Looking back on the alternative complement pathway

The alternative pathway of complement originated from the Properdin pathway originally described by the Pillemer laboratory in the 1950s. This work generated great controversy and it took several decades for a consensus on its components, its reaction sequence and its functions to emerge. This paper reviews this history and attempts to clarify some of the ambiguities that remain.     

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site,is a selective inhibitor of the alternative complement pathway

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b α chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.     

Inhibition of complement alternative pathway in mice with Fab antibody to recombinant adipsin/factor D

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r-adipsin) were produced in rabbits. Anti-r-adipsin IgG was shown to bind to radiolabeled r-adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of antir-adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4-h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti-r-adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti-r-adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.     

Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles

Liposomes are becoming increasingly important as drug delivery systems, to target a drug to specific cells and tissues and thereby protecting the recipient from toxic effects of the contained drug. Liposome preparations have been described to activate complement. In this study, we have investigated complement activation triggered by neutral dimyristoyl-phosphocholine (DMPC) liposomes in human plasma and whole-blood systems. Incubation in plasma led to the generation of complement activation products (C3a and sC5b-9). Unexpectedly, investigations of surface-bound C3 revealed contact activated, conformationally changed C3 molecules on the liposomes. These changes were characterized by Western blotting with C3 monoclonal antibodies, and by incubating liposomes with purified native C3 and factors I and H. Quartz crystal microbalance analysis confirmed binding of C3 to planar DMPC surfaces. In addition, we demonstrated that DMPC liposomes bound to or were phagocytized by granulocytes in a complement-dependent manner, as evidenced by the use of complement inhibitors. In summary, we have shown that C3 is activated both by convertase-dependent cleavage, preferentially in the fluid phase, by mechanisms which are not well elucidated, and also by contact activation into C3(H₂O) on the DMPC surface. In particular, this contact activation has implications for the therapeutic regulation of complement activation during liposome treatment.     

A small fragment of factor B as a potential inhibitor of complement alternative pathway activity

BackgroundThe complement system is a key player in innate immunity and a modulator of the adaptive immune system. Among the three pathways of complement, the alternative pathway (AP) accounts for most of the complement activation. Factor B (FB) is a major protease of the AP, making it a promising target to inhibit the AP activity in conditions of uncontrolled complement activation.MethodsBased on the data obtained from sequence analysis and conformational changes associated with FB, we expressed and purified a recombinant FB fragment (FBfr). We tested the inhibitory activity of the protein against the AP by in vitro assays.ResultsFBfr protein was proven to inhibit the complement AP activity when tested by C3b deposition assay and rabbit erythrocyte hemolytic assay.ConclusionOur recombinant FBfr was able to compete with the native human FB, which allowed it to inhibit the AP activity. This novel compound is a good candidate for further characterization and testing to be used in complement diagnostic tests and as a drug lead in the field of complement therapeutics.     

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.     

Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis

We have shown previously that complement factor H (CFH) and complement factor B (CFB) are constitutively expressed by retinal pigment epithelial cells and their production is regulated by inflammatory cytokines, suggesting that the alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study, we further investigated the role of the AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Mice with EAU show increased levels of C3d deposition and CFB expression in the retina. Retinal inflammation was suppressed clinically and histologically by blocking AP‐mediated complement activation with a complement receptor of the Ig superfamily fusion protein (CRIg‐Fc). In line with reduced inflammation, C3d deposition and CFB expression were markedly decreased by CRIg‐Fc treatment. Treatment with CRIg‐Fc also led to reduced T‐cell proliferation and IFN‐γ, TNF‐α, IL‐17, and IL‐6 cytokine production by T cells, and reduced nitric oxide production in BM‐derived macrophages. Our results suggest that AP‐mediated complement activation contributes significantly to retinal inflammation in EAU. CRIg‐Fc suppressed retinal inflammation in EAU by blocking AP‐mediated complement activation with probable direct effects on C3/C5 activation of macrophages, thus leading to reduced nitric oxide production by infiltrating CRIg^? macrophages.     

