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11.10D: The Complement System - Biology

11.10D: The Complement System - Biology


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The complement system helps antibodies and phagocytic cells clear pathogens from an organism.

Learning Objectives

  • Describe the function of the complement system

Key Points

  • The complement system has originally been identified as the part of the immune system called the innate immune system.
  • The complement system can also be recruited and brought into action by the adaptive immune system.
  • The three biochemical pathways that activate the complement system are the classical complement pathway, the alternative complement pathway, and the lectin pathway.
  • The complement system consists of small proteins found in the blood, generally synthesized by the liver, and normally circulating as inactive precursors. When stimulated by a trigger, proteases in the system cleave specific proteins to release cytokines that amplify further cleavages.
  • The end-result of this activation cascade is the massive amplification of the response and activation of the cell-killing membrane attack complex.

Key Terms

  • opsonization: the process of an antigen bound by antibody or complement to attract phagocytic cells.

The Complement System

The serum complement system, which represents a chief component of innate immunity, not only participates in inflammation but also acts to enhance the adaptive immune response. Specific activation of the complement via innate recognition proteins or secreted antibody releases cleavage products that interact with a wide range of cell surface receptors found on myeloid, lymphoid, and stromal cells. This intricate interaction among complement activation products and cell surface receptors provides a basis for the regulation of both B and T cell responses.

The complement system plays a crucial role in the innate defense against common pathogens. Activation of the complement leads to robust and efficient proteolytic cascades, which terminate in opsonization and lysis of the pathogen as well as in the generation of the classical inflammatory response through the production of potent proinflammatory molecules. More recently, however, the role of the complement in the immune response has been expanded due to observations that link complement activation to adaptive immune responses. It is now understood that the complement is a functional bridge between innate and adaptive immune responses that allows an integrated host defense to pathogenic challenges.

Activation of the Complement System

The complement system can be activated through three major pathways: classical, lectin, and alternative. Initiation of the classical pathway occurs when C1q, in complex with C1r and C1s serine proteases (the C1 complex), binds to the Fc region of complement-fixing antibodies (generally IgG1and IgM) attached to pathogenic surfaces. Autocatalytic activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments. The larger fragments associate to form C4bC2a on pathogenic surfaces, and the complex gains the ability to cleave C3 and is termed the C3 convertase.

Generation of the C3 convertase, which cleaves C3 into the anaphylatoxin C3a and the opsonin C3b, is the point at which all complement activation cascades converge. When C3 is cleaved into C3b, it exposes an internal thioester bond that allows stable covalent binding of C3b to hydroxyl groups on proximate carbohydrates and proteins. This activity underpins the entire complement system by effectively “tagging” microorganisms as foreign, leading to further complement activation on and around the opsonized surface and terminating in the production of anaphylatoxins and assembly of membrane attack complexes.

Functions of the Complement System

The functions of the complement system, oposonization, lysis, and generation of the inflammatory response through soluble mediators, are paradigmatic and represent a well-characterized component of an innate host defense. It has become increasingly understood that complement functions in host defense extend beyond innate immune responses. The finding that B lymphocytes bound C3 raised the question as early as in the 1970s as to whether the complement system was involved in adaptive immune responses. Subsequent work demonstrated that depletion of C3 impaired humoral immune responses and provided direct evidence that efficient adaptive responses were contingent on an intact complement system in some cases.

Further study in animals bearing natural complement deficiencies implicated the classical pathway as a crucial mechanism for efficient antigen trapping and retention in lymphoid tissues (e.g., splenic follicles), suggesting that a major function of the complement system was to localize foreign antigens into immune sites important for lymphocytes responses.


Complement and the Regulation of T Cell Responses

The complement system is an evolutionarily ancient key component of innate immunity required for the detection and removal of invading pathogens. It was discovered more than 100 years ago and was originally defined as a liver-derived, blood-circulating sentinel system that classically mediates the opsonization and lytic killing of dangerous microbes and the initiation of the general inflammatory reaction. More recently, complement has also emerged as a critical player in adaptive immunity via its ability to instruct both B and T cell responses. In particular, work on the impact of complement on T cell responses led to the surprising discoveries that the complement system also functions within cells and is involved in regulating basic cellular processes, predominantly those of metabolic nature. Here, we review current knowledge about complement's role in T cell biology, with a focus on the novel intracellular and noncanonical activities of this ancient system.


Complement System Overview

The complement system is one of the major mechanisms by which pathogen recognition is converted into an effective host defense against initial infection. Complement is a system of plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody, leading to a cascade of reactions that occurs on the surface of pathogens and generates active components with various effector functions.

