The classical pathway is, in general, activated by clusters of IgG or IgM, whose Fc regions are bound by the recognition molecule C1q. C1q has a hexameric structure, which exhibits low affinity for monomeric IgG but binds much stronger to aggregated IgG [77]. There is an IgG subclass difference in activation, i.e. IgG3 is the most reactive, followed by IgG1 and IgG2. C1q does not bind to IgG4 [11]. C1q also does not bind to free IgM, but antigen-bound IgM exposes new sites in the CH3 domain for C1q recognition and complement activation [111]. Although the antigen-bound IgG/IgM is viewed as the traditional target for C1q binding and classical pathway activation, C1q is a pattern recognition molecule, and a growing number of structures are now acknowledged as C1q ligands. These ligands include pentraxins (C-reactive protein) [2], abnormal endogenous proteins (prions, β-amyloloid fibers) [131], structures associated with cell death (phosphatidylserine, DNA) [108, 109] and glycosaminoglycans/proteoglycans [38, 53].

While not every ligand will activate complement, C1q binding may induce clearance of particles via direct opsonization, implicated especially in the removal of apoptotic cells [103, 155]. The majority of C1q forms a calcium-dependent C1 complex with C1r₂ and C1s₂ [169]. Upon activation, a conformational change within C1q occurs, which leads to enzymatic cleavage and thereby activation of the serine protease C1r, and this in turn cleaves and activates the serine protease C1s. C1s cleaves C4 into C4a and C4b, with C4b covalently attaching to the adjacent activation site (e.g. a cell coated with antibodies) and the small C4a fragment diffusing into the fluid phase. C2 associates with C4b and is cleaved by C1s into C2a and C2b. The C2a and C4b form on the target cell surface the classical pathway C3 convertase (C4bC2a), which cleaves C3 into C3a and C3b. C3a is released into the fluid phase as an anaphylatoxin and C3b is deposited on the target cell where it serves as an opsonin [capable of binding to the complement receptor (CR)1 on phagocytes and erythrocytes] or is further cleaved to iC3b as another opsonin [capable of binding to the CR3 receptor (CD11b/CD18) on phagocytes]. Moreover, C3b interacts with C4bC2a to form the C5 convertase of the classical pathway (C3bC4bC2a).

The surface-bound C3b is also a trigger for direct alternative pathway activation, which underlines the importance of the classical pathway for initiating an activation that subsequently is amplified within the alternative pathway. Even when the trigger of complement activation is solely dependent on the classical or lectin pathway, a large fraction of C3 cleavage can actually arise from an amplification loop within the alternative pathway. This pathway is initiated spontaneously by a certain amount of constant hydrolysis of the C3 thiol-ester bond, thus rendering C3(H₂O), which is “C3b-like” and binds factor B [55]. All three complement activation pathways (classical, lectin and alternative pathway) share the same terminal C5-C9 activation sequence (Fig. 8.1).

Complement deficiencies within the classical pathway have highlighted the importance of this pathway in the elimination of pyogenic encapsulated bacteria, especially Streptococcus pneumoniae and Haemophilus influenzae strains. Classical pathway activation effectively opsonizes these bacteria for phagocytosis by myeloid cells. Furthermore, classical pathway deficiencies increase the susceptibility for autoimmune disorders, especially for SLE or an SLE-like disease; this is particularly evident for C1q deficiency [141]. The mechanism for this increased susceptibility is not yet fully understood, but lack of C1q leads to impaired clearance of immune complexes and apoptotic cells, thereby increasing the exposure of autoantigens (the waste-disposal hypothesis) to the immune system [92]. The importance of C1q for the maintenance of B-cell tolerance against self-antigens and C1q-dependent cytokine response may also play a role in the pathogenesis of SLE upon C1q deficiency [156, 165].

