New perspectives on mannan-binding lectin-mediated complement activation

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Immunobiology 212 (2007) 301–311 www.elsevier.de/imbio

New perspectives on mannan-binding lectin-mediated complement activation Søren E. Degn, Steffen Thiel, Jens C. Jensenius Department of Medical Microbiology and Immunology, University of Aarhus, Denmark Received 10 August 2006; received in revised form 24 November 2006; accepted 5 December 2006

Abstract The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP is the most recently discovered, and least characterized. The CP and the LP are generally viewed as working through the generation of the C3 convertase, C4bC2b, and are here referred to as the ‘‘standard’’ pathways. In addition to the standard CP and LP, so-called bypass pathways have also been reported, allowing C3 activation in the absence of components otherwise believed critical. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging picture of the complement system is more that of a small ‘‘scale-free’’ network where C3 acts as the main hub, than that of three linear pathways converging in a common terminal pathway. r 2007 Elsevier GmbH. All rights reserved. Keywords: C3 convertase; Lectin pathway; MBL; Complement bypass pathway

Introduction The complement system is a defense mechanism comprising around 30 different soluble and membranebound proteins. It is part of the innate immune system and proceeds via controlled limited proteolysis and Abbreviations: AP, alternative pathway; CP, classical pathway; CRD, carbohydrate-recognition domain; FBG, fibrinogen-like domain; IGFBP-5, insulin-like growth factor-binding protein 5 ; LP, lectin pathway; MAC, membrane attack complex; MASP, MBL-associated serine protease; MBL, mannan-binding lectin ; PAMP, pathogen-associated molecular pattern; PRM, patternrecognition molecule; SIGN-R1, specific intercellular adhesion molecule-3 grabbing nonintegrin-related 1; SP, serine protease Corresponding author. Tel.: +45 89 42 17 78. E-mail address: [email protected] (S.E. Degn). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.12.004

conformational changes of constituent proteins through three activation pathways, the classical pathway (CP), the alternative pathway (AP) and the lectin pathway (LP), which converge in a common lytic cascade. The CP is initiated by recognition of immune complexes containing IgG or IgM, and C-reactive protein, as well as some pathogen-associated molecular patterns (PAMPs), by the pattern recognition molecule (PRM) C1q. Activation of the LP occurs through recognition of PAMPs by either mannan-binding lectin (MBL) or ficolins. Finally, activation of the AP occurs constitutively by spontaneous hydrolysis of the thioester bond in C3, and specificity is achieved through inhibition on non-activating self-surfaces, and lack of inhibition on non-self (e.g., bacteria) and altered self (e.g., cancer cells). The AP also serves as an amplification loop for

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describe the recently discovered lectin bypass pathway in the context of the classical bypass pathways.

the other two activation pathways (Harboe et al., 2004; Brouwer et al., 2006). All three activation pathways converge in the common lytic cascade, which terminates in the formation of the membrane attack complex (MAC), also known as the terminal complement complex (TCC). The MAC inserts into cell membranes and forms pores leading to osmotic lysis of the target cells. An overview of the three activation pathways is given in Fig. 1. The CP is believed to be the most recent of the activation pathways, having evolved around the same time as the adaptive immune system (Fujita, 2002), approximately 500 million years ago. The interaction between the CP and the adaptive immune system creates an interesting and important cross-bridge between the innate and the adaptive defense systems. The LP has features in common with the CP but is evolutionarily much older (Fujita, 2002; Dodds, 2002). The AP is believed to be as old as the LP, possibly having appeared first as an amplification mechanism of this, and later evolved into an independent pathway (Dodds, 2002). Although the LP is a primordial defense mechanism, it is the most recently discovered of the three activation pathways, and thus also the least characterized. This review will give an overview of the LP, as well as

The standard LP of complement activation Activation of the LP occurs through recognition of PAMPs by either MBL or ficolins in association with MBL-associated serine proteases (MASPs). Upon binding to the target, the MASPs are activated allowing them to generate the C3 convertase, C4bC2b. MBL, the better-known PRM of the LP, is a plasma protein of hepatic origin, belonging to a family of proteins known as the collectins. Collectins are oligomers of polypeptide chains containing a C-type lectin carbohydrate-recognition domain (CRD) attached to a collagen-like region. In the case of MBL, three identical polypeptide chains assemble to form each structural subunit, which then associate into higher oligomeric forms, ranging from dimers to hexamers and even higher oligomers (Dahl et al., 2001). The overall structure of MBL resembles that of C1q, being sertiform (Holmskov et al., 2003). The PAMPs recognized by MBL comprise various

