Complement in multiple sclerosis: its role in disease and potential as a biomarker

Abstract

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system with a poorly defined and complex immunopathogenesis. Although initiated by reactive T cells, persistent inflammation is evident throughout the disease course. A contribution from complement has long been suspected, based on the results of pathological and functional studies which have demonstrated complement activation products in MS brain and biological fluids. However, the extent and nature of complement activation and its contribution to disease phenotype and long-term outcome remain unclear. Furthermore, functional polymorphisms in components and regulators of the complement system which cause dysregulation, and are known to contribute to other autoimmune inflammatory disorders, have not been investigated to date in MS in any detail. In this paper we review evidence from pathological, animal model and human functional and genetic studies, implicating activation of complement in MS. We also evaluate the potential of complement components and regulators and their polymorphic variants as biomarkers of disease, and suggest appropriate directions for future research.

Introduction

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults in northern European Caucasian populations, with an approximate lifetime risk of one in 400 [1]. Its exact aetiology remains unclear, but it is likely that diverse immunological factors interact with those from genes and the environment in individual patients to contribute to its characteristically variable pathology, phenotypic presentation, disease course and outcome [1,2]. The precise impact of these components of disease aetiology on clinical features is not well understood and largely unpredictable at disease onset [3], which creates considerable problems in disease management. Objective evidence from simple, easily repeatable, sensitive biomarkers are largely unavailable but would be invaluable in providing significant help to clinicians with diagnosis and assessment of prognosis, as well as the monitoring of treatment response in this complex disorder.

The fundamental concept underlying the perceived pathogenesis of MS is of wide arrays of inflammatory mediators interacting to cause both demyelination and axonal damage which, in turn, results in a variety of neurological deficits [4] (Fig. 1). However, this simplified overview may not explain entirely the pathogenic mechanism of progressive disease, attributed originally to multiple acute inflammatory events causing chronic axonal degeneration, and considered more recently to be an independent degenerative process possibly secondary to low levels of chronic background inflammation [5].

Fig. 1.

The pathogenic cascade in multiple sclerosis (MS): the initiation of inflammation central to the pathogenesis of MS depends on the peripheral activation of T cells via foreign antigens. Activated T cells express integrins which bind to up-regulated cell adhesion molecules on the blood–brain barrier (BBB) endothelial surface allowing diapedesis of T cells into the central nervous system (CNS). Within the CNS T cells re-encounter specific antigen and are reactivated setting up an inflammatory cascade which results in conduction block and demyelination. Myelin and axon damage is dependent on various effector mechanisms; the extent of involvement of each is thought to vary between individuals. Activated T cells bind directly to myelin epitopes leading to activation of macrophages (Mθ) which can phagocytose myelin directly or, along with T cells and glial cells, can release cytotoxic products and mediators such as proteases [122], inflammatory cytokines, e.g. tumour necrosis factor-α, interferon-γ[123], soluble reactive oxygen (0₃) and nitrogen (NO) intermediates [124,125], free radicals and glutamic acid. Further attack to myelin comes from antibodies and complement components – mainly the terminal complement complex – C5b–9 [126] triggered principally by infiltrating B cells and myelin antibodies.

Understanding of the role of complement (C) in the central nervous system (CNS) and in systemic inflammatory disorders is now evolving at a considerable pace. Although first suggested in the early 1970s to play a role in the pathogenesis of MS [6], the extent of C activation in subgroups of MS patients, the mechanisms of activation and the role of C in inflammation, axonal damage and neuroprotection remain unclear. In particular, the involvement of inflammatory mediators, including C, to the progressive phase of disease caused by axonal injury remains a point for debate and, without good animal models of axonal injury, remains difficult to investigate.

In part because of limited access to pathological human tissue, the animal model of MS, experimental autoimmune encephalomyelitis (EAE), has proved useful in developing insights into the role of C in MS. Indeed, in a recent review it has been suggested that animal models of MS may provide the best and broadest understanding of underlying disease processes and should be the primary focus of research [7]. However, there are many well-rehearsed problems in extrapolating models of animal disease to the human context, and pathological, functional and genetic studies in man will continue to provide a valuable contribution that should not be overlooked.