There are three pathways of complement activation: the classical pathway, which is triggered directly by pathogen or indirectly by antibody binding to the pathogen surface the MB-lectin pathway and the alternative pathway, which also provides an amplification loop for the other two pathways.

All three pathways can be initiated independently of antibody as part of innate immunity. The early events in all pathways consist of a sequence of cleavage reactions in which the larger cleavage product binds covalently to the pathogen surface and contributes to the activation of the next component.

The pathways converge with the formation of a C3 convertase enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement components, especially bound C3b and its inactive fragments, are recognized by specific complement receptors on phagocytic cells, which engulf pathogens opsonized by C3b and its inactive fragments.

The small cleavage fragments of C3, C4, and especially C5, recruit phagocytes to sites of infection and activate them by binding to specific trimeric G protein-coupled receptors. Together, these activities promote the uptake and destruction of pathogens by phagocytes. The molecules of C3b that bind the C3 convertase itself initiate the late events, binding C5 to make it susceptible to cleavage by C2b or Bb.

The larger C5b fragment triggers the assembly of a membrane attack complex/MAC, which can result in the lysis of certain pathogens. The activity of complement components is modulated by a system of regulatory proteins that prevent tissue damage as a result of inadvertent binding of activated complement components to host cells or spontaneous activation of complement components in plasma.


Complement beyond the cascade

The effector mechanisms of complement contribute directly to the elimination of undesired particles however, full capacity of the complement system is only achieved through its extensive collaboration with other defence systems 1,5 . Indeed, a plethora of crosstalk mechanisms have been described and extensively reviewed over the past few years 1,5,10 . Although the extent of its molecular and cellular connections could suggest an indispensable role for complement in the coordination of immunological responses, it is important to remember its primary role as a first-in-line threat detector. The crosstalk between effectors generated after upstream complement-mediated sensing of PAMPs and DAMPs with other defence systems is important for translating the threat message into cellular signals that modulate a downstream response 1,8 . Whereas a reduction in complement crosstalk, for example due to deficiencies in complement components, could influence the responsiveness of the system to certain insults, the overall effect on the functioning of the connected systems might often be small. Conversely, as discussed later, inadvertent triggering of the complement system will often have downstream inflammatory consequences. The following section describes important examples and emerging concepts related to complement-mediated crosstalk. Some of these mechanisms might be highly context-specific and many mechanisms that have been described in animal models await confirmation in human systems however, these examples illustrate how tightly embedded the complement system is in many aspects of immune surveillance and homeostasis.

Crosstalk with coagulation systems

Cooperation between the complement and coagulation systems has many potential implications for health and disease 31 . Both cascades are driven by tiered, serine protease-mediated activation steps, and examples of each pathway activating the other have been reported. For instance, coagulation enzymes such as thrombin and kallikrein can activate C3 and/or C5, whereas certain MASPs can cleave fibrinogen, prothrombin and factor XIII, among others. However, these cross-activation activities are typically low when compared to their normal routes of activation, and the (patho) physiological implications of this crosstalk remain to be explored. Independent of their mode of generation, anaphylatoxins influence coagulation through direct effects on platelets, neutrophils and endothelial cells or by stimulating pro-coagulant cytokines. For example, C5aR1 can induce expression of tissue factor (TF), thereby triggering the extrinsic coagulation pathway 32 . Conversely, thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase (carboxypeptidase B2), the active form of which desarginates anaphylatoxins and tames their effects. von Willebrand factor (vWF) has a complex modulatory role in complement𠄼oagulation interplay. Ultra-large multimeric forms of vWF, as observed after tissue injury, can provide a binding platform for C3b to trigger complement activation. Concurrently, vWF interacts with FH and enhances its cofactor activity for the FI-mediated degradation of C3b. Conversely, FH seems to interfere with the hydrolysis of vWF multimers by ADAMTS13. This bidirectional modulation has been proposed to strengthen platelet aggregation while keeping complement activation in control 33 .

The connection between complement and platelet activation has garnered considerable interest in the past few decades, but remains obscure 34 . Platelets seem largely unaffected by complement when in their quiescent form upon activation, however, platelets engage with complement through a complex interplay. For example, chondroitin sulfate exposed on the platelet surface is recognized by both the activator C1q and the regulators FH and C4BP. Exposed gC1qR can trigger additional C1q binding to platelets, and P-selectin, acting as a ligand for C3b, can initiate convertase formation on the surface of activated platelets. A 2015 study demonstrated that C3 can adhere to activated platelets whereupon it is transformed to a hydrolysed-like state that is capable of forming convertases and binding to CR3 (REF. 35). The release of complement components such as FD from the α-granules of activated platelets might fuel convertase-mediated complement turnover. Platelets themselves are well-protected from complement damage through the expression and engagement of complement regulators 31,34 however, the generation of complement effectors could be important in enhancing platelet activation. Soluble MAC (that is, sC5b𠄹) can trigger the secretion of the platelets’ α-granules, and C1q induces the expression of P-selectin, among other effects 34 . Anaphylatoxins have also been described as platelet activators. Although C3aR and C5aR1 are barely detectable on resting platelets, both are strongly upregulated upon platelet activation. The physiological effects of complement–platelet crosstalk on the defensive actions of complement or on platelet clearance remains debated, but current observations suggest that tempered complement activation on activated platelets by effector-mediated stimulation serves to sustain or amplify platelet activation 31 .