Lectin Pathway

Alternative Pathway

Pathogen-Associated Molecular Patterns

B

C4 C4a

MAp19 MASP-1 MBL MASP-2 MASP-3

C3(H 2O)

C3(H 2O)B

C3(H 2O)Bb

MASP-1 MBL MASP-2

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C4b

C2 r

Activating Surface No Inhibition

D

s

C1q s r

r

C3

s

C1q s r

Ba

B

C4b2

C3bB D

C2a

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C3a C4b2b

C3b

C3bBb

Immune Complexes C5

Classical Pathway C4b3b2b

C3b3bBb C5a C5b

MAC Formation

Fig. 1. The three activation pathways of the complement system. The names of the pathways and their main activators are shown in square boxes, proteins of the pathways in rounded boxes, and released fragments as plain text. Blue arrows denote the initiating events. Red letters denote the active serine proteases of the pathways, and red arrows the cleavages they mediate. The dashed arrows progressing from MASP-1 indicate activities on which there is not yet consensus. Curved black arrows indicate associations, broken black arrows dissociations, and straight black arrows progression to subsequent steps. The ficolin/MASPs can initiate the lectin pathway in a manner similar to MBL/MASPs.

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simple and complex carbohydrate motifs. MBL does not selectively bind only mannose or its multimers, as the often used name, mannose-binding lectin, implies, but rather in general recognizes sugars with 3- and 4-OH groups placed in the equatorial plane of the sugar ring structure (Weis et al., 1992). For example, the CRDs bind with similar affinity to mannose, N-acetylmannosamine (ManNAc), N-acetylglucosamine (GlcNAc), and fucose, in a Ca2+-dependent manner. Appropriate spacing of ligand sugars, allowing concomitant binding of multiple CRDs, is a requirement for high-avidity binding of MBL. More recently it has been discovered that the LP can also be activated by a group of proteins known as the ficolins. Three are found in humans: H-ficolin, L-ficolin, and M-ficolin, while the mouse has only two, ficolin A and ficolin B, orthologous to L- and M-ficolin, respectively (Endo et al., 2004). Ficolins are structurally similar to MBL and the other collectins, but instead of C-type lectin domains they possess fibrinogen-like domains (FBG) for PAMP recognition (Matsushita et al., 1996). H- and L-ficolin are serum proteins, synthesized in the liver, although H-ficolin is also found in secretions, i.e., bile, bronchiolar, and alveolar fluids (Matsushita et al., 1996; Akaiwa et al., 1999). M-ficolin (Lu et al., 1996) is found on the surface of monocytes (Teh et al., 2000; Frederiksen et al., 2005), although it lacks a transmembrane domain, and also in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung (Liu et al., 2005). L-ficolin has been demonstrated to activate complement upon binding to Salmonella typhimurium (Matsushita et al., 2000a) and lipoteichoic acids (Lynch et al., 2004), and was shown to bind some capsulated serotypes of Streptococcus pneumoniae and Staphylococcus aureus (Krarup et al., 2005; Aoyagi et al., 2005). H-ficolin has also been shown to activate complement (Matsushita et al., 2002) and to inhibit the growth of an Aerococcus viridans strain (Tsujimura et al., 2002). Less is known about M-ficolin, but a recent study showed binding to Staphylococcus aureus (Liu et al., 2005). The ligands for the ficolins were initially suggested to be acetylated monosaccharides like GlcNAc and GalNAc (Le et al., 1998; Sugimoto et al., 1998), in line with their evolutionary relationship to the tachylectins (Gokudan et al., 1999). However, it has been shown for L-ficolin that the motives recognized are more generally acetylated compounds, including non-sugars such as N-acetylglycine, N-acetylcysteine and acetylcholine (Krarup et al., 2004). Recently it was demonstrated that M-ficolin is able to bind acetylated compounds in a manner similar to L-ficolin (Frederiksen et al., 2005; Liu et al., 2005). Based on this selectivity L- and M-ficolin are not easily defined as lectins, making the term ‘‘lectin pathway’’ somewhat problematic. So far, the ligand specificity of H-ficolin has not been well defined,

