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Molecular Immunology 46 (2009) 2753–2766

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

eview

he role of the anaphylatoxins in health and disease

ndreas Klosa,1, Andrea J. Tennerb,1, Kay-Ole Johswicha, Rahasson R. Agerb, Edimara S. Reisc, Jörg Köhlc,d,∗

Institute of Medical Microbiology and Hospital Epidemiology, Medical School Hannover (MHH), Hannover, GermanyDepartment of Molecular Biology and Biochemistry, Institute for Immunology, Institute for Brain Aging and Dementia, University of California, Irvine, USAInstitute for Systemic Inflammation Research, University of Lübeck, Lübeck, GermanyDivision of Molecular Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, USA

r t i c l e i n f o

rticle history:eceived 9 April 2009ccepted 28 April 2009vailable online 28 May 2009

a b s t r a c t

The anaphylatoxin (AT) C3a, C5a and C5a-desArg are generally considered pro-inflammatory polypep-tides generated after proteolytic cleavage of C3 and C5 in response to complement activation. Theirwell-appreciated effector functions include chemotaxis and activation of granulocytes, mast cells andmacrophages. Recent evidence suggests that ATs are also generated locally within tissues by pathogen-,cell-, or contact system-derived proteases. This local generation of ATs is important for their pleiotropic

eywords:omplementnaphylatoxins

nflammationepsisllergy

biologic effects beyond inflammation. The ATs exert most of the biologic activities through ligation ofthree cognate receptors, i.e. the C3a receptor, the C5a receptor and the C5a receptor-like, C5L2. Here, wewill discuss recent findings suggesting that ATs regulate cell apoptosis, lipid metabolism as well as innateand adaptive immune responses through their impact on antigen-presenting cells and T cells. As we willoutline, such regulatory functions of ATs and their receptors play important roles in the pathogenesis of

urod
lzheimer diseasedaptive immunity
allergy, autoimmunity, ne

. Introduction

The complement system is an ancient danger sensing systemhat recognizes exogenous threats such as conserved micro-ial motifs as well as endogenous threats including altered-selfolecules (e.g. following injury or hypoxia, after virus-infection or

umor-related) and apoptotic cells (Köhl, 2006b). Danger sensingolecules that activate the complement system comprise solu-

le C-type lectins such as Mannan-binding-lectin (MBL), ficolins,he C-type lectin-like molecule C1q and, as recent evidence sug-ests, properdin (Kemper and Hourcade, 2008). In addition to thisirect recognition of conserved danger motifs, C1q binds stronglyo another class of innate danger sensors, i.e. natural immunoglob-lins (Ciurana et al., 2004). Once bound to danger motifs, C1q,BL, ficolins and properdin initiate activation of proteolytic cas-

ades that result in the cleavage of the central molecule of the
omplement system, C3, followed by cleavage of C5. Together withther cleavage products of C3 (i.e. C3b, iC3b, C3dg, C3d and C3c),he smaller cleavage products of C3 and C5, C3a and C5a, form aet of soluble mediators that bind distinct cell surface receptors
∗ Corresponding author. Institute for Systemic Inflammation Research, Universityf Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Tel.: +49 451 3067;ax: +49 451 3069.

E-mail address: [email protected] (J. Köhl).1 These authors contributed equally to this work.

161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2009.04.027

egenerative diseases, cancer and infections with intracellular pathogens.© 2009 Elsevier Ltd. All rights reserved.

expressed on a variety of target cells. The interaction of C3a andC5a with their cognate receptors induces pleiotropic effector func-tions, translating the danger information from the fluid phase intodefined cellular responses. Here, we will review recent findings thatprovide a better understanding of the mechanisms and pathwaysunderlying the various pro-inflammatory and regulatory functionsof the anaphylatoxin (AT) mediated through the three distinct ATreceptors.

2. Anaphylatoxins

C3a and C5a are small polypeptides consisting of 77 and 74amino acids, respectively. With 36% amino acid identity, they sharehigh similarity, but only C5a harbors an N-linked carbohydrate moi-ety. Furthermore, C5a and C3a are both highly cationic molecules(pI 9.0), the core structure of which is based on a 4-helix bun-dle that is stabilized by three disulphide bonds (Nettesheim et al.,1988). Interestingly, these features render C3a, but not C5a, a highlypotent antimicrobial peptide (Nordahl et al., 2004). Both AT com-prise highly conserved C-terminal pentapeptide sequences that arerequired for activation of their cognate receptors. For C3a, it is
LGLAR (Caporale et al., 1980); for C5a, it is MQLGR (Fernandez andHugli, 1976). Especially for C5a, cyclic C-terminal-derived peptideanalogs have been generated such as AcF-(OPdChaWR) that are nowin clinical trials for the treatment of several inflammatory diseases(Köhl, 2006a).
http://www.sciencedirect.com/science/journal/01615890

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The ATs are potent inflammatory mediators targeting a broadpectrum of immune and non-immune cells. C3a and C5a regu-ate vasodilation, increase the permeability of small blood vessels,nd induce contraction of smooth muscles (Ember et al., 1998).n macrophages (Murakami et al., 1993), neutrophils (Elsner et al.,994b), and eosinophils (Elsner et al., 1994a) C3a and C5a can trig-er oxidative burst. Basophils (Kretzschmar et al., 1993) and mastells (el Lati et al., 1994) react upon AT stimulation with release ofistamine. In eosinophils, C3a and C5a regulate the production ofosinophil cationic protein, their adhesion to endothelial cells asell as their migration (Takafuji et al., 1996; DiScipio et al., 1999).3a further promotes serotonin release from guinea pig plateletsFukuoka and Hugli, 1988) and modulates synthesis of IL-6 andNF-� from B cells and monocytes (Fischer and Hugli, 1997; Fischert al., 1999). C5a is a powerful chemoattractant for macrophagesAksamit et al., 1981), neutrophils (Ehrengruber et al., 1994), acti-ated B (Ottonello et al., 1999) and T cells (Nataf et al., 1999a),asophils (Lett-Brown and Leonard, 1977) and mast cells, the lat-er of which also migrate towards a C3a gradient (Hartmann et al.,997).

In addition to their pro-inflammatory properties ATs regulateissue regeneration (Mastellos et al., 2001; Strey et al., 2003) andissue fibrosis (Hillebrandt et al., 2005; Addis-Lieser et al., 2005;trey et al., 2003) as well as brain development (see Section 4.2)Benard et al., 2004). Further, C3a has been shown to enhance SDF-

(CXCL12)-induced homing of hematopoietic stem cells into theone marrow and their retention in this compartment (Ratajczakt al., 2004).

It is obvious that control mechanisms have evolved that reg-late the activity of these powerful bioactive molecules. Indeed,T activity in the circulation and in tissues is tightly controlled byarboxypeptidases which rapidly cleave off a C-terminal arginineesidue (Bokisch and Müller-Eberhard, 1970; Matthews et al., 2004).

hile the resulting C5a-desArg retains 1–10% of the inflamma-ory activity of C5a, C3a-desArg is devoid of any pro-inflammatoryctivity (Sayah et al., 2003).

Intriguingly, C3a-desArg, which is also called acylation stim-lating protein (ASP), has been described to possess metabolicormone activity that drives triglyceride synthesis and glucoseptake in adipose tissue in numerous studies from the Cianflone

aboratory (reviewed in Cianflone et al., 2003). Surprisingly, ASPoncentrations in the range from 1 to 10 �M, which is 2–3 logsbove the concentrations usually needed for biological functionsf AT, are required for such metabolic activity. In most studies,erum-purified but not recombinant C3a-desArg has been used.3a-analog C-terminal peptides, which bind and stimulate C3aR,id not cause any changes in lipid metabolism. These findingsay point toward a different mode of binding, a different bind-

ng partner or may indicate ASP-independent effects, for examplenduced by co-purified serum proteins. Even more importantly,he in vivo relevance of ASP remains elusive as it still needs toe demonstrated that high micromolar ASP levels can be achieved

n adipose tissue. Further, no specific ASP receptor has yet beenescribed on adipocytes (for review see also (Johswich and Klos,007)).

