The Role of the Complement System and the Activation Fragment C5a ...

Neuromol Med (2010) 12:179–192 DOI 10.1007/s12017-009-8085-y

ORIGINAL PAPER

The Role of the Complement System and the Activation Fragment C5a in the Central Nervous System Trent M. Woodruff Æ Rahasson R. Ager Æ Andrea J. Tenner Æ Peter G. Noakes Æ Stephen M. Taylor

Received: 31 March 2009 / Accepted: 25 August 2009 / Published online: 11 September 2009 Ó Humana Press Inc. 2009

Abstract The complement system is a pivotal component of the innate immune system which protects the host from infection and injury. Complement proteins can be induced in all cell types within the central nervous system (CNS), where the pathway seems to play similar roles in host defense. Complement activation produces the C5 cleavage fragment C5a, a potent inflammatory mediator, which recruits and activates immune cells. The primary cellular receptor for C5a, the C5a receptor (CD88), has been reported to be on all CNS cells, including neurons and glia, suggesting a functional role for C5a in the CNS. A second receptor for C5a, the C5a-like receptor 2 (C5L2), is also expressed on these cells; however, little is currently known about its potential role in the CNS. The potent immune and inflammatory actions of complement activation are necessary for host defense. However, if over-activated, or left unchecked it promotes tissue injury and contributes to brain disease pathology. Thus, complement activation, and subsequent C5a generation, is thought to play a significant role in the progression of CNS disease. Paradoxically, complement may also exert a neuroprotective role in these diseases by aiding in the elimination of aggregated and toxic proteins and debris which are a principal hallmark of many of these

T. M. Woodruff (&)
P. G. Noakes
S. M. Taylor School of Biomedical Sciences, University of Queensland, St. Lucia, Brisbane 4072, Australia e-mail: [email protected] P. G. Noakes Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane 4072, Australia R. R. Ager
A. J. Tenner Department of Molecular Biology and Biochemistry, University of California, Irvine 92697, USA

diseases. This review will discuss the expression and known roles for complement in the CNS, with a particular focus on the pro-inflammatory end-product, C5a. The possible overarching role for C5a in diseases of the CNS is reviewed, and the therapeutic potential of blocking C5a/CD88 interaction is evaluated. Keywords Complement
Central nervous system
C5a
Inflammation
Neurodegeneration

Introduction: Activation of Complement The complement system is a phylogenetically ancient innate immune pathway comprised of numerous serum and membrane bound components, which activates a cascade of molecular events leading to multiple effector mechanisms to protect the host in response to a wide variety of stimuli. Its primary function is to provide a rapid response to infection and injury by opsonising pathogens, recruiting immune and inflammatory cells, stimulating the immune system, and ultimately aiding in the destruction of invading organisms (Liszewski et al. 1996). Complement is activated by three major pathways: the classical, lectin, and alternative pathways; but may also be activated by a recently discovered fourth extrinsic protease pathway (Huber-Lang et al. 2006). The classical pathway is primarily activated in response to the complement factor C1q binding to antigen–antibody complexes. C1q may also directly bind to pathogen surfaces and to non-pathogen surfaces such as b-amyloid and liposomes (Jiang et al. 1994; Marjan et al. 1994). The lectin pathway is activated following the binding of the mannanbinding lectin protein to specifically arranged mannose residues and other sugars located on pathogens, but not on vertebrate cells; whereas the alternative pathway is

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activated by foreign surfaces. Activation by all three pathways results in the formation of C3 and C5 convertase enzymes which cleave their respective inactive complement factors (C3 and C5) into their active fragments (C3a, C3b and C5a, C5b). The final step in the complement cascade is the formation of the membrane attack complex (MAC) through C5b association with complement factors C6–C9 (C5b–9) on pathogenic cell membranes, which forms a transmembrane pore, resulting in cell lysis (Podack et al. 1982). A fourth pathway for complement activation, termed the extrinsic protease pathway, has been recently discovered. This pathway is initiated by proteases such as thrombin that directly cleave C5, even in the absence of C3 (Huber-Lang et al. 2006). As a result, synthesis of C5 by inflammatory cells can produce C5a via cleavage of C5 with cell-derived proteases (Huber-Lang et al. 2002). These pathways represent a model whereby local, extracellularly released C5, may be potentially cleaved directly into C5a and C5b, providing a source of complement activation factors in the absence of upstream complement activation. To prevent complement-mediated damage to host cells, the system is tightly regulated by a number of complement-regulatory molecules. These molecules, expressed either in the fluidphase or membrane-bound, inhibit the assembly of the C3 convertase enzymes or the insertion of the terminal MAC into host cell membranes (Liszewski et al. 1996). For a comprehensive review of these complement activation and regulation pathways, see (Ricklin and Lambris 2007). The C3a and C5a produced following complement activation are fluid-phase inflammatory mediators termed anaphylatoxins. C5a is a 74 amino acid protein, and is the most potent of the anaphylatoxins. It exerts its major effects through binding to the membrane-bound G-protein coupled first-identified C5a receptor (CD88 or C5aR) (Gerard and Gerard 1991). The C5a-like receptor 2 (C5L2 or GPR77) has also been identified, though less is known about its role, in part due to its inability to couple to G-proteins (Cain and Monk 2002). The major function of the anaphylatoxins is to recruit immune cells to the site of infection or injury through chemotaxis and the subsequent activation of these cells, thereby promoting inflammation and aiding in host defense (Huber-Lang et al. 2002). Despite being immunologically separate from the systemic serum complement system, the central nervous system (CNS) is able to internally express all complement factors (see below). However, it is not known if all of the same activation pathways occurring in the periphery also occur in the intact CNS. Certainly, in the case of CNS infections, some complement activation pathways are involved (Mehlhop and Diamond 2006). Necrotic and late apoptotic neuronal cells also readily activate complement through C1q binding and C3 activation likely through a

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loss of complement inhibitory/regulatory surface molecules from the dying cell, and exposure of complement activating intracellular constituents such as phosphatidyl serine and DNA (Cole et al. 2006)—a mechanism potentially occurring in neurodegenerative diseases. Furthermore, in Alzheimer’s Disease (AD), b-amyloid protein can activate alternative and classical pathways (Jiang et al. 1994; Bradt et al. 1998). C-reactive protein can also bind C1q to activate classical complement pathways (Biro et al. 2007). It is also possible that endogenous synthesis and release of C5 by CNS cells and subsequent proteolytic cleavage by cellderived proteases to generate C5a may occur in the CNS, as has been shown to occur in inflammatory cells (HuberLang et al. 2002). However, this has yet to be unequivocally demonstrated. Regardless, it is clear that complement factors can be abundantly expressed in the CNS, particularly in response to injury. This review will discuss the expression and functional roles of these complement activation products in the CNS, and explore the available evidence indicating a contribution of complement activation in CNS disease. The complement activation fragment C5a and its major receptor, CD88, are given particular emphasis.

Complement Expression in the CNS Outside the CNS, a primary site of complement synthesis is in liver by hepatocytes (Li et al. 2007) which provide a sufficient source of circulating complement factors necessary to fight most infections. However, unlike peripheral tissues, the CNS does not receive the same composition of blood-borne protein factors as the blood–brain barrier (BBB) restricts the passage of cells and large molecules from the blood into the brain parenchyma (Kleine and Benes 2006). The restricted immunosurveillance created by the BBB has led scientists to designate the brain as an immune-privileged organ, and to propose that in order for the brain to generate an immune response in the absence of BBB damage, cells of the CNS must generate and secrete most of their own immune molecules (Kleine and Benes 2006). This is a relatively new concept and the unraveling of the CNS from the adaptive immune system is forcing a new appreciation of specific and local immune mechanisms that co-exist with the general body systems, which is a challenge with clear therapeutic implications. Over the past few decades, extensive studies have demonstrated various syntheses of complement components by various cells of the CNS (Gasque et al. 2000; Van Beek et al. 2003). The first study showing complement synthesis by brain cells was performed using both primary and cell-line derived rodent astrocytes (Levi-Strauss and Mallat 1987). In subsequent years, both primary and cell

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Table 1 A wide variety of complement components are synthesized by cells of the human CNS Pathway/cell group

Component

Cell type

Reference

Neurons Classical

C1q, C2, C3

IMR-32, SK-SH, SK-MC

Walker and McGeer 1993; Walker et al. 1995a

C4, C1-INH

SH-SY5Y and KELLY neuroblastoma

Veerhuis et al. 1998; Thomas et al. 2000

Alternative

C3, fB, fH

SK-SH, SK-MC, and SH-SY5Y neuroblastoma

Walker and McGeer 1993; Gasque et al. 1996a; Thomas et al. 2000

Terminal

C5, C6, C7, C9

SK-MC, SK-SH, SH-SY5Y

Walker and McGeer 1993; Gasque et al. 1996a

Sp, clusterin

KELLY neuroblastoma

Thomas et al. 2000

C3aR CD59, MCP

SK-MC neuroblastoma IMR-32 and SK-SH neuroblastoma

Davoust et al. 1999 McGeer et al. 1991; Gasque et al. 1996a

C1q, C1r, C1s

Primary microglia

Receptors Inhibitors, cell bound Microglia Classical

Walker et al. 1995a, b

C3, C4, C1-INH

Veerhuis et al. 1999

Alternative

C3

Primary Microglia

Walker et al. 1995a, b

Receptors

CR2, C3aR

Primary microglia/THP-1 and U937 monocyte cell lines

Gasque et al. 1998

Inhibitors, cell bound

DAF, CD59

THP-1 and U937 monocyte cell lines

Gasque et al. 1998

C1q, C1r, C1s, C2

Primary astrocytes/D54-MG, U105-MG, CB193,

Barnum et al. 1992a, b, 1993; Gasque et al. 1992

C3, C4, C1-INH

U118-MG, T193, T98G, and U373-MG astroglioma

Gasque et al. 1993; Walker and McGeer 1993; Veerhuis et al. 1998; Walker et al. 1998