Coordination with other immune pathways

Complement activation products, particularly C3a and C5a, can modulate the activation state of most immune cell types, including neutrophils, monocytes, macrophages, and dendritic cells 19,36,37 ( FIG. 2 ). For example, a positive feedback mechanism between complement and neutrophil activation has been described in vitro: C5a can activate neutrophils to secrete properdin that, in turn, activates complement on released neutrophil extracellular traps to generate more C5a. This perpetuation of neutrophil-derived inflammation signals might enhance defence mechanisms but also have pathological consequences in diseases such as antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (see below) 38,39 . By contrast, C3a-mediated signalling demonstrated a protective effect in a mouse model of ischaemia–reperfusion injury (IRI), constraining neutrophil mobilization in response to injury 40 . Complement has also been linked to the development and recruitment of myeloid-derived suppressor cells (MDSCs), which are increasingly recognized as important players in the immune response to cancer or transplants. For example, the induction of MDSC development by hepatic stellate cells in cultured murine dendritic cells was strongly dependent on C3, and addition of exogenous iC3b to dendritic cell cultures influenced MDSC differentiation 41 . In the tumour microenvironment, complement activation leads to the recruitment and/or activation of MDSCs in a C5a-dependent manner, as shown in a syngeneic cervical cancer model 42 .

The generation of complement effectors stimulates a broad spectrum of downstream immune, inflammatory, and procoagulative responses. The anaphylatoxin C5a, for example, exerts strong proinflammatory effects by acting as a chemoattractant and stimulator of various immune cells via C5aR1-mediated signalling, thereby influencing priming and activation with release of mediators (for example, cytokines, neutrophil extracellular traps (NETs)), differentiation, and functional activity. C3a has a distinct spectrum from C5a and has, for example, been shown to activate mast cells. Activation of professional phagocytes induces the expression of complement receptors, which enable complement-mediated phagocytosis, whereas crosstalk between C5aR, FcγR, and dectin-1 also affects antibody-mediated uptake. Adherence of opsonins to CR1 on erythrocytes is an important mechanism that directs immune complexes to the liver and spleen. Complement activation also modulates adaptive immune responses by lowering the threshold of B-cell stimulation (via iC3b or C3dg interaction with CD21) or by influencing T-cell activation (for example, by binding of C3b to CD46), differentiation, and homeostasis. Complement effectors such as C5a, sublytic membrane attack complex (MAC), and MASP-1, can directly activate endothelial cells and, for example, increase expression of tissue factor (TF) as an inducer of coagulation. Serine proteases of the complement and coagulation systems might cross-activate under certain circumstances to contribute to thrombo-inflammation. Concomitantly, the release of complement proteins and binding of both complement activators and regulators to platelets might amplify the platelet response and contribute to clearance of platelets and pathogens alike. BCR, B-cell receptor NK, natural killer TLR, Toll-like receptor.

Interactions with antigen presenting cells

Complement has a dual role in the activation of antigen presenting cells (APCs), depending on the target receptor and cell type involved. Activation of macrophages by C1q or iC3b induces the production of IL-10 and their participation in the clearance of apoptotic cells and damaged molecules — physiologic mechanisms that are not associated with an inflammatory process. Conversely, macrophage stimulation with C3a, C5a or C5b𠄹 usually induces a proinflammatory phenotype with the production of iNOS, TNF, and IL-1β driving pathogen elimination 37 . Similarly, engagement of C3aR or C5aR1 on dendritic cells is associated with their activation via PI3K/AKT, ERK, and NF-㮫 signalling, whereas C1q supports the differentiation of monocytes toward dendritic cells by engaging the leukocyte-associated immunoglobulin-like receptor 1 (REFS 43 , 44 ).

Given the broad effects of complement on APC responses, it is reasonable to assume that APCs primed in the presence or absence of complement-derived signals will differentially modulate T-cell responses. A role for C5a in APC activation, with consequent polarization of CD4 + T cells, has long been appreciated 45 . C3a and C5a modulate T-cell responses by regulating the expression levels of major histocompatibility complex class II and co-stimulatory molecules, and the production of cytokines by APCs 46 .