.1. Anaphylatoxin receptors

The ATs bind to a family of three receptors, so-called ATeceptors, which belong to the superfamily of G-protein-coupledeceptors (GPCR). The AT receptor family comprises the C3a
eceptor (C3aR), C5a receptor (C5aR) and C5a receptor-like 2C5L2). They share high sequence homology (Lee et al., 2001)nd are closely related to other chemotactic receptors such ashe N-formyl-methionine-leucine-phenylalanine (fMLP) receptor,hemR23 (Samson et al., 1998) and the chemokine receptors CXCR1
logy 46 (2009) 2753–2766

and CXCR2. Despite their similarity, the AT receptors differ in ligandspecificity, signal transduction capacity and function.

2.1.1. C3aRThe C3aR specifically binds C3a with a Kd of about 1 nM, but

does not recognize its desarginated form or C5a (Crass et al., 1996;Wilken et al., 1999). It is a 54 kDa molecule comprising 482 aminoacids with two glycosylation sites located at asparagines 9 and 195and a sulfated tyrosine at position 174 (Crass et al., 1999; Ameset al., 1996). Compared to the other AT receptors, C3aR possesses aremarkable large second extracellular loop that accounts for almosta third of its size and is indispensable for ligand binding. Also, thesulfated tyrosine 174 that resides within this loop plays a pivotal rolefor the interaction of C3aR with C3a (Gao et al., 2003). However, thereceptor N-terminus seems not to be involved in C3a binding (Crasset al., 1999). Upon C3a binding to the C3aR, intracellular signaltransduction is promoted via heterotrimeric G-proteins. In neu-trophils, C3aR signals through pertussis-toxin-sensitive G-proteins(most likely G�i) and mobilizes calcium fluxes from the extracellu-lar medium but not from intracellular stores, suggesting that it doesnot activate phosphatidylinositol-bisphosphate-3-kinase gamma(PI3K-�) (Norgauer et al., 1993). However, C3aR may also encounterpertussis-toxin-insensitive G�16 for signal transduction (Crass etal., 1996). In endothelial cells, C3aR may couple to pertussis-toxin-insensitive G�12 or G�13 (Schraufstatter et al., 2002). Downstreamsignaling events include activation of protein kinase C by phospho-lipase C and, in astrocytes, the mitogen activated protein (MAP)kinases Erk1 and Erk2 (Langkabel et al., 1999; Sayah et al., 2003).Furthermore, in mast cells, C3a promotes cytokine expression bysignaling pathways that require activation of PI3K and subse-quent Akt-phosphorylation, as well as MAP kinases Erk1 and Erk2(Venkatesha et al., 2005).

The C3aR is expressed on cells of myeloid origin like neutrophils,basophils, eosinophils, mast cells, monocytes/macrophages, den-dritic cells (DC) and microglia (Glovsky et al., 1979; Daffern et al.,1995; Klos et al., 1992; Zwirner et al., 1998a,b; Gutzmer et al., 2004).Additionally, non-myeloid cells express C3aR. These include astro-cytes from inflamed brain (Gasque et al., 1998; Ischenko et al.,1998), endothelial cells (Monsinjon et al., 2003), epithelial cells,smooth muscle cells, submucosal and parenchymal vessels of thelung from patients suffering from asthma (Fregonese et al., 2005)and activated but not naïve human T cells (Werfel et al., 2000).Other studies have confirmed the absence of C3aR on unstimulatedhuman T cells and on naïve or activated human B cells (Zwirner etal., 1999; Martin et al., 1997). More recently, minor C3aR expressionhas been described on murine CD4+ T cells which was upregulatedupon DC stimulation (Strainic et al., 2008). Furthermore, Northernblot analysis suggests expression of C3aR mRNA in tissues of lung,liver, kidney, brain, heart, muscle and testis (Hsu et al., 1997).

2.1.2. C5aRThe C5aR (CD88) binds C5a with high affinity (Kd ∼ 1 nM) and

C5a-desArg with somewhat lower affinity (Kdk ∼ 660 nM) whereasC3a and C3a-desArg are not recognized (Gerard and Gerard, 1991;Gerard et al., 1989; Okinaga et al., 2003). C5aR is a 42 kDa proteinconsisting of 350 amino acids, of which asparagine at position 5 isglycosylated. Additional posttranslational tyrosine sulfation occursat positions 11 and 14 (Farzan et al., 2001). The essential structuresrequired for ligand binding of C5aR have been elaborated in numer-ous studies (Mery and Boulay, 1993; DeMartino et al., 1994; Crasset al., 1999; Siciliano et al., 1994; Baranski et al., 1999; Gerber et
al., 2000; Geva et al., 2000; Klco et al., 2005, 2006; Matsumoto etal., 2007). The bottom line is that C5a binding involves two distinctsites at the C5aR. First, the aspartate-rich acidic N-terminus of C5aRinteracts with the basic core of C5a. Then, the agonistic C-terminusof C5a interacts with a binding pocket formed by hydrophobic

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esidues from the transmembrane domains and charged residues athe base of the extracellular loops. The latter step is indispensableor receptor activation.

C5aR signal transduction depends on heterotrimeric G-proteins.t is mainly achieved by the pertussis-toxin-sensitive alpha units�i2 (Skokowa et al., 2005) or the pertussis-toxin-insensitive�16 (Monk and Partridge, 1993; Amatruda et al., 1993) which isxpressed by cells of the hematopoietic lineage. A unique featuref the C5aR is precoupling to G-proteins in the absence of ligands,hereby apparently lacking a low affinity conformation found inther GPCRs (Siciliano et al., 1990). When C5aR is uncoupled fromts G-protein by GTP�S, its affinity is dramatically decreased. Inact, the binding affinity of C5a to the C5aR lacking G-protein cou-ling is too low for detection by competitive ligand binding assaysRaffetseder et al., 1996). C5a binding to C5aR causes calcium fluxesrom both, intracellular stores as well as from extracellular medium.fter activation, �-arrestins 1 and 2 bind to C5aR, targeting it foreceptor internalization via clathrin coated pits (Braun et al., 2002).inding of arrestins depends on phosphorylation of the C-terminusf the receptor by G-protein-coupled receptor kinases (GRKs). Inddition to their function as kinases, GRKs can also interact withther components of intracellular signaling such as Akt, MAPK/ERKinase (MEK) and PI3K-� (for review see Ribas et al., 2007). It haseen shown that C5aR activation leads to downstream activationf several components of different signaling pathways like PI3K-�inase (Perianayagam et al., 2002; la Sala et al., 2005), phospho-ipase C �2 (Jiang et al., 1996), phospholipase D (Mullmann et al.,990) and Raf-1/B-Raf mediated activation of MEK-1 (Buhl et al.,994). Another intracellular molecule that binds to the C-terminusf activated C5aR is the Wiskott–Aldrich syndrome protein (WASP)Tardif et al., 2003). This interaction is significantly increased in theresence of cell division cycle 42 (cdc42), a GTP binding proteinhich is thought to induce a conformational change of WASP to its

ctive state. WASP is a multifunctional protein that regulates actinynamics and therefore might play an important role in the C5aependent chemotaxis.

C5aR is expressed in various cell types. It is most abundantlyxpressed in neutrophils, eosinophils and basophils, mono-ytes/macrophages, mast cells and DCs (Chenoweth and Goodman,983; Chenoweth and Hugli, 1978; Gerard et al., 1989; Werfel etl., 1997; Morelli et al., 1996). The expression of C5aR in cells ofymphoid origin is not generally accepted. Several reports havehown expression in human and mouse T cells (Nataf et al., 1999a;onnelly et al., 2007; Strainic et al., 2008; Lalli et al., 2008). Fur-her, C5a-mediated migration has been demonstrated for B and Tells (El-Naggar et al., 1980). In contrast, in another study bind-ng of C5a has been solely detected in a small subpopulation of 6%f lymphocytes (Van-Epps and Chenoweth, 1984) and anti-C5aRntibodies failed to demonstrate receptor expression in murineymphoid cells (Soruri et al., 2003). In addition to immune cells,body of evidence has accumulated that the C5aR is expressed inbroad range of non-immune tissue cells (reviewed in Monk et al.,007). These include endothelial cells (Laudes et al., 2002a), neu-ons (Farkas et al., 1998), astrocytes and microglia (Gasque et al.,997) as well as cells from kidney, lung, liver, spleen, intestine, skinnd heart (Fayyazi et al., 2000; Wetsel, 1995). However, data regard-ng its expression in epithelial cells of lung, liver and intestine wereegatively re-evaluated (Fayyazi et al., 1999, 2000) and found to beisleading due to cross-reactivity of some anti-C5aR antibodies to

esmosomal antigens (Werfel et al., 1996).