Alternative

C3, fB, fD, fI, fH, fP

Primary astrocytes/D54-MG, U105-MG

Barnum et al. 1992a, b, 1993; Gordon et al. 1992a

CB193 astroglioma

Gasque et al. 1992; Avery et al. 1993

Terminal

C5, C6, C7, C8, C9

T98G, CB193, U373-MG

Gasque et al., 1993; Walker and McGeer 1993; Gasque et al. 1995a, b

Primary astrocytes/T98G, CB193, HSC2, T193, T109

Gasque et al. 1996b

U118-MG astroglioma

Gasque et al. 1998; Ischenko et al. 1998; Davoust et al. 1999

DAF, MCP, CD59

Primary astrocytes/D54-MG astroglioma

Gordon et al. 1992b; Yang et al. 1993; Gasque and Morgan 1996

Classical

C1q, C2, C3, C4, C1-INH, C4bp

Primary oligodendrocytes/HOG cells

Gasque and Morgan 1996; Hosokawa et al. 2003

Alternative

C3, fH

HOG cells

Terminal

C5, C6, C7, C8, C9, Sp, Primary oligodendrocytes clusterin

Gasque and Morgan 1996; Hosokawa et al. 2003 Gasque and Morgan 1996; Hosokawa et al. 2003

Inhibitors, cell bound

CD59, MCP, DAF

Astrocytes Classical

Sp, clusterin Receptors

Inhibitors, cell bound

C3aR, CR1, CR2

Walker et al. 1998

Oligodendrocytes

Primary oligodendrocytes/HOG cells

line-derived human CNS cells were also shown to produce components of the complement system under the appropriate stimulatory conditions (Table 1). Complement synthesis in the human brain, however, was reported to be generally low or non-detectable under normal conditions (Walker and McGeer 1992). Interestingly, during an infection (Stahel and Barnum 1997) or in response to pathogenic self-stimuli (Singhrao et al. 1999), cells of the

Gasque and Morgan 1996; Scolding et al. 1998; Zajicek et al. 1995

CNS can be induced to express a wide variety of complement components, as seen in human brain and experimental animal models. It has therefore been concluded that cells of the CNS can be a source of endogenously synthesized complement components, under the proper stimulatory conditions. In addition to synthesizing components of the complement pathway, cells of the CNS have also been reported to

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Table 2 The receptors for C5a are expressed within the CNS Receptor

Protein/message

Cell/tissue type

Species

Reference

CD88

Both

Fetal brain astrocytes, astrocyte cell lines

H

Gasque et al. 1995a

CD88

Both

Adult spinal cord astrocytes, microglia

H

Lacy et al. 1995

CD88

Message

Most regions throughout the brain

H

Ames et al. 1996

CD88

Protein

Fetal and adult brain astrocytes, microglia

H

Gasque et al. 1997

CD88

Protein

Neuroblastoma TGW cells

H

Farkas et al. 1999

CD88

Protein

Cortical neurons, hippocampal pyramidal and granular neurons

H

Farkas et al. 2003

C5L2

Message

Frontal cortex, hippocampus, hypothalamus, pons

H

Lee et al. 2001

CD88

Message

SH-SY5Y, LAN-5 neuroblastoma; cortical and hippocampal pyramidal neurons, dentate gyrus granular neurons, cerebellar Purkinje cells

H, M

O’Barr et al. 2001

CD88

Both

Spinal cord motor neurons

H, M

Humayun et al. 2009

CD88

Both

Cortical and hippocampal pyramidal neurons and Purkinje cells

M

Stahel et al. 1997a

CD88

Message

Adult cortical, hippocampal and striatal neurons; neonatal astrocytes; neuroblastoma N2A, microglia BV2 cells

M

Osaka et al. 1999a

CD88

Message

Fetal cortico-hippocampal neurons

M

Osaka et al. 1999b

CD88

Both

Cortical, cerebellar and hippocampal neurons, astrocytes and microglia

M

Stahel et al. 2000

CD88

Both

Cortical neurons, astrocytes

M

Van Beek et al. 2000

CD88

Protein

Primary adult hippocampal neural stem cells; adult transit-amplifying progenitors, migrating neuroblasts

M, R

Rahpeymai et al. 2006

CD88

Protein

Neuroblastoma Neuro-2A and differentiated PC12 cells

M, R

Humayun et al. 2009

CD88

Message

Cortical pyramidal neurons, Purkinje cells

R

Stahel et al. 1997b

CD88

Message

Spinal cord astrocytes, microglia, ventral motor and dorsal neurons

R

Nataf et al. 1998

CD88

Both

Oligodendrial cell line, fetal brain oligodendrocytes, astrocytes

R

Nataf et al. 2001

CD88

Both

Juvenile cerebellar granular neurons

R

Benard et al. 2004

C5L2

Both

Neonatal astrocytes; adult cortical, hippocampal, cerebellar neurons and astrocytes

R

Gavrilyuk et al. 2005

CD88, C5L2 CD88

Protein Both

Striatal neurons Spinal cord dorsal horn microglia

R R

Woodruff et al. 2006a Griffin et al. 2007

CD88, C5L2

Both

Spinal cord astrocytes, motor neurons

R

Woodruff et al. 2008a

H human, M mouse, R rat

express receptors for complement activation products, such as the anaphylatoxin C5a receptors (see Table 2). The classic C5a receptor, CD88, though first characterized on cells of myeloid lineage, has been reported to be expressed by many different cell and tissue types (Haviland et al. 1995; Gasque et al. 1997; Schieferdecker et al. 1997). The same holds true for the alternative receptor for C5a, C5L2 (Ohno et al. 2000; Gao et al. 2005). Not surprisingly, several different reports have shown that both microglia and astrocytes constitutively express CD88 (Lacy et al.

1995) and that astrocytes express C5L2 (Gavrilyuk et al. 2005). Similar reports on CD88 expression also demonstrate receptor functionality on these different cell types (Nolte et al. 1996). Interestingly, both neuroblastoma and subsets of neurons in vivo express the receptors for C5a (Table 2). Specifically, human and rodent CD88 expression has been observed on subsets of cortical, hippocampal, and cerebellar neurons within the brain and alpha motor neurons in the spinal cord (refer to Table 2). As observed for other members of the complement cascade, the relative

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levels of C5a receptor expression in the CNS may also be influenced by the inflammatory state; generally with an increase in receptor expression seen in diseased states (Stahel et al. 1997a; Stahel et al. 2000; Van Beek et al. 2000; Woodruff et al. 2006a; Woodruff et al. 2008a).

Physiological Role of Complement in the CNS The Complement System: Sensor and Orchestrator of the Innate Immune Response The complement system has been historically regarded as a supplementary system of the humoral immune response to bacterial infections. However, modern work has demonstrated that the complement system plays a crucial role in host defense, including the recognition of dangerous materials (both foreign and self), and the translation of danger signals into both an innate and an adaptive immune response (Mastellos et al. 2005). The primary immune cells of the CNS, microglia and astrocytes, have been demonstrated to utilize components of the complement system, to provide the brain with immune sensors and response capabilities. In addition, recent findings provide evidence of a complement-mediated role in peripheral and nervous tissue development and regeneration (Daveau et al. 2004; Mastellos et al. 2005; Rahpeymai et al. 2006; Stevens et al. 2007; Benard et al. 2008). Thus, a more modern view of the complement system has arisen which recognizes the role of complement in bridging the gap between the innate and adaptive immune responses as well as mediating tissue homeostasis. It now seems clear that complement has multiple roles in the body, and that it is naively simplistic to ascribe its roles or functions as being only associated with host defense. The various roles of complement in the body are clearly multifarious, requiring a paradigm reset, and nowhere is this better exemplified than in the CNS. C1q, the recognition component of the classical pathway, belongs to a group of soluble molecules known as the defense collagens, and it has been demonstrated to bind to many different molecular substrates including bacterial cell wall components (Tenner et al. 1984; Loos and Clas 1987) and components of host cellular and subcellular membranes (Storrs et al. 1983; Peitsch et al. 1988) including neuronal cell membranes (Van Beek et al. 2005). In addition, C1q has also been observed to bind to apoptotic cells, including neurons (Cole et al. 2006), and may therefore target damaged or dying neurons to be removed by microglia, thus preventing the release of toxic components that may result in additional insult or injury. Neurons have also been shown to produce C1q in response to disease or injury (Gasque et al. 2000) and recent study has demonstrated that C1q alone can provide neuroprotection against b-amyloid in

