Complement activation in autoimmune demyelination: Dual role in neuroinflammation and neuroprotection

Journal of Neuroimmunology 180 (2006) 9 – 16 www.elsevier.com/locate/jneuroim

Review article

Complement activation in autoimmune demyelination: Dual role in neuroinflammation and neuroprotection Horea Rus a,d,⁎, Cornelia Cudrici a , Florin Niculescu b , Moon L. Shin c a

Department of Neurology, Baltimore MD, USA Division of Rheumatology and Clinical Immunology, Department Medicine, Baltimore MD, USA c Department of Pathology, University of Maryland School of Medicine, Baltimore MD, USA Veterans Administration Maryland Health Care System Multiple Sclerosis Center of Excellence, Baltimore MD, USA b

d

Received 26 April 2006; received in revised form 5 July 2006; accepted 7 July 2006

Abstract Multiple sclerosis and its animal model experimental allergic encephalomyelitis are inflammatory demyelinating diseases of the central nervous system mediated by activated lymphocytes, macrophages/microglia and the complement system. Complement activation and the C5b-9 terminal complex contribute to the pathogenesis of these diseases through its role to promote demyelination. C5b-9 was also shown to protect oligodendrocytes from apoptosis both in vitro and in vivo. Our findings indicate that activation of complement and C5b-9 assembly plays a pro-inflammatory role in the acute phase, but may also be neuroprotective. © 2006 Elsevier B.V. All rights reserved. Keywords: Complement; C5b-9; Neuroprotection; Apoptosis; EAE; MS

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complement system: activation and assembly of the terminal pathway . Role of complement activation in neuroinflammation . . . . . . . . . . 3.1. In vitro demyelination by C5b-9 . . . . . . . . . . . . . . . . . 3.2. Activation of complement by myelin and oligodendrocytes. . . . 3.3. Role of complement in inflammatory demyelination during EAE. 4. Role of complement activation in neuroprotection . . . . . . . . . . . . 4.1. Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 . . . . . 4.2. Neuronal protection by complement. . . . . . . . . . . . . . . . 4.3. Contribution of complement activation to neuroprotection in EAE 5. Complement activation in multiple sclerosis . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MBL, mannose-binding lectin; MASP, MBL associated serine protease; MS, multiple sclerosis; OLG, oligodendrocytes; MOG, myelin oligodendrocyte glycoprotein; PNS, peripheral nervous system. ⁎ Corresponding author. University of Maryland, School of Medicine, Department of Neurology, 655 W. Baltimore Street, BRB 12-016, Baltimore, MD 21201. Tel.: +1 410 706 3170; fax: +1 410 706 0186. E-mail address: [email protected] (H. Rus). 0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.07.009