Although C activation is not specific to MS, patterns of activation, assessed in combination with other inflammatory and immune markers, may be of value as biomarkers in MS subgroups. Here we review the evidence from pathological work, animal model studies and human functional and genetic studies, implicating C in the pathogenesis of MS, and discuss the use of C activation products, components, regulators and their polymorphic variants as biomarkers of disease.

Complement activation and regulation

Complement plays a central role in the innate immune system, providing an important defence against infection and immune complex disease. The system consists of approximately 30 circulating and membrane expressed proteins which collaborate to provide protection from infection [8,9]. The physiological actions of C are mediated through production of opsonins (molecules that enhance the ability of macrophages and neutrophils with C receptors to phagocytose material – C3b, iC3b, C4b, etc.), anaphylatoxins (peptides that induce local and systemic inflammatory responses, increasing the permeability of blood vessels and attracting neutrophils through their chemotactic properties – C3a, C4a and C5a) and through direct killing of organisms by the terminal complement complex (TCC/C5b–9) which disrupts and forms pores in the phospholipid bilayer of a target cell. Although the C components in plasma are synthesized mainly by hepatocytes in the liver, within the CNS it is clear that glial cells and neurones can produce the majority of C proteins and expression is increased in response to inflammation [10–12].

The C system is activated via three initiating pathways (classical, alternative and lectin), all of which converge on a common effector pathway with formation of the TCC (Fig. 2). Inappropriate activation of C leading to depletion of the C components is normally prevented by C inhibitors present either in the fluid-phase or membrane-bound [13,14] (Fig. 2). Regulation in the alternative pathway is particularly important because the pathway is characterized by a constant ‘tick-over’ activation of C3, which can be amplified in pathology. Regulation in plasma is provided by factor H (fH) and deficiency of fH allows unregulated amplification with breakdown of C3 leading to C3 deficiency.

Fig. 2.

Activation and regulation of the complement (C) system: activation through both classical and alternative pathways results in the formation of C5 convertases, cleaving C5 and eventually forming the membrane attack complex (C5b–9). They also result in the formation of anaphylatoxins which cause inflammation and chemotaxis. The C system is under tight regulation by soluble (in italics) and membrane-bound (in bold) proteins (C1-inh, C1 inhibitor; C4bp, C4 binding protein; fI, factor I; MCP, membrane co-factor protein; CR1, C receptor 1; DAF, decay accelerating factor; fH, factor H; fB, factor B).

Humoral immunity in neuromyelitis optica and MS

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease affecting predominantly the optic nerves and spinal cord. Traditionally seen as a variant of MS, it has been redefined recently according to new criteria using a combination of phenotypic subtyping along with a newly developed biomarker of disease, NMO-immunoglobulin G (IgG) (reported sensitivity of 58–76% and specificity of 85–99% for NMO) [15,16]. The clinical differences between NMO and MS are mirrored by pathological differences. NMO is typified by abundant Ig and C deposits surrounding blood vessels in a distinctive rim or rosette fashion (corresponding with aquaporin-4 expression) [17], and is thought to be driven by humoral processes [18]. Therapeutic plasma exchange, known to deplete plasma proteins non-selectively, has been reported as having a beneficial role in NMO [19,20] and is thought to exert its therapeutic effect by reducing circulating levels of relevant autoantibodies.

Until recently, studies of MS pathogenesis and treatment have focused on the role of T cells; however, it is now clear that humoral immune mechanisms also play an important role which has been reassessed recently [21,22]. Circumstantial evidence for a B cell contribution to disease came initially from the demonstration of central clonal expansion of B cells and production of oligoclonal cerebrospinal fluid IgG (used routinely as a diagnostic biomarker in MS) [23]. More recently, several studies have demonstrated antibodies to myelin and other CNS autoantigens in MS patients but without convincing disease specificity, and the specific antigens responsible for initiating the immune response in MS remain unidentified [24]. Although Ig and C deposits are described in MS lesions, the localization of infiltrate is different from that in NMO [25] and has been suggested to be present only in a histological subgroup of patients (pattern II lesions) [26].

Therapies targeting humoral mechanisms, such as plasma exchange [19,27,28] and rituximab [a B cell-depleting monoclonal antibody (mAb)][29], have been tested with variable success in acute demyelinating events and it has been suggested that patients with pattern II histological lesions, showing prominent Ig and C deposition, are more likely to have a favourable response to this intervention [30]. The recognition of specific biomarkers for MS patients with extensive C activation may therefore be important in identifying patients who may benefit most from humoral-mediated therapies [31].