.1.3. C5a receptor-like 2C5L2 was discovered in 2000 as putative orphan receptor

GPR77) (Ohno et al., 2000). It is a 37 kDa protein consisting of 337mino acids with Asparagine 3 as potential glycosylation site. Inhe conserved transmembrane regions, C5L2 shares 58% sequence

logy 46 (2009) 2753–2766 2755

identity with C5aR and 55% with C3aR (Lee et al., 2001). C5L2is expressed in various tissues of myeloid and non-myeloid ori-gin and transcripts were detected in brain, placenta, ovary, testis,spleen and colon (Gavrilyuk et al., 2005; Lee et al., 2001). Surfaceexpression of C5L2 was detected in lung, liver, heart, kidney (Gaoet al., 2005), in adipose tissue and in skin fibroblasts (Kalant et al.,2005), in neutrophils (Huber-Lang et al., 2005) and in immature,but not in mature DCs (Ohno et al., 2000). In combination witha C5aR antagonist, binding of C5a could be demonstrated on dif-ferentiated myeloblastic HL-60 and U937 and epithelial HeLa-cells(Johswich et al., 2006). C5L2 and C5aR seem to be frequently co-expressed in most cells or tissues (Okinaga et al., 2003; Gao et al.,2005).

C5a binds with high affinity (Kd: 2.5 nM) to C5L2. Suggestinga slightly different function, C5a-desArg binds with a 20–30 foldhigher affinity to C5L2 than to C5aR (Cain and Monk, 2002). More-over, binding kinetics of the two receptors differ for the same ligand:the on-rate of C5aR is about 100 fold faster than that of C5L2. It isimportant to consider that only a small percentage of the split prod-ucts of C5 and C3 (close to the nucleus of complement activation)are available as C5a and C3a in the circulation as they are rapidlydegraded to their desArg products. C5L2 appears to bind C5a andC5a-desArg by different mechanisms. Unlike C5aR, C5L2 uses criti-cal residues in its N-terminal domain for binding only to C5a-desArg(Scola et al., 2007).

In addition to C5a and C5a-desArg binding, C5L2 has been con-sidered a binding partner for C3a, C3a-desArg, C4a and C4a-desArg(Cain and Monk, 2002; Kalant et al., 2003; Okinaga et al., 2003).Re-evaluation of such data revealed “unspecific” binding mainly toplastic surfaces due to the highly cationic nature of the ligands C3aand C3a-desArg; a binding which mimics in the absence of any cellssaturable, specific, high-affinity binding to the C3aR (Johswich et al.,2006). Similar problems had already been reported before with ratmast cells (Kajita and Hugli, 1991). Indeed, using assay conditionsthat take the cationic nature of the ligands into account, no bindingto the recombinant human C5L2, at least not in the low or mediumnanomolar range, has been observed (Johswich et al., 2006). Thisdoes not exclude a biological role for C3a-desArg (ASP) in lipid andglucose metabolism in fat cells and fibroblasts (Kalant et al., 2005).Yet, it makes it highly unlikely that C5L2 is the suggested bindingpartner for C3a-desArg or C3a.

C5L2 is an enigmatic receptor as a couple of reports suggestopposing functions. On the one hand, it has been described as anon-signaling scavenger receptor for C5a and C5a-desArg. In con-trast, at least two reports indicate that C5L2 alone, or in conjunctionwith other receptors, serves as a signaling receptor. In support ofa role as a decoy receptor, no mobilization of intracellular Ca2+

occurs in C5L2-transfected cells after AT administration (Cain andMonk, 2002; Okinaga et al., 2003). Moreover, no calcium fluxes havebeen observed in neutrophils from C5aR−/− mice after stimulationwith C5a (Hopken et al., 1996) or in endogenously C5L2 express-ing cell lines (Johswich et al., 2006). In heptahelical receptors, ahighly conserved DRY motif in the third transmembrane domainis important for its interaction with the corresponding G-proteins.The DRY motif is DRF in C5aR and DRC in C3a, but DLC in C5L2.The central arginine residue plays a key role in coordinating thetransmembrane domains 3 and 6 and mutagenesis of this residuein C5aR leads to loss of function (Kolakowski et al., 1995). WhenDLC in C5L2 is restored to DRC, C5L2 couples weakly to intracellu-lar Ca2+ fluxes in HEK293 cells co-expressing G�16 (Okinaga et al.,2003). Rat basophilic leukaemia cells (RBL.2H3)-cells are frequently
used to study functional intracellular coupling and signaling of ATreceptors. Intriguingly, no Ca2+ fluxes occurred in these cells usinga C5L2-mutant where the DRY motif and two additional regionstypically involved in G-protein coupling were replaced by the corre-sponding C5aR-sequences (Scola et al., 2009). Taken together, these

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ndings strongly suggest that C5L2 on its own is uncoupled from-proteins.

In functional studies, C5L2 failed to promote biologic activ-ties. Preloading of the tyrosine kinase coupled IgE-receptorsFc�RI) with DNP-specific IgE resulted in a small increase in �-exosaminidase release upon AT pretreatment which was absent

n C5L2-transfected RBL cells (Cain and Monk, 2002). Further, C5ainding to C5L2 in bone marrow cells derived from C5aR−/− miceailed to promote any changes in mRNA expression (Okinaga etl., 2003). Thus, C5L2 has been considered to belong to the groupf decoy receptors negatively regulating primary inflammatoryhemokines (reviewed in Locati et al., 2005). Decoy receptors suchs US28, CCX CKR, DARC and D6 not only lack the DRY motif andunctional coupling, but also constantly recycle giving rise to a highroportion of intracellular receptors combined with highly efficient

igand internalization and degradation.Rapid ligand-induced receptor internalization is a central fea-

ure of functional G-protein-coupled receptors. C5aR and C3aR areo exception (Bock et al., 1997; Settmacher et al., 1999). Expressed

n RBL.2H3-cells, C5aR rapidly internalizes upon C5a stimulationhereas C5L2 does not (Scola et al., 2009). This view is supported

y two independent studies which found no internalization of C5L2pon stimulation with ATs (Cain and Monk, 2002; Okinaga et al.,003). On the other hand, C5L2 but not C5aR constitutively recycles

n transfected RBL cells leading to major intracellular expression.or example, in transfected CHO cells C5L2 is more efficient than5aR at internalizing and retaining C5a and C5a-desArg. Addition-lly, in human neutrophils and HeLa-cells, C5L2 is responsible fornternalization and degradation of C5a and C5a-desArg (Scola et al.,009).

The phosphorylation of C-terminal serine or threonine residuesy GRKs, protein kinase A or protein kinase C, followed by their asso-iation with arrestins to the receptor is a prerequisite for receptornternalization. C5L2-transfected mouse L1.2 cells elicited only aery basal level of phosphorylation upon C5a stimulation despiteserine/threonine rich C-terminus (Okinaga et al., 2003). In con-

rast, using transfected HEK293 cells, C5L2 was found to be robustlyhosphorylated after stimulation with very high concentrationsf C3a-desArg (2 × 10−5 M). Further, a cotransfected �-arrestin-FP fusion protein translocated from the plasma membrane tondocytic vesicles after stimulation with C5a, C3a or C3a-desArg,hereby suggesting functionality of C5L2 (Kalant et al., 2005). How-ver, this result has recently been questioned, as no translocationf �-arrestin has been found after C5a-stimulation of transfectedBL.2H3-cells (Scola et al., 2009).