Classical pathway deficiencies (C1, C4, and C2) are inherited in an autosomal recessive pattern. Deficiencies of C1 and C4 are rare, and the genetics are complex, involving multiple genes for each component. C1q (encoded by C1QA, OMIM*120550, C1QB, OMIM*120570 and C1QC OMIM*120575), C1r (C1R, OMIM*613785), and C1s (C1S, OMIM*120580) are required for C1 function. Thus, if any one of these subunits is missing, the functional complex cannot form. Deficiency of C1q is either due to a failure in synthesis or to the synthesis of a non-functional low-molecular weight C1q [88]. Twelve causative mutations within C1QABC have been described, in 64 confirmed cases from 38 families (2011) [126]. Fourteen cases of C1r and C1s deficiency have been reported (2010) [88]. The C1r and C1s genes are closely linked, and patients deficient in C1r are usually low in C1s as well [88]. C4 exists in humans in two forms, C4A (acidic) and C4B (basic), encoded by two adjacent coexisting genes (C4A, OMIM*120810 and C4B, OMIM*120820). All four alleles must be deleted or defective to cause total C4 deficiency [125]. Moreover, both genes may be present in up to seven copies and contain numerous polymorphisms. Complete C4 deficiency is extremely rare and has only been described in 28 cases (2010) [88].

C2 deficiency (C2, OMIM*613927) is the most common classical pathway component deficiency, with an incidence of 1 homozygous case per 20,000 in Western white populations [114]. C2 deficiencies are classified into two types: type I representing absence of C2 synthesis and type II representing failure in protein secretion [67]. Nine out of ten cases are of type I, with a majority caused by a 28-bp deletion in exon 6 [146].

Patients with a deficiency of an early classical pathway component (C1, C4, or C2) are predisposed to infections with pyogenic encapsulated bacteria, but the infections are usually milder than those observed in patients with a deficiency of properdin, C3, or a terminal component. A study of 40 C2-deficient patients in Sweden reported 57 % of them to have invasive infections, of which 30 % had repeated infections [68]. Most significant, deficiencies of classical pathway components are frequently associated with the development of autoimmune syndromes, particularly SLE. Individuals with total deficiency of any component of the C1 complex or C4 display a higher occurrence of SLE and usually more severe manifestations of the disease compared to a deficiency of C2 [88]. The onset is in general early, and the female to male ratio in C1/C4 deficiency is 1:1, in contrast to deficiency of C2, in which the epidemiology of SLE has a later onset and with a female predominance. Of the 64 described cases of C1q deficiency, 88 % exhibited SLE or SLE-like disease [126].

The corresponding fractions of patients with SLE in C1s/r and C4 deficiency are 57 % and 75 %, respectively [114]. Also partial C4 deficiency is associated with the disease, primarily regarding C4A. Homozygous C4A deficiency is up to 5 times more prevalent in the SLE patient cohort compared to the healthy population [88]. Approximately 10 % of the C2-deficient subjects develop SLE [114]. The highly significant relation between classical pathway deficiencies and the development of an SLE-like disease underline the critical function of the classical pathway, primarily connected with the clearance of immune complexes and apoptotic cells [9]. C2 deficiency is also associated with an increased prevalence of atherosclerosis and cardiac disease [68].

Classical pathway deficiency can be detected by the traditional CH50 assay, based on hemolysis of sensitized sheep erythrocytes, or by the novel ELISA screening assay for all pathways [129]. An absence or a decrease of CH50 in the presence of normal AH50 indicates that at least one of the early components of the classical pathway is missing or low [115]. Complete deficiencies of C1q, C1r or C1s will induce a low classical pathway activity, but a normal lectin and alternative pathway activity in the ELISA screening test. A complete deficiency of C4 or C2 will lead to a low classical and lectin pathway activity, whereas the alternative pathway activity will be normal.

The identification of the missing component follows from the recovery of complement activity in either of these tests by the addition of a purified component, by immunochemical tests, or by gene sequencing of the component in question.

Treatment of patients with complement deficiencies is based on treatment of the clinical manifestations. Identification of autoimmune diseases in patients with deficiencies of early components of the classical pathway is necessary, anti-inflammatory therapies should be used for management of the autoimmune disease. Recurrent infections in these patients should be managed with appropriate antibiotic therapy and vaccination [162]. Allogenic Hematopoietic stem cell transplantation (HSCT) has recently been applied for a few number of patients with C1q deficiency that were resistant to medical therapy to cure SLE [107]. While autoimmune disease is often the dominant clinical picture, death is most often due to infection. Therefore, education and strategies to ensure ready access to medical care and antibiotics is critical. Furthermore, studies suggest an important role for complement in atherosclerosis, and aggressive management of cardiac risks is important.