Complement in MS – evidence from pathology

A role for C in MS was proposed initially following the demonstration of C component C3 deposited in the brains of MS patients [6]. Immunocytochemical localization of the TCC in five of seven MS patients was reported in 1989; TCC was demonstrated in association with capillary endothelial cells, predominantly within plaques and adjacent white matter [32]. Localization of C activation to areas of active myelin destruction was demonstrated in a case report showing TCC deposited exclusively in such areas [33]. In a study of 23 cases of secondary progressive MS, the opsonin C3d was shown deposited in association with short segments of disrupted myelin in plaques with low-grade active demyelination and provided evidence for a C contribution to disease progression as well as acute inflammation [34].

More recently a complex and heterogeneous pathological model for MS has been proposed by Luchinetti et al., who have described four distinct patterns of demyelination in a group of 83 patients, with specimens collected either at autopsy (n = 32) or biopsy (n = 51) [26,35]. The pathological classification was based mainly on the presence of Ig and activated C (pattern II only), remyelination (patterns I and II) and primary oligodendrocyte (OGD) damage (patterns III and IV). The authors suggested that the four different patterns of disease provided evidence for different mechanisms of immune-mediated demyelination, with C playing a role in a distinct but large subgroup of patients.

When considering the pathological subgroups and their relation to disease phenotype, it must noted that the frequencies of the different patterns seen may be skewed heavily by patient selection for biopsy (possibly more aggressive disease). Further, the specimens collected in the study by Luchinetti et al. were from a cohort of patients with very early disease (the mean disease duration before autopsy or biopsy was 39 and 9 months respectively) and therefore may not represent accurately a global picture of MS. In addition, the study showed C and Ig depositions only in pattern II lesions, represented exclusively in more than 50% of the patients and in all subgroups of disease (relapsing–remitting, secondary and primary progressive). However, a more recent study has failed to demonstrate this heterogeneity, showing C and Ig deposition present consistently in areas of demyelination [25]. These authors have suggested that the heterogeneity described by Luchinetti et al. may have been a phenomenon of early disease and was therefore not seen in their samples, in which the mean disease duration was 22 years.

Serum or CSF biomarkers that identify any pathological subgroups accurately would help considerably with clarifying these issues and would provide direction for future research.

Complement in MS – animal models

The EAE, most commonly in rodents, has long been used as an animal model for MS [36,37], but was constructed initially to examine the monophasic illness: acute disseminated encephalomyelitis [38]. However, since its introduction numerous variations to the initial concept have evolved. Disease induction with a known CNS antigen [such as myelin basic protein (MBP) [39], proteolipoprotein [40], myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein and S-100 protein [41]] causes paralysis in animals with varying degrees of demyelination depending on the method of EAE induction and species used [42]. The two main models employed are the MBP model (resulting in a monophasic, non-demyelinating disease) and the antibody-mediated demyelinating EAE model (ADEAE) (where MBP immunization is followed by administration of anti-MOG antibodies). Although the MBP-induced model of EAE is thought by some to bear the closest resemblance to MS, it is likely that different models will have relevance in the understanding of different immunopathological subtypes or phases of disease. Information gained from these studies must be tempered by the knowledge that EAE, like most animal models of disease, is an incomplete and not entirely accurate reflection of the human situation. Models reflecting the axonal damage seen in MS have still to be used to analyse disruption of the CNS, but may yet provide a clearer understanding of progressive disease [43]. Despite all these problems, much information on C pathology, potential C biomarkers and therapeutics has been obtained from EAE studies in normal animals and transgenic mouse models [44], and these models have provided a test bed for studies of treatment with C suppressors [31].

Anti-C agents in EAE

The first studies to examine the role of C in EAE used the C activator cobra venom factor (CVF) as a treatment to reduce serum C, reducing the severity of EAE markedly [45–48]. Despite initial success, it was soon realized that response to this treatment was transient and repeated injections were highly immunogenic; further, CVF-induced C activation led to production of vast quantities of the proinflammatory anaphylatoxins C3a and C5a, sufficient to cause a shock syndrome in recipients. It was also reported that, although CVF treatment in MBP-induced EAE caused suppression of disease, in the ADEAE model CVF had no effect on disease severity despite C9 deposition being abolished [49].