Taken together, there is accumulating evidence that C5L2 is aecoy receptor in primary cells and transfected cell lines suggest-

ng that it acts as a functional antagonist of the C5aR in vitro andn vivo. Thus, biological effects mediated by C5aR can be expectedo be stronger in the absence and weaker in the presence of C5L2.ndeed, neutrophils and macrophages from C5L2−/− mice produce

ore TNF-� and IL-6 in response to combined stimulation with5a and LPS than their wildtype littermates. Further, C5a and LPStimulation drive more IL-6 production from rat neutrophils when5L2 is blocked (Gao et al., 2005). In vivo, C5L2−/− mice suffer fromugmented inflammatory responses (IL-6 and TNF-�) and higherumbers of infiltrating neutrophils when compared to their wild-ype littermates in a model of pulmonary immune complex injuryGerard et al., 2005). The anti-inflammatory role of C5L2 is furtherupported by a study in which LPS-injected C5L2−/− mice showedigher IL-1� levels and decreased survival rates (Chen et al., 2007)
nd a report in which C5L2−/− mice displayed higher serum con-entrations of IL-6 as compared to wildtype and C5aR−/− mice in aodel of septic peritonitis (Rittirsch et al., 2008).However, under the same conditions, a strong reduction of other
nflammatory mediators, such as IL-1�, MIP-1� and MIP-2 was

logy 46 (2009) 2753–2766

observed in C5L2−/− mice compared to wildtype mice. Indeed, theconcentrations of these mediators were comparable to those ofC5aR−/− mice. Furthermore, C5L2−/− mice, like C5aR−/− mice, oranimals in which each of the receptors was blocked by antibod-ies, showed a higher survival rate in mid-grade sepsis (Rittirsch etal., 2008). Contrasting the in vitro findings that the C5a/LPS-drivenIL-6 production of mouse neutrophils is increased when C5L2 isblocked by antibodies (see above), Chen et al. found decreased IL-6 release from C5L2−/− neutrophils (Chen et al., 2007). Moreover,C5a + LPS-induced Mac-1 surface expression on neutrophils wasalso diminished. Likewise, whereas C5a or C5a + LPS led to strongERK1/2- and AKT-phosphorylation in neutrophils from wildtypemice, there was only a weak effect in the absence of C5L2. Addi-tionally, in C5L2−/− macrophages, there was an impaired inductionof co-stimulatory molecules (CD40, CD86). Even effects which aremediated by C3a such as ERK1/2- and AKT-phosphorylation or F-actin formation on neutrophils were impaired in C5L2−/− mice(Chen et al., 2007). In addition to these in vitro findings, inflam-matory responses were reduced in vivo in models of thioglycollateinduced peritonitis, thioglycollate induced migration into dorsal airpouches, or OVA induced airway hyperresponsiveness. Thus, thestudies of Rittirsch et al. and of Chen et al. point to a more complexrole of C5L2 in inflammation with C5L2 acting not only as a decoyreceptor but also as positive modulator of C5a or even C3a. AlthoughC3a-desArg (ASP) does not bind directly to C5L2 (Johswich et al.,2006), over-expression of C5L2 or its downregulation by antisenseoligonucleotides influenced effects of C3a-desArg (ASP) (Kalant etal., 2005), suggesting that C5L2 can modulate signaling pathways ofother receptors. As GPCRs, including C5aR, tend to homo- or hetero-oligomerize (Klco et al., 2003; Rabiet et al., 2008) it is tempting tospeculate that such oligomerization may be one mechanism under-lying the positive modulatory effect of C5L2 on C5a or C3a effectorfunctions (Rabiet et al., 2008).

3. Anaphylatoxins in acute and chronic inflammation

ATs have been shown to promote inflammatory responses dur-ing the effector phase of allergic, infectious and autoimmunediseases (reviewed in Guo and Ward, 2005; Köhl, 2001). In the fol-lowing paragraphs, we will focus on recent findings related to therole of C5a in sepsis and the impact of AT receptor signaling on thedevelopment of adaptive immune responses in allergy, transplan-tation, tumor biology and infection.

3.1. Anaphylatoxins in sepsis

The sepsis syndrome is still the leading cause of death in inten-sive care units in the Western world. It is well accepted thatinvading microorganisms induce the release of a large number ofhumoral and cellular pro-inflammatory mediators causing a sys-temic inflammatory response syndrome which defines the patients’outcome. Most microbes activate the complement system throughtheir interaction with C1q, MBL/ficolins, or properdin leading tolocal and/or systemic complement activation with subsequentgeneration of C3a and C5a. Indeed, high plasma or serum concen-trations of C3a-desArg and C5a-desArg (Bengtson and Heideman,1988; Weinberg et al., 1984; Hack et al., 1989; Stove et al., 1996;Selberg et al., 2000) have been observed in septic patients and inexperimental models of sepsis (Ward, 2008a). Importantly, C3a-desArg plasma levels have been associated with fatal outcome
(Hack et al., 1989; Selberg et al., 2000) suggesting a causal role ofthe ATs in sepsis pathogenesis. The Ward laboratory has contributedtremendously to our understanding of the role of the ATs, and in par-ticular C5a, in experimental sepsis. Most of the work has recentlybeen reviewed (Ward, 2008a,b, 2004) and will be summarized only

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ery briefly. Based on the initial observation that interruption of the5a/C5aR interaction in a model of septic peritonitis significantly

mproved survival (Czermak et al., 1999), several mechanisms haveeen worked out underlying the detrimental effects of C5a in sep-is. C5a paralyzes immune functions of neutrophils (Huber-Langt al., 2001), promotes septic cardiomyopathy (Niederbichler et al.,006), drives apoptosis of thymocytes (Guo et al., 2000) and adrenaledullary cells (Flierl et al., 2008) and contributes to consumptive

oagulopathy (Laudes et al., 2002b). Importantly, C5aR is markedlypregulated in several organs including lung, liver, kidney, hearts well as in the thymus (Huber-Lang et al., 2002b). Initially, thedark side of C5a” has been exclusively attributed to the activa-ion of the C5aR. However, assessing the septic peritonitis model in

ore detail, it turned out that in addition to C5aR, C5L2 is involvedn C5a-mediated sepsis. In “mid-grade” sepsis with mortality ratesf 60–70% absence of either C5aR or C5L2 improves survival. Inhigh-grade” sepsis (mortality rate 100%) only the combined inhi-ition of C5aR and C5L2 is protective, suggesting that C5aR and5L2 synergistically promote septic inflammation (Rittirsch et al.,008). Importantly, C5L2 but not C5aR was found to regulate theroduction of the high mobility group box 1 protein (HMBG1)rom phagocytes, the ablation of which protects from organ dam-ge and failure in experimental sepsis (Wang et al., 1999). Further,PS-induced HMBG1 production from C5L2−/− macrophages wasignificantly reduced as compared with WT controls suggestinghat C5L2 regulates LPS-driven Toll-like receptor (TLR) 4 activa-ion. Indeed, cross-talk between C5a and TLR signaling has beenhown before, although related to C5aR signaling (Hawlisch et al.,005; Zhang et al., 2007), indicating complex interactions betweenhe complement system and other danger sensing systems (Köhl,006b) (see also Section 3.2.5). In support of this view, MBL andLR2/6 signaling cooperate in Staphylococcus aureus infection topecify and amplify host defense (Ip et al., 2008).

.2. Anaphylatoxins regulate adaptive immune responses

It is now well appreciated that complement contributes to theegulation of adaptive immune responses (for review see Carroll,004; Köhl, 2006b; Kemper and Atkinson, 2007). Initially, com-lement and particularly C3-derived cleavage products have beenonsidered to regulate B cell immunity (Fearon and Locksley, 1996).n a series of elegant studies, it was demonstrated that CD21CR2) and CD35 (CR1) are critical for the elimination of self-eactive B cells, selection and and/or maintenance of B1 cells, andhe amplitude of humoral responses to thymus-dependent andhymus-independent antigens (summarized in Carroll, 2004). Dur-ng the past 10 years, we have learned that in addition to CD21nd CD35, signaling through other complement receptors includ-ng the AT receptors regulates adaptive immune responses. Inontrast to CD21 and CD35, AT-mediated regulation focuses oncell-dependent immune responses promoting inflammation in

llergy, autoimmunity, transplantation, tumors and infection withntracellular microorganisms. In the following paragraphs, we willummarize recent findings in these areas.