The overall structure of the lectin pathway (Fig. 8.1) is similar to that of the classical pathway, and both pathways share C4 and C2 during downstream activation. The pattern recognition molecules of the lectin pathway comprise five lectins capable of initiating activation: mannan-binding lectin (MBL), ficolin 1, 2 and 3 (also termed M-, L-, H-ficolin) and collectin 11 (also termed collectin kidney 1) [24, 25, 54, 58]. Collectin 11 is the most recently characterized member of the complement-activating lectins, but has until now only been shown to activate complement in vitro [58, 90]. Currently, the data describing in which context and to what extent collectin 11-dependent opsonization and activation is significant, are limited.

Activation of the lectin pathway is initiated by any of these lectins binding specific carbohydrate structures (MBL) or patterns of acetyl groups (ficolins). Analogously to C1s/C1r in the classical pathway, serine proteases are associated to the recognition molecules of the lectin pathway. These proteases are termed MASP-1, MASP-2 and MASP-3. MASP-2 is ubiquitous for any lectin pathway activation; it becomes activated after lectin binding and cleaves both C4 and C2 [3, 56]. Recent data suggest MASP-1 to be equally important for lectin pathway activation, acting as a crucial activator of MASP-2 [56]. MASP-1 is not able to cleave C4 but has been indicated to substantially contribute to C2 cleavage [56]. Currently, the relevance of MASP-3 in complement activation is not clear [22]. The cleavage of C2 and C4 results in the formation of a C3 convertase (C4bC2a), which is identical to that of the classical pathway. The same is true for the subsequent downstream events.

Deficiency of MBL (MBL2, OMIM*154545) is predominantly caused by one of three SNPs in exon 1 of the MBL2 gene, coding for the allelic variants referred to as B, C and D, with A being wild-type MBL [40, 87, 91, 147]. Any of these mutations affect the essential assembly of a functional MBL oligomer from MBL monomers [39, 40]. There are also additional SNPs in the promoter region of MBL2 that affect the serum MBL level [39, 40]. These variants of MBL2 are quite common, and thus, MBL deficiency is seen in many populations. MBL deficiency is not a complete deficiency but is related to a serum “cut-off” value, often <100 ng/mL (normal levels range from 0.5 to 5 μg/mL). Referring to this value, approximately 10 % of Caucasians are MBL deficient, and up to 30 % exhibit levels below the normal range [124, 151]. Thus, MBL deficiency is one of the most common protein deficiencies in humans. It has been claimed that the genetic evolution of MBL2 represents heterosis, implying that there is an advantage of being heterozygous [57]. According to this concept, a complete deficiency of MBL might increase infection susceptibility, whereas high levels of the protein may lead to host tissue damage caused by excessive complement activation (“the double-edged sword”) [99].

In contrast to MBL, deficiencies in the ficolins and in collectin 11 are rare. The ficolin genes are polymorphic, since both ficolin 1 (FCN1, OMIM*601252) and ficolin 2 (FCN2, OMIM*601624) present polymorphisms in the promoter and structural parts that affect the serum levels, but no cases of complete ficolin 1 or 2 deficiency have been described to date. In 2009, a complete ficolin 3 (FCN3, OMIM*604973) deficiency was described in a patient suffering from recurrent severe pulmonary infections [101]. A frameshift mutation (FCN3 + 1637delC) in exon 5 of FCN3 caused a complete ficolin 3 deficiency in the homozygous patient. The allele frequency of this mutation was reported to be about 0.01 [101, 102]. Collectin 11 (COLEC11, OMIM*612502) is highly conserved, and the function within the complement system is to date largely unknown. Recently, two reports by Sirmaci et al. and Rooryck et al. linked mutations within the MASP1 (OMIM*600521) and COLEC11 genes to developmental disorders classed in the 3MC syndrome [119, 134]. Mutations within the well-conserved carbohydrate recognition domain of COLEC11, with premature termination or deletion of exon 1–3, were found in seven families. Collectin 11 was further linked to embryonic development in zebrafish, in which a functional loss of collectin 11 leads to craniofacial abnormalities that could be rescued by injection of human collectin 11 mRNA [119].

MASP1 codes for the three splice variants MASP-1, MASP-3 and MAP44, the latter lacking the serine protease domain. Mutations within the exon specific for MASP-3 have been suggested to be causative for the 3MC syndrome, found in six families. MASP1 was also linked to the embryonic development in the zebrafish [119]. MASP-2 (MASP2, OMIM*605102) deficiency was first described in 2003 by Stengaard-Pedersen et al. in a patient with non-functional lectin pathway as a result of a homozygous missense mutation (p.D120G) in exon 3 of MASP2 [142]. Later studies have shown MASP-2 deficiency to be fairly common, caused by several different polymorphisms, of which the frequent p.D120G has an allelic frequency of 0.01–0.04 in Caucasians [13, 153].