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1. Introduction Multiple sclerosis (MS) is considered an autoimmune disease in which myelin and the myelin-producing oligodendrocytes (OLG) are the targets of immune attack. The essential requirements for initiating central nervous system (CNS) inflammation characteristic of MS are the expression of encephalitogenic antigens, generation of chemotactic signals in the CNS, expression and up-regulation of adhesion molecules on endothelial cells, and activation of antigen-specific CD4+ T cells (Steinman, 2001). CD4 T cells are primed in the periphery and then enter the CNS. In the perivascular space they encounter myelin antigen expressed by local antigen-presenting cells, microglia, and possibly dendritic cells (Greter et al., 2005; McMahon et al., 2005). The reactivated CD4 T cells then invade the parenchyma of the CNS and release proinflammatory cytokines and activate microglia (Heppner et al., 2005). Subsequent demyelination may occur through activation of macrophages and cytotoxic T cells (Lucchinetti et al., 2001, 2000). Activation of complement by antibodies against myelin proteins found in MS patients (Egg et al., 2001; Reindl et al., 1999) or by myelin and OLG themselves (Vanguri et al., 1982; Wren and Noble, 1989) can also contribute to myelin damage. Recent observations have indicated that inflammation has protective effects. In the context of inflammatory demyelination, these protective effects lead to axon preservation/ repair, limitation of glial scarring, remyelination, and protection from cell death within the CNS. A protective role has been shown for T-lymphocytes and macrophages, which also secrete growth factors involved in regulating survival and differentiation of neurons (Hohlfeld et al., 2000) and differentiation of OLG progenitors (Diemel et al., 1998). Several cytokines, such as TNFα, IL-1β, and IFNγ were also shown to have protective effects (Correale and Villa, 2004; Diemel et al., 2003). Neuroprotective effects of complement activation have recently been documented, suggesting that the C5b-9 complement complex plays also an important role in the repair process (Weerth et al., 2003). In this review we present experimental and clinical evidence that point to the involvement of complement activation and the subsequent assembly of the C5b-9 complement complex in both neuroinflammation and neuroprotection in MS and in experimental autoimmune encephalomyelitis (EAE). 2. Complement system: activation and assembly of the terminal pathway Complement is a system of soluble proteins, receptors, and regulators that protect the host from infection and function as immune effectors and regulators (Frank et al., 1995; Shin et al., 1996). The complement cascade is initiated by one of three pathways: the classical, alternative, or lectin pathways (Fig. 1). Antigen–antibody complexes primarily activate the classical pathway, although a number of other structures are capable of activating it, such as bacterial surfaces, viruses, C-reactive protein, and cell debris. It is activated when the binding of C1q

to the antibody–antigen complex allows activation of two molecules each of C1r and C1s. Activated C1r and C1s possess serine esterase activity, and this activity cleaves C4 into the products C4a and C4b. The C1rC1s complex also cleaves C2 to generate the products C2a and C2b. C4a and C2b function as anaphylatoxins, while C4b and C2a form the C3 convertase, C4b2a. The alternative pathway is activated when factor B is associated with either the spontaneously hydrolyzed C3 (H2O) or C3b formed from the classical pathway. Factor B is then cleaved by factor D to generate C3Bb or C3 (H2O) Bb, the alternative pathway C3 convertase. The lectin pathway is initiated when mannose-binding lectin (MBL) binds to monosaccharides on the surfaces of bacteria, fungi, and parasites. MBL then interacts with MBL associated serine proteases (MASP)-1, 2 and 3 that in turn become active and form C3 convertase by cleaving C2 and C4 (Wallis, 2002). All three pathways converge at the level of C3 cleavage and proceed to the assembly of the terminal, membrane attack pathway. C3 and C5 cleavage generates the anaphylatoxins, C3a and C5a. C5a is also a chemotactic factor (Frank et al., 1995). The activation of C5–C9 and assembly of C5b-9 starts when C5-convertase generating C5a and C5b cleave C5. The terminal pathway leads to assembly of the terminal complement complexes C5b-7, C5b-8, and C5b-9, as depicted in Fig. 1. C5b-7 is cytolytically inactive and does not form pores (Morgan and Campbell, 1985; Shin and Carney, 1988), while C5b-8 and C5b-9 are pore-forming complexes. C5b-8 pores range from 0.4 nm to 3 nm in diameter and are required in large numbers to lyse erythrocytes (Gee et al., 1980) or nucleated cells (Shin et al., 1996). The diameter of C5b-9 pores can range from 1 to 11 nm, as up to 16 molecules of C9 polymerize and insert into the cytoplasmic membrane (Dalmasso and Benson, 1981; Ramm et al., 1985). 3. Role of complement activation in neuroinflammation 3.1. In vitro demyelination by C5b-9 The requirement for anti-myelin antibodies and serum complement in demyelination was studied in vitro by using myelinated CNS explant cultures (Seil, 1977). Treatment of myelinated explants with IgG or IgM anti-myelin antibodies and fresh serum induced extensive demyelination. To study the possible role of C5b-9, serum complement was activated using the IgM fraction of anti-guinea pig spinal cord antiserum and human serum depleted in late component C8 (C8D) with and without C8 to assemble C5b-7 and C5b-9 (Liu et al., 1983) respectively. Exposure to antibody and C8D failed to induce demyelination, in contrast C8 addition to C8D to complete C5b-9 assembly induced extensive demyelination and eventual myelin loss after 20 h. Removal of antibody and complement after 2 h failed to inhibit myelin vesiculation (Liu et al., 1983) suggesting that molecular changes of myelin damage were initiated by C5b-9 within 2 h, and continuous activation of complement may not be required. These findings unequivocally demonstrated the C5b-9 complex as an effector