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vitro (Pisalyaput and Tenner 2008). Thus, C1q, expressed by neurons in response to infection or injury, could play a protective role by enhancing the clearance of apoptotic cells and providing direct protective effects on neurons in addition to its historic role in recognizing foreign materials and initiating of complement activation. In the classical viewpoint, once the complement system has identified a potential perturbation in tissue homeostasis, the next step is to direct the proper immune response. The complement system possesses an array of bioactive cleavage fragments and receptors necessary to carry out such a response. Once the defense collagens play out their roles as system activators, they, along with the cleavage products of C3 (e.g., C3b/iC3b) can act as opsonins to trigger the ingestion of their bound targets by phagocytes (Bohlson et al. 2007). This is accomplished through association with an array of different receptors, like CR1 and CR3, expressed on various phagocytic cells including microglia (Akiyama et al. 2000). As a result of C3 cleavage, there is also the generation of the biologically active fragment, C3a. C5 cleavage similarly results in the generation of a second distinct biologically active protein fragment known as C5a. Both of these molecules (C3a and C5a), have been demonstrated to be important in the recruitment/chemotaxis of glia to the site of infection and/ or damage, through their respective anaphylatoxin G-protein coupled receptors (C3aR and CD88) (Yao et al. 1990; Miller and Stella 2009). Historically, C5a has been demonstrated to be the more potent and pro-inflammatory of the two. A second receptor for C5a, C5L2, binds C5a and its degradation product C5a-desArg with comparable affinities (Cain and Monk 2002). The cellular expression pattern of C5L2 is similar to that seen for CD88; however, unlike CD88, C5L2 is unable to couple to G-proteins, and little has been agreed upon as to the exact function of this receptor. Recent evidence implicates both a possible antiinflammatory decoy receptor role (Gerard et al. 2005), or a synergistic, inflammatory role (Rittirsch et al. 2008). Both roles are not mutually exclusive, but there is no general agreement over how C5L2 regulates CD88-mediated homeostatic functions (or vice versa). While only minimal studies have so far been performed exploring the role of C5L2 in the CNS, C5L2 expression on astrocytes has been shown to be regulated by noradrenaline, and this interaction may act to restrict inflammation elicited by these cells (Gavrilyuk et al. 2005). The role of C5L2, within and beyond the CNS, will be a major area of complement research in the next decade. The activation of the complement system ultimately leads to the formation of the MAC, through the association of the terminal pathway components (C5b, C6, C7, C8, and C9), which can cause the lysis of a target cell by forming a pore in the phospholipid bilayer. In the experimental

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allergic encephalomyelitis model, mice which are deficient for C5 are demonstrated to have greater axonal preservation and myelin formation, which has been partially suggested to be due to the lack of MAC formation (Cudrici et al. 2006). However, most host cells are normally protected by the complement regulator CD59, which is upregulated upon stimulation with sublytic levels of MAC (Mason et al. 1999). In contrast, sublytic levels of MAC may act as an anti-apoptotic signal to oligodendrocytes, as demonstrated in vitro (Rus et al. 2005). Complement in the CNS: Beyond Immune Surveillance With the discovery of complement factors and their receptors being expressed by a multitude of CNS cell types, it should come as no surprise that some investigators have demonstrated that components of the complement system contribute to physiological processes outside of immune surveillance. Although generally regarded as a potent proinflammatory mediator (and therefore being a promoter of inflammatory tissue damage), several investigators have published conflicting data suggesting a protective role for C5a within the CNS. Recent study on the role of C5a in glia show that microglia, in response to C5a stimulation, can upregulate the glutamate transporter, GLT-1, and increase their glutamate uptake which may provide protection against glutamate-mediated excitotoxicity (Persson et al. 2009). In addition to a possible glial-mediated protective effect, C5a has also been observed to provide direct protection to differentiated SH-SY5Y neuroblastoma cells against b-amyloid (O’Barr et al. 2001) and primary mouse neurons against kainic acid (Osaka et al. 1999b). C5a, when administered with kainic acid intraventricularly for 24 h prior to glutamate treatment in primary neuronal mouse cultures, was also shown to be neuroprotective against glutamate-mediated caspase-3 activation (Osaka et al. 1999b). It was subsequently hypothesized that the C5a/ CD88-mediated protection may be dependent on the modulation of Ca2?/calmodulin-dependent protein kinase and MAP-kinase activity (Mukherjee and Pasinetti 2000, 2001). By contrast to the neuroprotective effects observed for C5a, it has also been demonstrated that C5a receptors are involved in neuronal apoptosis and cell death in TGW and Neuro-2A neuroblastoma cells, differentiated PC12 cells, and primary rat cortical neurons (Farkas et al. 1998, 2003; Humayun et al. 2009). These reports amply illustrate the enigma of the role of C5a in the CNS: it could well be either neuropathologic and/or neuroprotective. Future studies are required to carefully define whether there are anatomically regional and/or functional activities of CNS C5a receptors. In particular, previous studies were conducted without knowledge or mention of C5L2, which may act as a potential ‘‘anti-inflammatory’’ decoy receptor. This along

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with the knowledge that G-protein coupled receptors can assemble as both homo- and hetero-multimers, means that some of the effects of C5a may be modulated by CD88– C5L2 heterometric receptor complexes. In any event, the existence of two receptors for C5a that likely are involved in balancing local inflammation provides additional opportunity for fine-tuning of therapeutic targeting. Thus, it is essential to have knowledge of the expression and cellular localization of these receptors in normal and neurodegenerative CNS disorders. Complement products have also been observed to take part in other physiological processes including the developmental refinement of synapses within the visual system (Stevens et al. 2007). In this report, the projection of retinal ganglionic cells (RGCs) axons to the dorsal lateral geniculate nucleus (dLGN) was assessed during the development of the visual system. It was found that mRNA for C1q was induced in RGCs during development in response to astrocyte-initiated signals, and that translated C1q protein was targeted specifically to RGC–dLGN synapses. Finally, it was shown that mice deficient in C1q or C3 had failed to develop the mature RGC to dLGN innervation patterns, and that this was due to a failure to prune out developmentally inappropriate RGC–dLGN synapses. These data suggest that members of the complement system can play a role in the refinement of synaptic connections during neural development. In addition to developmental synapse elimination, members of the complement system have been recently shown to play a role in neurogenesis. For example, C3a and C5a, have been implicated in basal and ischemia-induced neurogenesis (Rahpeymai et al. 2006). In this study, both clonally derived rat hippocampal neural stem cells and murine neural progenitor cells expressed C3aR and CD88. Mice lacking C3, C3aR, or in which C3aR activity was inhibited, were shown to have reduced numbers of newly formed neurons in areas of adult neurogenesis. Although these studies do not provide a mechanism behind the C3aR activation that would lead to the creation of new neurons, nor a source for the generation of C3a or C5a, the results are consistent with a significant contribution by complement activation fragments in neurogenesis. The contribution of the anaphylatoxin receptors to CNS development is not restricted to neurogenesis within the hippocampus. Both the classic anaphylatoxin receptors C3aR and CD88 have been shown to be expressed by granule neurons of the cerebellum (Benard et al. 2004), and this expression was shown to increase during cellular maturation. When granule neurons were subjected to apoptotic conditions in vitro, C5a receptor stimulation using a C5a agonist promoted cell survival through the inhibition of caspase-3, suggesting the increased CD88 expression during maturation may help provide anti-apoptotic signaling to

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granule neurons during development. More recent data supports the proposed role of CD88 activation in the development of the cerebellum in the rat (Benard et al. 2008). Young rats receiving a C5a receptor agonist within the cerebellum had an enlarged external granule cell layer (EGL) that was shown to be due to increased proliferation of immature granule neurons (Benard et al. 2008). In addition, a C3aR agonist was also shown to increase the size of the internal granule cell layer (IGL) when injected to the cerebellum, thus providing evidence that both anaphylatoxin receptors may play a role in cerebellar development (Benard et al. 2008). These studies clearly provide new impetus for the role of complement in the developing CNS.