To combat the problems of CVF, soluble C receptor 1 (sCR1) (which blocks the action of C3 and C5 convertases) was trialled as a treatment to decrease C activity and reduce the severity of EAE [50]. Treatment with sCR1 worked to some extent, but showed much less reduction in clinical severity scores when compared with treatment with CVF, perhaps reflecting an incomplete blockade of C activation [51].

Gene deletion models in EAE

Gene-deleted (knock-out) animal models have been used to enhance our understanding of the roles of the pathways of C activation in EAE. A study of C3 and factor B (fB) knock-out mice (the former unable to activate C, the latter alternative pathway-deficient) demonstrated normal incidence of MOG-induced EAE but attenuated severity of chronic disease in both mouse strains, with decreased numbers of infiltrating macrophages and T cells [52]. This work showed unequivocally that C contributed to the development of EAE, and also that the alternative pathway was vital in the development of disease. The role of C3 has since been questioned, with C3 knock-out mice showing similar disease to wild-type mice in one study [53], but attenuated disease in others [54,55]. The differences here may be explained by the use of different mouse models. Evidence that the alternative pathway is the main player is supported by studies in C4-deficient (classical pathway absent) animals, which showed EAE onset and progression virtually identical to wild-type animals [56,57].

Once activated, C has multiple roles in the EAE inflammatory lesion, causing demyelination [58], axonal damage and OGD injury [45]. The mechanisms by which this damage is mediated have been examined in detail. The membrane attack complex (MAC), the cytolytic end-product of the C cascade, is thought to play a significant role in demyelination, with evidence from pathological studies (as discussed), in vitro studies (deficiency of the MAC inhibitor CD59 was shown to be the cause of rat OGD susceptibility to lysis in culture [59] and in vivo studies. C9/MAC deposition was shown to correlate closely with demyelination in the spinal cord of rats in which EAE was induced with anti-MOG antibodies [58], while C6-deficient rats (unable to produce MAC), when immunized with MBP and given anti-MOG antibodies to induce ADEAE, failed to develop demyelination, axonal damage or paralysis, but when reconstituted with C6 went on to develop pathology and clinical disease similar to wild-type rats [60]. Despite these findings, the role of MAC has been questioned [54]; C5 knock-out mice were not protected from MBP-induced EAE, suggesting that the terminal pathway was not essential for disease [61,62]. The extent of the role of MAC in demyelination and remyelination in models and MS thus remains uncertain.

Anaphylatoxins C3a and C5a are important inflammatory mediators and were considered initially to play an important role in CNS inflammation, based predominantly on evidence showing the expression of their receptors (C3aR and C5aR) on neuronal cells [63]. C3aR expression on microglia and infiltrating macrophages was increased during EAE but neuronal expression was largely unchanged [64]. The expression of C5aR on microglia and hypertrophic astrocytes within the spinal cords of Lewis rats was highly up-regulated in EAE [65], leading to the hypothesis that C5a is an important inflammatory mediator in EAE. Since these initial studies, it has been demonstrated that C5a plays no role in demyelination in EAE; C5aR-deficient animals are fully susceptible to EAE [66], and treatment with a C5aR antagonist failed to protect against development of EAE [67]. In contrast to this, C3a-deficient mice in which EAE was induced exhibited an attenuated clinical course, and immunohistochemical analysis revealed reduced demyelination and infiltration of macrophages and T cells compared with control mice, indicating that anaphylatoxin C3a plays a role in inflammatory cell recruitment and demyelination [68]. Interestingly, these effects were more apparent in the chronic phase of disease, corresponding with results from C3-deficient mice [52].

Summary

Animal models have been useful in clarifying the role of C in EAE. It is clear that the alternative pathway contributes to disease while the roles of the classical and terminal pathways are less clear, with the latter reported to be vital for demyelination in some, but not all, EAE models. The anaphylatoxin C5a probably contributes little to disease, while C3a plays a larger part – predominantly through recruitment of inflammatory cells. The alternative pathway plays a dominant role in EAE, as has been shown in many other disease models [69,70], impacting particularly in chronic disease. While direct translation of these findings into human disease is not possible, they may suggest that the alternative pathway is important in disease perpetuation after initiation by a primary source. The roles of C in mediating axonal damage need to be investigated further and require animal models that represent axonal damage and chronic disease more clearly.

