Cover Page. Author: Kotimaa, Juha Title: Analysis of systemic complement in experimental renal injury and disease Issue Date:

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1 Cover Page The handle holds various files of this Leiden University dissertation Author: Kotimaa, Juha Title: Analysis of systemic complement in experimental renal injury and disease Issue Date:

2 chapter 1 8

3 1 Chapter 1 General introduction Adapted from Croonian Lecture On Immunity with Special Reference to Cell Life, Paul Erlich read 22 March Proceedings of the Royal Society, January 1899, London 9

4 chapter 1 contents 1. Introduction A Brief history of complement discoveries The complement system Complement pathways The classical pathway The lectin pathway The alternative pathway Bypass pathways Terminal pathway Regulators of complement activation Effectors of complement activation Biosynthesis Complement in disease and injury Complement in renal disease Complement and renal ischemia/reperfusion injury Therapeutic inhibition of complement Complement diagnostics and detection methods Scope of the thesis

5 1 1. INTRODUCTION Chronic kidney disease (CKD) is a progressive disease with prevalence of 11% in United States and Europe [1]. The severity of CKD correlates well with the probability of end stage renal disease (ESRD), where renal function is lost and the patients require renal replacement therapy (RRT) of either dialysis or transplantation [2]. CKD results in ESRD in 8 68/ patients per year depending on care level and ethnic background [1]. The underlying causes for CKD are numerous, including diabetes mellitus, hypertension, and glomerulonephritis which can be caused by exposure to toxic chemicals, infection, autoimmune diseases, or genetic abnormalities. The complement system has been shown to contribute, or in some cases be the primary cause of renal injury and disease. This is true for some autoimmune diseases, glomerulonephritides and for the inevitable ischemia/reperfusion injury (I/RI) during organ transplantation. Experimental animal models have been used extensively for complement research, revealing intriguingly diverse modes of activation and mechanisms of injury. Genetically engineered or natural complement-deficient rodents have been central in studying the contribution of complement in experimental disease models and for studying therapeutic intervention of complement activation. However, unlike for humans, only few standardised or practical methods are available for studying the systemic serum complement in rodents. This thesis is focused around development of advanced methodologies for analysis of complement system in preclinical mouse and rat models of renal disease and injury. In this introductory chapter basic concepts of complement are introduced, the role of the complement system in several renal diseases is discussed and the current status of methods of complement measurement is presented. 11

6 chapter 1 2. A BRIEF HISTORY OF COMPLEMENT DISCOVERIES The complement system was described more than a century ago by Hans Büchner and his colleagues who described a serum based system which could kill bacteria independent of phagocytes [3, 4]. Soon after, Belgian scientist Jules Bordet described that the bactericidal system consisted of two components: a heat-stable factor which sensitised bacteria towards killing, and a heat-labile factor which caused bacteriolysis [5]. Paul Ehrlich renamed the Alexin system, a term coined by Büchner and Bordet, as the complement system and integrated it as part of his broader theory on humoral immunity which described how antibody specificity directs the complement system to lyse bacteria [6, 7]. During the decades to follow, seminal complementologists, such as Louis Pillemer and Hans Müller-Eberhard, identified the key factors involved in the classical [8, 9], alternative [10], and terminal pathway of complement [11 14]. The last two decades of complement research have been exceptionally productive. The field has revisited old paradigms, expanded the understanding of the molecular mechanisms of complement activation, and broadened the scope of complement involvement in health and disease. For example the alternative pathway regulator properdin was found already in 1954 by Louis Pillemer who proposed that properdin could specifically target and initiate alternative pathway (AP). However, it was not until 2007 that the first definitive proof of properdin directed activation of complement was shown [15, 16]. A whole new area of complement research was initiated through discovery of a new pathway of complement activation, the lectin pathway (LP). Although it was originally identified in 1976 from children who could not effectively opsonise yeast and had recurrent pyogenic, or pus forming, infections [17], it was not until discovery of mannan binding lectin (MBL) [18] and MBL-associated serine proteases (MASPs) that definitive proof of LP was established [19, 20]. Further studies on LP-mediated complement activation identified three 12

7 1 other initiators called ficolins, that each interact with unique sugar moieties and activate complement via MASPs [21 24]. In recent decades it has become evident that the complement system is more dynamic and multifaceted than previously postulated. Although the liver is the most prominent source of most complement factors in circulation, it is now understood that most tissues and inflammatory cells, such as neutrophils and macrophages, are potent sources of complement factors. Upon activation both cells in peripheral tissues as well as immune cells can produce complement proteins by secreting them either locally or into circulation [25 29]. Furthermore, the auxiliary role of complement has been expanded through recent studies, showing that anaphylatoxin-mediated signalling and complement deposition on the activating surface are essential for mounting adaptive immune responses [30 32]. 3. THE COMPLEMENT SYSTEM The complement system comprises of more than 30 abundant soluble and integral membrane proteins which can be classified as: initiation factors, complement cascade factors, regulators of complement activation (RCAs), and soluble or cell surface complement effectors. The complement system consists of three major pathways: antibody activated classical pathway (CP), lectin pathway (LP), which activates predominantly on sugar moieties, and alternative pathway (AP) which can activate on unprotected surfaces and amplifies complement activation of other pathways. Activation pathways converge at the level of C3 to activate C5 and initiate the terminal pathway (TP) which conveys some of the main effector functions of the complement system, including formation of anaphylatoxin C5a and lytic terminal complement complex (TCC, C5b-9), also known as membrane attack complex (MAC) [33, 34]. 13

8 chapter 1 Figure 1. Central pathways of the complement system. Classical (CP) and Lectin (LP) share a C4/C2 dependent mode of C3, and later, C5 activation. Alternative pathway (AP) operates either through autoactivation of C3, or specific fixation of C3 through properdin. CP and LP mediated activation is further amplified as activated C3 can trigger AP activation. All three pathways converge at the level of C5 activation and initiation of terminal pathway (TP) which results in generation of major complement effectors C5b-9 and C5a. 14