p38/ATF-2通路参与C反应蛋白诱导的内皮细胞活化

目的:考察p38 MAPK/ATF-2通路在C反应蛋白(CRP)诱导的内皮细胞活化中的作用。方法:采用培养的人冠状动脉内皮细胞(HCAEC)第3~7代用于实验。CRP刺激诱导内皮细胞活化,给予p38抑制剂SB203580和SB202190干预。免疫印迹法检测p-e NOS、p-p38和p-ATF2的水平;ELISA法测定HCAEC分泌的黏附分子ICAM-1、VCAM-1和MCP-1的变化。结果:CRP呈浓度依赖性地抑制p-e NOS水平,CRP诱导HCAEC分泌ICAM-1、VCAM-1和MCP-1;CRP激活p38/ATF-2通路;SB203580和SB202190部分恢复p-e NOS水平和抑制CRP诱导的ICAM-1、VCAM-1和MCP-1分泌。结论:p38 MAPK/ATF-2通路参与CRP诱导的HCAEC活化。     

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9.     

Trichosanthin, an initiator of the alternative complement activation pathway.

Trichosanthin (TCS), a protein purified from the plant Trichosanthes Kirilowii Maxim, activates normal human serum complement via the alternative pathway, as shown by TCS-induced C3 conversion in normal serum and its prevention by depletion of factor B, but not with the addition of EGTA. Injection of TCS to BALB/c mice consumed the complement alternative pathway (APC) activity in serum, implying in vivo activation of the alternative pathway by TCS. Elevation of peripheral blood leucocyte count as well as protein exudation and neutrophil accumulation in the peritoneal cavities could be induced by peritoneal injection of TCS. The main effect of complement activation by TCS was demonstrated to be induction of neutrophil accumulation.     

补体经典激活途径C3转化酶的体外组装及活性观察

目的：体外组装包含人C4分子的补体经典激活途径C3转化酶，并对其转化酶活性及衰变特性进行观察。方法：利用豚鼠血清功能纯C1、C2及溶血中间体EAC4^hu体外组装经典途径C3转化酶，观察不同C1、C2用量及孵育温度对C3转化酶形成和自发性衰变的影响，以及人红细胞膜抽提蛋白对C3转化酶衰变化的影响。结果：高剂量和低剂量的C1均会影响C3转化酶的形成，增加C2用量可增加C3转化酶的形成数量，C3转化酶的自发性衰变随孵育温度的升高而加速，人红细胞膜抽提蛋白可抑制C3转化酶的自发性衰变过程，结论：C1、C2用量及孵育温度是影响C3转化酶形成和自发性衰变的主要因素，体外组装的补体经典激活途径C3转化酶可应用于相关补体调控蛋白的活性检测。     

Interactions between mannose-binding lectin and MASPs during complement activation by the lectin pathway

The lectin pathway of complement performs a key role within the immune system by recognising pathogens through patterns of sugar moieties displayed on their cell surfaces and neutralising them via an antibody-independent reaction cascade. While particularly important during early childhood before the adaptive immune system is established, or when adaptive immunity is compromised, it has a protective function throughout life, neutralising invading pathogens directly and helping to stimulate and direct an effective immune response. Complement activation is initiated when complexes comprising mannose-binding lectin (MBL) or serum ficolins and MBL-associated serine protease-2 (MASP-2) bind to pathogens. Binding induces conformational changes in these complexes, leading to autoactivation of the MASPs, which in turn activate the downstream reaction cascade. A major goal in complement research is to understand the molecular events that trigger complement activation. Over the last few years, structure-function studies have improved our knowledge of the way in which MBL binds to MASPs by defining the portions of these proteins that interact and by solving the structures of key protein fragments. In this review, I will summarise the main findings of these studies and describe current theories to explain how the components combine to initiate the reaction cascade.     