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although H-ficolin has been shown to bind PSA, a capsular polysaccharide produced by Aerococcus viridans (Tsujimura et al., 2002; Matsushita et al., 2002). Liu et al. (2005) also indicate binding of M-ficolin to sialic acid. Sialic acid is usually the terminal sugar of the oligosaccharides that decorate mammalian glycoproteins. Neither MBL, nor H- or L-ficolin, appears to bind this sugar. If M-ficolin has this specificity it would indicate that it might be able to recognize self-structures, depending on the ligand pattern requirement. As mentioned, MBL and the ficolins are associated with so-called MASPs. In humans there are three of these, MASP-1, -2, and -3, along with an alternative splicing fragment of MASP-2 called MAp19 or sMAP (Schwaeble et al., 2002). The three enzymes have the same modular buildup as C1r and C1s of the CP, i.e., they are composed of well-described domains in the order: CUB1-EGF-CUB2-CCP1-CCP2-SP. The first five domains constitute the A-chain, and the serine protease SP domain constitutes the B-chain. MASP-1 and -3 are alternative splicing products of the MASP1/-3 gene (Dahl et al., 2001; Nonaka and Miyazawa, 2002). They share the same A-chain, apart from the 15 C-terminal residues, which constitute the linker or activation peptide, but have distinct B-chains. MAp19 and MASP-2 are also alternative splicing fragments of one gene, the MASP-2 gene, with MAp19 being a smaller product consisting of only CUB1-EGF with an additional four unique amino acids at the C-terminal (Stover et al., 1999; Takahashi et al., 1999). The three MASPs and MAp19 are all able to homodimerize and associate with MBL, H- and L-ficolin, in the presence of Ca2+, but apparently do not form heterodimers (Thielens et al., 2001; Chen and Wallis, 2001). The roles played by the three MASPs have not been resolved yet. Initially, it was thought that MBL mediated the formation of the C3 convertase using C1r and C1s (Ikeda et al., 1987; Ohta et al., 1990; Lu et al., 1990). When MASP was discovered it appeared to activate both C4 and C2 (Matsushita and Fujita, 1992), but then another MASP appeared on the scene, MASP2 (Thiel et al., 1997). One could now imagine a scenario similar to that of the CP where MASP-1 autoactivated and then cleaved MASP-2, which then cleaved C4 and C2. However, later studies found that both MASP-1 and -2 were able to autoactivate whereas MASP-3 does not appear to have this capability (Zundel et al., 2004). The prevailing view today is that MASP-2 cleaves both C4 and C2, thus on its own mediating activation of the LP (Vorup-Jensen et al., 2000). This leaves the function of MASP-1 and MASP-3 unaccounted for. MASP-1 has been reported to activate C2 (Matsushita et al., 2000b; Chen and Wallis, 2004; Møller-Kristensen et al., 2006). Some studies also find that MASP-1 has direct C3 activating capacity (Matsushita and Fujita, 1995; Matsushita et al., 2000b). However, this is much