Deficiency of MBL generally increases the risk of any type of infection but is mostly related to an increased frequency of pyogenic infections, including pneumococcal infection and sepsis, particularly in neonates/infants or in patients undergoing immunosuppressive treatment. The increased risk in infants can be explained by the “window” period from 6 to 18 months of life, in which the mother’s antibodies have disappeared and the levels of those of the child are still insufficient, resulting in reduced immune defense. Normally, MBL deficiency is not associated with an increased incidence of infections, but MBL may be of importance as a redundancy protein in immunosuppressed patients, e.g. those treated with cytostatics, irradiation for malignancy or after HIV infection. MASP-2 deficiency is associated with an increased susceptibility to infection and autoimmunity disease [142]. Lung infections and cystic fibrosis have been reported in patients with MASP-2 deficiency, but homozygous mutations are also found in the healthy population [37, 106, 153]. Ficolin 3 deficiency has been described in one patient suffering from recurrent respiratory disease and in two premature infants with severe necrotizing enterocolitis [101, 127]. A deficiency in the lectin pathway may lead to autoimmune diseases [114], but may also modify the progress of a disease to be more benign, as seen in rheumatoid arthritis patients [41], or to be more serious, as seen in cystic fibrosis [42]. A 2- to 3-fold increased incidence in MBL deficiency has been reported in patients with lupus erythematosus [75]. Some studies have shown that MBL deficiency is related to cardiovascular disease, in which a deficiency might be either beneficial or detrimental [105, 121]. Thus, at present, any conclusion with respect to the clinical consequences of MBL deficiency can hardly be drawn.

Whole exome sequencing has revealed a link between mutations within the MASP1 gene and the cause of the 3MC syndrome [134]; this was later extended also to mutations within the COLEC11 gene. Patients with the 3MC syndrome experience a spectrum of developmental disorders, of which the most common are growth and mental retardation, facial dysmorphism (e.g. hypertelomerism, cleft lip and palate, high arched eyebrows) and hearing loss [154]. Complement has, in experimental animal models, been implicated in embryonic development [4, 143, 157], but this is the first link in humans in which mutations within complement genes are related to the pathogenesis of a developmental disorder.

MBL-dependent activation of the lectin pathway can be assessed by a screening ELISA [129]. MBL deficiency and MASP-2 deficiency will cause low lectin pathway activity, whereas classical and alternative pathway activities will be normal. Moreover, a functional assay of MBL binding and capacity to activate C4 has also been described, in which the amount and function of later complement components are not of importance [112]. Further analysis includes immunochemical quantitation of MBL and genotyping of MBL and MASP-2.

Patients with recurrent infections may benefit from antibiotic prophylaxis and immunization with a polyvalent pneumococcal vaccine [162]. Purified MBL for therapeutic use as a substitution therapy is under development [158], but the indications for such treatment remains at the moment unclear.

In contrast to the classical and lectin pathways, the alternative pathway is triggered primarily not by the exposure of molecular patterns of pathogenic or damaged cells, but instead by any surface that lacks sufficient complement activation regulation. The trigger of the alternative pathway is C3b and C3(H₂O), the latter being a C3b-like molecule that at a slow rate is produced in plasma by spontaneous hydrolysis of native C3 (a process termed “C3 tick-over”) (Fig. 8.1) [110]. Once C3b/C3(H₂O) is exposed on surfaces or in the fluid phase, respectively, it associates with factor B, which subsequently is cleaved by factor D into Ba and Bb. Ba is released into the fluid phase, and Bb forms, together with C3b/C3(H₂O), the C3 convertase of the alternative pathway C3bBb [27]. Analogous to the C4b2a, C3bBb cleaves C3 into fluid phase-released C3a and surface-deposited C3b. C3b of any origin (classical, lectin or alternative pathway) triggers further alternative pathway activation. In this way, the alternative pathway is responsible for a potent amplification of complement activation, referred to as the “amplification loop” [55, 82]. Alternative pathway activation/amplification is efficiently counteracted by membrane-bound and fluid-phase regulators present at or in close proximity to host cells, of which a majority is focused at the level of convertase formation and C3b degradation. Properdin is a positive regulator of the alternative pathway, as it acts by stabilizing the C3bBb complex. The properdin-stabilized C3bBbP has a 10-fold higher stability than C3bBb [26]. Properdin has been proposed to be a pattern recognition molecule of the alternative pathway, suggested to bind directly to surfaces (e.g. to LPS) and attract C3b, thereby triggering alternative pathway activation [76, 139]. This may explain the increased susceptibility to neisserial infection in cases of properdin deficiency. An alternative pathway C5 convertase is formed when C3b associates to C3bBb, thus activating the terminal pathway as in the classical and lectin pathways.