9 1 Although initially focussed on pathogen recognition, it has become clear that the initiation factors of the complement system also recognise a wide variety of modified-self molecular patterns. The non-self-structures on pathogens are also known as pathogen-associated molecular patterns (PAMPs), whereas the modified-self belongs to the family of danger-associated molecular patterns (DAMPs). Initiation of complement cascades via DAMPs, PAMPs, or antibodies is essential for the disposal and killing of infectious pathogens, development of humoral immunity, homeostatic clearance of damaged cells or immune complexes, and orchestration of the cell mediated inflammatory response [35 38]. Clinical studies on complement deficiency or dysregulation results in complement mediated disease, infections, autoimmune disease, or exacerbation of underlying injury and disease, as is the case in sepsis and ischemia/reperfusion injury (Table 4.). Together these studies have established that the complement system has a multifaceted role in health, injury, and disease [39, 40]. 4. COMPLEMENT PATHWAYS 4.1. the classical pathway The classical pathway (CP) is phylogenetically the most recent component of the complement system, arisen in tandem with antibodies [41]. The CP links and augments antibody mediated immunity through binding of C1-complex to naturally pentameric IgM or membrane bound hexamerised IgGs to activate the complement system [42, 43]. The C1 complex can also bind a wealth of other targets, such as different phospholipids, including phosphatidyl serine (PS), which are present on apoptotic cell surfaces, pentraxin 3 (PTX3), C-reactive protein (CRP), and serum amyloid-p (SAP) [44 47]. CP activation has been described in detail in Figure 2 and 3, and the main components in Table 1. 15

10 chapter 1 Figure 2. Complement activation pathways. The complement system can be initiated through Classical (red), Lectin (yellow) and Alternative (blue) pathways. CP and LP converge at the level of C4/C2 activation (orange) forming C3-convertase C4bC2a. AP can activate spontaneously via C3 hydrolysis (tickover), through properdin fixation and via CP/LP C3 convertase (amplification loop), all incorporating Bb with C3b to form AP C3 convertase. All three pathways continue to form C5 convertases through cleavage of C3 and incorporation of C3b to form C5-convertases, and subsequent activation of the Terminal pathway (purple). Main effectors of complement activation are depicted in blue boxes and include anaphylatoxins C3a and C5a, as well as terminal pathway activation products C5b-9 and soluble C5b-9. 16

11 1 The C1q is a protein complex formed from 18 peptide chains arranged into three hexameric subunits which have pairs of peptide chains encoded by C1QA, C1QB, and C1QC genes. The functional C1 complex is comprised of one C1q complex with two pairs of C1r and C1s serine proteases (C1qr 2 s 2 ) [48]. The C1 complex has three distinct functions: 1) the six globular heads have calcium dependent binding capacity towards a number of targets including multimeric immunoglobulins, 2) the collagenous stalk of C1q promotes phagocytosis [49], and 3) the C1 complex can cleave C4 to C4b and C2 to C2a initiating the complement cascade via the classical pathway [50]. The cleavage of C4 results in the potent opsonin C4b, which will be covalently bound to the activation site. The C4b can bind the C2a fragment, forming the Mg 2+ -dependent CP/ LP C3-convertase [19]. The CP/LP C3-convertase binds C3 to cleave it to C3b and C3a. C3a is a weak anaphylatoxin, whereas C3b becomes covalently bound to the activating surface and serves both as an opsonin and as an initiation factor for the terminal pathway (TP) through the formation of CP/ LP C5-convertase (C4bC2aC3b) [33]. Genetic abnormalities within the CP underline the diverse roles of classical pathway and its importance for the normal function of immune system. Impaired classical pathway activation predisposes to recurrent infections, especially by capsulated bacteria such as H. inluenza and N. meningides, which is analogous to hypo- and dysgammaglobulinemias where the complement system is intact but antibody levels are low or non-existent [39]. Lack of C1q, C1r, C1s, low copy number or complete deficiency of C4 all predispose to autoimmune diseases, such as systemic lupus erythematosus (SLE) [39, 51 53]. Although complete deficiency of C2 does exhibit similar symptoms, they are generally less severe [54]. The mechanism of CP-specific deficiency and autoimmune disease has been attributed to impaired homeostatic clearance of apoptotic bodies and immune complexes from the body [55]. 17

12 chapter 1 pattern recognition Abbreviation, Factor Ligands alternative name C1q IgG, IgM CP Mannan binding Lectin MBL IgA, carbohydrate moieties LP Ficolin-1 FCN1, M-Ficolin carbohydrate moieties LP Ficolin-2 FCN2, L-Ficolin carbohydrate moieties LP Ficolin-3 FCN3, H-Ficolin carbohydrate moieties LP Properdin fp LPS, DAMPs, PAMPs AP complement serine proteases Abbreviation, Factor Role alternative name C1r C1 complex, cleaves C1s CP C1s C1 complex, cleaves C4 and C2 CP MASP-1 MASP-2 MASP-3 MBL-associated serine protease 1 MBL-associated serine protease 2 MBL-associated serine protease 3 Associates with MBL and FCN1-3, cleaves MASP- 2, C3, pro-fd Associates with MBL and FCN1-3, cleaves C2 and C4 Unknown C2 Source of C2a, cleaves C3/C5 with the help of C4b CP/LP fb CFB Bb cleaves C3/C5 AP fd CFD Cleaves C3bB to C3bBb, not consumed AP fi CFI Cleaves and inactivates C3b and C4b AP LP LP LP complement cascade components Factor Abbreviation, alternative name C3 Complement component 3 C4 Complement component 4 C5 Hc, haemolytic complement, complement component 5 Role Part of C5 convertase, can autoactivate AP through ticover, source for opsonin C3b, anaphylatoxin C3a and Cr1/Cr2 ligands (ic3b, C3c, C3d, C3dg) Part of CP/LP C3 and C5 convertases, source of opsonin C4b and anaphylatoxin C4a Source of anaphylatoxin C5a, initiator of C5b-9 complex CP/ LP/ AP CP/LP TP C6 Complement component 6 Part of C5b-9 complex TP C7 Complement component 7 Part of C5b-9 complex TP C8 Complement component 8 Insertion of C5b-9 to lipid bilayer TP C9 Complement component 9 C5b-9 pore formation through polymerisation TP Table 1. Central components of the complement system. Complement cascades are usually initiated through specific pattern recognition molecules. Serine proteases initiate and mediate the activation of downstream complement cascade 18 and are responsible for initiation of terminal pathway (TP). Cleavage of C5 by C5-convertases is the final serine protease mediated even in complement cascade, and results in assembly of the C5b-9 complex.