Modulation of T cell activity

In the past 5 years, new concepts relating to the direct modulation of T-cell responses by complement have emerged. Local activation of T cell-derived autocrine C3 and C5 has been suggested to upregulate C3aR and C5aR1 expression on both APCs and T cells, resulting in direct activation of CD4 + T cells 46 . However contradictory findings, such as a reported inability to detect C5aR1 on T cells in C5aR-GFP reporter mice 47 , suggest further research is needed to determine the conditions under which anaphylatoxins might affect T cells 47,48 . Signalling via CD46, which serves as a regulator of and a receptor for C3b and other ligands, represents another mechanism by which complement interacts with immune pathways. Activated T cells produce C3, which, when locally activated to C3b, can trigger CD46-derived signals that are critical for the induction of type 1 T helper (TH1) cell-mediated immunity via the Notch signalling pathway and IL-10 production 49,50 . Consistent with these findings, individuals who are deficient in CD46 or C3 have suboptimal TH1 responses in vitro 50,51 . A 2013 study demonstrated that intracellular C3 can be processed into C3a and C3b by the T cell-produced protease cathepsin L the resulting CD46 and C3aR-mediated signalling regulated the induction of TH1 and TH17 cell responses 52 .

Interactions with pattern recognition receptors

In addition to complement, DAMPs and PAMPs are sensed by several classes of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), leucine-rich repeat-containing receptors (NLRPs), nucleotide-binding oligomerization domain (NOD)-like receptors, RIG-I-like receptors, and C-type lectin receptors 53 . Although some physiological roles of these receptors are still under investigation, evidence indicates that crosstalk between complement and PRRs determines the quality and magnitude of innate responses and the polarization of adaptive immunity 1,5,46,53,54 . Notably, while PRRs are characterized by their tissue-specific expression, complement components are expressed by virtually all tissues but at differing levels 55 . The distribution of and interplay between different PRRs and complement is essential for host defence, particularly in exposed organs such as the kidneys, but might also drive adverse events. Indeed, PRRs and complement are involved in the pathology of the same kidney diseases and are critical factors that determine the outcome of kidney transplantation 56� .

A considerable body of work has demonstrated intense collaboration between responses mediated by complement and TLRs 1,5,54,61 . Complement effector signalling via C3aR and C5aR1, in particular, but also CR3 and gC1qR modulate TLR responses with effects on the production of proinflammatory cytokines by mouse and/or human APCs 54,62 . Of note, seemingly contrasting effects are observed depending on whether dendritic cells, monocytes, or macrophages are targeted 54 . This differential modulation by C5a was further explored in a 2013 study which showed that in response to lipopolysaccharide, C5a enhanced the secretion of proinflammatory cytokines by monocytes, but induced an anti-inflammatory response in macrophages, with production of anti-inflammatory IL-10 (REF. 63). Additional insight has been provided by the observation that CR3 positively modulates lipopolysaccharide-induced responses in myeloid dendritic cells, but not macrophages, by promoting endosome-mediated endocytosis of the TLR required for subsequent signalling 64 . In support of a bi-directional crosstalk between TLRs and complement, activation of TLR4 in a mouse model of sepsis leads to increased production of FB in tissues such as heart, lung, kidney, liver, and spleen the resulting increase in complement activation in turn correlates with increased TLR signalling 65 .

Tuning of inflammasome activity

Several lines of evidence suggest that complement activation modulates inflammasome function 66 . For example, cooperation between C3aR, TLR, and NLRP3 has been demonstrated in human monocytes, in which C3a seems to modulate the TLR4-mediated production of IL-1β with subsequent NLRP3 inflammasome activation and induction of TH17 responses by T cells 67 . Sublytic amounts of MAC deposited on cell surfaces, which could reflect improper regulation in disease states, increases cytosolic Ca 2+ and results in NLRP3 activation 68,69 . In addition, C5a has been indirectly implicated in the modulation of NLRP3 activation and inflammation induced by cholesterol crystals in atherosclerosis 70 . Finally, an inhibitory effect of C1q on inflammasome activation in response to apoptotic cells has been demonstrated in lipopolysaccharide-stimulated human macrophages 71 .