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Fig. 1. Activation pathways and assembly of complement system. Activation of classical, alternative and lectin pathways leads to the generation of C3 convertases (C4b2a, C3bBb) and C5 convertases (C4b2a3b, C3bBb3b). Cleavage of C5 to generate C5b initiates the terminal complex pathway and assembly of C5b-9 complex. There are several levels of regulation of the complement system. Membrane proteins CD55, CR1 and CD46 are important regulators inhibiting C3 and C5 convertases, while CD59 down-regulate both C8/C9 binding to C5b-7 and C9 polymerization. The life span of C3 and C5 convertases is greatly reduced by serum protein C4bp, factor H and factor I. Fluid phase assembly of C5b-9 is inhibited by S-protein and clusterin.

in myelin damage and demyelination. Although isolated myelin (Cyong et al., 1982; Silverman et al., 1984; Vanguri and Shin, 1986) and OLG (Scolding et al., 1989a; Wren and Noble, 1989) can activate C1 and generate C5b-9 in vitro, anti-myelin antibodies, including anti-galactocerebroside, are required for complement to produce significant demyelination in explants by increasing the level of C5b-9. Demyelination induced in explants, as shown by massive myelin vesiculation, may differ from that of in vivo demyelination since damaged myelin is opsonized by C4b/C3b and iC3b and will be actively removed by macrophages derived from circulating monocytes. 3.2. Activation of complement by myelin and oligodendrocytes Activation of complement by myelin was initially shown by the ability of purified myelin prepared from adult human spinal cord and rat brain to induce consumption of total hemolytic activity (Liu et al., 1983). CNS but not peripheral nervous system (PNS) myelin depleted serum C5 activity and generated C5b-9 and this effect is mediated by activation of C1 (Cyong et al., 1982; Silverman et al., 1984; Vanguri and Shin, 1986). The component of myelin that activates C1 was only partially characterized (Vanguri and Shin, 1986). Primary

OLG isolated from rat brain can activate complement through the classical pathway in the absence of antibody and lead to C5b-9 assembly in heterologous as well as homologous system (Scolding et al., 1989a; Wren and Noble, 1989). The lack of CD59 expression on rat OLG is thought to increase their lytic susceptibility, since incorporation of human CD59 in rat OLG increased the survival from human complement attack (Wing et al., 1992). This protection was reversed by antiCD59 antibody (Wing et al., 1992). In adult human OLG, CD59 expression is deficient only in a subpopulation, while deficiency of complement receptor 1 (CR1), membrane cofactor protein (MCP) or CD46, and clusterin is more general (Scolding et al., 1998). Only decay accelerating factor (DAF) was consistently expressed by human OLG. Comparative studies have shown that DAF offers weaker protection against complement attack than MCP or CD59 (Tandon et al., 1994). In contrast, human myelin is not deficient in CD59 expression, but lack MCP and DAF (CD55). These data suggest that that human OLG and myelin are vulnerable to complement attack because they are unable to inhibit the activation cascade. However the repair mechanisms from complement attack by eliminating membrane-inserted C5b-9 is intact in OLG (Scolding et al., 1989b). Thus myelin appears significantly more vulnerable to C5b-9 attack than OLG.