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debated and some believe that this activity is too weak to have any biological significance (Takahashi et al., 2000; Rossi et al., 2001; Ambrus et al., 2003). Two recent communications from the VIth International Workshop on the First Component of Complement C1 and Collectins, Seeheim, June 2006, point to a new understanding of a role for MASP-1. Teizo Fujita reported that MASP-1 activates MASP-2, indicating a sequential reaction as seen for the C1 complex. Jens Christian Jensenius also presented data involving MASP-1 in the generation of C3 convertase, but indicating a different mode of action. MASP-1 certainly increased the C3b deposition by MASP-2, but had no influence on C4b deposition mediated by MASP-2. The interpretation was that at the experimental conditions (i.e., serum diluted six-fold on a mannan surface) MASP-2 mainly worked on C4 to generate C4b, while MASP-1 provided an increased amount of C2b for the C3 convertase (Møller-Kristensen et al., 2006). The role of MASP-3 has not yet been elucidated, but a recent study demonstrated cleavage of insulin-like growth factor-binding protein 5 (IGFBP-5) (Cortesio and Jiang, 2006). However, this was done using recombinant catalytic domain (SP domain) and substrate in a 1:10 molar ratio, and required incubation at 37 1C for 4 h for full cleavage. Thus, the physiological role of this enzymatic activity is uncertain. In summary, it appears that MASP-1 and MASP-2 are both involved in the standard LP activation. Upon binding of MBL to its PAMP targets a conformational change probably occurs, causing autoactivation of associated MASP-1 and -2 (Ga´l et al., 2005). While MASP-2 is able to cleave C4 at a high efficiency, and C2 to a lower degree, on its own activating the LP, MASP-1 has also been reported to be able to cleave C2 (Chen and Wallis, 2004; Rossi et al., 2001; Matsushita et al., 2000b). Although one study has not seen a C2-cleaving activity of MASP-1 (Ambrus et al., 2003), and one paper discusses if the activity is too weak to be physiologically significant (Rossi et al., 2001), we find that MASP-1 and -2 cooperate in activation of the LP (Møller-Kristensen et al., 2006), as has also previously been suggested by others (Chen and Wallis, 2004). Although both H- and L-ficolin have been found to form complexes with the MASPs and to activate complement in vitro using purified components (Matsushita et al., 2000a, 2002), the physiological situation is somewhat unclear. Importantly, an in vitro study, employing a C4-deposition assay using full serum and exogenous C4, found that MBL bound to Staphylococcus aureus and H-ficolin bound to Aerococcus viridans activated complement, whereas L-ficolin bound to Streptococcus pneumoniae did not (Krarup et al., 2005). This raises the question about the use of purified components as opposed to serum, which would likely represent more physiological conditions. In line with the

results on L-ficolin and Streptococcus pneumoniae, an earlier study found that immediate defense against Streptococcus pneumoniae primarily requires the CP of complement (Brown et al., 2002). With regards to M-ficolin, two recent studies demonstrated activation of complement by recombinant M-ficolin in association with MASPs on acetylated surfaces (Frederiksen et al., 2005; Liu et al., 2005). Liu et al. (2005) found that M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in the lung. This led to the suggestion that M-ficolin is a regulated secretory protein, in agreement with its sequence, which contains an N-terminal hydrophobic signal sequence, but no transmembrane or membrane-anchor motif. However, M-ficolin has not been detected in serum and studies have shown that M-ficolin is found on the surface of monocytes (Teh et al., 2000; Frederiksen et al., 2005). Such surface-expression could occur through post-translational attachment of a GPI anchor or binding via ficolin receptor(s). Alternatively, or in addition, stimulated release of M-ficolin could occur locally. The question remains whether the in vitro complement activating activity observed with recombinant M-ficolin and recombinant or purified MASP-1 and -2 reflects the physiological situation. Activation on, or close to, the surface of host monocytes would appear detrimental. Perhaps complement regulatory mechanisms on the monocytes would be strong enough to avoid their destruction, while at the same time allowing complement deposition on a PAMP target. Alternatively, there might be a difference in the complement activating ability of membrane-bound and soluble M-ficolin, possibly as a consequence of a difference in the polymeric state of the molecule in these two different situations.

Control of activation The complement system is a powerful component of the innate immune system and an important modulator of the adaptive response. However, it also holds the potential to damage the host. Therefore, control of the complement system is crucial. Discrimination of self and non-self or altered self is the first, and most essential, point of control. Thus, in the case of MBL, the recognized targets are patterns of carbohydrates with 3- and 4-OH in the equatorial plane, whereas the terminal sugar that usually decorates mammalian glycoproteins is sialic acid. Thus, complement activation on self is normally avoided. However, after erroneous activation on self or correct activation on non-self or altered self, control of the complement system is also required to avoid explosive amplification. This is mediated by host-cell surface-associated regulators, as

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well as fluid-phase regulation. Specific control of the CP and LP is achieved through C1-inhibitor (C1-Inh), a member of the serpin family of inhibitors. C1-Inh forms a covalent 1:1 complex with both the activated C1r and C1s, resulting in the release of (C1-Inh)-C1r-C1s-(C1Inh) complexes. This prevents hyperactivation of the CP and also exposes determinants on C1q, allowing it to interact with a possible C1q receptor. A similar inhibitory effect of C1-Inh on activated MASP-1 and -2 limits activation of the LP (Matsushita et al., 2000b). Interestingly C1-Inh does not appear to inhibit MASP-3 (Zundel et al., 2004). In addition a2M has been reported to inhibit MASP-1 and -2 (Terai et al., 1995; Gulati et al., 2002), but these data are somewhat controversial. Although other studies have found that a2M associates with MBL–MASP complexes (Storgaard et al., 1995; Petersen et al., 2000), in one of these no inhibitory activity of a2M on the activation of C4 was seen (Petersen et al., 2000).