13 the lectin pathway The lectin pathway (LP) is initiated through calcium-dependent binding of oligomeric pattern recognition protein complexes and cleavage of C4 by complex-associated serine proteases. There are four known LP specific initiation complexes: mannose-binding lectin (MBL), ficolin-m (FCN1), ficolin-l (FCN2), and ficolin-h (FCN3). Complement activation is mediated by MBL associated serine proteases (MASPs) which facilitate the cleavage of C4 and C2 into C4b and C2a that form the Mg 2+ dependent CP/LP C3-convertase [19, 56]. MBL shares structural similarity with C1q, although MBL is formed from trimers of identical peptides, which can form higher form oligomers. MBL has a collagenous stalk and, depending on the oligomerisation, 6 18 globular domains responsible for the binding specificity. Main targets of MBL include terminal sugar residues including mannose, glucose, N-acetylglucosamine, and N- acetylmannosamine. MBL has a very low affinity for monosaccharides, but due to multiple lectin binding domains in one functional molecule, it has high avidity for pathogen-associated molecular patterns [57]. Total absence or deficiency of MBL has not been reported, but a number of MBL mutations affecting both promoter and the encoding gene are known. These result in highly variable serum concentration of MBL, from low nanogram range to mid-microgram range [58]. Mannan is a central component of fungal cell walls, therefore it is not surprising that low MBL serum concentration is a risk factor for fungal infections [59]. Insufficient serum MBL predisposes to meningococcal infections, although the effect is observed only with young children. This would suggest that MBL has a protective role against some pathogens before maturation of adaptive immune responses [20, 60]. Ficolins and MBL share a high degree of sequence and structural homology, and all three ficolins have MASP-mediated lectin pathway activation capability analogous to MBL [21, 61, 62]. 19

14 chapter 1 Figure 3. Classical and Lectin pathways in detail. Both pathways are initiated via specific pattern recognition molecules: C1q for CP and MBL or ficolins for LP. The pattern recognition molecules acquire specific serine proteases C1s and C1r for CP, and MASP1-3 for LP. When the initiation complex binds to its target surface, the proteases facilitate the activation of C4 and C2 to form the CP/LP C3 convertase C4bC2a and C5 convertase C4bC2aC3b. The activation of C3 feeds also to AP amplification loop, through binding of fb. Ficolins have a broad ligand repertoire; importantly they have affinity for different acetylated sugars predominantly expressed on invasive pathogens such as Group B streptococci and S. aureus [23, 61, 63]. The MBL-associated serine proteases MASP-1, MASP-2 and MASP-3 have a similar role for LP mediated complement activation as the C1 associate proteases C1r and C1s. 20

15 1 MASP-3 does not have known ligands in humans, but it may have a modulatory function as it is usually associated with higher oligomer forms of MBL together with MASP-2, effectively downregulating the complex activity [64, 65]. In vitro analysis suggests that MASP-2 alone is sufficient for the LP mediated activation of complement [66], whereas MASP-1 has an auxiliary role in C2 activation. In vivo evidence from MASP1/3 deficient patient sera contradicts the in vitro results showing clearly that MASP-1 is indeed essential for LP C4- activation through activation of MASP-2 and C2 in vivo [67]. Furthermore evidence from MASP1/3 knockout mice has showed that MASP1/3 activate MASP2 in analogy to C1r and C1s. Interestingly, it seems that MASP1/3 are also essential in activating profactor D and for normal function of AP in mice, but not in humans [67, 68]. Potential targets for MASP-1 include fibrinogen and factor XIII, which could potentially link the antimicrobial activity of LP to the coagulation system [69] the alternative pathway The alternative pathway (AP) is phylogenetically the most primitive component of the complement system and central in the overall function of the complement system [41, 70]. Schematic representation of the AP has been described in Figure 4 and the main components in Table 1. There are three major mechanisms that can trigger AP: autoactivation of C3 to C3(H 2 O) (C3i), properdin mediated activation, and amplification of CP- and LP-mediated activation. Complement factor C3 is an inherently labile protein which can spontaneously undergo hydrolysis or a tickover of a thioester bond, followed by a conformational change and formation of a metastable activated C3i or C3 (H 2 O). Formation of AP C3 convertase is similarly Mg 2+ dependent as with CP and LP C3 convertases [71]. The C3(H 2 O) and factor B (fb) bind together with Mg 2+, and fb is cleaved and activated by factor D (fd) to form short-lived, minor AP C3 convertase C3(H 2 O)Bb [72]. The minor convertase cleaves C3 to C3a and C3b, which in turn can covalently bind activation 21