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and maintain a normal brain homeostatic environment (see above). However, when uncontrolled, these powerful immune factors may also lead to host cell damage, with neurons being particularly susceptible to complementmediated damage (Singhrao et al. 2000). The imbalance of these complement-protective and complement-degenerative pathways potentially contributes to disease pathology in many CNS diseases (see Fig. 1). C5a: A Neuropathogenic Mediator in Numerous CNS Disease States

There are several lines of evidence indicating that overactivation and/or under-regulation of complement activity in the CNS can play a detrimental role in the pathogenesis of CNS disease. As stated above, complement activation results in opsonization by C1q and C3 cleavage products, the generation of the anaphylatoxins C3a and C5a, and the formation of the cell-destructive MAC (C5b–9). These factors, when controlled, protect the CNS from infection

Despite the availability of data demonstrating potential neuroprotective activities of C5a (Osaka et al. 1999b; Mukherjee and Pasinetti 2001; O’Barr et al. 2001; Mukherjee et al. 2008), the overall body of evidence accumulated to date suggests that C5a-induced CD88 activation plays a deleterious role in CNS disease. Experimental autoimmune encephalitis, an animal model of multiple sclerosis, may be the exception, where C3a (Boos et al. 2004) and the terminal MAC (Mead et al. 2002) have been shown to be a driver of pathology, with minimal involvement from C5a (Morgan et al. 2004; Reiman et al. 2005). Table 3 lists and summarizes the available data on the role of C5a/CD88 interactions in these diseases. In particular,

Fig. 1 Propagation of disease pathology by complement in CNS disease. In this proposed model, complement is activated by certain disease triggers, such as b-amyloid (Jiang et al. 1994), apoptotic and necrotic cells (Cole et al. 2006), and cell debris (Van Beek et al. 2005). This leads to deposition and opsonization of C1q and C3b on neuronal surfaces (Fonseca et al. 2004a; Van Beek et al. 2005), production of anaphylatoxins C3a and C5a, and formation of the MAC on these neurons (Webster et al. 1997; Cowell et al., 2003). In response to C5a, glia migrates to sites of complement activation (Armstrong et al. 1990; Yao et al. 1990). Astrocytes and microglia also upregulate CD88 (Gasque et al. 1997) and C3aR (Gasque et al.

1998) in these regions of neuronal death, and may be activated by C5a and C3a (Moller et al. 1997; Sayah et al. 1999). In response to opsonization, migrating and activated glia will phagocytose neuronal cells (Van Beek et al. 2005), and insertion of the MAC into neuronal membranes will lyse these cells (Itagaki et al. 1994). The combined outcome is the death of the neuron. C5a may also directly cause neuronal cell death through interaction with neuronally expressed CD88 (Farkas et al. 1998; Humayun et al. 2009). In this way, activation of complement following initial disease onset, propagates inflammation and neuronal cell death, leading to further complement activation and contributing to disease pathology

Role of Complement in CNS Diseases

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Table 3 Role of C5a and CD88 in CNS diseases Disease state

CD88 or C5a protein up-regulation in human samples

CD88 or C5a protein up- Protection following regulation in animal CD88 inhibition in models animal models

Reference

Neurodegenerative disease Alzheimer’s disease

CD88—around plaques

ND

Yes—PMX205

Farkas et al. 2003; Fonseca et al. 2009

Amyotrophic lateral sclerosis

CD88—motor neurons

CD88—motor neurons, astrocytes

Yes—PMX205

Woodruff et al. 2008a; Humayun et al. 2009

Huntington’s disease

CD88—astrocytes, microglia

CD88—striatal neurons

Yes—PMX205

Gasque et al. 1997; Woodruff et al. 2006a

Experimental CD88—astrocytes Autoimmune Encephalitis/Multiple Sclerosis

CD88—spinal cord tissue; mRNA only— microglia, astrocytes

No—PMX53; CD88-/- mice

Muller-Ladner et al. 1996; Gasque et al. 1997; Nataf et al. 1998; Reiman et al. 2002; Morgan et al. 2004

Acute CNS injuries Traumatic brain/closed ND head injury

C5a—serum; CD88 Yes—PMX53 mRNA only—neurons, glia

Stahel et al. 1997b; Stahel et al. 2000; Sewell et al. 2004; Leinhase et al. 2006

Ischemic Stroke

C5a—serum

CD88—endothelial cells, Yes—PMX53 astrocytes

Van Beek et al. 2000; Mocco et al. 2006; Kim et al. 2008

Intracerebral hemorrhage

C5a—serum

C5a—brain perihematomal expression

Yes—PMX53

Xi et al. 2001; Mack et al. 2007; Garrett et al. 2009

Encephalitis/meningitis C5a—CSF; CD88— astrocytes, microglia, endothelium

C5a—brain; CD88— cortical, hippocampal, cerebellar neurons

ND

Stahel et al. 1997a; Gasque et al. 1997; Chen and Reiss 2002

Cerebral malaria

C5a—serum

Yes—CD88 or C5a anti-serum

Roestenberg et al. 2007; Patel et al. 2008; Nyakoe et al. 2009

CNS infections

C5a—serum

ND not determined, PMX205/PMX53 cyclic peptide CD88 antagonists: hydrocinnamate-[OPdChaWR] (PMX205); AcF-[OPdChaWR] (PMX53), CSF cerebral spinal fluid

the neurodegenerative diseases, Alzheimer’s disease (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) all seem to indicate CD88 activation as a driver of CNS pathology. This has been primarily demonstrated with increased CD88 expression in human tissue, and protection in animal models using a specific CD88 antagonist termed PMX205 (Table 3). PMX205 (hydrocinnamate-[OPdChaWR]), a derivative of an earlier compound PMX53 (AcF-[OPdChaWR]), belongs to a class of cyclic peptide drugs developed at the University of Queensland (March et al. 2004). Both compounds are based on the linear CD88 antagonist, MeFKPdChaWR, (itself based on the C-terminal active ‘‘tail’’ of C5a), and the eventual cyclization to induce structural and metabolic stability (March et al. 2004). This new class of cyclic peptide macromolecules was shown to act as insurmountable, ‘‘pseudo-irreversible’’ CD88 antagonists, and importantly were found to display serum and gastric stability and oral activity (Woodruff et al. 2005), likely due to the cyclic nature of the drugs. They have no detectable affinity for C5L2 (Otto et al. 2004), making them ideal

candidates to explore the specific effects of C5a specifically interacting with CD88 (as opposed to C5a blocking antibodies, or other C5a inhibitory strategies). PMX53 has successfully completed three Phase 1 clinical trials in psoriasis and rheumatoid arthritis patients (Woodruff et al. 2006b), however, clear efficacy in these limited trials is lacking (Vergunst et al. 2007). We have shown that PMX205 displays increased potency over PMX53 in an acute rat model of inflammatory bowel disease (Woodruff et al. 2005), and provides greater efficacy than PMX53 in a model of striatal neurodegeneration, potentially due to increased CNS penetrance across the BBB (Woodruff et al. 2006a). As such, PMX205 is the recommended compound to examine the role of C5a/CD88 activation in diseases of the CNS (Woodruff et al. 2008b). Neurodegenerative Disease: A Crucial Role for CD88 Activation Over the past few decades, there has been substantial evidence demonstrating complement activation in association

Neuromol Med (2010) 12:179–192

with AD pathology (Eikelenboom et al. 1989; Afagh et al. 1996). In addition, in vitro evidence has shown b-amyloid can directly activate both the classical and alternative complement pathways (Jiang et al. 1994; Bradt et al. 1998). Complement components have been observed to associate with two of the disease’s most prominent features; thioflavine-positive fibrillar b-amyloid plaques (Afagh et al. 1996; Loeffler et al. 2008), and hyperphosphorylated tau comprised neurofibrillary tangles (Shen et al. 2001). Mouse models, in which overexpression of mutated forms of the human amyloid precursor protein (APP) results in b-amyloid deposition, display age-related AD pathology including the association of b-amyloid plaques with complement factors (Fonseca et al. 2004b; Zhou et al. 2008), thus providing additional evidence of the potential role of complement activation in AD. Since it has been shown that receptors for C5a are expressed in the brain (Table 2), and that as a result of complement activation the generation of C5a elicits an inflammatory response, it is conceivable that C5a/C5a receptor interactions participate in AD development. Conflicting results, however, have also been reported regarding CD88 expression in the AD brain with one group (O’Barr et al. 1998) seeing comparable CD88 expression in normal and AD brain while another group reported that neuronal CD88 expression is decreased in the AD brain (Farkas et al. 2003). Using both polyclonal and monoclonal antibodies against human CD88, this latter study reported seeing reduced neuronal staining in a broad range of areas, including the hippocampus. However, they did report increased CD88 immunoreactivity concentrated around b-amyloid plaques which were hypothesized to be due to dystrophic neurites (Farkas et al. 2003). In both studies, no glial specific CD88 reactivity was reported (O’Barr et al. 1998; Farkas et al. 2003). In support of a pathogenic role for C5a in AD, we have recently shown, using two distinct mouse models of AD, that oral treatment with the CD88 antagonist PMX205 significantly attenuates AD-like pathology after 12 weeks of treatment (Fonseca et al. 2009). When compared with untreated AD model mice, antagonist treatment results in a substantial reduction in activated glia and a decrease in thioflavine-positive b-amyloid plaques. The reductions in pathology were correlative with improvements in contextual memory (Fonseca et al. 2009). These results suggest that inhibition of the pro-inflammatory activities of C5a may be a potential target for reducing pathology and cognitive impairment associated with AD. Complement activation products, as well as CD88, are also upregulated in the striatum of HD patients, compared with tissue from non-HD individuals (Gasque et al. 1997; Singhrao et al. 1999). These components are seen specifically on neurons and proliferating glia in the degenerating regions of HD brains, suggesting an involvement of