Classical bypass pathways of complement activation It would appear that in addition to the standard pathways, several so-called bypass pathways of comple-

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ment activation exist, which can come into play when some components are missing, as is seen in various deficiency states, and which to some extent could compensate in such scenarios. The first suggestion of the existence of such a bypass pathway came almost 40 years ago, with the discovery that C4- and C2-deficient guinea pig sera could support lysis of sheep red blood cells. This required significantly higher quantities of sensitizing IgG or IgM than when using normal guinea pig serum (May et al., 1972; May and Frank, 1973a, b). C1 was found to be an absolute requirement, and the effect was thought to be mediated by the AP (May and Frank, 1973a, c). This novel pathway was initially dubbed the C1-bypass pathway because of the C1 and ‘‘bypass pathway’’ (an early name for the AP) requirement, but it has recently been renamed the C4-bypass pathway because it bypasses C4 (Wagner et al., 1999). A similar lysis of heavily sensitized sheep erythrocytes was later observed using C2-deficient human serum (Matsushita and Okada, 1986; Steuer et al., 1989). In this case, it was shown that C4b deposited on cells facilitated AP activation (Matsushita and Okada, 1986). This pathway was dubbed the C2-bypass pathway, because it requires C4 but bypasses C2. We will refer to these pathways as the classical C4-bypass pathway and the classical C2-bypass pathway, collectively as the classical bypass pathways, in order to distinguish them

Standard Pathways

Bypass Pathways → weaker responses

Faster kinetics → stronger responses

Slower kinetics No Factor B / D

No C2

Lectin Pathway with AP amplification

Lectin Pathway no AP amplification

Lectin C2-Bypass Pathway? AP dependent

No MASP-1/-2/-3 / C4 / C2

Lectin C4-Bypass Pathway AP dependent

MBL binds Factor I, H Alternative Pathway

do not bind

Surface -Type -Pattern -Density

Ab+C1q binds No C1s? / C1r? / C4 / C2

Classical Pathway with AP amplification

No C2

No Factor B / D

Classical C4-Bypass Pathway AP dependent Classical C2-Bypass Pathway AP dependent

Classical Pathway no AP amplification

Fig. 2. The bypass pathways of complement activation. The left-hand side shows the standard activation pathways. The right-hand side shows the proposed bypass pathways, as well as the standard classical and lectin pathways in the absence of alternative pathway amplification. The ficolins can substitute for MBL in the activation of the standard lectin pathway, but it is not known whether they play a role in bypass pathways. For further details, please refer to the text.