.2.1. Anaphylatoxins in allergic asthmaThe worldwide prevalence and severity of allergic asthma in

ndustrialized countries have increased dramatically in recentecades reaching epidemic proportions. In the US alone, 15 millioneople suffer from this chronic inflammatory disease of the lung.he mechanisms underlying the development of pulmonary allergy
n individuals from industrialized countries remain elusive. Onemportant observation is that asthmatics suffer from a dysregulateddaptive immune response which accounts for most of the patho-hysiological characteristics found in asthmatics including airflowbstruction, airway hyperresponsiveness (AHR) and airway inflam-
logy 46 (2009) 2753–2766 2757

mation. Specifically, environmental factors drive a T helper cell type2 (Th2)-biased immune response associated with strong produc-tion of cytokines such as IL-4, IL-5, and IL-13 which orchestratethe pulmonary inflammatory response, involving the recruitmentand the activation of eosinophils, the production of allergen spe-cific IgE, induction of AHR and mucus hypersecretion (Wills-Karp,2004). One of the current challenges is to understand the mecha-nisms that regulate tolerance towards environmental allergens atthe mucosal surface, preventing the development of the maladap-tive, Th2-biased immune response in healthy individuals.

ATs are generated in the lungs of both healthy individuals andasthmatics. Under physiologic or steady state conditions, ATs arelocally generated in the pulmonary tissue at low levels, while inthe inflamed asthmatic environment, high concentrations of ATsare produced (Krug et al., 2001). C3a and C5a play distinct rolesin the pathogenesis and the pathology, depending on the con-ditions under which they are generated and the cell types thatbecome activated. Under steady state conditions, C5aR signalingat the DC/T cell interface controls the development of the maladap-tive immune response towards innocuous aeroallergens (Karp et al.,2000; Köhl et al., 2006; Drouin et al., 2006; McKinley et al., 2006). Inan inflamed environment that promotes strong complement acti-vation, C3a (Bautsch et al., 2000; Humbles et al., 2000; Drouin etal., 2002, 2001; Baelder et al., 2005) as well as C5a (Abe et al., 2001;Baelder et al., 2005; Köhl et al., 2006; Peng et al., 2005) act mainlyon infiltrating cells such as eosinophils, mast cells and basophilsand promote a pro-inflammatory scenario. Interestingly, an exclu-sive role for C3a has recently been shown in the development ofanaphylactic shock in peanut allergy (Khodoun et al., 2009).

The mechanisms underlying the protective role of C5a duringallergen sensitization include control of the pulmonary accumu-lation of immunogenic myeloid DCs, increased release of theTh2 effector cell-homing chemokines CCL17 and CCL22 and theregulation of mDC susceptibility toward the suppressor activ-ity of naturally occurring regulatory T cells (Köhl et al., 2006;Lewkowich et al., 2005). Further insights into the regulatory roleof C5 on distinct pulmonary DC population result from a recentstudy comparing the mechanisms underlying asthma-susceptibleC5-deficient A/J mice and resistant C3H mice. A/J mice favor aller-gen uptake by mDCs leading to upregulation of co-stimulatorymolecules and production of a Th2 and Th17-promoting cytokineprofile. In contrast, in C3H mice, allergens are preferentially takenup by tolerogenic plasmacytoid DCs (Lewkowich et al., 2008). Thesedata suggest that C5 and possibly C5a regulate maladaptive immu-nity in asthma through an impact on pDC. In support of this view,C5aR blockade during allergen sensitization decreases expressionof the co-stimulatory molecules B7-H1 (pD-L1) and B7-DC (pD-L2)on pDCs but not on mDCs. Importantly, B7-H1 and B7-DC regulateTh2 cytokine production from CD4+ T effector cells (Zhang et al.,2009). Together, these data suggest that C5/C5a sets the thresholdfor the development of maladaptive immunity by defining the path-ways of allergen uptake (mDC vs. pDC) and the potential of pDCs tosuppress mDC-induced activation of T effector cells. In future stud-ies, it will be important to delineate the molecular mechanism bywhich C5a regulates mDC and/or pDC function. At this point, therole of C5L2 in this scenario is unclear. C5L2-deficiency results in adecreased allergic phenotype similar to that seen in C3aR-deficientmice (Chen et al., 2007).

3.2.2. AT and transplantationFirst evidence for an important contribution of local comple-

ment production to allograft survival came from the Sacks labshowing that the lack of C3 in donor kidneys is associated withlong-term graft survival in experimental transplantation (Pratt etal., 2002). The association of C3 polymorphisms with late graft fail-ure confirmed the importance of this finding (Brown et al., 2006)

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lthough these data were negatively re-evaluated in a larger cohortVaragunam et al., 2009). Searching for mechanisms contributingo this C3-dependent effect, the same lab found that APCs arehe source of C3 and that macrophages or DCs lacking C3 haven impaired ability to stimulate alloreactive T cells and to driveh1-biased adaptive immune responses (Zhou et al., 2006; Pengt al., 2006). In a follow up study, Peng et al. were able to assignhe contribution of C3 to the interaction of C3a with the C3aR onCs. They found that DCs not only synthesize C3 but also har-or the machinery to proteolytically cleave C3 into C3a leadingo autocrine C3aR signaling. Taking advantage of C3aR-deficientCs, they showed that such cells suffer from reduced MHC-II, co-

timulatory molecule expression and IL-12 production, which wasssociated with impaired allospecific T cell stimulation (Peng et al.,008). More recently, C3aR signaling in DCs was shown to decreaseAMP levels as one mechanism by which C3a regulates DC activa-ion and T cell responses (Li et al., 2008). In addition to C3a, C5a

ight be important in allograft rejection. Inhibition of the C5aR inurine model of life-supporting renal allotransplantation substan-

ially improved graft survival from 11 d to 12 weeks. In additiono reduced kidney inflammation and apoptosis, the authors foundttenuated priming of alloreactive T cells (Gueler et al., 2008).n support of these findings, pharmacological targeting of C5aRsuring organ preservation was found to improve kidney graft sur-ival (Lewis et al., 2008). Thus, C3aR and C5aR signaling appear toontribute significantly to the innate and adaptive inflammatoryesponse following solid organ transplantation. Given that severalompanies have potent C5aR antagonist in their pipeline; moreork is required in this area to define the role of AT receptors in

llotransplantation in particular with regard to the regulation ofD4+ and CD8+ T cell responses and long-term graft survival.

.2.3. Role of AT receptor signaling in T cell activationA couple of recent joint publications from the labs of Medof

nd Heeger suggest that the AT not only act on APCs but also on Tells. In an allogeneic setting or following antigen stimulation, theyound that upon APC–T cell interaction, T cells as well as APCs startroducing complement factors of the alternative pathway such as

actors B and D as well as C3 (Heeger et al., 2005). Further, theyemonstrated downregulation of the membrane-bound comple-ent regulator CD55 (decay accelerating factor; DAF). Importantly,AF-deficient APCs or T cells increased T cell proliferation and Th1ytokine production in vitro and in vivo. In follow up studies, theyere able to link the decreased expression of CD55 on APCs to

ncreased production of C5a which they found to be critical for dif-erentiation into Th1 effector cells (Lalli et al., 2007) confirming themportance of C5a signaling for Th cell differentiation (Hawlisch etl., 2005; Köhl et al., 2006). Extending their studies, they showedhat cognate APC–T cell interaction promotes C3 and C5 produc-ion from APCs and T cells and subsequent generation of C3a and5a. Further, they not only described C3aR and C5aR expression onCs and naïve, unstimulated CD4+ T cells but also found upreg-lation of both AT receptors upon antigen challenge (Strainic etl., 2008). Mechanistically, AT generation and AT receptor upreg-lation was linked to co-stimulation by B7-CD28 and CD40-CD40Lolecules. Importantly, the authors were able to define autocrine

nd paracrine feedback loops between AT production, AT receptornd co-stimulatory molecule expression suggesting that the ATsnd their receptors on APCs and T cells contribute to the networkf co-stimulation required for optimal T cell activation and differ-ntiation. Further, they showed that naïve T cells already produce
ow amounts of C3a and C5a under steady state conditions leadingo autocrine activation of AT receptors, which seems to promoteustained viability of naïve T cells. The latter finding was recentlyxtended to effector T cells. Here they demonstrated that C5aR sig-aling on effector T cells controls extrinsic and intrinsic pathways
logy 46 (2009) 2753–2766

of apoptosis by regulating T cell expression of the apoptosis regu-lator proteins Fas and Bcl-2 (Lalli et al., 2008). These data suggesta critical role for complement in the maintenance of naïve T cellsand the expansion of effector T cells. Whether this regulatory effecton T cell differentiation, expansion and apoptosis applies to all sub-populations of CD4+ T cell as well as to CD8+ T cells remains to bedetermined in future studies.