16 chapter 1 surfaces and form more stable major AP C3 convertase C3bBb. Properdin is a unique regulator of complement activation through its ability to stabilise and increase the half-life of the AP C3 convertase up to 5-10 fold through forming C3bBbP complex [73]. Figure 4. Alternative pathway in detail. The AP can be activated through three known mechanisms: First, autoactivation, or tickover, where an internal thiosulphide bridge is hydrolysed and fb can bind to form a metastable AP C3 convertase. Second, properdin can bind specific ligands and then recruit C3 and fix a C3 convertase at the binding site. It can also associate with C3 convertase independent of ligand binding extending the half-life of the convertase significantly. Third, the C3 activation via CP/LP results in generation of C3b, which can acquire fb and feed into the AP amplification loop. 22

17 1 It has been shown that properdin can independently initiate the AP. Properdin has separate domains for pattern recognition and C3 binding, allowing it to bind diverse range of targets, including zymosan and bacteria, while retaining the AP C3 convertase stabilisation capability [15]. The AP is central in amplifying CP and LP mediated complement activation through recruitment of fb to the C3b fragments generated by CP/LP C3 convertase. Indeed, it has been suggested that up to 80% of overall activation of CP may originate from AP mediated amplification loop [74] bypass pathways The first non-canonical, or bypass, pathway was found with C4-deficient guinea pig serum, which contrary to expectations presented low levels of erythrocyte lysing capability. The bypass mechanism requires C1, but not C4 or C2, and proceeds through the AP to the terminal pathway [75 77]. Similarly, a MBL-AP bypass pathway has been identified which activates complement independent of C4, C2, and MASP2 [78]. It has been hypothesised that C3b association with C1q and MBL allows evasion from AP regulators fi and fh, in analogy to IgG-C3b and C4b-C3b [79 81]. The in vivo mechanisms of LP C4 bypass are still under research, with proposed contribution of MASP-2, MASP1/3, and C2 [82]. There are two reported mechanisms for C2 bypass. Both are based on C4b and C3 interaction. C4b can form a hybrid classical/alternative C3-convertase (C4bC3bBb) [83] or alternatively fix C3bBbP AP C3-convertase on tissues [84, 85]. Both C2 bypass mechanisms can potentially explain why C2 deficiency results in milder symptoms than C1 and C4 deficiencies [40]. Finally, there are two mechanisms which can bypass C3 in the activation of complement. Inducible serine proteases from neutrophils and macrophages can cleave C5 to C5a and C5b, bypassing the need of local C3 [86]. However, 23

18 chapter 1 Figure 5. The bypass mechanisms of complement system activation. A) C2 bypass occurs in both CP and LP pathways after C4b forms a hybrid C3 convertase with C3b and fb (C4bC3bBb). MASP-2 may bypass C4 and C2 altogether by cleaving C3 directly, initiating AP. B) When C1q and MBL are deposited on surfaces, C3 may autoactivate on them and subsequently avoid inhibition by fh and fi, effectively augmenting CP and LP mediated activation. C) Certain cells such as macrophages and polymorphonuclear cells (PMNs), such as neutrophils, have C5 serine proteases on their surface. These cleave surrounding C5, amplifying C5a generation locally. D) Last, upon activation of coagulation system several coagulation system factors, such as thrombin and plasmin, have been shown to cross-activate complement factors at the level of C3 and C5 activation, effectively linking these two systems together. 24

19 1 it is not clear whether this activation of C5 alone is sufficient to activate the terminal pathway cascade and induce C5b-9 formation. The coagulation system, through similarities of the respective serine proteases of complement and coagulation, forms the second C3-bypass pathway. Recent studies have shown that most central coagulation factors such as thrombin, plasmin, FIXa, FXa, and FXIa cleave C3 and C5, promoting AP mediated activation of complement and generation of C5a promoting localised inflammation [87 89] terminal pathway Activation of the terminal pathway (TP) results in formation of C5a and C5b- 9. C5a is a potent anaphylatoxin, which recruits inflammatory cells to the site of activation and modulates local inflammation. The terminal complement complex (TCC, membrane attack complex, MAC), or C5b-9, is a poreforming complex that can directly lyse microbes and cells lacking sufficient ability to modulate their intracellular osmotic pressure. The C5 convertase initiates the terminal pathway activation by association of C3b to the CP/LP and AP C3-convertases to form C4bC2aC3b and C3bBb(P)C3b. The cleavage of C5 to C5a and C5b induces a conformational shift in C5b allowing binding of C6 [90], which then allows sequential binding of C7, C8, and C9. Each terminal pathway component undergoes a conformational shift upon binding allowing the binding of the next factor, culminating in C9 polymerisation and lytic pore formation [91, 92]. Association of C7 to C5bC6 results in a lipophilic complex allowing weak interaction with surfaces. C8 is the first MAC component to breach cell membranes after incorporating C5b-C7, resulting in weak lytic complex [93, 94]. The binding of C8 to C5b-7 provides an oligomerisation site for up to 18 C9 proteins which form a membrane spanning pore capable of lysis of cells [92, 95, 96]. 25

20 chapter 1 5. REGULATORS OF COMPLEMENT ACTIVATION The regulators of complement activation (RCAs) or complement control proteins (CCPs) are soluble, membrane bound or transmembrane proteins which modulate complement activation. RCAs are essential in limiting the complement activation on healthy host cells and in directing the activation on damaged host cells or unprotected pathogen surfaces. List of known RCAs have been compiled in Table 2 and their targets in the complement system in Figure 6. Figure 6. Regulators of complement activation. Schematic presentation of key complement regulators and their targets in the complement system. 26