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from the recently proposed MBL-dependent C2-bypass pathway (discussed below, also see Fig. 2). At the time when these studies were performed the LP had not yet been discovered. Although the observation that higher amounts of sensitizing antibody were required indicates the function of a CP bypass mechanism, a possible contribution of a similar LP bypass mechanism, or a more direct LP mechanism, was thus obviously not excluded. However, when showing that EAC4b1 were lysed by C2-deficient human serum, Matsushita and Okada (1986) used serum in an Mg2+-EGTA buffer for the lysis step, thus Ca2+ was not present and this excludes a role for the LP. Furthermore, they treated the C4b-coated erythrocytes with 1 mM PMSF to inhibit any residual C1r/C1s, and since this is a general inhibitor of SPs it would also have inhibited the homologous MASPs, excluding any role of these proteases in the observed activation. In the study by Steuer et al. (1989), mentioned above, the observed effects were abolished in the presence of either EDTA or Mg2+-EGTA, suggesting that Ca2+ and probably C1 were required, and thus that the AP alone could not be responsible. Dependence on both C1 and C4 was confirmed by depletion experiments. They also depleted for factor B, properdin or factor D, and saw that this had no effect on lysis by normal sera but abolished lysis in C2-deficient sera. The conclusion was that AP activity was not required under C2-sufficient conditions. This curiously contrasts with a recent study, which found that AP amplification accounts for more than 80% of CP activity (Harboe et al., 2004). The latter study was conducted under more physiological conditions with 50% serum, whereas for the former the normal serum was diluted 1/80, and the C2-deficient sera 1/7.5–1/15. Diluting serum more than about 1/10 effectively abolishes AP activity, and this may explain the discrepancy. Selander et al. (2006) found that no AP amplification was required for full activation through the CP in their experimental setup, but this could be because their assay (carried out in serum 1/4) got saturated. Interestingly, the classical bypass pathways have also been observed to play a role in the lysis of IgM-sensitized Giardia lamblia trophozoites by both C2deficient human serum and C4-deficient guinea pig serum (Deguchi et al., 1987). Furthermore, the C4bypass pathway has been implicated in human pathologic situations, such as chronic urticaria and angioedema (Ballow et al., 1975), as well as hemolytic uremic syndrome (Nolin et al., 1979). Recent studies have suggested that the C2-bypass pathway may have a function in the prevention of immune complex disease in C2-deficient individuals (Traustadottir et al., 1998; Klint

et al., 2000). The in vivo relevance was also tested in a Forssman shock model in guinea pigs (Wagner et al., 1999). Injection of rabbit IgG anti-Forssman antibody i.v. into normal guinea pigs leads to a cataclysmic reaction, with rapid pulmonary edema and hemorrhage leading to death. This effect cannot be induced in C4-deficient guinea pigs, indicating that CP function is required. However, Wagner et al. (1999) found that when C2-deficient guinea pigs were challenged, pulmonary shock occurred with pathologic findings that resembled those in normal guinea pigs. Further experiments indicated that this effect was mediated by the C2-bypass pathway. It has been shown that C4b can bind the AP C3 convertase, C3bBb, via a weak interaction with C3b (Farries et al., 1990a). Furthermore, nascent C3b attaches covalently with high efficiency to C4b, and C3b in C4bC3b complexes is protected from inactivation by factors H and I (Meri and Pangburn, 1990), an observation that was also previously made for C3b attached to IgG (Fries et al., 1984). This might explain the C2-bypass pathway function, as well as more generally the AP recruitment following CP and LP activation and the stability of the C5 convertase on surfaces which do not themselves provide protection for C3b from factors H and I. With regards to the C4bypass pathway, the scenario may be the same, i.e., C1 provides a protected site for the AP C3 convertase, albeit to a much lower extent than C4b. Finally, since C3b bound to IgG is also protected, one could imagine that the increased antibody sensitization required for bypass pathway activity not only provides binding sites for C1q but also adds to the protecting effect of C1 and C4b. One peculiar feature of the C4-bypass pathway is that intact C1 appears to be required for the protective effect. That C1 is required in the case of the C2-bypass pathway makes sense, since in this case the enzymatic activity of C1r and C1s is required for cleavage of C4. On the contrary, there is no obvious reason for the requirement for the enzymatic components of C1 in the case of C4-bypass. Yet, this is what has been observed, as shown by the Ca2+-dependence mentioned above. It is generally accepted that the Ca2+-dependence of C1 function is due to a Ca2+-requirement of C1r and C1s, whereas C1q does not require Ca2+ to bind most of its targets, although studies have shown that the heterotrimeric globular head of C1q contains one Ca2+ (Villiers et al., 1980; Gaboriaud et al., 2003; Roumenina et al., 2005).

Lectin bypass pathway of complement activation 1 EAC4b denotes AbC1C4b-coated sheep erythrocytes, stripped of C1 complex by treatment with an EDTA-containing buffer; C4b remains covalently bound.