Complement as a serological or CSF biomarker of MS

In light of the evidence implicating C in MS, various C proteins have been considered as biomarkers of disease activity. These are summarized in Table 1 and discussed below.

Table 1.

Studies examining concentrations of complement (C) components in multiple sclerosis (MS) patients compared with control subjects.

Reference	C component measured	Sample type	Significant alteration in concentration compared with controls
Kuwert et al. 1965 [127]	C3/C4	CSF	Reduced
Link 1972 [128]	C3/C4	CSF	No difference
Dube et al. 1973 [129]	C3/C4	CSF	Increased
Yam et al. 1980 [130]	C3/C4	CSF	C3: increased
			C4: no difference
Price et al. 1980 [131]	C3/C4	CSF	In relapse: no difference
			In remission: decreased
Jans et al. 1984 [71]	C3/C4	CSF/serum	Serum and CSF C3: no difference
			Serum C4: increased (P = 0·05)
			CSF C4: reduced (P < 0·01)
Jongen et al. 2000 [72]	C3/C4	CSF/serum	Serum C3: reduced in RRMS and SPMS
			CSF C3: reduced in RRMS
			Serum and CSF C4: no difference
Sellebjerg et al. 1998 [76]	C3 and TCC	CSF/plasma	C3: no difference
			CSF TCC: increased and levels correlated with disability (P = 0·003)
Morgan et al. 1984 [73]	C9	CSF/plasma	Reduced in CSF
			No difference in plasma
Compston et al. 1986 [132]	C9	CSF/plasma	Reduced in CSF
			No difference in plasma
Sanders et al. 1986 [78]	TCC	CSF	Increased
Mollnes et al. 1987 [77]	TCC	CSF/serum	Increased in CSF
			No difference in serum
Rodriguez et al. 1990 [74]	C9	CSF	No difference
Halawa et al. 1989 [75]	C9	CSF	No difference

CSF, cerebrospinal fluid; RRMS, relapsing–remitting multilpe sclerosis; SPMS, secondary progressive multiple sclerosis; TCC, terminal complement complex.

C3 and C4

C3 and C4 are the most abundant of the C proteins and are measured routinely in many laboratories. Like most of the C components, both C3 and C4 are acute-phase reactants and increased synthesis in response to inflammation can mask even quite marked consumption. Perhaps, as a consequence, studies of C3 and C4 levels in MS serum and CSF have shown inconsistent and conflicting results [71,72]. Levels in serum and CSF from patients with neuroinflammatory diseases are likely to be influenced both by the presence of active inflammation and the integrity of the blood–brain barrier, both highly fluctuant in MS. Indeed, positive correlation of C3 and C4 CSF : serum ratios with albumin CSF : serum ratios has been identified in controls but not in MS patients [72].

Terminal pathway components and the TCC

Measurements of the terminal component, C9, and of the TCC have been performed in MS, although results have again been inconsistent. Low levels of C9 in MS CSF have been demonstrated in two studies, including one in which patients were subclassified into acute relapsing, remission and progressive with lowered C9 levels in all groups, implicating prior activation of the terminal C pathway [32,73]. Other studies have shown no difference in C9 levels compared with controls [74,75]. Measurement of TCC in CSF in three studies has consistently shown raised levels in MS patients [76–78], one study showing good correlation of CSF TCC concentration with disability [76]. There were no changes in C9 or C5b–9 levels in plasma in any of these studies, suggesting that CSF changes were a much better indicator of CNS disease.

Complement regulators

To date, little work has been conducted looking specifically at C regulators in MS. A recent study using proteomic analyses to examine C proteins in MS CSF found significant reduction in one unspecified isoform of fB, and of the alternative splice product of the fH gene, fHL-1, in MS CSF [79]. The authors suggested that measurement of specific C component and regulator protein isoforms would be most informative as biomarkers. Although there have been no follow-up studies, this work highlights the potential of C measurements as biomarkers in MS.