Crosstalk with adaptive immune systems

Complement participates in multifaceted collaborations between the innate and adaptive immune systems. This 𠆋ridging’ ability seems to have been conserved throughout evolution in fish, among the most primitive species with both innate and adaptive immune systems, C3b has a role in the uptake of antigens by APCs, increasing the efficacy of antigen presentation and proliferation of B and T cells 72� . As mentioned earlier, iC3b and C3dg bind to CR2 (CD21), which is part of the B cell co-receptor complex ( FIG. 1 ). The resulting co-ligation of CR2 and the B-cell receptor by iC3b/C3dg-coated antigens largely augments B-cell responses, especially at the outset of an immune response when limited amounts of antigen are available 75,76 . Moreover, opsonization of particles by iC3b and C3dg mediates the shuttling of antigens between B cells and follicular dendritic cells in the lymph nodes and is important for memory B-cell induction and maintenance in the germinal centres 5,75,77 . Importantly, robust antibody production against pathogens improves the innate immune response by facilitating C1q-mediated activation of complement.

Extensive collaboration also occurs between complement components and antibodies in providing effector functions. For example, complement receptors and Fc receptors (FcRs) coordinate to regulate phagocytosis and modulate various immune responses 78,79 . A 2012 study described an intricate crosstalk mechanism, whereby immune complexes carrying galactosylated IgG1, FcγRIIB, and the C-type lectin receptor Dectin-1 suppressed C5aR-derived inflammation in neutrophils, offering new mechanistic insights into the regulation of inflammation by immune complexes 80 .

In summary, complement determines the type and magnitude of immune responses in different tissues by communicating with other defence pathways and immune cells. Many of these events are likely to be context-specific, and the physiological relevance of each pathway remains to be defined. Ideally, such crosstalk should facilitate the rapid elimination of microbial intruders, damaged cargo, and injurious agents, and contribute to repair and the maintenance of homeostasis. However, as described below, these protective mechanisms can have deleterious effects when inappropriately activated.


Complement and the Regulation of T Cell Responses

The complement system is an evolutionarily ancient key component of innate immunity required for the detection and removal of invading pathogens. It was discovered more than 100 years ago and was originally defined as a liver-derived, blood-circulating sentinel system that classically mediates the opsonization and lytic killing of dangerous microbes and the initiation of the general inflammatory reaction. More recently, complement has also emerged as a critical player in adaptive immunity via its ability to instruct both B and T cell responses. In particular, work on the impact of complement on T cell responses led to the surprising discoveries that the complement system also functions within cells and is involved in regulating basic cellular processes, predominantly those of metabolic nature. Here, we review current knowledge about complement's role in T cell biology, with a focus on the novel intracellular and noncanonical activities of this ancient system.


Pathways of Complement System | Immunology

In this article we will discuss about the classical and alternative pathways of complement system.

1. The Classical Pathway of Complement:

The classical pathway of complement is ini­tiated by the interaction of antibody with antigen directly (soluble antigen-antibody complexes or immune complexes).

The gradual progress of classical pathway can be mediated by these successive stages called:

(i) Activation of C1 component

(ii) Production of C3 convertase

(iii) Production of C5 convertase and

(iv) Action of membrane attack complex (MAC)

(i) Activation of CI component:

The initial stage of activation involves C1, C2, C3 and C4. The soluble antigen-antibody complex induces a conformational changes in the fragment crystalized (Fc) portion of the antibody molecule that exposes a binding site for the C1 component of the complement system.

1. C1 is a complex macromolecular protein present in serum in inactive condition. It is a com­plex of three proteins named—C1q, C1r and C1s, out of which C1q recognizes and binds to the Fc region of the antibody and C1r and C1s remain as inactive proteases with their two subunits each. C1q and two molecules of each C1r and C1s held together is a complex called C1qr2s2 which is stabilized by Ca 2+ ions.

2. The structure of C1 is mainly exhibited by C1q a large molecule composed of 18 polypeptide chains that associate in such a way that forms six collagen-like triple helical arms. The amino-terminal two-thirds of the polypep­tides form the stalk and the carboxy-terminal one-third of the polypeptides form the globular flower, which contains the binding site for anti­body.

3. Normally, C1r2s2 complex remains in inactive form and never binds with C1q at that time and shows the configuration ‘S’. Each C1r and C1s includes two domains named catalytic domain and interaction domain. Due to action of interaction domain in presence of antigen- antibody complex in the serum it binds with C1q.

4. C1q binds to an antibody Fc region by its globular heads, in terms, activates serine pro­teases C1r and C1s which are proteolytic enzymes gives serine residues at the active site after being activated.

On binding to antibody, one molecule of C1r is induced to cleave itself, becomes enzymatically active. Gradually it cleaves and acti­vates the second C1r and both C1s molecules. The activated serine protease C1s binds, cleaves and activates the next two components of the classical pathway i.e. serine protease C4 and C2. Ultimately active CI component is called C1qr2s2 (Fig. 7.9 and 10).