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The pro-inflammatory activities associated with demyelination may also be generated by OLG through C5b-9mediated mobilization of arachidonic acid. Metabolic products of arachidonic acid, such as leukotriene E4, leukotriene B4, and 15-hydroxyeicosatetraenoic acid are released from OLG during C5b-9 attack (Shirazi et al., 1987). Only during sublytic C5b-9 attack, when OLG remain alive, is leukotriene B4 production observed. Hydrolysis of phospholipids induced by C5b-9 is through the activation of phospholipases via Pertussis toxin-sensitive Gi- and Ca2+dependent pathways, which are also involved in activating PKC by C5b-9 (Niculescu et al., 1993; Seeger et al., 1986). These lipid-derived inflammatory mediators may enhance demyelination by amplifying the inflammatory response. 3.3. Role of complement in inflammatory demyelination during EAE The involvement of complement in demyelination in vivo has been investigated in EAE by inhibiting complement activation and by using rodents deficient in particular complement components. In rats, depletion or inhibition of complement using cobra venom factor or soluble CR1 has been shown to ameliorate EAE (Linington et al., 1989; Piddlesden et al., 1994). In an antibody-mediated model of EAE induced with Myelin Oligodendrocyte Glycoprotein (MOG) complementfixing anti-MOG antibody was found to be essential in inducing demyelination (Piddlesden et al., 1991, 1993). In C3 knockout mice, MOG-induced EAE has produced conflicting results: one group has reported lower clinical scores with reduced inflammation, indicating a requirement of C3 in demyelination (Nataf et al., 2000), while another group, using a higher dose of MOG, has seen similar clinical signs in EAE knockout and control mice (Calida et al., 2001). Deletion of C3aR has been found to be protective in MOG-induced EAE (Boos et al., 2004). Factor B knockout mice developed less severe disease than did control mice, indicating a role for the alternative pathway in complement activation in EAE (Nataf et al., 2000). Blockade of C5a receptors and C5a receptor deficiency in mice failed to protect against EAE (Morgan et al., 2004; Reiman et al., 2002). Thus, C5aR might not play an important role in mediation of inflammation in EAE. The role of C5b-9 in demyelination and axonal damage has been reported in an EAE model in C6-deficient rats (Mead et al., 2002; Tran et al., 2002). In these rats, which are unable to form C5b-9, disease activity and demyelination, as well as T-cell and macrophage infiltrates, were significantly reduced (Mead et al., 2002; Tran et al., 2002). Axonal fragmentation and swelling was seen in demyelinated area of the C6 sufficient rats, compared with the normal axonal structure in the C6 deficient rat. Strong staining for C9, indicative of C5b-9 deposition, correlates well with the area of axonal degeneration and demyelination in the C6 sufficient rats. The expression of P-selectin is also lower on endothelial cells in C6-deficient rats than in normal animals, but no differences are seen in the profile of Th1 or Th2 cytokines (Tran et al., 2002). These data