3.2.4. C5a and cancerAnother example of C5a-mediated regulation of adaptive immu-

nity has recently been uncovered in tumor immunology. The fateof tumors depends on the balance between innate and adaptiveimmune responses and the potential of the malignant cells toevade immune surveillance. Of particular importance is the inter-play between myeloid-derived suppressor cells (MDSC) and CD8+

cytotoxic T cells. MDSC are a heterogeneous population of regu-lar myeloid cells trapped in intermediate stages of differentiationtoward mononuclear cells such as monocytes, macrophages or DCsor polymorphonular granulocytes (Marigo et al., 2008). Markiewskiet al. found that MDSC express C5aR and that C5a attracts MDSCsto TC-1 tumor cells in vivo. Further, activation of the C5aR resultsin increased production of reactive oxygen and nitrogen speciesfrom MDSC, which are known mediators of MDSC that suppressCD8+ T cell functions. Importantly, in a model of cervical cancer,genetic or pharmacological C5aR targeting resulted in decreasednumbers of MDSCs within the tumor, associated with high numbersof CD8+ effector T cells and decreased tumor growth (Markiewskiet al., 2008). These data underscore the view of C5a as an impor-tant immunoregulatory molecule of adaptive immunity. Of note,C5a may not only be generated in the tumor environment throughtumor sensing by C1q-mediated mechanisms. In fact, C5a can begenerated from C5 by cell-derived proteases (Huber-Lang et al.,2002a), serine proteases of the clotting and the fibrinolysis sys-tem (Huber-Lang et al., 2006; Markiewski and Lambris, 2007) andseveral pathogens (see Section 3.2.5) which fits well with the obser-vation that inflammation and infection can promote and enhancetumor growth. In line with this view, C5a cooperates with pathogen-induced TLR activation to drive IL-6 and IL-1� production frommononuclear cells, which are pro-inflammatory mediators thatalso increase the accumulation and activation of MDSCs in tumors(Ostrand-Rosenberg, 2008).

3.2.5. Cross-talk of AT receptors with TLRs and its impact onadaptive immune responses

The complement system and TLRs are ancient danger sens-ing systems that have co-evolved over hundreds of millions ofyears. As might have been expected, cross-talk between comple-ment proteins and TLRs has been described that suggest rathercomplex mechanisms of pathogen sensing. Soluble complementproteins such as C1q, MBL/ficolins and properdin (Kemper andHourcade, 2008) recognize conserved microbial motifs resultingin the activation of the classical, lectin or the alternative pathwayof complement. Similarly, conserved microbial patterns are rec-ognized by cellular pattern recognition receptors (PRR) includingTLRs. Direct cooperation between the soluble complement-derivedsensing molecule MBL and membrane-bound TLR2 has been foundin S. aureus infection which specifies and amplifies the hostresponse. Importantly, this cooperation takes place in the specialenvironment of the phagosome, suggesting that soluble comple-ment proteins are multifunctional molecules that promote theirantimicrobial defense functions not only in the fluid phase but in
defined cell compartments (Ip et al., 2008). Another example is thecooperation between TLR2 and CR3 in periodontopathic bacterialinfection. Porphyromonas gingivalis exploits the host danger sensingsystem to shut down adaptive immunity allowing the bacterium toenter host cells and survive within phagosomes. P. gingivalis fim-

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riae are recognized by TLR2 which then stimulates in-side-outignaling to promote clustering of CR3, providing a safe and effi-ient entry pathway for the pathogen. This CR3 activation furthereads to suppression of IL-12 production which would otherwiseesult from TLR2 activation of the phagocyte (Hajishengallis et al.,008). Interestingly, P. gingivalis can degrade C5 to release a C5a-

ike fragment which is biologically active (Wingrove et al., 1992).s C5a can also promote clustering of CR3 (Jones et al., 1998),

his may serve as an additional mechanism of how the microbexploits host defense. Further, C5a has been shown to cross-talkith TLR2, TLR4 and TLR9. In peritoneal macrophages, C5a (and

o a lesser extent also C3a) downregulates TLR4-induced produc-ion of IL-12 family cytokines including IL-12, IL-27 and IL-23 byI3K-� and ERK1/2-dependent pathways (Hawlisch et al., 2005).mportantly, this suppressive effect of C5a also applies to CD40-D40L-induced amplification of IL-12 production in macrophages.he in vivo importance of this effect was demonstrated in a modelf cutaneous leishmaniasis. BALB/c mice, which are sensitive to L.ajor infection, become resistant when C5aR signaling is ablated

n C5aR-deficient mice. This protection is associated with increasedroliferation of CD4+ and CD8+ T cells and high IFN-� production.f note, Leishmania major uses a similar entry mechanism as P.ingivalis, i.e. through CR3 and shuts down IL-12 production uponacrophage infection suggesting that this pathway is an Achilles

eel that serves intracellular survival of several pathogens (Lieset al., 2008). In fact activation of CR3 by several ligands has beenhown to shut down IL-12 (Marth and Kelsall, 1997). The negativeegulatory impact of C5a and C3a on TLR-driven IL-12 productionas been confirmed in vivo injecting TLR ligands into mice (Zhangt al., 2007). The bottom line of this study is that the ATs downreg-late IL-12 production but act synergistically with TLRs 2, 4 and 9

n promoting TNF-�, IL-1�, IL-6 and IL-10 production from bloodells.

On the other hand, C5aR blockade has been shown to suppress. aureus-induced production of IL-12 from human monocytes ando enhance the production of IL-10 (Karp et al., 2000) suggesting aynergistic role for C5aR signaling in microbial-derived IL-12 pro-uction. Similarly, mice lacking C3aR and C5aR die rapidly followingoxoplasma gondii infection which is associated with a markedlyeduced ability to mount protective Th1 immunity (Strainic et al.,008). Taken together, these data suggest that the impact of C5aRnd C3aR signaling on microbial-induced production of IL-12 andh1 protective immunity is complex. The emerging model from thevailable data is that C5aR and C3aR signaling in macrophages andonocytes suppresses TLR-induced IL-12 production. In DCs andcells, C3aR and C5aR signaling may act synergistically with TLR

ignaling or activation of other PRR such as NOD-like receptors inase of S. aureus infection. More studies are needed that delineatehe fascinating cross-talk between the distinct AT receptors andRRs in APCs and T cells in infection in particular with regard tontracellular pathogens.

.3. Cross-talk between C5aR and immunoglobulin G receptorFc�R) in autoimmune diseases

Autoimmune diseases are chronic disabling disorders in whichaladaptive, self-directed immune responses drive severe inflam-ation. More than 80 autoimmune diseases have been identified,

ncluding systemic lupus erythematosus (SLE), multiple sclerosis,ype I diabetes and rheumatoid arthritis (RA). Immune complexesICs) are integral to the pathogenesis of several autoimmune dis-
ases, including SLE and RA. They activate the classical and thelternative pathway of the complement system and thus inter-ct with both receptors for Fc of immunoglobulin G (Fc�R) andvariety of complement receptors (Köhl, 2001). When clearanceechanisms are overwhelmed, ICs can become an important cause
logy 46 (2009) 2753–2766 2759

of tissue damage. In such settings, ICs promote pro-inflammatoryprocesses characterized by activation of myeloid cells at sites of ICdeposition.

In mice, four different Fc�Rs have been described, the activat-ing Fc�Rs I, III and IV and the inhibitory Fc�RIIB (Nimmerjahn andRavetch, 2006). IC binding to activating Fc�Rs promotes inflamma-tion through an immunoreceptor tyrosine-based activation motif(ITAM) whereas ligation of the inhibitory Fc�RIIB blocks the inflam-matory response upon co-ligation with activating Fc�Rs through animmunoreceptor tyrosine-based inhibitory motif (ITIM). This cou-pling of activating and inhibitory signals from cellular receptorswhich recognize similar ligands has emerged as a general princi-ple during evolution in which the overall response is determinedby the relative contribution of each signaling pathway (Ravetchand Lanier, 2000). In line with this view, Fc�RIIB deficient micesuffer from augmented inflammation in experimental models of IC-disease, systemic anaphylaxis, and show enhanced IgG-mediatedclearance of pathogens and tumor cells (Takai, 2002).