The present view of the LP is that it is analogous to the CP in generating the C3 convertase C4bC2b, but an

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early study indicated that MBL was able to activate the alternative complement pathway and enhance serum bactericidal activity on a mannose-rich isolate of Salmonella montevideo (Schweinle et al., 1989). Curiously, these investigators did not see any activity that might be attributed to the standard LP, although, according to the purification procedure used (Ezekowitz et al., 1989), we would expect them to have employed MBL–MASP complex and not ‘‘pure’’ MBL. Instead, what they observed was a relatively weak, dosedependent activation of the AP by MBL, which could be inhibited by mannan. Serum was used at only 2.5% in their experiments, which normally prevents AP function, possibly contributing to the very weak, but significant, activation of C3 observed. It seems plausible though that MBL, if it has AP activating activity, would be able to induce this activation at lower serum concentration than what is required for conventional AP activation. The faster kinetics of C3-deposition observed in the presence of MBL supports this hypothesis. Importantly, Schweinle et al. (1989) carried out some of their experiments in pre-adsorbed serum (preincubation with the bacteria used should remove any MBL and antibodies able to bind), in serum with Mg2+EGTA, in C1q-deficient serum, or in C2-deficient serum. Together this means that the observed effect could not be due to presence of MASPs on the purified MBL, or CP activation (in this connection it is curious that the effect is not abolished in serum with Mg2+-EGTA, since our experience is that Ca2+ is absolutely required for both MBL binding and retention). Dependence on the AP, on the other hand, was determined using factor Ddepleted serum, as well as by using MBL and purified AP components only. To our knowledge, this is the first hint of an MBL-dependent bypass pathway, but this has not been well recognized. A likely explanation is the subsequent delineation of the more powerful standard MBL pathway, emerging with the discovery of the MASPs, withdrawing attention from these results. Recently, however, an investigation by Selander et al. (2006) has refocused our attention by suggesting the existence of exactly such an MBL-dependent C2-bypass pathway, analogous to the classical bypass pathways. They found that MBL, bound to a surface of LPSderived O antigen-specific oligosaccharides from Salmonella thompson (a ligand of MBL), efficiently supported C3 deposition at high serum concentration (1/4 or 1/5) in the absence of C2 and MASPs. C4 also did not appear to be required, making it more like the classical C4-bypass pathway than the classical C2-bypass pathway. Thus, a more appropriate name might be ‘‘MBLdependent C4-bypass pathway’’, or shorter ‘‘lectin C4-bypass pathway’’ (see Fig. 2). In analogy with the requirement for higher concentrations of sensitizing antibodies required for hemolysis by the classical bypass pathways, as compared to the

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standard CP, one could imagine that higher concentrations of MBL are required for the MBL-dependent bypass pathway, as compared to the standard LP. Indeed, the capacity to support C3 deposition in C2deficient sera was correlated with the concentration of MBL in the study performed by Selander et al. (2006). As, mentioned, the results of Schweinle et al. (1989) also fit with this. Furthermore, Harboe et al. (2006) observed a dose-dependent increase of C3 deposition after activation of C2-deficient serum diluted 1:2 with increasing amounts of mannan on the solid phase. Mannan itself can be an activator of the AP (Roos et al., 2003) but the dramatic effect could in part be explained by increased MBL binding at higher mannan coating concentrations. In fact, Selander et al. (2006) also saw an increase in AP activation on a mannan coat when adding rMBL to C2-deficient serum. This points to a mechanism, which could be imagined to be similar to that for the classical bypass pathways, i.e., protection of C3b. Analogous to the classical C4- and C2-bypass pathways, one could envisage that not only a lectin C4-bypass pathway exists, but also a more efficient lectin C2-bypass pathway, which would then be dependent on C4 and the C4-cleaving MASP-2 (see Fig. 2). This, however, did not appear to be the case in the experiments by Selander et al. (2006). In addition to the possible C3b-protective role of bound MBL, it has been suggested that MBL alters the nature of the C3 acceptor bond, resulting in an amide linkage between C3 and putative bacterial acceptor molecules, possibly by altering the bacterial surface and allowing access of C3 to bacterial outer membrane proteins (Schweinle et al., 1989). A note in this connection is that the change from hydroxyl acceptor to amine acceptor could be caused directly by the binding of MBL to the sugar compounds containing hydroxyl groups, thus ‘‘forcing’’ C3 to react with the available amines. A recent study found activation of the classical complement pathway through another lectin, specific intercellular adhesion molecule-3 grabbing nonintegrinrelated 1 (SIGN-R1), a cell surface C-type lectin receptor with homology to DC-SIGN (Kang et al., 2006). This receptor is expressed on marginal-zone macrophages (Geijtenbeek et al., 2002) and is important in resistance to pneumococcal infection (Lanoue et al., 2004). As mentioned earlier, another study found that defense against Streptococcus pneumoniae requires the CP of complement (Brown et al., 2002). These previous findings are now integrated by the observation that SIGN-R1 activates the CP of complement on the surface of marginal-zone macrophages, independently of antibodies (Kang et al., 2006; Roozendaal and Carroll, 2006). SIGN-R1 binds both Streptococcus pneumoniae polysaccharide and C1q, leading to complement activation on the captured bacterium. In this connection, the same consideration applies as for

