biological significance in inflammatory and immune-mediated demyelination. Apoptosis of OLG has been observed in EAE and MS (64, 65). IFN-{gamma}, cuprizone, and HTLV-1, known to induce demyelination in vivo, also induce OLG apoptosis (66, 67, 68). Therefore, an understanding of mechanisms leading to and preventing apoptosis of OLG and its progenitor cells is critically important to develop rational approaches to enhance OLG survival and remyelination.


   Acknowledgments
 
We thank Drs. E. Podack and S. Korsemyer who generously provided bcl-2 and bax cDNA, respectively. We also appreciate the excellent technical help of Dr. M. Chi in preparing primary OLG and O-2A progenitors and the preparation of the manuscript by N. Dehghan.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS36231 and NS15662. Back

2 Address correspondence and reprint requests to Dr. Moon L. Shin, University of Maryland School of Medicine, Department of Pathology, 10 South Pine Street, MSTF 600-E, Baltimore, MD 21201. E-mail address: Back

3 Abbreviations used in this paper: MS, multiple sclerosis; C7D, normal human serum immunochemically depleted of C7; DEVD-CHO, Asp-Glu-Val-Asp-Cho; EAE, experimental allergic encephalomyelitis; ERK, extracellular signal-related kinase; FGF, fibroblast growth factor; GC, galactocerebroside; MBP, myelin basic protein; MTS, methyl tetrazolium salt; NHS, normal human serum; O-2A, OLG progenitor cells identified by mAb A2B5; OLG, oligodendrocyte; PARP, poly(ADP-ribose) polymerase; PDGF, platelet-derived growth factor; PI-3, phosphatidylinositol-3; PLP, proteolipid protein; RT, room temperature; TCC, terminal complement complexes representing C5b-7, C5b-8, and C5b-9. Back

Received for publication June 15, 1999. Accepted for publication September 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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