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complement in the progression of this disease. Our laboratory has also performed experiments indicating that C5a may play a deleterious role in HD (Woodruff et al. 2006a). We utilized an acute model of 3-nitropropionic acid (3-NP)-induced striatal degeneration in rats. Toxin infusion resulted in upregulation of complement factors C3, C9 and the receptors for C5a (CD88 and C5L2) on striatal neuronal cells in the areas of degenerating tissue (Woodruff et al. 2006a). Pre- or post-treating 3-NP infused animals with the CD88 antagonist, PMX205, significantly reduced striatal lesion size, apoptosis, neutrophil infiltration, hemorrhage, astrocyte proliferation, and improved neurological deficits, indicating a pathogenic role for C5a in this disease model (Woodruff et al. 2006a). Similar to AD and HD, complement activation productions are upregulated in the CSF and degenerating spinal cord in ALS patients (Woodruff et al. 2008b). Three independent studies have demonstrated that motor neurons from four different SOD1 transgenic mouse models of ALS had significant increases in C1q and C4 compared to wildtype animals (Ferraiuolo et al. 2007; Fukada et al. 2007; Lobsiger et al. 2007). Our laboratories have also recently shown a pathogenic role for C5a in a rat SOD1G93A transgenic model of ALS (Woodruff et al. 2008a), with C3b deposition in the vicinity of degenerating motor neurons in the lumbar spinal cord of diseased SOD1G93A rats, accompanied by a significant upregulation of CD88 in these regions localized to proliferating astrocytes. By contrast to CD88, we found an early increase in C5L2, followed by a decrease in later-stage diseased animals. Finally, we demonstrated that treating SOD1G93A rats with the CD88 antagonist PMX205 was able to significantly reduce motor deficits and extend survival (Woodruff et al. 2008a). More recently, CD88 was also shown to be upregulated in degenerating motor neurons from NFL-/mice, and similarly upregulated on motor neurons from ALS patients (Humayun et al. 2009), indicating an overarching role for CD88 upregulation in ALS.

Concluding Remarks The complement system is a powerful component of the innate immune system. Its functions are wide and varied and include the opsonization of pathogens, the recruitment and stimulation of immune and inflammatory cells, and the lysis of cells, ultimately aiding in the destruction of invading organisms. In the CNS, it plays similar functions to protect the brain from infection, but recent studies show that complement also plays roles in the development and homeostasis of the CNS (Stevens et al. 2007). However, there are now many lines of evidence to indicate that improper complement activation or regulation, and in

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particular, the generation of the potent proinflammatory molecule, C5a, plays a deleterious role in many CNS diseases. In particular, the neurodegenerative diseases AD, ALS and HD all seem to involve prolonged complement activation as part of their disease pathology, and CD88 activation seems to be a key pathogenic driver of these pathologies. Targeting complement inhibition, with a particular focus on C5a, may therefore be a useful treatment strategy in CNS diseases. To date there are very few complement inhibitors clinically available. A specific monoclonal antibody that inhibits C5 cleavage (to produce C5a and the MAC) has been approved by the US Food and Drug Administration for the treatment of paroxysmal nocturnal hemoglobinuria (Rother et al. 2007). However, due to its proteinaceous nature (precluding facile penetration into the CNS), and potential immunosuppressive actions when administrated chronically, this drug is unlikely to be effective in chronic CNS diseases. As such, specific inhibition of individual complement factors or receptors (such as CD88), with small molecule drugs which are both orally active and can penetrate the BBB, are more ideal CNS disease therapeutics. For example, the cyclic peptide CD88 antagonist PMX205 has shown clear therapeutic efficacy in several animal models of neurodegenerative disease (Woodruff et al. 2006a, 2008a; Fonseca et al. 2009). One aspect of chronic blockade of CD88 is whether this will compromise host defenses, especially against invading pathogens. It is worth noting that to date, there is no evidence of immune suppression in animals chronically treated for several months with the cyclic peptidic CD88 antagonists PMX53 and PMX205 (Woodruff et al. 2002; Fonseca et al. 2009), although the corresponding situation in humans awaits exploration. However, considering all available lines of evidence, there is a strong and clear rationale for future clinical trials with these or other small molecule CD88 antagonists in neurodegenerative and other disease states of the CNS. Acknowledgments Supported by the National Health and Medical Research Council of Australia (Project Grant #455856 to P. G. Noakes and S. M. Taylor and Career Development Award fellowship #519700 to T. M. Woodruff), and the Motor Neuron Disease Research Institute of Australia. We also thank Dr. M. L. Manchadi for editorial assistance, and Ms. Marianna Shek for graphical support.

References Afagh, A., Cummings, B. J., Cribbs, D. H., Cotman, C. W., & Tenner, A. J. (1996). Localization and cell association of C1q in Alzheimer’s disease brain. Experimental Neurology, 138, 22–32. Akiyama, H., Arai, T., Kondo, H., Tanno, E., Haga, C., & Ikeda, K. (2000). Cell mediators of inflammation in the Alzheimer disease brain. Alzheimer Disease and Associated Disorders, 14(Suppl 1), S47–S53.

Neuromol Med (2010) 12:179–192 Ames, R. S., Li, Y., Sarau, H. M., Nuthulaganti, P., Foley, J. J., Ellis, C., et al. (1996). Molecular cloning and characterization of the human anaphylatoxin C3a receptor. Journal of Biological Chemistry, 271, 20231–20234. Armstrong, R. C., Harvath, L., & Dubois-Dalcq, M. E. (1990). Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. Journal of Neuroscience Research, 27, 400–407. Avery, V. M., Adrian, D. L., & Gordon, D. L. (1993). Detection of mosaic protein mRNA in human astrocytes. Immunology and Cell Biology, 71(Pt 3), 215–219. Barnum, S. R., Ishii, Y., Agrawal, A., & Volanakis, J. E. (1992a). Production and interferon-gamma-mediated regulation of complement component C2 and factors B and D by the astroglioma cell line U105-MG. Biochemical Journal, 287(Pt 2), 595–601. Barnum, S. R., Jones, J. L., & Benveniste, E. N. (1992b). Interferongamma regulation of C3 gene expression in human astroglioma cells. Journal of Neuroimmunology, 38, 275–282. Barnum, S. R., Jones, J. L., & Benveniste, E. N. (1993). Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells. Glia, 7, 225–236. Benard, M., Gonzalez, B. J., Schouft, M. T., Falluel-Morel, A., Vaudry, D., Chan, P., et al. (2004). Characterization of C3a and C5a receptors in rat cerebellar granule neurons during maturation. Neuroprotective effect of C5a against apoptotic cell death. Journal of Biological Chemistry, 279, 43487–43496. Benard, M., Raoult, E., Vaudry, D., Leprince, J., Falluel-Morel, A., Gonzalez, B. J., et al. (2008). Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum. Molecular Immunology, 45, 3767–3774. Biro, A., Rovo, Z., Papp, D., Cervenak, L., Varga, L., Fust, G., et al. (2007). Studies on the interactions between C-reactive protein and complement proteins. Immunology, 121, 40–50. Bohlson, S. S., Fraser, D. A., & Tenner, A. J. (2007). Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Molecular Immunology, 44, 33–43. Boos, L., Campbell, I. L., Ames, R., Wetsel, R. A., & Barnum, S. R. (2004). Deletion of the complement anaphylatoxin C3a receptor attenuates, whereas ectopic expression of C3a in the brain exacerbates, experimental autoimmune encephalomyelitis. Journal of Immunology, 173, 4708–4714. Bradt, B. M., Kolb, W. P., & Cooper, N. R. (1998). Complementdependent proinflammatory properties of the Alzheimer’s disease beta-peptide. Journal of Experimental Medicine, 188, 431–438. Cain, S. A., & Monk, P. N. (2002). The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). Journal of Biological Chemistry, 277, 7165–7169. Chen, N., & Reiss, C. S. (2002). Innate immunity in viral encephalitis: Role of C5. Viral Immunology, 15, 365–372. Cole, D. S., Hughes, T. R., Gasque, P., & Morgan, B. P. (2006). Complement regulator loss on apoptotic neuronal cells causes increased complement activation and promotes both phagocytosis and cell lysis. Molecular Immunology, 43, 1953–1964. Cowell, R. M., Plane, J. M., & Silverstein, F. S. (2003). Complement activation contributes to hypoxic–ischemic brain injury in neonatal rats. Journal of Neuroscience, 23, 9459–9468. Cudrici, C., Niculescu, T., Niculescu, F., Shin, M. L., & Rus, H. (2006). Oligodendrocyte cell death in pathogenesis of multiple sclerosis: Protection of oligodendrocytes from apoptosis by complement. Journal of Rehabilitation Research and Development, 43, 123–132. Daveau, M., Benard, M., Scotte, M., Schouft, M. T., Hiron, M., Francois, A., et al. (2004). Expression of a functional C5a