Early alternative pathway deficiencies of factor D (CFD, OMIM*134350) and properdin (CFP, OMIM*300383) are rare. No factor B (CFB, OMIM + 138470) deficient individuals have been found to date. Total homozygous deficiency of factor D has been described in three families [5, 59, 140], of which two were defined at the molecular level [5, 137]. Properdin deficiency is found more frequently than factor D deficiency. The properdin gene is located on the X-chromosome, therefore the properdin deficiency is generally present only in males, whereas female carriers typically have half of the normal plasma level [32]. Properdin deficiency is classified into three types [135]; type I representing complete absence in protein, type II with diminished protein levels (typically 1–10 % of normal levels) but still functionally active and type III presenting with properdin in normal levels but not functional. Type I is the most frequent phenotype of properdin deficiency, predominantly caused by frameshift mutations and premature stop codons, leading to transcription of a truncated protein, which is rapidly degraded [32].

Alternative pathway component (properdin, factor D) deficiency is associated with severe, fulminant infections by Neisseria gonorrhoeae or N. meningitidis, with a high mortality rate [32]. All index patients in factor D-deficient families presented with Neisseria meningitides infection [140]. Properdin deficiency is significantly linked to meningococcal disease, the risk of fulminant and fatal meningococcal infection was estimated to be 250 times higher in the type I-deficient patients compared to the general population [31]. The disease is often seen with sepsis, and the lethality is higher in deficient patients compared to patients with normal properdin levels [31]. The deficiency of alternative pathway components is not associated with autoimmune diseases [9].

The traditional AH50, based on the lysis of rabbit or guinea pig erythrocytes, has been used for screening of alternative pathway-component deficiencies but is hampered by not always detecting properdin deficiency. This problem does not occur with the ELISA screening assay [129]. A defect in alternative pathway components (factor B, factor D or properdin) will yield low alternative pathway activity with normal classical and lectin pathway activity. Alternative pathway components can be further evaluated by functional and immunochemical tests, and by gene sequencing.

Patients with alternative pathway-component deficiencies should be vaccinated with the tetravalent meningococcal vaccine, particularly important in properdin-deficient persons [136]. Early antibiotic treatment is mandatory.

Present at approximately 1.3 mg/mL in blood, complement component 3 (C3) is the most abundant complement protein in plasma. C3, together with factor B and MASP, is thought to be among the most ancient complement proteins [166]. By displaying a variety of conformations and fragments, it exerts multiple functions in the cascade, including alternative pathway activation, convertase formation, opsonization (C3b/iC3b), stimulation of inflammatory cells (C3a), and B-cell stimulation (C3d).

Recent crystal structure determination of native C3, C3c [66] and C3b in complex with factor B and D [33] has led to new insights into this complex component. Native C3 is a rather inert molecule, consisting of an alpha-chain and a beta-chain held together with a disulfide bond [122]. Upon proteolytic cleavage by a C3 convertase, C3a is split from the alpha chain. The remaining C3b undergoes a major conformational change that leads to the exposure of a previously hidden reactive thioester. In the vicinity of a surface, this thioester mediates the covalent attachment of C3 to hydroxyl groups or amines, whereas in the absence of an accepting surface, it hydrolyzes within fractions of a second [49]. Surface-bound C3b is able to interact with factor B in an alternative pathway C3 convertase as well as with complement receptors (CR) 1, 3, and 4. Following surface attachment, C3b is prone to inactivation by factor I, in which case the alpha chain is cleaved into iC3b, which has even higher affinity for the important CR3 phagocytic receptor. iC3b can then be further degraded to C3c and C3dg [19]. Apart from being proteolytically activated, C3 can adopt a C3b-like conformation through the spontaneous hydrolysis of the thioester of fluid phase C3. C3(H₂O) still retains C3a in the alpha chain but has C3b-like functions, including alternative pathway C3 convertase formation and receptor interactions [49].