Specific antibodies enhance Sephadex-induced activation of the alternative complement pathway in human serum

Sephadex beads, which resemble cellulose in their basic chemical structure, were used to study the molecular mechanisms by which cellulosic dialysis membranes activate the alternative complement pathway in normal human serum. Sera from different individuals were found to vary in the extent of activation which occurred following incubation with a fixed amount (surface area) of polymer. Preadsorption of serum with an excess of Sephadex at 2°C resulted in loss of activation when the adsorbed serum was interacted with fresh Sephadex beads. Acid eluted proteins from adsorbed Sephadex restored the capacity of preadsorbed serum to activate complement in the presence of fresh Sephadex. Adsorption of the immunoglobulin G (IgG) fraction and of F(ab′)2 fragments from IgG prepared from the plasma of a normal individual with Sephadex, resulted in the specific binding of some IgG and F(ab′2) molecules to the particles. IgG and F(ab′)2 coated beads activated complement in Sephadex-adsorbed serum. Thus, specific anti-dextran IgG antibodies trigger activation of the alternative complement pathway by Sephadex in human serum. The effect is independent of the Fc region of IgG. These results suggest that specific antibodies could be important in determining complement activation in vivo in patients undergoing haemodialysis with cellulosic membranes.     

The phylogeny of the complement system and the origins of the classical pathway

The origins of the complement system have now been traced to near to the beginnings of multi-cellular animal life. Most of the evidence points to the earliest activation mechanism having been more similar to the lectin pathway than to the alternative pathway. C1q, the immunoglobulin recognition molecule of the classical pathway of the vertebrates, has now been shown to predate the development of antibody as it has been found in the lamprey, a jawless fish that lacks an acquired immune system. In this species, C1q acts as a lectin that binds MASPs and activates the C3/C4-like thioester protein of the lamprey complement system. The classical pathway can, therefore, be regarded as a specialised arm of the lectin pathway in which the specificity of C1q for carbohydrate has been recruited to recognise the Fc region of immunoglobulin.     

Trypanosoma cruzi calreticulin inhibits the complement lectin pathway activation by direct interaction with L-Ficolin

Trypanosoma cruzi, the agent of Chagas’ disease, the sixth neglected tropical disease worldwide, infects 10–12 million people in Latin America. Differently from T. cruzi epimastigotes, trypomastigotes are complement-resistant and infective. CRPs, T-DAF, sialic acid and lipases explain at least part of this resistance. In vitro, T. cruzi calreticulin (TcCRT), a chaperone molecule that translocates from the ER to the parasite surface: (a) Inhibits the human classical complement activation, by interacting with C1, (b) As a consequence, an increase in infectivity is evident and, (c) It inhibits angiogenesis and tumor growth. We report here that TcCRT also binds to the L-Ficolin collagenous portion, thus inhibiting approximately between 35 and 64% of the human complement lectin pathway activation, initiated by L-Ficolin, a property not shared by H-Ficolin. While L-Ficolin binds to 60% of trypomastigotes and to 24% of epimastigotes, 50% of the former and 4% of the latter display TcCRT on their surfaces. Altogether, these data indicate that TcCRT is a parasite inhibitory receptor for Ficolins. The resulting evasive activities, together with the TcCRT capacity to inhibit C1, with a concomitant increase in infectivity, may represent T. cruzi strategies to inhibit important arms of the innate immune response.     

Antibody-mediated activation of the classical pathway of complement may compensate for mannose-binding lectin deficiency

Deficiency of mannose-binding lectin (MBL), a recognition molecule of the lectin pathway of complement, is associated with increased susceptibility to infections. The high frequency of MBL deficiency suggests that defective MBL-mediated innate immunity can be compensated by alternative defense strategies. To examine this hypothesis, complement activation by MBL-binding ligands was studied. The results show that the prototypic MBL ligand mannan can induce complement activation via both the lectin pathway and the classical pathway. Furthermore, antibody binding to mannan restored complement activation in MBL-deficient serum in a C1q-dependent manner. Cooperation between the classical pathway and the lectin pathway was also observed for complement activation by protein 60 from Listeria monocytogenes. MBL pathway analysis at the levels of C4 and C5b-9 in the presence of classical pathway inhibition revealed a large variation of MBL pathway activity, depending on mbl2 gene polymorphisms. MBL pathway dysfunction in variant allele carriers is associated with reduced MBL ligand binding and a relative increase of low-molecular-mass MBL. These findings indicate that antibody-mediated classical pathway activation can compensate for impaired target opsonization via the MBL pathway in MBL-deficient individuals, and imply that MBL deficiency may become clinically relevant in absence of a concomitant adaptive immune response.     