In one study, antibodies to two membrane C regulators (CD46 and CD59) were identified in MS serum, present in the acute phase of relapsing–remitting MS but not in chronic MS or control groups [80]. The antibodies were directed against the active site of the C regulator, inactivating their regulatory function and leading to excess activation of C. The authors suggest this as a mechanism for damage caused during acute relapse, but so far no one has either replicated this very small study, performed with less than 20 patients in each subgroup, or investigated these antibodies as possible biomarkers of disease. Nevertheless, the study highlights the need for the subclassification of MS on clinical grounds at the point of study design.

Summary

Although showing some variation in MS, C components and activation products have not been established as biomarkers of disease, in part because results obtained from different studies measuring the same C component have often been conflicting. These differences could be due to lack of standard techniques of measurement or to low patient numbers leading to underpowered studies; however, universally there has been poor stratification of study groups according to phenotype. In a relapsing immunologically heterogeneous inflammatory disease, clinical phenotype (such as aggressive or benign disease or intercurrent relapse) is likely to have a large effect on levels of C components. It is the view of the authors that much information can be gained from further analysis of C components and regulators in serum, plasma and CSF employing correlation of levels with clinical phenotype and other modalities of disease activity and severity such as magnetic resonance imaging.

Role of C in neuroprotection in MS

Neuroprotection from sublytic MAC

The neurotoxic effects of MAC are concentration-dependent and it has been suggested that partial deficiency of terminal components has evolutionary benefits [81–83]. Several groups have shown that a sublytic dose of MAC can be cytoprotective, with prior exposure leading to accumulated resistance to a subsequent lytic attack [84,85].

In one study in EAE, C5-deficient mice showed greater inflammatory demyelination and axonal loss [61], with more OGD apoptosis compared with controls [62]. Studies in vitro suggested strongly that this was a consequence of sublytic levels of MAC conferring protection from OGD apoptosis [86–89].

It is known that OGD apoptosis is associated with activation of caspases 3, 8, 9 and 11 [88–91] and inhibition with caspase-3 and -8 inhibitors can prevent OGD apoptosis in vitro[89]. Sublytic MAC has been shown in vitro to inhibit cytochrome c activation of caspase-3 and -9 and induce phosphorylation of BAD which, when phosphorylated, can bind to cytoplasmic 14-3-3 protein and increase cell survival [86]. Sublytic MAC has also be shown to inhibit Fas ligand and tumour necrosis factor-α-induced OGD apoptosis by inhibition of caspase-8 processing [87,89]. This provides in vitro and in vivo evidence of neuroprotection mediated by low levels of MAC, acting primarily via regulation of caspases [92,93].

Neuroprotection by anaphylatoxins

In vitro studies of anaphylatoxins C3a and C5a have shown potential neuroprotective effects. Pretreatment of primary murine corticohippocampal neurones with human or mouse recombinant C5a reduced glutamate neurotoxicity and prevented glutamate-induced neuronal apoptosis [94]; these events were signalled through mitogen-activated protein kinase-mediated regulation of caspase cascades [95]. C3a was neuroprotective against N-methyl-d-aspartate toxicity in the presence of astrocytes, but did not protect against serum deprivation-induced apoptotic neuronal death, or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate-mediated excitotoxicity [96]. In animal models, C5a receptor-deficient mice were more susceptible to apoptotic injury in vivo because of increased excitotoxicity [97].

Genetic findings implicating C in MS

Epidemiological studies have long recognized the important role of genetic factors in susceptibility to MS, with a number of largely unidentified genes contributing to disease risk [98–100]. More recently, genome-wide association studies have confirmed that single nucleotide polymorphisms (SNPs) in inflammatory cytokine receptors interleukin (IL)-2Rα and IL-7R are associated with susceptibility to MS [101–103].

A more significant role for genetic variability influencing C in MS may be signalled by recent findings in other CNS disorders with a significant inflammatory component. Studies in age-related macular degeneration (AMD) have recently identified a SNP in the locus encoding the C regulator fH (Y402H); homozygosity for the minor allele conferred a sixfold increased risk of disease [104–106]. This common polymorphism is located in short consensus repeat 7, a region implicated in binding multiple ligands, and has been suggested to alter affinity of fH for surfaces, thereby influencing capacity to inhibit C [107]. Other identified polymorphisms in the alternative pathway component fB and the classical pathway component C2 may contribute further to disease risk [108]. These minor changes in the C proteins causing subtle dysregulation in C have been shown to be associated strongly with the disease and are suggested to be global markers for AMD [109]. The Y402H polymorphism in fH described in AMD has also been identified as a risk allele for Alzheimer's disease (AD) in some, but not all, studies [110,111].