Neuromol Med (2010) 12:179–192 receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat. Journal of Immunology, 173, 3418–3424. Davoust, N., Jones, J., Stahel, P. F., Ames, R. S., & Barnum, S. R. (1999). Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells. Glia, 26, 201–211. Eikelenboom, P., Hack, C. E., Rozemuller, J. M., & Stam, F. C. (1989). Complement activation in amyloid plaques in Alzheimer’s dementia. Virchows Archiv B, Cell Pathology Including Molecular Pathology, 56, 259–262. Farkas, I., Baranyi, L., Kaneko, Y., Liposits, Z., Yamamoto, T., & Okada, H. (1999). C5a receptor expression by TGW neuroblastoma cells. Neuroreport, 10, 3021–3025. Farkas, I., Baranyi, L., Liposits, Z. S., Yamamoto, T., & Okada, H. (1998). Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells. Neuroscience, 86, 903–911. Farkas, I., Takahashi, M., Fukuda, A., Yamamoto, N., Akatsu, H., Baranyi, L., et al. (2003). Complement C5a receptor-mediated signaling may be involved in neurodegeneration in Alzheimer’s disease. Journal of Immunology, 170, 5764–5771. Ferraiuolo, L., Heath, P. R., Holden, H., Kasher, P., Kirby, J., & Shaw, P. J. (2007). Microarray analysis of the cellular pathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS. Journal of Neuroscience, 27, 9201–9219. Fonseca, M. I., Ager, R. R., Chu, S. H., Yazan, O., Sanderson, S. D., LaFerla, F. M., et al. (2009). Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer’s disease. Journal of Immunology, 183, 1375–1383. Fonseca, M. I., Kawas, C. H., Troncoso, J. C., & Tenner, A. J. (2004a). Neuronal localization of C1q in preclinical Alzheimer’s disease. Neurobiology of Diseases, 15, 40–46. Fonseca, M. I., Zhou, J., Botto, M., & Tenner, A. J. (2004b). Absence of C1q leads to less neuropathology in transgenic mouse models of Alzheimer’s disease. Journal of Neuroscience, 24, 6457–6465. Fukada, Y., Yasui, K., Kitayama, M., Doi, K., Nakano, T., Watanabe, Y., et al. (2007). Gene expression analysis of the murine model of amyotrophic lateral sclerosis: Studies of the Leu126delTT mutation in SOD1. Brain Research, 1160, 1–10. Gao, H., Neff, T. A., Guo, R. F., Speyer, C. L., Sarma, J. V., Tomlins, S., et al. (2005). Evidence for a functional role of the second C5a receptor C5L2. FASEB Journal, 19, 1003–1005. Garrett, M. C., Otten, M. L., Starke, R. M., Komotar, R. J., Magotti, P., Lambris, J. D., et al.. (2009). Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage. Brain Research. Gasque, P., Chan, P., Fontaine, M., Ischenko, A., Lamacz, M., Gotze, O., et al. (1995a). Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes. Journal of Immunology, 155, 4882–4889. Gasque, P., Chan, P., Mauger, C., Schouft, M. T., Singhrao, S., Dierich, M. P., et al. (1996b). Identification and characterization of complement C3 receptors on human astrocytes. Journal of Immunology, 156, 2247–2255. Gasque, P., Dean, Y. D., McGreal, E. P., VanBeek, J., & Morgan, B. P. (2000). Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology, 49, 171–186. Gasque, P., Fontaine, M., & Morgan, B. P. (1995b). Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines. Journal of Immunology, 154, 4726–4733. Gasque, P., Ischenko, A., Legoedec, J., Mauger, C., Schouft, M. T., & Fontaine, M. (1993). Expression of the complement classical pathway by human glioma in culture. A model for complement

189 expression by nerve cells. Journal of Biological Chemistry, 268, 25068–25074. Gasque, P., Julen, N., Ischenko, A. M., Picot, C., Mauger, C., Chauzy, C., et al. (1992). Expression of complement components of the alternative pathway by glioma cell lines. Journal of Immunology, 149, 1381–1387. Gasque, P., & Morgan, B. P. (1996). Complement regulatory protein expression by a human oligodendrocyte cell line: Cytokine regulation and comparison with astrocytes. Immunology, 89, 338–347. Gasque, P., Singhrao, S. K., Neal, J. W., Gotze, O., & Morgan, B. P. (1997). Expression of the receptor for complement C5a (CD88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system. American Journal of Pathology, 150, 31–41. Gasque, P., Singhrao, S. K., Neal, J. W., Wang, P., Sayah, S., Fontaine, M., et al. (1998). The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. Journal of Immunology, 160, 3543–3554. Gasque, P., Thomas, A., Fontaine, M., & Morgan, B. P. (1996a). Complement activation on human neuroblastoma cell lines in vitro: Route of activation and expression of functional complement regulatory proteins. Journal of Neuroimmunology, 66, 29–40. Gavrilyuk, V., Kalinin, S., Hilbush, B. S., Middlecamp, A., McGuire, S., Pelligrino, D., et al. (2005). Identification of complement 5alike receptor (C5L2) from astrocytes: Characterization of antiinflammatory properties. Journal of Neurochemistry, 92, 1140– 1149. Gerard, N. P., & Gerard, C. (1991). The chemotactic receptor for human C5a anaphylatoxin. Nature, 349, 614–617. Gerard, N. P., Lu, B., Liu, P., Craig, S., Fujiwara, Y., Okinaga, S., et al. (2005). An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2. Journal of Biological Chemistry, 280, 39677–39680. Gordon, D. L., Avery, V. M., Adrian, D. L., & Sadlon, T. A. (1992a). Detection of complement protein mRNA in human astrocytes by the polymerase chain reaction. Journal of Neuroscience Methods, 45, 191–197. Gordon, D. L., Sadlon, T. A., Wesselingh, S. L., Russell, S. M., Johnstone, R. W., & Purcell, D. F. (1992b). Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation. Journal of Neuroimmunology, 36, 199– 208. Griffin, R. S., Costigan, M., Brenner, G. J., Ma, C. H. E., Scholz, J., Moss, A., et al. (2007). Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. Journal of Neuroscience, 27, 8699–8708. Haviland, D. L., McCoy, R. L., Whitehead, W. T., Akama, H., Molmenti, E. P., Brown, A., et al. (1995). Cellular expression of the C5a anaphylatoxin receptor (C5aR): Demonstration of C5aR on nonmyeloid cells of the liver and lung. Journal of Immunology, 154, 1861–1869. Hosokawa, M., Klegeris, A., Maguire, J., & McGeer, P. L. (2003). Expression of complement messenger RNAs and proteins by human oligodendroglial cells. Glia, 42, 417–423. Huber-Lang, M., Sarma, J. V., Zetoune, F. S., Rittirsch, D., Neff, T. A., McGuire, S. R., et al. (2006). Generation of C5a in the absence of C3: A new complement activation pathway. Nature Medicine, 12, 682–687. Huber-Lang, M., Younkin, E. M., Sarma, J. V., Riedemann, N., McGuire, S. R., Lu, K. T., et al. (2002). Generation of C5a by phagocytic cells. The American Journal of Pathology, 161, 1849–1859.

190 Humayun, S., Gohar, M., Volkening, K., Moisse, K., Leystra-Lantz, C., Mepham, J., et al. (2009). The complement factor C5a receptor is upregulated in NFL-/- mouse motor neurons. Journal of Neuroimmunology, 210, 52–62. Ischenko, A., Sayah, S., Patte, C., Andreev, S., Gasque, P., Schouft, M. T., et al. (1998). Expression of a functional anaphylatoxin C3a receptor by astrocytes. Journal of Neurochemistry, 71, 2487–2496. Itagaki, S., Akiyama, H., Saito, H., & McGeer, P. L. (1994). Ultrastructural localization of complement membrane attack complex (MAC)-like immunoreactivity in brains of patients with Alzheimer’s disease. Brain Research, 645, 78–84. Jiang, H., Burdick, D., Glabe, C. G., Cotman, C. W., & Tenner, A. J. (1994). Beta-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain. Journal of Immunology, 152, 5050–5059. Kim, G. H., Mocco, J., Hahn, D. K., Kellner, C. P., Komotar, R. J., Ducruet, A. F., et al. (2008). Protective effect of C5a receptor inhibition after murine reperfused stroke. Neurosurgery, 63, 122–125; discussion 125–126. Kleine, T. O., & Benes, L. (2006). Immune surveillance of the human central nervous system (CNS): Different migration pathways of immune cells through the blood–brain barrier and blood–cerebrospinal fluid barrier in healthy persons. Cytometry A, 69, 147–151. Lacy, M., Jones, J., Whittemore, S. R., Haviland, D. L., Wetsel, R. A., & Barnum, S. R. (1995). Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia. Journal of Neuroimmunology, 61, 71–78. Lee, D. K., George, S. R., Cheng, R., Nguyen, T., Liu, Y., Brown, M., et al. (2001). Identification of four novel human G protein-coupled receptors expressed in the brain. Molecular Brain Research, 86, 13–22. Leinhase, I., Holers, V. M., Thurman, J. M., Harhausen, D., Schmidt, O. I., Pietzcker, M., et al. (2006). Reduced neuronal cell death after experimental brain injury in mice lacking a functional alternative pathway of complement activation. BMC Neuroscience, 7, 55. Levi-Strauss, M., & Mallat, M. (1987). Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation. Journal of Immunology, 139, 2361–2366. Li, K., Sacks, S. H., & Zhou, W. (2007). The relative importance of local and systemic complement production in ischaemia, transplantation and other pathologies. Molecular Immunology, 44, 3866–3874. Liszewski, M. K., Farries, T. C., Lublin, D. M., Rooney, I. A., & Atkinson, J. P. (1996). Control of the complement system. Advances in Immunology, 61, 201–283. Lobsiger, C. S., Boillee, S., & Cleveland, D. W. (2007). Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proceedings of the National Academy of Sciences of the United States of America, 104, 7319–7326. Loeffler, D. A., Camp, D. M., & Bennett, D. A. (2008). Plaque complement activation and cognitive loss in Alzheimer’s disease. Journal of Neuroinflammation, 5, 9. Loos, M., & Clas, F. (1987). Antibody-independent killing of gramnegative bacteria via the classical pathway of complement. Immunology Letters, 14, 203–208. Mack, W. J., Ducruet, A. F., Hickman, Z. L., Garrett, M. C., Albert, E. J., Kellner, C. P., et al. (2007). Early plasma complement C3a levels correlate with functional outcome after aneurysmal subarachnoid hemorrhage. Neurosurgery, 61, 255–260; discussion 260–261. March, D. R., Proctor, L. M., Stoermer, M. J., Sbaglia, R., Abbenante, G., Reid, R. C., et al. (2004). Potent cyclic antagonists of the