Several studies of experimental IC-disease have shown thatboth activating Fc�Rs (Sylvestre and Ravetch, 1994), as well asthe C5aR (CD88) (Heller et al., 1999a,b; Baumann et al., 2000,2001), drive effector responses in IC-mediated inflammation andthat cross-regulation exists between the two receptor signalingpathways (Shushakova et al., 2002; Godau et al., 2004). The emerg-ing paradigm is that activating Fc�Rs provide IC-mediated cellulareffector responses, while C5a sets the threshold for Fc�R activationby upregulation of activating Fc�Rs and downregulation of Fc�RIIB.C5aR signaling through G�i2-dependent mechanisms (Skokowa etal., 2005) that activate PI3K-� and � (Konrad et al., 2008) are crit-ical for the regulatory impact of C5a on Fc�R expression. Indeed,contribution of both Fc�Rs and C5aR has been shown in modelsof IC-alveolitis and peritonitis (Baumann et al., 2000; Heller et al.,1999a), rheumatoid arthritis (Ji et al., 2002), autoimmune vitiligo(Trcka et al., 2002), and autoimmune hemolytic anemia (AIHA)(Kumar et al., 2006). Further, a positive amplification loop has beendescribed between Fc�R signaling and the complement system ina model of AIHA in which activating Fc�Rs promote the productionof C5 from tissue macrophages for its subsequent cleavage into C5a(Kumar et al., 2006).

Taken together, these data suggest that the interplay betweenFc�Rs and C5aR contributes significantly to the effector phase ofIC diseases, i.e. the enhancement of Fc�R-mediated clearance ofICs by macrophages and the pro-inflammatory scenario throughthe recruitment and activation of neutrophils. Given that Fc�Rs,in particular Fc�RIIB, contributes to the development of autoim-munity (Takai, 2002), it will be important to delineate in futurestudies whether the regulatory link between C5aR and Fc�RIIB willalso affect afferent immunity and the development of autoimmunediseases such as SLE.

4. Anaphylatoxins and their receptors in CNS

4.1. Evidence for a role for C5a/C5aR in neurodegenerative disease

Degenerative diseases are becoming a larger issue in the modernage in which lifespan has been prolonged. One example of age-related neurodegenerative disorders is Alzheimer’s disease (AD)with over 4.5 million individuals in the US alone afflicted with thedisease, most above the age of 65 (Hebert et al., 2003). Symptomsof the disease include: loss of memory, decreased reasoning andcommunication ability and loss of independence. The characteris-
tic neuropathological changes include synaptic and neuronal loss,extracellular amyloid beta (A�) plaques and neurofibrillary tanglesof hyperphosphorylated tau mainly within the hippocampus andcortex of the brain (Terry et al., 1999; Trojanowski and Lee, 2005;Aizenstein et al., 2008). The observation that individuals who take

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on-steroidal anti-inflammatory drugs (NSAIDs) show decreasedusceptibility to AD onset (Pasinetti, 2002), as well as a plethoraf in vitro data (Bales et al., 2000; Shaftel et al., 2008; Meda etl., 2001; Veerhuis et al., 2003), have led many to speculate thatnflammation may play a substantial role in AD pathogenesis. Inontrast to diffuse plaques seen in brain tissue from non-dementedndividuals, the A� peptide in plaques in AD brain is in a �-sheettructure conformation (can be stained with a dye thioflavine) andas been termed fibrillar in contrast to diffuse. Components ofhe complement system have been observed almost exclusivelyn association with these fibrillar A� plaques (Afagh et al., 1996;oeffler et al., 2008). Some positive complement immunostain-ng has been shown in thioflavine-negative, cognitively normalrains, but to a far lesser extent (Lue et al., 2001). In vitro stud-

es have shown that these �-sheet rich A� assemblies activate bothhe classical and alternative complement pathways (Rogers et al.,992; Jiang et al., 1994; Bradt et al., 1998; Watson et al., 1997),hus generating complement activation products, including C3b,3a and C5a. Prolonged complement activation triggered by fib-illar A� plaques or activation in the absence of proper regulationay contribute to many of the manifestations present in the dis-

ase (Cotman et al., 1996) due to C5a-recruited and -activated gliahat promote inflammatory events (Akiyama et al., 2000; Grammasnd Ovase, 2001; Yao et al., 1990; O’Barr and Cooper, 2000). Fur-hermore, studies have suggested that C5aR activation may benvolved in neuronal apoptosis (Farkas et al., 2003) and decreasedell viability (Humayun et al., 2009). Similar scenarios may applyo Parkinson disease (McGeer and McGeer, 2004), Huntington’s dis-ase (Singhrao et al., 1999) and age-related macular degenerationAMD) (Hageman et al., 2005). For example, complement com-onents, glia and complement AT receptor mRNAs are stronglyxpressed in caudate in Huntington’s disease. Complement com-onents are found in drusen in AMD, and over 70% of the risk ofMD can be attributed to polymorphisms predominantly in thendogenous complement pathway regulator, Factor H, with someontributions from polymorphisms in C2, C3, and Factor B (Maller etl., 2007; Jakobsdottir et al., 2008; Spencer et al., 2008, 2007). Com-lement activation is strongly implicated in cerebral ischemia andemorrhagic stroke, although the precise contributions and mech-nisms of complement-dependent neuronal degeneration are yeto be unequivocally defined. The evidence for, and consequencesf, C3a and C5a generated as a result of this injury has recentlyeen thoroughly reviewed by Arumugan et al. and thus will not beiscussed here further (Arumugam et al., 2009).

The role of the complement system in neurodegenerative dis-ase progression appears to be a complex one, with evidenceemonstrating both complement-dependent detrimental effectsnd protective effects. Indeed, C3a and C5a are generated duringany/most pathological events in the CNS (Bonifati and Kishore,

007). A vital tool in exploring the possible mechanisms and over-ll contribution of complement initiated pathogenesis has beenhe use of animal models of disease. Murine models are particu-arly important as mice genetically deficient in specific complementomponents such as C1q, C3, C5, and Factor B are available for usen dissecting the role of complement in general and/or the functionf the individual component. In addition, novel approaches such asectopic” expression of C3a or C5a may also prove useful in validat-ng the direct role of these mediators in disease (Boos et al., 2004).ince it has been demonstrated that receptors for C5a are expressedn the brain (Gasque et al., 1997, 1998; Singhrao et al., 1999; O’Barr etl., 2001; Nataf et al., 1999b) and that CNS cells do respond to C5a
Sayah et al., 2003), the complement activation product C5a haseen the focus of several recent CNS studies. For example, a lackf C5/C5a rendered mice resistant to cerebral malaria (Patel et al.,008), and significantly reduced neutrophil recruitment and CNSamage in a traumatic brain injury model (Sewell et al., 2004), sup-
logy 46 (2009) 2753–2766

porting the hypothesis that C5a can exacerbate inflammation in theCNS, with involvement of either mononuclear phagocytes or infil-trating neutrophils. Interestingly, C3a but not C5a, had a dominanteffect in accelerating experimental autoimmune encephalomyelitis(EAE) (Boos et al., 2004).