Activation of classical pathway via IgM and IgG:

The cascade reaction of complement system is only initiated when antibody binds to multiple sites on a cell surface, normally that of a pathogen. When IgM (pentameric) is bound to antigen on a target surface, it requires at least three binding sites for C1q attachment.

In case of IgG molecule, it contains a single C1q binding site in the CH2 domain of the Fc. As C1q globular head requires at least two Fc sites for a stable C1-antibody reaction, it indicates that two IgG are required to be present on a target surface.

The structural differences between IgM and IgG exert the effect on their activation level. At the acti­vation of Clq binding, IgG requires less amount of time but a good number of IgG molecules are to be present. Whereas IgM activation is delayed one but it is more efficient, even a single IgM molecule can initiate the process (Fig. 7.11).

(ii) Production of C3 convertase:

Active serine protease enzyme C1qr2s2 has two distinct substrates, C4 and C2. C4 component is a large globular glycoprotein containing three polypeptide chains named α, β and γ. C4 is activated when C1s hydrolyzes a small fragment C4a from the amino terminus of the chain, exposed a binding site on the larger fragment C4b. The C4b fragment attaches to the target surface of the C1 bound to antibody on the pathogen surface.

Besides, active C4 component, the activated C1s protease acts on C2 serine protease, as a result the smaller fragment C2b will be cleaved away from the site of action and C2a larger fragment will remain active at the active site. After that C4b2a active complex is formed which in turn act on the substrate C3 component. C4b2a is called C3 convertase of the classical pathway.

(iii) Production of C5 convertase:

C3 is almost very similar to C4. C3 compo­nent is with two types of polypeptide chains — α and β. C3 convertase (C4b2a) helps to cleave the smaller fragment C3a from the amino terminus of the a chain of C3 component.

Even a single C3 convertase molecule can accelerate the production of more than 200 molecules of C3b, and the result is amplification. In due course produced C3b binds with C4b2a to form a tri-molecular complex called C4b2a3b i.e. C5 convertase.

(iv) Action of membrane attack complex (MAC):

C5 convertase acts on C5 protein component, cleaves C5a from the action site and C5b attaches to the antigenic surface. This bound C5b initiates formation of membrane-attack complex (MAC) by taking participation of C6, C7, C8 and C9 compo­nents gradually and ultimately forms C5b6789 (MAC) which makes a large pore in the membrane of the antigen and accelerates lysis of it (Fig. 7.12).

2. The Alternative Pathway:

Besides the classical pathway, complement system can be initiated by another method called alternative pathway. Unlike classical pathway the alternative pathway is initiated by the cell-wall constituents of both gram-positive and gram- negative bacteria as foreign particles.

Microbial surfaces directly affect the serine protease C3, gradually cleaving of C3 into C3a and C3b. This conformational change extends its effect on another factor i.e. factor B. In turn Ba removed from active site keeping Bb towards the C3b in presence of Mg ++ forms C3bBb, and consi­dered as C3 convertase of alternative pathway.

Binding of C3b exposes a site on factor B that again serves as the substrate for an enzymatically active serum protein called factor D. Actually factor D cleaves the C3b bound factor B, and helps to form C3bBb. The action of C3bBb is very unsta­ble, becomes stabilized by the presence of another exclusive serum protein properdin in this pathway, helps to increase the convertase activity period.

Formation of C3bBb accelerates the auto- catalyse of more C3 component and forms C3bBb3b as C5 convertase. Though structural basis of C3 and C5 convertase vary in these two path­ways of complement system but their mode of action is alike.

Here, C3bBb3b subsequently hydro­pses the bound C5, C6, C7, C8 and C9 respectively, resulting in Membrane Attack Complex (MAC) formation which binds to the antigenic surfaces of microbes (antigen). MAC gradually displaces the membrane phospholipids, forms a large trans­-membrane channel and gradually destroys the membrane and lysis of the antigen occurs.

The Lectin mediated pathway:

The third pathway of complement system is lectin-mediated pathway. Lectin-mediated path­way is activated by the binding of mannose-binding protein present in blood plasma to mannose containing proteoglycans on the surfaces of the bacteria and yeast, it forms MBP-MASP (Mannose-binding protein-mannose-associated serum protease). In lectin pathway MBP-MASP acts on the substrate C4 and C2 component protein.

Three different pathways of complement acti­vation is shown in the Fig. 7.13.