Given the role of C in neuroinflammation and the reports of C polymorphisms contributing to other inflammatory conditions, a candidate gene approach in MS to identify association with C components and regulators would seem logical. Although no studies have yet identified susceptibility genes within C components or receptors (Table 2), evidence for linkage in MS in the region around the centromere of chromosome 5 (containing genes for C components C6, C7 and C9) has been identified in four independent linkage studies [112–115].

Table 2.

Studies examining complement (C) component and regulator single nucleotide polymorphisms (SNPs) in relation to multiple sclerosis (MS) disease susceptibility.

Study	Gene tested	No. SNPs tested from gene	Cohort of patients	Result
Chataway et al. 1999 [133]	C6	1	227 multiplex families	No association
	C7	3	466 affected siblings	No distortion of transmission
			112 unaffected siblings
Simon et al. 2007 [134]	CR2	9	Not disclosed	No association
Bulman et al. 1991 [135]	C3	1 allele	129 patients	No association
			69 controls
Unpublished	FH	3	480 patients	No association

All the studies conducted so far looking at candidate genes have been underpowered to detect significant differences where the risk proportioned to each identified allele is very small (odds ratios < 1·3) [116], therefore negative studies can not be deemed reliable. Also, work to date has focused on disease susceptibility risk and has not examined phenotype, which may be more likely to be altered by C dysregulation. Further studies with large groups of patients are needed in order to uncover genetic biomarkers for MS within the C system, and to direct functional studies.

Complement activation and dysregulation in neurological disorders

Complement has been implicated firmly in the pathology of a number of diseases of the CNS and its role in NMO and AMD has been discussed. In addition, there have been reports of the alternative pathway regulator fH being a marker in AD, elevated significantly in AD patients compared with controls [117]. Further evidence of a role for fH in AD pathology has been provided by Honda and co-workers, who developed a mAb that specifically stained senile plaques and demonstrated subsequently that the mAb bound fH, present both in the senile plaques and in CSF in AD [118]. The Y402H polymorphism in fH described in AMD has also been identified as a risk allele for AD in some, but not all, studies [110,111]. Further genetic and functional work is being conducted to establish this C regulator as a clinically useful biomarker in AD.

Complement activation is also described in several other CNS conditions, including Guillain–Barré syndromes in which antibody and C-mediated mechanisms have been shown to be important in pathogenesis [119], and ischaemia–reperfusion injuries such as trauma and stroke in which C inhibition has been shown to be neuroprotective [120,121] (reviewed in [10]. These disorders demonstrate how, although not being the primary pathogenic factor, C dysregulation can contribute significantly to disease in the presence of other disease-initiating mechanisms.

Conclusion

This review summarizes compelling evidence from pathological, animal model and functional studies that C plays an important role in the immunopathogenesis of MS. Prior understanding of the immunology of MS and knowledge gained from animal studies leads us to conclude that C does not initiate disease, but propagates ongoing disease with increased contribution over the course of the illness. Severity may vary between individuals according to the extent of C dysregulation, contributed to possibly by functional polymorphisms in C components and regulators. Despite detailed information from animal model studies regarding the activation and regulation of C in demyelination, questions still remain as to the role of the terminal pathway, the extent of the neuroprotective function of C in disease, the role of C in axonal damage and the translation of these studies into the more complex human disease of MS.

This review has highlighted the importance of clinical classification of disease subgroups at the point of study design for functional and genetic work. In particular, studies looking at concentrations of C in serum and CSF have often been complicated by the disease heterogeneity in which acute relapse, stable and chronic disease are highly likely to have distinctly varying C contributions. In recent years advances in technology, in particular the use of proteomic and genetic analytical techniques, have led to better opportunities for finding sensitive and specific biomarkers of disease, as proven in other complex disorders such as AD, AMD and NMO. Use of these techniques, along with better clinical stratification, will bring us closer to finding useful biomarkers for MS; however, given the genetic, pathological, immunological and clinical heterogeneity of MS, it is highly unlikely there will be a single effective surrogate biomarker for disease and a combination of markers, possibly including C components and polymorphisms, are more likely to be informative.