21 1 CP and LP regulators include C1-inhibitor (C1INH) and C4b-binding protein (C4BP). Although decay accelerating factor (DAF, CD55) and factor I are predominantly AP inhibitors, they also have specificity for C4b and can contribute to CP and LP regulation. C1INH is a multifunctional serpin family protease inhibitor that can irreversibly bind and deactivate C1r, C1s, and MASPs [97, 98]. C1-INH deficiency or dysfunction results in hereditary angioedema (HAE), which is not strictly a complement-mediated disease. HAE episodes are characterised by marked decrease in key complement components in serum, but the extensi ve swelling is a result of unchecked activation of plasma kallikrein and subsequent activation of bradykinin. Although the complement system is impacted by HAE episodes, the loss of C1 control results invariably to consumption of key complement factors C3 and C4 which can predispose to SLE due to hypocomplementemia and aggravate acute HAE episode [99]. C4b-binding protein (C4BP) is the second major regulator of CP and LP, with no known deficiencies. C4BP has a natural binding capacity for apoptotic cells, together with protein S, CRP, and DNA, and a central role in facilitating clearance of damaged tissues. C4BP has decay acceleration activity towards the C4bC2a C3-convertase, thus limiting the activation of both classical and lectin pathways [100]. Most importantly it facilitates the factor I-mediated inactivation of C3b and C4b as an essential cofactor [101]. The AP can activate independently, but is essential also for amplification of CP and LP. Therefore AP RCAs are central in modulating complement activation in vivo. Factor H (fh) is a multifunctional soluble serum AP RCA which can bind to host cell surfaces through glycosaminoglycans. It inhibits alternative pathway activation through cofactor activity with factor I (fi) and has a decay accelerating activity specific for AP C3-convertase C3bBb [102]. Factor I facilitates the cleavage and deactivation of cell surface C3b into ic3b, C3c, C3d, and C3dg, with similar activity towards C4b [103]. Cleavage of C3b to ic3b inhibits binding of fb and therefore formation of AP C3-convertase 27

22 chapter 1 and AP-amplification. Factor I can use number of other regulatory proteins as cofactors to enable cleavage of C3b and C4b into inactive forms [101]. These include DAF and fh which facilitate processing ic3b further to soluble C3c and membrane bound C3d and C3dg [91]. Deficiency, unnatural regulation, or acquired impairment of the AP RCAs invariably results in injurous (auto-) activation of the complement system and severe diseases including atypical haemolytic uremic syndrome (ahus), Shiga-like toxin-producing E. coli haemolytic uremic syndrome (STEC-HUS), and sepsis [ ]. On cell surfaces the complement activation is modulated by membrane proteins, such as complement receptor 1 (CR1, CD35), decay accelerating factor (DAF, CD55), MAC-IP (CD59), and membrane cofactor protein (MCP, CD46). CR1 and MCP both have a cofactor activity with fi, accelerating the decay of C3- and C5-convertases, whereas DAF has only C3-convertase specificity [36]. Activation of the terminal pathway results in assembly of lytic C5b-9 complex either in solution or on membranes [95]. The inadvertent lysis by these complexes is regulated by soluble serum proteins, such as vitronectin and clusterin, and membrane protein MAC-IP (CD59). All of the C5b-9 regulators inhibit the pore formation and C9 polymerisation by binding the forming complex [13, 107, 108]. Excessive or unchecked complement activation can result in potentially life threatening conditions, such as ahus and paroxysmal nocturnal haematuria (PNH) [105, 109]. 28

23 complement receptors Name Alternative name Ligands Function 1 CR1 Complement receptor 1, CD35 C3b, ic3b Phagocytosis, C3/C5 convertase inhibition CR2 Complement receptor 2, CD21 ic3b, C3dg B-cell stimulation CR3 Complement receptor 3, CD11b/CD18 ic3b Phagocytosis CR4 Complement receptor 4, CD11c/CD18 ic3b Phagocytosis C3aR C3a receptor C3a, C3a des-arg Proinflammatory signalling C5aR C5a receptor, CD88, C5aR1 C5a, C5a des-arg Proinflammatory signalling C5L2 C5a receptor 2, C5aR2 C5a, C5a des-arg (Possibly C3a, C3a des-arg) C5a signalling, pro/antiinflammatory soluble regulators of complement activation Name Alternative name Ligands Function C1s, C1r, C1-INH SERPIN1, C1 esterase inhibitor CP/LP activation inhibition MASP1/2 CP/LP C3 convertase C4BP C4 binding protein C4bC2a inhibition MAP-1 MAp19, MBL associated protein 19 MBL MASP competitor smap MAp44, MBL associated protein 44 MBL MASP competitor fh CFH C3b, C3(H 2 O), self-surfaces, CRP FHR-1 Factor H-related protein C5, self-surfaces fi CFI C3b, C4b Binds self-surfaces, inhibition of C3 activation C5a, C5b-9 assembly inhibition Degradation of C3/C5 convertases VTN Vitronectin, S-protein C5b and C5b-8 C5b-9 assembly inhibition CLU Clusterin, Apolipoprotein J C7, C8, C9 C5b-9 assembly inhibition CPN Carboxypeptidase-N C3a, C5a Removal of C-terminal arginine from anaphylatoxin (desarginylation) membrane regulators of complement activation Name Alternative name Ligands Function MCP CD46, membrane cofactor protein Cofactor of fi Degradation of C3/C5 convertases DAF CD55, decay accelerating factor C3 and C5 convertase Terminal pathway inhibition MAC-IP CD59, protectin C8, C9 C5b-9 assembly inhibition CR1 CD35 complement receptor 1 C3b, ic3b Phagocytosis, C3/C5 convertase inhibition Crry Complement receptor 1-related gene/protein Y C3b, C4b Mouse and rat homologue of human MCP and DAF Table 2. Overview of complement regulators and receptors. Complement and anaphylatoxin (C3a/C5aR) receptors bind complement activation fragments triggering intracellular signalling cascades and acting as co-receptors. Soluble regulators of complement are present in circulation and interstitial fluids acting against unchecked complement activation. Membrane RCAs are universally expressed by all cells and they are essential in inhibiting unchecked complement activation. 29 The expression and total composition of complement receptors and membrane RCAs exhibit major differences depending on the tissue and cell type.