Complement system in adaptive immunity: B-cell regulation and humoral immunity

The aforementioned functions of the complement system, oposonization, lysis, and generation of the inflammatory response through soluble mediators, are paradigmatic and represent a well-characterized component of an innate host defense. It has become increasingly appreciated that complement functions in host defense extend beyond innate immune responses. The finding that B lymphocytes bound C3 raised the question as early as in the 1970s as to whether the complement system was involved in adaptive immune responses 84 . Subsequent work demonstrated that depletion of C3 impaired humoral immune responses and provided direct evidence that efficient adaptive responses were contingent on an intact complement system in some cases 85 . Further study in animals bearing natural complement deficiencies implicated the classical pathway as a crucial mechanism for efficient antigen trapping and retention in lymphoid tissues (e.g., splenic follicles), suggesting that a major function of the complement system was to localize foreign antigens into immune sites important for lymphocytes responses 86, 87, 88 .

The humoral arm of the adaptive immune response is tasked with protecting extracellular spaces through the generation of effector and memory B cells, and B-cell-produced antibodies, leading to neutralization and opsonization of pathogen and providing immunological memory against reinfection. The potency of this response stems from a complex interplay of immune mechanisms, contingent on the strength of antigenic stimuli and the presence of helper T-cell assistance, among many other factors 2 . Complement effectors are engaged with humoral immunity at multiple stages of B-cell differentiation and can influence B-cell biology on several levels 89, 90 . As alluded to previously, complement enhances B-cell immunity principally through CRs, CR1 (CD35) and CR2 (CD21), expressed on B lymphocytes and follicular dendritic cells (FDCs), and binding to the complement opsonins in a concerted effort with the phagocytic system 75, 90, 91 . CR2 forms a receptor complex with the signaling protein CD19 and the tetraspan protein CD81 to form the B-cell coreceptor complex (CD21-CD19-CD81), which supports an enhanced signal via the B-cell receptor (BCR e.g., surface immunoglobulin) when it encounters antigen coated with complement opsonins (e.g., C3d), resulting in the reduction of B-cell activation threshold by several orders of magnitude 92, 93 . Thus, complement can be viewed as a 'natural adjuvant' and as an instructor of the humoral immune response 94 .

The functional consequence of this modulation of B-cell signaling can be observed in multiple settings. B cells first express the CD21-CD19-CD81 coreceptor as they migrate from the bone marrow into the periphery, generally referred to as the transitional stage that has important implications in the elimination of self-reactive B cells and in the positive selection of B1 cells 95 . B1 cells, which are the chief sources of natural antibody with repertoires that are highly biased toward conserved antigens (e.g., nuclear antigens), are a long-lived and physiologically distinct population of B cells 2 . Complement seems to function in the selection and maintenance of B1 cells, as CR2-deficient mice have an altered repertoire of natural antibody, which can be observed by a marked reduction in injury following ischemia/reperfusion despite normal levels of IgM 96, 97 . These mice also have reduced numbers of B1a cells and show impaired generalized antibody production 98 .

In addition to modulating B1 activity and the production of natural antibodies, cross-linking of the CD21-CD19-CD81 coreceptor complex with BCR enhances B-cell immunity in later stages of B-cell differentiation as well. Coupling C3d to low-affinity antigen, which (if uncoupled) would cause B-cell death, results in not only survival but also B-cell activation and production of antibody, suggesting a role of complement in the 'instruction' of naive B cells in the periphery 99 . Similarly, activation of mature peripheral and follicular B cells by complement-opsonized antigen leads to their migration to the lymphoid T-cell:B-cell boundary, where helper T cells provide costimulation via CD40, leading to B-cell activation and expansion. Subsequently, activated B cells initiate the formation of germinal centers (GCs), where CRs on B cells enhance BCR signaling, leading to effective differentiation into plasma and memory B cells 89, 90 . This is supported by the observation that antigen-specific B cells lacking CR1/CR2 fail to survive within a GC when put in competition with WT B cells, insinuating that coreceptor signaling is vital to clonal selection of B cells and in the absence of this complement-assisted cosignaling, B cells fail to compete and undergo cell death 100 . FDCs are central to this process as they are specialized stromal cells that secrete the B-lymphocyte chemoattractant, help to organize GCs, and provide effective means of trapping and retaining antigen within B-cell follicles and displaying them to both naive and GC B cells 101 . FDCs express relatively high levels of CR1 and CR2 and effectively retain C3-coated immune complexes within the lymphoid follicles, promoting the antigen selection of high-affinity GC B cells 92 . Furthermore, post-GC B cells require complement on FDCs for an efficient maintenance of long-term memory B cells, affinity maturation, and effective recall responses 102 .

In addition to the CRs, CR1 and CR2, some evidence suggests a role of anaphylatoxins in the modulation of B-cell biology. B cells have been reported to express C3aR and both ligands, C3a and C3adesArg, have been shown to negatively regulate the polyclonal immune response, as well as limit the secretion of TNF-α and IL-6 103, 104 . Conversely, C5a has been reported to play a role in the trafficking and migration of various B-cell populations, including GC B cells and tonsillar memory and naїve B cells 105, 106, 107 .