indicate that C5b-9 plays a pro-inflammatory role in the acute phase of EAE. 4. Role of complement activation in neuroprotection 4.1. Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 Differentiation of OLG in serum free medium is associated with apoptotic cell death. Serum withdrawal induced apoptosis in OLG is associated with caspase activation that include caspase-8, caspase-9 and caspase-3 (Cudrici et al., 2006; Rus et al., 2004; Soane et al., 2001, 1999). Apoptosis of OLG is inhibited by a specific caspase-3 inhibitor, DEVD-CHO (Soane et al., 1999) and the caspase-8 inhibitor, Z-IETDFMK in a dose-dependent manner (Cudrici et al., 2006). We have also found that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is down-regulated during OLG differentiation and induced in response to sublytic C5b-9. In addition, BID cleavage, cytochrome c release, and a markedly reduced expression of c-FLIPL are seen in serum deprived OLG. Furthermore, we showed that sublytic C5b-9 was effective in inhibiting cytochrome c release, activation of caspase-9 and caspase-3, thus rescuing OLG from serum deprivation-induced apoptotic cell death. C5b-9-mediated activation of the PI3K/Akt signaling pathway led also to the phosphorylation of BAD (Soane et al., 2001). Association of BAD with BCL-XL is thought to produce mitochondrial damage by allowing oligomerization of proapoptotic BAX and BAK. On the other hand, dissociation of BAD from BCL-XL and binding to cytoplasmic 14-3-3 proteins increase cell survival and require phosphorylation of BAD at Ser112, Ser136, and possibly Ser156 (Zha et al., 1996). C5b-9 has been shown to stimulate phosphorylation of BAD at Ser112 and Ser136 and cause dissociation of the BAD/BCL-XL complex (Soane et al., 2001). The PI3K inhibitor, LY 240092, can reverse both processes. Therefore, sublytic C5b-9 appears to increase OLG survival in part by activating signaling pathways that phosphorylate BAD and reduce its association with BCL-XL. To identify initiators of apoptosis in OLG, we evaluated the ability of TNFα and FasL to induce apoptosis and examined the effect of C5b-9 on these pathways. Both TNFα and FasL were able to induce apoptosis of OLG and C5b-9 inhibited FasL and TNF-α-induced cell death. This effect is most probably mediated through inhibition of caspase-8 processing by C5b-9 (Cudrici et al., 2006). Since cleavage of BID by caspase-8 has been shown to directly trigger the release of cytochrome c from mitochondria (Esposti, 2002), we monitored BID cleavage as well as the levels of c-FLIP, an endogenous inhibitor of caspase-8 (Thome and Tschopp, 2001). Exposure to C5b-9 inhibited BID cleavage and caused a significant increase in c-FLIPL expression (Cudrici et al., 2006). These results suggest that C5b-9 prevent further caspase-8 processing, through a c-FLIPL-dependent mechanism. All these C5b-9 effects on OLG apoptosis are mediated through activation of PI3K. Thus, our data indicate that C5b-9,

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acting through PI3K signaling, is able to rescue OLG apoptosis by regulating caspase-8 activation and preventing mitochondrial insertion of proapoptotic proteins BAD and BID. 4.2. Neuronal protection by complement In addition to the role in neurodegeneration, complement activation has neuroprotective effects (van Beek et al., 2003). Complement anaphylatoxins C3a and C5a have multiple effects relevant to neuronal survival. C5a protects against glutamate-induced neurotoxicity (Osaka et al., 1999) and apoptosis in neurons through MAPK-mediated regulation of caspase activation (Mukherjee and Pasinetti, 2001). C3a also has protective effects against NMDA-induced neuronal cell death (van Beek et al., 2001). Both C3a and C5a induced NGF mRNA expression in astrocytes and in association with IL-1β increased NGF secretion (Jauneau et al., 2006). These data suggest that C3a and C5a participate in modulation of neuronal survival. 4.3. Contribution of complement activation to neuroprotection in EAE We have recently analyzed the influence of C5 on inflammatory demyelination during the course of EAE in C5deficient (C5-d) and C5-sufficient (C5-s) mice (Weerth et al., 2003). Both groups of mice displayed early-onset EAE, with a

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short recovery phase followed by a stable chronic course. During acute EAE, the lesions were more diffuse and extensive in C5-s than in C5-d mice. More striking differences attributed to the presence of C5 were found in the chronic lesions. In the absence of C5, EAE was associated with greater inflammatory demyelination, axonal loss, and progression to gliosis (Weerth et al., 2003). On the other hand, C5-s mice showed prominent myelin repair and axon preservation during the chronic phase of EAE. We have next analyzed the effect of complement C5 on the apoptosis of OLG during EAE (Niculescu et al., 2004). In acute EAE, C5-d and C5-s mice had similar numbers of total apoptotic cells; during recovery, however, C5-s mice showed significantly fewer apoptotic cells than did C5-d mice. In addition, although both groups of mice displayed TUNEL(+) OLG, there were significantly fewer in C5-s than in C5-d during both acute EAE and recovery (Niculescu et al., 2004). These data also point to a dual role for C5: enhancing inflammatory demyelination in acute EAE and promoting remyelination and repair during recovery. In vivo evidences also indicate that complement activation is protective for neurons. Mice deficient in C5 are more susceptible to kainic acid excitotoxicity than normal mice (Tocco et al., 1997). Decay accelerating factor (CD55) is expressed by neurons in response to chronic but not acute EAE in the presence of C3b deposition on neuronal cell bodies and axons. Levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46) and CD59 were