24 chapter 1 6. EFFECTORS OF COMPLEMENT ACTIVATION There are three main types of complement effectors: covalently bound opsonins, soluble anaphylatoxins, and the lytic C5b-9 complex. Opsonins enable receptor mediated endocytosis via complement receptors expressed on various phagocytic cells. Anaphylatoxins act through specific receptors (C3aR, C5aR and C5L2) which promote inflammation and chemotaxis. The C5b-9 complex can directly lyse unprotected cells and promote apoptosis of injured cells. List of complement effectors and their ligands have been described in Table 3. The C3 and C4 activation generates covalently-bound opsonins C3b and C4b, which are further processed to inactive ic3b, C3d, and C3dg, which are membrane bound, and C3c which is soluble. Opsonins are ligands for complement receptors predominantly expressed by peripheral blood mononuclear cells (PBMCs), such as macrophages, dendritic cells, and lymphocytes. CR1 binds predominantly C3b and C4b [110, 111], whereas CR2 preferably binds only C3dg [112]. The CR1 receptors have also inhibitory function through suppression of C3 and C5 convertase activity [113]. The binding of CR1 and CR2 is essential for in phagocytosis of C3b/C3b opsonised immunocomplexes and cells [114], as well as in mounting effective adaptive immune response [32, 114]. Complement receptors CR3 and CR4 are heterodimeric membrane receptors formed with integrin α M (CD18) and either integrin β 2 (CD11b, CR3) or integrin β x (CD11c, CR4). CR3 is expressed on monocytes, dendritic and natural killer cells, whereas CR4 is present predominantly on dendritic cells and macrophages [115]. Both have specificity towards ic3b, with affinity towards many natural microbial components, such as LPS and β-glucan, effectively augmenting pathogen clearance [116]. CR3 and CR4 can influence inflammatory cell adhesion through ICAM-1 and take part in modulation of inflammatory cell activation [ ]. Importantly, C3 is important factor in modulation of oxidative burst from granulocytes upon activation [119]. 30

25 1 Activation of C2, C3, C4, and C5 produces small peptides C2b, C3a, C4a, and C5a called anaphylatoxins. The C2b has no known function whereas C4a has a weak impact on macrophage chemotaxis [120]. C3a and its metabolite C3a des-arg are also weak anaphylatoxins, but due to their high physiological concentrations they have central pro-inflammatory and modulatory roles [30, 121]. C5a is one of the most potent inflammatory mediators, and together with C3a they promote cellular and local inflammation and chemotactic recruitment of inflammatory cells via universally expressed C3aR and C5aR receptors [30, 122]. The anaphylatoxin receptors C3aR and C5aR (CD88) are abundantly expressed on PBMCs and are responsible for immunomodulatory functions, including DC- mediated priming of specific T-cell responses in tandem with Toll-like receptors (TLRs) [35]. The C5L2 receptor is specific for C5a, but its role is ambiguous and may be cell type, tissue, and disease condition specific [123]. Both C3a and C5a have been studied as potential targets for therapeutic intervention due to their involvement in many inflammatory and autoimmune diseases [124]. The lytic C5b-9 has a significant role in killing of bacteria, underlined by the high prevalence of meningococcal infections in individuals with terminal pathway deficiencies [125]. The C5b-9 formation is kept under control by vitronectin, clusterin, and CD59. Therefore under normal conditions the impact of C5b-9 on host cells is sublytic and pro-apoptotic rather than lytic and necrotic. The controlled or sublytic formation C5b-9 induces cellular upregulation of caspases and endoplasmic reticulum stress proteins and therefore facilitates controlled clearance of damaged cells through apoptosis [108, ]. 31

26 chapter 1 complement activation products and effector molecules Name Alternative name Generated by Function C4b C1, MBL, FCN1-3 Opsonin, CP/LP serine protease, cleaves C3 C4a Anaphylatoxin C4a C1, MBL, FCN1-3 Very weak chemotactic peptide C4d fi (with cofactors MCP, DAF, fh, CR1) Not known C2a C1, MBL, FCN1-3 CP/LP serine protease, cleaves C3 C2b Anaphylatoxin C2b C1, MBL, FCN1-3 Not known Bb fd Binds C3(H 2 O) or C3b to form AP C3 convertase, cleaves C3 to C3b, B-lymphocyte proliferation Ba fd Inhibition of B-lymphocyte proliferation C3a C3a des- Arg Anaphylatoxin C3a Acylation stimulating protein (ASP) MASP2, C4b, C4bC2a, C3b(P)Bb Carboxypeptidase-N C5a Anaphylatoxin C5a C4bC2aC3b, C3b(P)BbC3b C5a des- Arg Carboxypeptidase-N C3(H 2 O) C3u Spontaneous Weak chemotactic peptide, proinflammatory Immunomodulatory Strong chemotactic peptide, proinflammatory Weak and stable variant of C5a Spontaneously hydrolysed thioester allows transient binding to surfaces, fb binding and AP activation C3b ic3b, C3c, C3d, C3dg MASP2, C4b, C4bC2a, C3b(P)Bb fi (with cofactors MCP, DAF, fh, CR1) Large C3 activation fragment Opsonisation, phagocytosis mc5b-9 MAC, TCC C5b Pore formation, apoptosis SC5b-9 Soluble C5b-9, ic5b-9 C5, clusterin, vitronectin Soluble (inhibited) TCC, weakly proinflammatory Table 3. The activation products of the complement system. The established naming convention of complement rules that the physically smaller fragment is denoted with a and larger fragment with b, the only exception being with C2 where opposite naming is used. Further cleavage products of the large fragment are denoted with c, d, etc. Enz ymatic modification of small fragments, such as anaphylatoxins, result in des-arginylation of the C-terminal arginine which is denoted as C3a des-arg or C5a des-arg. 32