The roles of complement in humoral immunity can be illustrated by the characterization of mice bearing deficiencies in both complement components and CRs 90 . Studies have demonstrated the importance of an intact complement classical pathway (C1q, C3, or C4) in humoral response to both thymus-dependent and thymus-independent antigens 108 . In many cases, mice deficient in CR1/2 (a single gene Cr1/2 encodes both proteins in mice) exhibit similar impairment, suggesting that pro-humoral responses are mediated by these receptors 89 . For example, mice deficient in CR1/2 and C3 exhibited markedly reduced IgM (and IgG) levels, failure in isotype switch to IgG, and decreased antigen uptake in response to T-independent type II polysaccharide antigens 109, 110 . Similar results were established for T-dependant antigens, such as keyhole limpet hemocyanin and bacteriophage ΦX174, as well as viral and bacterial pathogens, such as herpes simplex virus, West Nile virus, and Streptococcus pneumoniae 98 , 111, 112, 113, 114 . These and other studies highlight the critical role complement plays in the generation of robust antibody response at several levels of B-cell biology.


The Complement System: An Unexpected Role in Synaptic Pruning During Development and Disease

An unexpected role for the classical complement cascade in the elimination of central nervous system (CNS) synapses has recently been discovered. Complement proteins are localized to developing CNS synapses during periods of active synapse elimination and are required for normal brain wiring. The function of complement proteins in the brain appears analogous to their function in the immune system: clearance of cellular material that has been tagged for elimination. Similarly, synapses tagged with complement proteins may be eliminated by microglial cells expressing complement receptors. In addition, developing astrocytes release signals that induce the expression of complement components in the CNS. In the mature brain, early synapse loss is a hallmark of several neurodegenerative diseases. Complement proteins are profoundly upregulated in many CNS diseases prior to signs of neuron loss, suggesting a reactivation of similar developmental mechanisms of complement-mediated synapse elimination potentially driving disease progression.


Complement System and Cancer

Complement has been considered since a long time as an immune surveillance system against cancer, because complement is activated on the surface of tumor cells. Nevertheless, tumor cells develop inhibitory mechanisms for the terminal steps of the complement cascade, thus preventing complement-mediated cytotoxicity. Surprisingly, recent studies demonstrated that complement activation within the tumor microenvironment can promote tumor growth. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. The mechanisms of these phenomena are not fully understood. Prolonged complement activation supports chronic inflammation, promotes an immunosuppressive microenvironment, induces angiogenesis, and activates cancer-related signaling pathways.

Several lines of evidence indicate a role for molecules of the complement system in tumor growth and metastasis. C3, C4, or C5aR deficiencies prevent tumor growth in mice, potentially via inhibition of the classical pathway and the generation of C5a, which has a potent inflammatory potential. In mouse models, the presence of C5a in the tumor microenvironment enhances tumor growth by recruitment of myeloid-derived suppressor cell / MDSC and increasing T cell-directed suppressive abilities. In a breast cancer model, C5aR facilitated metastasis in the lungs through different immune mechanisms in the metastatic niche, including the suppression of effector CD8(+) and CD4(+) T cell responses, the recruitment of immature myeloid cells and the generation of Tregs and a Th2-oriented response.

Cancer cells also secrete complement proteins that stimulate tumor growth upon activation via a direct autocrine effect through C3aR and C5aR signaling. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. In addition, patients with non-small cell lung cancer have elevated C5a plasma levels.

C3a and C5a seem to have opposing effects during tumor development and in case of anti-tumor radiotherapy. While C3a and especially C5a promote tumor growth, radiotherapy-induced tumor cell death and transient local complement activation with production of C3a and C5a. The latter appeared crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity.

Overexpression of FH has been described in non-small cell lung cancer cell lines and on non-small cell lung cancer biopsies (but not in small cell lung carcinoma and carcinoid cell lines), in bladder tumor cells, in cutaneous squamous cell carcinoma (cSCC) and cell lines, and in hepatocellular carcinoma tumors. Low titer anti-FH antibodies were also found in sera from patients with non-small cell lung cancer. Recent studies demonstrated that FH binds to pentraxin 3 (PTX3) in the tumor microenvironment, thus preventing local complement overactivation and generation of pro-tumorigenic C5a.

These examples clearly indicate that complement is indispensable immunosurveillance system, which needs to function with the right force when and where is needed. Therefore, therapeutic strategies are needed to adjust the level of complement activation in pathological conditions.


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