Neuromol Med (2010) 12:179–192 complement C5a receptor on human polymorphonuclear leukocytes. Relationships between structures and activity. Molecular Pharmacology, 65, 868–879. Marjan, J., Xie, Z., & Devine, D. V. (1994). Liposome-induced activation of the classical complement pathway does not require immunoglobulin. Biochimica et Biophysica Acta, 1192, 35–44. Mason, J. C., Yarwood, H., Sugars, K., Morgan, B. P., Davies, K. A., & Haskard, D. O. (1999). Induction of decay-accelerating factor by cytokines or the membrane-attack complex protects vascular endothelial cells against complement deposition. Blood, 94, 1673–1682. Mastellos, D., Germenis, A. E., & Lambris, J. D. (2005). Complement: An inflammatory pathway fulfilling multiple roles at the interface of innate immunity and development. Current Drug Targets. Inflammation and Allergy, 4, 125–127. McGeer, P. L., Walker, D. G., Akiyama, H., Kawamata, T., Guan, A. L., Parker, C. J., et al. (1991). Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain. Brain Research, 544, 315–319. Mead, R. J., Singhrao, S. K., Neal, J. W., Lassmann, H., & Morgan, B. P. (2002). The membrane attack complex of complement causes severe demyelination associated with acute axonal injury. Journal of Immunology, 168, 458–465. Mehlhop, E., & Diamond, M. S. (2006). Protective immune responses against West Nile virus are primed by distinct complement activation pathways. Journal of Experimental Medicine, 203, 1371–1381. Miller, A. M., & Stella, N. (2009). Microglial cell migration stimulated by ATP and C5a involve distinct molecular mechanisms: Quantification of migration by a novel near-infrared method. Glia, 57, 875–883. Mocco, J., Wilson, D. A., Komotar, R. J., Sughrue, M. E., Coates, K., Sacco, R. L., et al. (2006). Alterations in plasma complement levels after human ischemic stroke. Neurosurgery, 59, 28–33; discussion 28–33. Moller, T., Nolte, C., Burger, R., Verkhratsky, A., & Kettenmann, H. (1997). Mechanisms of C5a and C3a complement fragmentinduced [Ca2?]i signaling in mouse microglia. Journal of Neuroscience, 17, 615–624. Morgan, B. P., Griffiths, M., Khanom, H., Taylor, S. M., & Neal, J. W. (2004). Blockade of the C5a receptor fails to protect against experimental autoimmune encephalomyelitis in rats. Clinical and Experimental Immunology, 138, 430–438. Mukherjee, P., & Pasinetti, G. M. (2000). The role of complement anaphylatoxin C5a in neurodegeneration: Implications in Alzheimer’s disease. Journal of Neuroimmunology, 105, 124–130. Mukherjee, P., & Pasinetti, G. M. (2001). Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinasedependent inhibition of caspase 3. Journal of Neurochemistry, 77, 43–49. Mukherjee, P., Thomas, S., & Pasinetti, G. M. (2008). Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo. J Neuroinflammation, 5, 5. Muller-Ladner, U., Jones, J. L., Wetsel, R. A., Gay, S., Raine, C. S., & Barnum, S. R. (1996). Enhanced expression of chemotactic receptors in multiple sclerosis lesions. Journal of the Neurological Sciences, 144, 135–141. Nataf, S., Davoust, N., & Barnum, S. R. (1998). Kinetics of anaphylatoxin C5a receptor expression during experimental allergic encephalomyelitis. Journal of Neuroimmunology, 91, 147–155. Nataf, S., Levison, S. W., & Barnum, S. R. (2001). Expression of the anaphylatoxin C5a receptor in the oligodendrocyte lineage. Brain Research, 894, 321–326. Nolte, C., Moller, T., Walter, T., & Kettenmann, H. (1996). Complement 5a controls motility of murine microglial cells in

Neuromol Med (2010) 12:179–192 vitro via activation of an inhibitory G-protein and the rearrangement of the actin cytoskeleton. Neuroscience, 73, 1091–1107. Nyakoe, N. K., Taylor, R. P., Makumi, J. N., & Waitumbi, J. N. (2009). Complement consumption in children with Plasmodium falciparum malaria. Malaria Journal, 8, 7. O’Barr, S. A., Caguioa, J., Gruol, D., Perkins, G., Ember, J. A., Hugli, T., et al. (2001). Neuronal expression of a functional receptor for the C5a complement activation fragment. Journal of Immunology, 166, 4154–4162. O’Barr, S., Yu, J. X., & Cooper, N. R. (1998). Neuronal expression of the C5a receptor. Molecular Immunology, 35, 26. Ohno, M., Hirata, T., Enomoto, M., Araki, T., Ishimaru, H., & Takahashi, T. A. (2000). A putative chemoattractant receptor, C5L2, is expressed in granulocyte and immature dendritic cells, but not in mature dendritic cells. Molecular Immunology, 37, 407–412. Osaka, H., McGinty, A., Hoepken, U. E., Lu, B., Gerard, C., & Pasinetti, G. M. (1999a). Expression of C5a receptor in mouse brain: Role in signal transduction and neurodegeneration. Neuroscience, 88, 1073–1082. Osaka, H., Mukherjee, P., Aisen, P. S., & Pasinetti, G. M. (1999b). Complement-derived anaphylatoxin C5a protects against glutamate-mediated neurotoxicity. Journal of Cellular Biochemistry, 73, 303–311. Otto, M., Hawlisch, H., Monk, P. N., Muller, M., Klos, A., Karp, C. L., et al. (2004). C5a mutants are potent antagonists of the C5a receptor (CD88) and of C5L2: Position 69 is the locus that determines agonism or antagonism. Journal of Biological Chemistry, 279, 142–151. Patel, S. N., Berghout, J., Lovegrove, F. E., Ayi, K., Conroy, A., Serghides, L., et al. (2008). C5 deficiency and C5a or C5aR blockade protects against cerebral malaria. Journal of Experimental Medicine, 205, 1133–1143. Peitsch, M. C., Tschopp, J., Kress, A., & Isliker, H. (1988). Antibodyindependent activation of the complement system by mitochondria is mediated by cardiolipin. Biochemical Journal, 249, 495– 500. Persson, M., Pekna, M., Hansson, E., & Ronnback, L. (2009). The complement-derived anaphylatoxin C5a increases microglial GLT-1 expression and glutamate uptake in a TNF-alphaindependent manner. European Journal of Neuroscience, 29, 267–274. Pisalyaput, K., & Tenner, A. J. (2008). Complement component C1q inhibits beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms. Journal of Neurochemistry, 104, 696–707. Podack, E. R., Tschoop, J., & Muller-Eberhard, H. J. (1982). Molecular organization of C9 within the membrane attack complex of complement. Induction of circular C9 polymerization by the C5b–8 assembly. Journal of Experimental Medicine, 156, 268–282. Rahpeymai, Y., Hietala, M. A., Wilhelmsson, U., Fotheringham, A., Davies, I., Nilsson, A. K., et al. (2006). Complement: a novel factor in basal and ischemia-induced neurogenesis. EMBO Journal, 25, 1364–1374. Reiman, R., Campos Torres, A., Martin, B. K., Ting, J. P., Campbell, I. L., & Barnum, S. R. (2005). Expression of C5a in the brain does not exacerbate experimental autoimmune encephalomyelitis. Neuroscience Letters, 390, 134–138. Reiman, R., Gerard, C., Campbell, I. L., & Barnum, S. R. (2002). Disruption of the C5a receptor gene fails to protect against experimental allergic encephalomyelitis. European Journal of Immunology, 32, 1157–1163. Ricklin, D., & Lambris, J. D. (2007). Complement-targeted therapeutics. Nature Biotechnology, 25, 1265–1275.