27 1 7. BIOSYNTHESIS Serum forms the systemic pool of the complement system, and most complement factors are predominantly produced by the liver. However, there are some exceptions, like properdin and C1q which are predominantly produced by certain peripheral blood mononuclear cells (PBMCs); factor D which is produced by adipocytes; and C7 which is produced by both liver and bone marrow cells [28, ]. The hepatic complement synthesis of most complement factors is enhanced during an acute-phase response, increasing the total serum complement activity in response to injury and infection [28]. A number of physiological and genetic factors can have a major impact on the functionality of the complement system, including age and gender. The complement system matures during the early development of the newborn, and it is striking that neonates have a very low total complement activity [ ]. Similar maturation of the complement system has been observed with other mammals, including mice and rats (Ong and Mattes, 1989). A comprehensive study on the gender dimorphism of the complement system in humans remains to be published in a peer reviewed journal. The most comprehensive human study has been published as part of doctoral thesis by Marc Seelen, which shows that functional AP activity, MBL, and C3 serum concentrations are significantly lower in adult Caucasian females compared to males (Chapter 6, Seelen, 2005). In rodents, it has been known for decades that male mice possess higher total complement activity, which has been attributed to hormonal control of several complement factors [ ]. Studies on female rodent complement suggests that some complement factors and total complement activity would be lower in female mouse sera, although compelling evidence has been shown only for C4 and C5 (Beurskens et al., 1999; Cinander et al., 1964; Ong and Mattes, 1989). Evidence suggests that also female rats exhibit lower total complement activity at least with Wistar females [143]. 33

28 chapter 1 The traditional view of the complement system as a system of liver-produced serum proteins still holds true. However, the role of extrahepatic complement expression, namely by parenchymal, interstitial, or inflammatory cells have been shown to have major roles in homeostasis, injury, and disease. Indeed, most organs can produce their own set of complement factors upon suitable stimulation, resulting in complement activation independent of the serum complement proteins [28, 144, 145]. Although PBMCs and PMNs do not have a major contribution to systemic complement pool, they are capable of producing and secreting most complement factors locally [28, 146]. Importantly, the local milieu and inflammatory conditions are important for determining what complement factors and regulators will be secreted [ ]. 8. COMPLEMENT IN DISEASE AND INJURY The complement system is intimately involved in cellular and humoral immune responses to injury and disease, acting as a robust first line defence and effector arm of adaptive immunity against invading micro-organisms. The potent serum complement is kept in check with combination of vascular integrity and soluble and cell membrane RCAs. Organ specific extrahepatic complement expression is usually selective, resulting in a partial local pool of complement, that is further inducible with exogenous inflammatory stimuli such as DAMPs and PAMPs [28, 36]. Breakdown of the complement control due to excessive injury, infection, or deficiency of regulators lead to complement mediated injury or aggravation of underlying disease or trauma. Most notable examples of the destructive effects of the complement system are sepsis [151], prominent organ trauma such as ischemia/reperfusion damage [152], and rare complement factor deficiencies which can result in severe diseases, 34

29 1 such as atypical haemolytic uremic syndrome (ahus) and paroxysmal nocturnal haematuria (PNH) [153]. For reasons not yet completely understood, kidneys are particularly susceptible for complement mediated damage, especially via the AP [154]. One potential explanation might be the physiological role of kidneys in filtrating waste from plasma and the large volume of blood processed daily. Glomeruli filter up to 180 litres of pro-urine daily, with 99% recovery back to circulation by the proximal and distal tubules every day complement in renal disease The complement system is the direct cause or aggravating factor in several inflammatory and autoimmune diseases outlined in Table 4. Notable examples of autoimmune diseases impacting kidneys are anti-glomerular basement membrane disease (anti-gbm, Goodpasture disease), C3-nephritic factor related nephropathy, IgA nephropathy (IgAN, Berger s disease), ANCAglomerulonephritis, and systemic lupus erythematosus (SLE) [105, 155]. A common denominator for most renal autoimmune and non-autoimmune diseases is that they impact the glomerulus. Probable causes for this susceptibility are direct contact with complement and antibodies in circulation, intraglomerular complement expression, and high filtration rate [156, 157]. C3 glomerulopathy is a new diagnostic definition for a group of renal diseases which are characterised by prominent complement activation due to dysregulation of AP, autoantibodies, or rare genetic mutations [154]. C3 glomerulopathy is the new umbrella classification that encompasses previous classifications membranoproliferative glomerulonephritis type I (immune complex involvement), type II (dense deposit disease, DDD), and type III (familial glomerulonephritis) (Dell Omo et al., 2002; Pickering et al., 2013; West and McAdams, 1998). In addition to clinical observations, experimental animal models have been used extensively to investigate the role and mechanism of 35

30 chapter 1 complement in renal disease. For example aggregated IgA has been shown to induce MBL mediated complement activation in vitro and in an experimental model of IgA-nephropathy in rats [161, 162]. Complement involvement in SLE and LN is paradoxical. As with other immune complex autoimmune diseases, the CP, activated through autoantibodies, contributes to the inflammation and injury. However, the hereditary deficiency of any of the CP components predisposes to the development of SLE [179]. The CP is central in homeostatic clearance of apoptotic material, and impairment of this system can result in accumulation of potentially immunogenic necrotic material in tissues and development of autoimmune disease [180]. Based on experimental models, SLE and particularly LN are both CP and LP mediated. In the case of hereditary CP deficiency, MBL-mediated LP activation can compensate the observed complement mediated injury [181]. The susceptibility of kidneys to complement-mediated injury is emphasised by the SLE and LN. SLE without LN shows relatively minor evidence of systemic complement activation, whereas prominent SLE with consumption of C3 and C4 is a predictor of LN [179, 182]. Antibodies directed against glomerular basement membrane (GBM) components are found in patients suffering from classical Goodpasture disease [183]. ANCA and anti-gbm overlap significantly with their renal impact, as is shown by the finding that 20 30% of anti-gbm patients have also autoantibodies against classical ANCA proteins, such as MPO [169]. Based on the experimental mouse models, both alternative and terminal pathways are central in development of both anti-gbm and ANCA. Both C1q and C4 deficient mice show only partial protection, whereas fb and C5 deficiency or blockade of C5aR significantly ameliorates the disease [170, 176, 184]. 36