Multiple routes of complement activation by Mycobacterium bovis BCG

Mycobacterium tuberculosis is the leading cause of infectious disease in humans in the world. It evades the host immune system by being phagocytosed by macrophages and residing intracellularly. Complement-dependent opsonisation of extracellular mycobacteria may assist them to enter macrophages. This work examines in detail the mechanisms of complement activation by whole mycobacteria using Mycobacterium bovis BCG as a model organism. M. bovis BCG directly activates the classical, lectin and alternative pathways, resulting in fixation of C3b onto macromolecules of the mycobacterial surface. Investigation into the classical pathway has shown direct binding of human C1q to whole mycobacteria in the absence of antibodies. Most human sera contain IgG and IgM-anti-(M. bovis BCG), and pre-incubation with human immunoglobulin enhances C1q binding to the bacteria. Therefore classical pathway activation is both antibody-independent and dependent. The bacteria also activate the alternative pathway in an antibody-independent manner, but Factor H also binds, suggesting some regulation of amplification by this pathway. For the lectin pathway we have demonstrated direct binding of both MBL and L-ficolin from human serum to whole mycobacteria and subsequent MASP2 activation. H-ficolin binding was not observed. No M. bovis BCG cell surface or secreted protease appears likely to influence complement activation. Together, these data provide a more detailed analysis of the mechanisms by which M. bovis BCG interacts with the complement system.     

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.     

Carbamylation of immunoglobulin abrogates activation of the classical complement pathway

Post‐translational modifications of proteins significantly affect their structure and function. The carbamylation of positively charged lysine residues to form neutral homoitrulline occurs primarily under inflammatory conditions through myeloperoxidase‐dependent cyanate (CNO^?) formation. We analyzed the pattern of human IgG₁ carbamylation under inflammatory conditions and the effects that this modification has on the ability of antibodies to trigger complement activation via the classical pathway. We found that the lysine residues of IgG₁ are rapidly modified after brief exposure to CNO^?. Interestingly, modifications were not random, but instead limited to only few lysines within the hinge area and the N‐terminal fragment of the CH2 domain. A complement activation assay combined with mass spectrometry analysis revealed a highly significant inverse correlation between carbamylation of several key lysine residues within the hinge region and N‐terminus of the CH2 domain and the proper binding of C1q to human IgG₁ followed by subsequent complement activation. This severely hindered complement‐dependent cytotoxicity of therapeutic IgG₁. The reaction can apparently occur in vivo, as we found carbamylated antibodies in synovial fluid from rheumatoid arthritis patients. Taken together, our data suggest that carbamylation has a profound impact on the complement‐activating ability of IgG₁ and reveals a pivotal role for previously uncharacterized lysine residues in this process.     

Ambivalent effect of immunoglobulins on the complement system: activation versus inhibition

Pathogen and self-specific antibodies are known for their ability to trigger generation of active complement fragments that then serve as effectors of cell damage. The remainder of the immunoglobulin pool of the host has the capacity to quench harmful effects of activated complement cascade by preventing active complement fragments from binding to their specific receptors. This scavenging function is mediated by different acceptor sites within the immunoglobulin molecule. Large fragments, such as C3b and C4b are preferentially bound to the Fc region, while biologically potent C3a and C5a anaphylatoxins get neutralized by low-affinity interaction with the constant domain of the F(ab)^′_2. The ambivalent effect of immunoglobulins on the complement system implies their role in homeostasis as well as expansion of use of high-dose intravenous immunoglobulins in diseases and states mediated by inappropriate complement activation.