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activation of complement by monocyte cell-surface associated M-ficolin, i.e., that control mechanisms must be powerful enough to thwart complement activation on the host cells. Further studies approaching the in vivo situation should address this question. The existence of the classical bypass pathways of complement activation was suggested to reflect the evolution of the complement system (Farries et al., 1990b). The discovery of a lectin bypass pathway has expanded this (Atkinson and Frank, 2006), since this pathway is believed to be the oldest of the three pathways (Fujita, 2002; Dodds, 2002). One could imagine that opsonization was the primary function of the primitive humoral immune system. This could be mediated directly by MBL-like molecules or indirectly by cleavage and activation of an ancient thioestercontaining protein (C3-like) by bacterial surface proteases causing ‘‘C3-like’’ to deposit on the bacterial surfaces (Dodds, 2002; Zhu et al., 2005; Dishaw et al., 2005). We speculate that these mechanisms ‘‘merged’’, coupling the PRM with the thioester ‘‘tagging’’, and that an evolutionary remnant of this is seen in the lectin bypass pathway. An important step could have been the development of MBL/MASP, which could directly cleave ‘‘C3-like’’. This system may have been improved and elaborated by the evolution of intermediate steps employing C4 and later C2. Concomitantly, the control mechanisms keeping the somewhat unstable C3 molecule in check were probably developed, forming the basis of the AP. A C1q-like molecule with carbohydratebinding activity and associated SPs has been found in lamprey (Matsushita et al., 2004). The origins of the classical bypass pathways could be imagined to go further back than the standard CP, e.g., to such a primordial C1q molecule. When the adaptive immune system appeared, a C1q-like molecule could have developed antibody-recognition properties, thus connecting antibodies and complement activation. It is important to note that the bypass pathways are all relatively weak compared to the standard pathways, and have slower kinetics. Thus, their effects are only observable at close to physiological conditions (low serum dilution) and when the effects of the complete pathways are absent (deficiencies or surfaces activating only parts of complement), but of course these are exactly the conditions where they should be operational from a teleological point of view. The role of the bypass pathways has not been examined in humans, but absence of their effects may explain the observation that C1 or C4 deficiency usually causes more severe clinical symptoms than C2 deficiency (Morgan and Walport, 1991; Traustadottir et al., 1998; Klint et al., 2000). In general, they move the focus towards the PRMs, indicating that these are essential. In this connection, it will be exciting to obtain more data on the role played by the ficolins. A schematic overview of

the activation pathways of complement, according to the discussion above, is presented in Fig. 2.

Conclusion and future perspectives Instead of seeing complement as three distinct linear pathways converging in a common terminal pathway, one could, in analogy with the recent suggestions that many biological networks are ‘‘scale-free’’, e.g., networks of metabolic activity and protein interactions (Jeong et al., 2000, 2001; for an overview see Baraba´si and Bonabeau, 2003), consider the complement system a small ‘‘scale-free’’ network where C3 acts as the main hub integrating the other parts.2 The interplay of the three activation pathways confers on the system a greater degree of resilience to the removal of any component by providing bypass pathways. The one exception is of course C3, which is absolutely crucial, even though some effector functions of the pattern recognition receptors of complement are still working in its absence. It is important to bear in mind that the bypass pathways are much less efficient than the standard pathways, both in terms of degree of activation and kinetics. In the fully functional complement system, they are expected to play little role, but in the absence of one or more components they may come into play. Although the recent studies have attempted to approach more physiological conditions, e.g., by using low serum dilution, they are still quite far from the in vivo situation, thus little is known about the true physiological importance. Studies using knock-out animals for various components might shed some light on this in the future.

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