Fig. 2. Role of complement activation in the pathogenesis of multiple sclerosis. The immune response is initiated in the peripheral lymphoid tissue by myelin antigens or cross-reactive foreign antigens presented by antigen-presenting cells (APC) to T cells. In the CNS these T cells re-encounter antigens that are presented by microglial cells through the MHC class II molecules. CD8 + T cells can directly affect OLG by Fas–FasL specific interaction leading to OLG apoptosis. After B cell migrate into the brain and encounter their specific antigen they clonally expanded and mature into plasma cells. Antibodies produced by plasma cells will induce demyelination by antibody-dependent cell-mediated cytotoxicity and by activation of the complement system with subsequent assembly of C5b-9. Complement can also be activated directly by myelin. Complement activation products like C3b also play an important role in opsonization of myelin fragments and uptake by macrophages through complement receptors. C5b-9 is known to be involved in demyelination and was also found to protect oligodendrocytes from apoptosis induced by Fas–FasL pathway.

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unaffected in both acute and chronic EAE. These data suggest that increased CD55 expression by neurons might represent an important protective mechanism in an inflammatory milieu.

drugs that modulate rather than inhibit the activation of the complement system may represent a new direction in therapy for demyelinating disorders.

5. Complement activation in multiple sclerosis

Acknowledgments

In the MS white matter lesions deposition of C1q, C3d, and C5b-9 was detected on and within macrophages/microglia, astrocytes and in blood vessel walls (Barnett and Prineas, 2004; Compston et al., 1989; Lucchinetti et al., 2001; Storch et al., 1998) . The MS lesions have been have been confined to four patterns. High levels of immunoglobulins and C5b-9 deposition at sites of active myelin destruction were found in pattern II and C5-9 deposits were present only in this type of demyelinating lesions (Lucchinetti et al., 2001, 2000). Although apoptosis of OLG may occur in all four types of lesions, only pattern III generally shows a significant number of apoptotic OLG. While incidence of patterns II and III lesions were similar in acute MS, the latter become rare in chronic MS indicating that apoptosis of OLG is an early event in MS lesion evolution. Based on this classification it is tempting to speculate that apoptotic OLG are less frequently seen in pattern II as a result of the activation of complement and subsequent C5b-9 assembly, leading to the rescue of OLG from apoptosis (Fig. 2). When only acute MS lesions were examined, extensive OLG apoptosis and microglial activation were observed in tissues containing few or no lymphocytes or myelin phagocytes (Barnett and Prineas, 2004). In acute lesions, within hours OLG throughout the affected tissue appear apoptotic, myelin sheaths stain positively for activated complement components C3d and C5b-9, while immunoreactivity for 2′, 3′-cyclic nucleotide 3′-phosphodiesterase and myelin-associated glycoprotein is diminished, and ramified microglia with thickened processes appear in increased numbers (Barnett and Prineas, 2004). Engulfed myelin positive for complement activation products was present in macrophages. These data suggest that complement activation also plays a role in the clearance of vacuolated myelin by macrophages (Barnett and Prineas, 2004). In the purely cortical lesions, the extent of complement deposition was generally low and suggest that the role of complement in the pathogenesis of MS lesion is also location-dependent (Brink et al., 2005). Increased levels of SC5b-9 have been detected in the spinal fluid of MS patients during relapses (Mollnes et al., 1987; Sanders et al., 1988; Sellebjerg et al., 1998), and these levels have been shown to correlate with neurological disability as measured by Expanded Disability Status Scale (Sellebjerg et al., 1998). These findings suggest that complement activation and C5b-9 assembly take place also in the MS spinal fluid when a breakdown has occurred in the blood–brain barrier. Taken together, these data indicate that complement activation can play both pro-inflammatory and neuroprotective roles in MS. Because activation of complement has many beneficial effects, long-term inhibition of complement activation may have undesirable consequences. Therefore, designing

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