Mouse models expressing human mutated forms of the amyloidprecursor protein (APP) have been shown to develop age-relatedaccumulation of A� in the brain, and have been reported tomimic certain other features associated with human age-related ADpathology, including the association of complement components,such as C1q, C3 and C4 (Fonseca et al., 2004; Zhou et al., 2005, 2008),as well as reactive/inflammatory astrocytes and microglia, with thefibrillar A� plaques. Treatment of one AD mouse model (APP23)with a low molecular weight heparin, enoxaparin, which is aninhibitor of A� mediated classical complement activation reducedA� load and astrocyte activation (Bergamaschini et al., 2004), thussuggesting complement inhibition may result in reduced AD asso-ciated pathology. Further and more direct evidence for complementinvolvement in AD pathogenesis was demonstrated by crossingan AD mouse model (Tg2576) to a C1q knock out to generate anAD mouse lacking the ability to activate the classical complementpathway. These C1q-deficient mice had significant reductions inglial activation as well as increased neuronal integrity in contrastto C1q-sufficient Tg2576 mice (Fonseca et al., 2004). Interestingly,however, the genetic deficiency of C5 has been shown to be oneof a limited number of genetic differences that are associated withdecreased amyloid deposition in DBA/2J mice vs. C57Bl6 mice trans-genic for the human APP gene (Ryman et al., 2008). As is the case forperipheral monocytes, C5a has been shown to be chemotactic formicroglia and astrocytes (Miller and Stella, 2009; Yao et al., 1990),and thus in AD may provide a significant signal for glial recruitmentto the plaque vicinity. In addition, C5a can synergize with otherligands to induce pro-inflammatory cytokine and/or chemokineproduction (O’Barr and Cooper, 2000), and thus can contribute toa secondary/synergistic inflammatory reaction to the plaques, par-ticularly since fibrillar amyloid may bind TLR2 and TLR4 (Jana etal., 2008; Lotz et al., 2005) and TLR and C5aR have been shown tosynergize in other tissues (Zhang et al., 2007; Hawlisch and Köhl,2006). This exacerbated inflammatory nidus likely contributes toneurotoxicity and the subsequent cognitive loss symptomatic of AD.

Since many of the detrimental effects of complement can resultfrom the influx and activation of inflammatory myeloid-derivedcells (neutrophils, macrophages, microglia) or synergistic signal-ing with other receptors, such as P2Y6 (Flaherty et al., 2008)or TLRs (Zhang et al., 2007; Hawlisch et al., 2005), develop-ing inhibitors of C5a activation of myeloid cells and/or receptorantagonists of C5a has been targeted as major mechanism forinhibiting acute C5a-induced inflammatory disorders as well aschronic debilitating disorders (Konteatis et al., 1994). The C5areceptor antagonist PMX-205, a derivative of PMX-53 (Köhl, 2006a),which is an orally active CD88-specific receptor antagonist, hasbeen shown to reduce disease activity in several animal models,including models of CNS disease such as brain trauma (Sewell et al.,2004), Amyotrophic Lateral Sclerosis (ALS) (Woodruff et al., 2008)and Huntington-like neurodegeneration (Woodruff et al., 2006).Mouse models of AD, treated with PMX-205, have also shown sig-nificant decreases in both fibrillar A� and inflammatory glia incortex and hippocampus (Fonseca et al., 2009). These reductionsin pathology were correlated with improvements in cognitive per-formance. These observations provide experimental evidence fora role for C5aR/CD88 in neurodegenerative disease progression.
In addition, the results suggest that blockade of a receptor forcomplement-mediated inflammation reduces neuroinflammationand behavioral deficits, and may serve as a potent therapeutic targetfor those afflicted with AD and other neurodegenerative diseasesthat are accelerated by complement mediated inflammation.

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.2. Neurogenesis and development

Interestingly, seemingly contradictory results were reportedn other studies of the influence of the complement component5 on inflammation. C5a, when given with kainic acid intra-entricularly or 24 h prior to glutamate treatment in neuronalouse cultures, was shown to be neuroprotective against gluta-ate mediated caspase-3 activation (Osaka et al., 1999). It was

ubsequently hypothesized that the C5a-mediated protection maye dependent on the modulation of Ca2+ and MAP-kinase activ-

ty (Mukherjee and Pasinetti, 2000, 2001). In other systems, C5a,s well as C3a, provided direct neuroprotection (Van Beek et al.,001; Mukherjee and Pasinetti, 2001; O’Barr and Cooper, 2000);owever, these were cell lines and/or neurons perhaps at differ-nt stages of maturation which may align with the studies ofontaine and co-workers in newborn rat brain. These researchersemonstrate that in the developing cerebellar cortex brain, C5aRtimulation triggered increased BrdU incorporation by granule neu-ons and a C3aR agonist promoted migration of cells to their properocation (Jauneau et al., 2006; Benard et al., 2004, 2008). How-ver, since defects in cerebellum have not been reported in C3-,3aR-, C5- or C5aR-deficient animals, further studies will be nec-ssary to determine if these systems are redundant, residual ornvolved in facilitating survival and development during infec-ion. The underlying basis for the differences in outcome due to5a/C3a engagement of their receptors is likely different differen-iation states of the cells and/or the cell signaling resulting from

ixed cell interaction. Another example of the complexity of theseesponses is the report that C5a (but not C3a) drives up expres-ion of microglial (but not astrocyte) glutamate receptor (GLT-1)hich should provide increased glutamate uptake and thus protecteurons in the environment against glutamate toxicity (Humayunt al., 2009). Since the two C5aR (CD88 and C5L2) may “cooper-te” with other receptors (see Sections 3.2.5 and 3.3), it is possiblehat a diverse, but precise set of responses to a changing envi-onment could be orchestrated depending on the repertoire ofnteracting receptors available in the sensing cell. Clearly a “sys-ems” approach to these responses will facilitate the clarification ofhese pathways and identification of targets for therapeutic inter-entions.

The ATs have been shown to be important for both hepato-yte proliferation and regeneration (Reca et al., 2003; Daveau et al.,004; Mastellos et al., 2001; Markiewski et al., 2004). Recent reports

n addition to those mentioned above involving development of raterebellum, have implicated C3a (and possibly C5a) in neural stemell regeneration and directed migration of stem cells. After demon-trating that both clonally derived rat hippocampal neural stemells and murine neural progenitor cells expressed receptors for3a and C5a, mice lacking C3, C3aR, or had C3aR activity inhibitedith an antagonist, showed reductions in migrating neuroblasts

nd newly formed neurons in areas of basal adult neurogenesisRahpeymai et al., 2006). A similar reduction in neuroblast migra-ion and number of newly formed neurons was observed afterschemic injury in the deficient or C3aR-inhibited mice. Althoughheir study did not provide a mechanism behind the C3a receptorctivation that would lead to the creation of new neurons nor aource for the generation of C3a or C5a (their results did not ruleut a contribution of C5a), their results do provide evidence thatomplement components can play a role in neurogenesis. Inter-stingly, it has been reported that C3a induces or synergizes withL-1� to enhance NGF expression (Heese et al., 1998; Jauneau et
l., 2006) in human microglial cell cultures, which implicates theossible involvement/cross-talk between these two systems. Takenogether, it will be important to try to preserve and/or enhancehe potential protective/reparative effects of specific ATs in the CNShen designing therapeutic targets.
logy 46 (2009) 2753–2766 2761

5. Conclusions

Since the discovery of the ATs almost 40 years ago andtheir respective receptors 10–15 years ago, our view of the ATsas mere pro-inflammatory mediators generated in response tocomplement activation has changed. Today we consider ATs asimmunoregulatory molecules with pleiotropic biologic functions,the development of which can be complement system-dependentor independent through serine proteases of different origins. Exceptthe ancient antimicrobial activity and the metabolic activity ofC3a/C3adesArg, ATs exert their biologic activities through ligationof their cognate receptors, belonging to the most abundant recep-tor family in vertebrates, i.e. the GPCRs. Depending on the localenvironment and the circumstances under which ATs are gen-erated, they can contribute to cell and tissue homeostasis, thebenefit or burden of inflammation as well as to tissue regenera-tion or fibrosis. Based on the strong pro-inflammatory properties,C5a and the C5aR in particular have been considered attractivepharmacological targets. Several companies have C5aR antagonistsin their pipelines to attenuate inflammation in allergic asthma,ischemia/reperfusion injury, rheumatoid arthritis or age-relatedmacular degeneration. While C5aR targeting appears an attractiveapproach in acute inflammation, long-term approaches should con-sider direct or indirect effects of AT receptor signaling on adaptiveimmune responses, cell apoptosis and tissue regeneration/fibrosisthat may impact on such treatments.

Acknowledgements

The authors thank the following institutions for providingsupport for the work cited in this review: National Instituteof Allergy and Infectious Diseases (NIAID) grants AI059305 andAI057839 to JK. NS-35144 (AJT), P50 – AG 00538 (AJT), NIH Train-ing Grant NS-007444 (RRA). DFG grant of the SFB 566 (AK). TheMinistry of Science and Culture of Lower Saxony through a Georg-Christoph-Lichtenberg Scholarship (K-OJ). We thank Una Dohertyfor proofreading and excellent assistance in the preparation of themanuscript.

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