31 1 diseases and injuries with complement involvement Disease or injury Cause(s) Atypical hemolytic uremic syndrome a ahus a Mutations in fh, fi, fb, MCP AP [104] Shiga-like toxin producing E. coli HUS b STEC- HUS b Infection AP [106, 163] Paroxysmal nocturnal hematuria a PNH a CD59 deficiency AP [164] Hereditary angioedema a HA a C1-INH deficiency CP, LP [165] Systemic lupus erythematosus b SLE b C1q deficiency, autoantibodies CP, LP Lupus nephritis b LN b C1q deficiency, autoantibodies CP, LP [99, 166, 167] [99, 166, 167] Rheumatoid arthritis b RA b Autoimmune disease CP, AP [168] Sepsis b Extensive infection, loss of complement regulation and C5a production AP [151] Recurrent bacterial infections a C-deficiency (fp, fd, C1q, MBL etc ) CP, LP, AP [39] Goodpasture disease b anti-gbm b Autoantibodies directed against glomerular basement membrane IgA Nephropathy, Berger's disease b IgAN b LP CP, AP [169, 170] [162, 171] C3 Glomerulonephropaty a C3GN a Impaired C3 activation control, C3 nephritic factor, fh mutations, AP [160, 172] Dense deposit disease a DDD (MPGN II) a Impaired C3 activation control, C3 nephritic factor, fh mutations, AP [160, 173] Membranoproliferative IgM, IgG deposition in glomeruli with MPGN glomerulonephritis b b C1 and C3 Age related macular degeneration a AMD a Complement regulator SNP polymporphism association CP, AP [154] AP [174, 175] Thrombotic thrombocytopenic purpura b TTP b ADAMTS13 deficiency AP [163] Anti-neutrophil cytoplasmic antibody vasculitis b ANCA b Autoantibodies against MPO, PR3 CP, AP [176] Ischemia/reperfusion Injury b I/RI b Transplantation, hypovolemia, radiocontrast induced I/RI CP, LP, AP Graft versus host, rejection b Alloantibodies CP, AP [152] [177, 178] Table 4. Diseases with complement indications. Diseases with primary complement cause ( a ), diseases with active complement contribution ( b ) 37

32 chapter complement and renal ischemia/ reperfusion injury Renal ischemia/reperfusion injury (I/RI) is a multifactorial and progressive condition present after transplantation, radiocontrast infusion, major surgery, and severe infections [185, 186]. The hallmarks of renal I/RI include acute tubular necrosis (ATN), complement activation at the corticomedullary junction, infiltration of PMNs, and impaired renal function resulting in accumulation of nitrogen metabolites (urea, creatinine) in circulation [187]. The I/RI is initiated by occlusion of blood flow which results in cellular hypoxia, depletion of cellular energy (ATP) and accumulation of metabolic waste [188]. However, the majority of the cascades responsible for the damage are initiated after reperfusion. The reperfusion phase is characterised by early release of inflammatory cytokines such as IL-6 [189], formation of oxygen radicals [190, 191], and microvascular coagulation which can prolong ischemia locally [192]. Damaged vascular endothelium compromises vascular integrity [192] promoting infiltration of inflammatory cells [186] and allowing extravasation of serum components to the interstitium [193]. The contribution of the complement system to renal I/RI has been established with complement-deficient animals (either natural mutants or genetically engineered) lacking specific activators, effectors, and regulators. Further information on the contribution of complement has been acquired with targeted systemic depletion of key complement factors, systemic inhibition of complement activation processes and preventing signalling. Together the studies suggest that AP (properdin, fb), LP (MBL- MASP1/2), and terminal pathway (C3, C5-C9, C5a) contribute to the experimental renal I/RI in mice [ ]. In case of experimental rat renal I/RI, results suggest that in rats the MBL-MASP1/2 complex is responsible for majority of the injury, without prominent contribution from terminal pathway effectors C5a and C5b-9 [198]. 38

33 1 Analogous mouse studies with MBL-A/C deficient mice confirm and expand the importance of MBL to experimental renal I/RI [199]. Together with evidence that C4 deficient mice are not protected, it seems that MBL alone can induce tubular epithelial cell damage as proposed by van der Pol et al [195]. Alternative explanation may be that a MASP-2 mediated C4 bypass mechanism has a role in renal I/RI, as has been described for myocardial and gastrointestinal intestinal I/RI [82]. Studies of systemic inhibition have shown that expression of intrarenal complement factors, receptors, and regulators has a major contribution in renal I/RI. Renal expression of complement factors is known to be inducible by inflammatory mediators, such as IFN γ and TNF α, and has a regionally dependent profile. Renal complement expression includes key factors known to be involved in inducing renal injury, including AP components C3 and fb, and also CP and LP components C1q, C2, and C4 [156]. Experimental transplantation studies have established the importance of extravascular and intrarenal synthesis of C3 to renal I/RI. Seminal work by Farrar et al. (2006) with transplantation model using C3 KO and normal mice showed that intrarenal expression of C3 alone was sufficient for complement mediated damage, with no protection in the case of transplanting wild type (WT) kidneys to C3 KO mice. Analogous results have been obtained with total or selective systemic inhibition of the complement system. Soluble Crry-Ig complex, a recombinant fusion protein of mouse Crry (MCP/DAF human homolog, Table 2.) and IgG Fc-region, is a potent inhibitor of systemic complement through inhibition of C3 and C5 convertase function. However, systemic administration of Crry-Ig and subsequent inhibition of complement is not protective, although renal expression of Crry has been shown to be central factor in limiting complement mediated damage in ischemic kidney [200, 201]. 39