191 Rittirsch, D., Flierl, M. A., Nadeau, B. A., Day, D. E., Huber-Lang, M., Mackay, C. R., et al. (2008). Functional roles for C5a receptors in sepsis. Nature Medicine, 14, 551–557. Roestenberg, M., McCall, M., Mollnes, T. E., van Deuren, M., Sprong, T., Klasen, I., et al. (2007). Complement activation in experimental human malaria infection. Transactions of the Royal Society of Tropical Medicine and Hygiene, 101, 643–649. Rother, R. P., Rollins, S. A., Mojcik, C. F., Brodsky, R. A., & Bell, L. (2007). Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nature Biotechnology, 25, 1256–1264. Rus, H., Cudrici, C., & Niculescu, F. (2005). C5b–9 complement complex in autoimmune demyelination and multiple sclerosis: Dual role in neuroinflammation and neuroprotection. Annals of Medicine, 37, 97–104. Sayah, S., Ischenko, A. M., Zhakhov, A., Bonnard, A. S., & Fontaine, M. (1999). Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: Specific increase in interleukin-6 mRNA expression. Journal of Neurochemistry, 72, 2426–2436. Schieferdecker, H. L., Rothermel, E., Timmermann, A., Gotze, O., & Jungermann, K. (1997). Anaphylatoxin C5a receptor mRNA is strongly expressed in Kupffer and stellate cells and weakly in sinusoidal endothelial cells but not in hepatocytes of normal rat liver. FEBS Letters, 406, 305–309. Scolding, N. J., Morgan, B. P., & Compston, D. A. (1998). The expression of complement regulatory proteins by adult human oligodendrocytes. Journal of Neuroimmunology, 84, 69–75. Sewell, D. L., Nacewicz, B., Liu, F., Macvilay, S., Erdei, A., Lambris, J. D., et al. (2004). Complement C3 and C5 play critical roles in traumatic brain cryoinjury: Blocking effects on neutrophil extravasation by C5a receptor antagonist. Journal of Neuroimmunology, 155, 55–63. Shen, Y., Lue, L., Yang, L., Roher, A., Kuo, Y., Strohmeyer, R., et al. (2001). Complement activation by neurofibrillary tangles in Alzheimer’s disease. Neuroscience Letters, 305, 165–168. Singhrao, S. K., Neal, J. W., Morgan, B. P., & Gasque, P. (1999). Increased complement biosynthesis by microglia and complement activation on neurons in Huntington’s disease. Experimental Neurology, 159, 362–376. Singhrao, S. K., Neal, J. W., Rushmere, N. K., Morgan, B. P., & Gasque, P. (2000). Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis. The American Journal of Pathology, 157, 905–918. Stahel, P. F., & Barnum, S. R. (1997). Bacterial meningitis: Complement gene expression in the central nervous system. Immunopharmacology, 38, 65–72. Stahel, P. F., Frei, K., Eugster, H. P., Fontana, A., Hummel, K. M., Wetsel, R. A., et al. (1997a). TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis. Journal of Immunology, 159, 861–869. Stahel, P. F., Kariya, K., Shohami, E., Barnum, S. R., Eugster, H., Trentz, O., et al. (2000). Intracerebral complement C5a receptor (CD88) expression is regulated by TNF and lymphotoxin-alpha following closed head injury in mice. Journal of Neuroimmunology, 109, 164–172. Stahel, P. F., Kossmann, T., Morganti-Kossmann, M. C., Hans, V. H., & Barnum, S. R. (1997b). Experimental diffuse axonal injury induces enhanced neuronal C5a receptor mRNA expression in rats. Molecular Brain Research, 50, 205–212. Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., et al. (2007). The classical complement cascade mediates CNS synapse elimination. Cell, 131, 1164– 1178.

192 Storrs, S. B., Kolb, W. P., & Olson, M. S. (1983). C1q binding and C1 activation by various isolated cellular membranes. Journal of Immunology, 131, 416–422. Tenner, A. J., Ziccardi, R. J., & Cooper, N. R. (1984). Antibodyindependent C1 activation by E. coli. Journal of Immunology, 133, 886–891. Thomas, A., Gasque, P., Vaudry, D., Gonzalez, B., & Fontaine, M. (2000). Expression of a complete and functional complement system by human neuronal cells in vitro. International Immunology, 12, 1015–1023. Van Beek, J., Bernaudin, M., Petit, E., Gasque, P., Nouvelot, A., MacKenzie, E. T., et al. (2000). Expression of receptors for complement anaphylatoxins C3a and C5a following permanent focal cerebral ischemia in the mouse. Experimental Neurology, 161, 373–382. Van Beek, J., Elward, K., & Gasque, P. (2003). Activation of complement in the central nervous system: Roles in neurodegeneration and neuroprotection. Annals of the New York Academy of Sciences, 992, 56–71. Van Beek, J., van Meurs, M., t Hart, B. A., Brok, H. P., Neal, J. W., Chatagner, A., et al. (2005). Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation. Journal of Immunology, 174, 2353– 2365. Veerhuis, R., Janssen, I., De Groot, C. J., Van Muiswinkel, F. L., Hack, C. E., & Eikelenboom, P. (1999). Cytokines associated with amyloid plaques in Alzheimer’s disease brain stimulate human glial and neuronal cell cultures to secrete early complement proteins, but not C1-inhibitor. Experimental Neurology, 160, 289–299. Veerhuis, R., Janssen, I., Hoozemans, J. J., De Groot, C. J., Hack, C. E., & Eikelenboom, P. (1998). Complement C1-inhibitor expression in Alzheimer’s disease. Acta Neuropathologica, 96, 287–296. Vergunst, C. E., Gerlag, D. M., Dinant, H., Schulz, L., Vinkenoog, M., Smeets, T. J., et al. (2007). Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation. Rheumatology (Oxford), 46, 1773–1778. Walker, D. G., Kim, S. U., & McGeer, P. L. (1995a). Complement and cytokine gene expression in cultured microglial derived from postmortem human brains. Journal of Neuroscience Research, 40, 478–493. Walker, D. G., Kim, S. U., & McGeer, P. L. (1998). Expression of complement C4 and C9 genes by human astrocytes. Brain Research, 809, 31–38. Walker, D. G., & McGeer, P. L. (1992). Complement gene expression in human brain: comparison between normal and Alzheimer disease cases. Molecular Brain Research, 14, 109–116. Walker, D. G., & McGeer, P. L. (1993). Complement gene expression in neuroblastoma and astrocytoma cell lines of human origin. Neuroscience Letters, 157, 99–102.

Neuromol Med (2010) 12:179–192 Walker, D. G., Yasuhara, O., Patston, P. A., McGeer, E. G., & McGeer, P. L. (1995b). Complement C1 inhibitor is produced by brain tissue and is cleaved in Alzheimer disease. Brain Research, 675, 75–82. Webster, S., Lue, L. F., Brachova, L., Tenner, A. J., McGeer, P. L., Terai, K., et al. (1997). Molecular and cellular characterization of the membrane attack complex, C5b–9, in Alzheimer’s disease. Neurobiology of Aging, 18, 415–421. Woodruff, T. M., Costantini, K. J., Crane, J. W., Atkin, J. D., Monk, P. N., Taylor, S. M., et al. (2008a). The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis. Journal of Immunology, 181, 8727–8734. Woodruff, T. M., Costantini, K. J., Taylor, S. M., & Noakes, P. G. (2008b). Role of complement in motor neuron disease: Animal models and therapeutic potential of complement inhibitors. Advances in Experimental Medicine and Biology, 632, 143–158. Woodruff, T. M., Crane, J. W., Proctor, L. M., Buller, K. M., Shek, A. B., de Vos, K., et al. (2006a). Therapeutic activity of C5a receptor antagonists in a rat model of neurodegeneration. FASEB Journal, 20, 1407–1417. Woodruff, T. M., Pollitt, S., Proctor, L. M., Stocks, S. Z., Manthey, H. D., Williams, H. M., et al. (2005). Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease. Journal of Pharmacology and Experimental Therapeutics, 314, 811–817. Woodruff, T. M., Proctor, L. M., Strachan, A. J., & Taylor, S. M. (2006b). Complement factor 5a as a therapeutic target. Drug Future, 31, 325–334. Woodruff, T. M., Strachan, A. J., Dryburgh, N., Shiels, I. A., Reid, R. C., Fairlie, D. P., et al. (2002). Antiarthritic activity of an orally active C5a receptor antagonist against antigen-induced monarticular arthritis in the rat. Arthritis and Rheumatism, 46, 2476– 2485. Xi, G., Hua, Y., Keep, R. F., Younger, J. G., & Hoff, J. T. (2001). Systemic complement depletion diminishes perihematomal brain edema in rats. Stroke, 32, 162–167. Yang, C., Jones, J. L., & Barnum, S. R. (1993). Expression of decayaccelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes. Journal of Neuroimmunology, 47, 123– 132. Yao, J., Harvath, L., Gilbert, D. L., & Colton, C. A. (1990). Chemotaxis by a CNS macrophage, the microglia. Journal of Neuroscience Research, 27, 36–42. Zajicek, J., Wing, M., Skepper, J., & Compston, A. (1995). Human oligodendrocytes are not sensitive to complement. A study of CD59 expression in the human central nervous system. Laboratory Investigation, 73, 128–138. Zhou, J., Fonseca, M. I., Pisalyaput, K., & Tenner, A. J. (2008). Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer’s disease. Journal of Neurochemistry, 106, 2080–2092.