Classical pathway complement activation initiated

In the first step, C1s cleaves C4 to produce C4b, which binds covalently to the surface of the pathogen.

The covalently attached C4b then binds one molecule of C2, making it susceptible, in turn, to cleavage by C1s. C1s cleaves C2 to produce the large fragment C2b, which is itself a serine protease. The complex of C4b with the active serine protease C2b remains on the surface of the pathogen as the C3 convertase of the classical pathway.

Its most important activity is to cleave large numbers of C3 molecules to produce C3b molecules that coat the pathogen surface. At the same time, the other cleavage product, C3a, initiates a local inflammatory response. These reactions, which comprise the classical pathway of complement activation, are shown in schematic form in Fig. The classical pathway of complement activation generates a C3 convertase that deposits large numbers of C3b molecules on the pathogen surface.

The steps in the reaction are outlined here and detailed in the text. The cleavage of C4 by C1s exposes a reactive more The proteins of the classical pathway of complement activation. The MB-lectin pathway uses a protein very similar to C1q to trigger the complement cascade. This protein, called the mannan-binding lectin MBL , is a collectin, like C1q. Mannan-binding lectin binds specifically to mannose residues, and to certain other sugars, which are accessible and arranged in a pattern that allows binding on many pathogens.

On vertebrate cells, however, these are covered by other sugar groups, especially sialic acid. Thus, mannan-binding lectin is able to initiate complement activation by binding to pathogen surfaces.

It is present at low concentrations in normal plasma of most individuals, and, as we will see in the last part of this chapter, its production by the liver is increased during the acute-phase reaction of the innate immune response.

Thus the MB-lectin pathway initiates complement activation in the same way as the classical pathway , forming a C3 convertase from C2b bound to C4b. People deficient in mannan-binding lectin experience a substantial increase in infections during early childhood, indicating the importance of the MB-lectin pathway for host defense.

Mannan-binding lectin forms a complex with serine proteases that resembles the complement C1 complex. MBL forms clusters of two to six carbohydrate-binding heads around a central collagen-like stalk. This structure, easily discernible under the electron more We have seen that the classical and MB-lectin pathways of complement activation are initiated by proteins that bind to pathogen surfaces.

During the triggered-enzyme cascade that follows, it is important that activating events are confined to this same site, so that C3 activation also occurs on the surface of the pathogen, and not in the plasma or on host cell surfaces.

This is achieved principally by the covalent binding of C4b to the pathogen surface. Cleavage of C4 exposes a highly reactive thioester bond on the C4b molecule that allows it to bind covalently to molecules in the immediate vicinity of its site of activation.

In innate immunity , C4 cleavage is catalyzed by a C1 or MBL complex bound to the pathogen surface, and C4b can bind adjacent proteins or carbohydrates on the pathogen surface. If C4b does not rapidly form this bond, the thioester bond is cleaved by reaction with water and this hydrolysis reaction irreversibly inactivates C4b Fig. This helps to prevent C4b from diffusing from its site of activation on the microbial surface and becoming coupled to host cells.

Cleavage of C4 exposes an active thioester bond that causes the large fragment, C4b, to bind covalently to nearby molecules on the bacterial cell surface. C2 becomes susceptible to cleavage by C1s only when it is bound by C4b, and the C2b serine protease is thereby also confined to the pathogen surface, where it remains associated with C4b, forming a C3 convertase. The activation of C3 molecules thus also occurs at the surface of the pathogen. Furthermore, the C3b cleavage product is also rapidly inactivated unless it binds covalently by the same mechanism as C4b, and it therefore opsonizes only the surface on which complement activation has taken place.

This pathway can proceed on many microbial surfaces in the absence of specific antibody , and it leads to the generation of a distinct C3 convertase designated C3b , Bb.

In contrast to the classical and MB-lectin pathways of complement activation, the alternative pathway does not depend on a pathogen-binding protein for its initiation; instead it is initiated through the spontaneous hydrolysis of C3, as shown in the top three panels of Fig. The distinctive components of the pathway are listed in Fig. A number of mechanisms ensure that the activation pathway will only proceed on the surface of a pathogen.

Complement activated by the alternative pathway attacks pathogens while sparing host cells, which are protected by complement regulatory proteins. The complement component C3 is cleaved spontaneously in plasma to give C3 H 2 O , which binds factor B and more The proteins of the alternative pathway of complement activation.

This occurs through the spontaneous hydrolysis of the thioester bond in C3 to form C3 H 2 O which has an altered conformation, allowing binding of the plasma protein factor B. This complex is a fluid-phase C3 convertase , and although it is only formed in small amounts it can cleave many molecules of C3 to C3a and C3b. Much of this C3b is inactivated by hydrolysis, but some attaches covalently, through its reactive thioester group, to the surfaces of host cells or to pathogens.

C3b bound in this way is able to bind factor B, allowing its cleavage by factor D to yield the small fragment Ba and the active protease Bb. This results in formation of the alternative pathway C3 convertase, C3b,Bb see Fig. When C3b binds to host cells, a number of complement -regulatory proteins, present in the plasma and on host cell membranes combine to prevent complement activation from proceeding.

These proteins interact with C3b and either prevent the convertase from forming, or promote its rapid dissociation see Fig. Thus, the complement receptor 1 CR1 and a membrane-attached protein known as decay-accelerating factor DAF or CD55 compete with factor B for binding to C3b on the cell surface, and can displace Bb from a convertase that has already formed. Convertase formation can also be prevented by cleaving C3b to its inactive derivative iC3b. This is achieved by a plasma protease, factor I , in conjunction with C3b-binding proteins that can act as cofactors, such as CR1 and membrane cofactor of proteolysis MCP or CD46 , another host cell membrane protein.

Factor H is another complement-regulatory protein in plasma that binds C3b and, like CR1, it is able to compete with factor B and displace Bb from the convertase in addition to acting as a cofactor for factor I.

Factor H binds preferentially to C3b bound to vertebrate cells as it has an affinity for the sialic acid residues present on these cells. By contrast, because pathogen surfaces lack these regulatory proteins and sialic acid residues, the C3b , Bb convertase can form and persist.

Indeed, this process may be favored by a positive regulatory factor, known as properdin or factor P , which binds to many microbial surfaces and stabilizes the convertase. Deficiencies in factor P are associated with a heightened susceptibility to infection with Neisseria species. Once formed, the C3b,Bb convertase rapidly cleaves yet more C3 to C3b, which can bind to the pathogen and either act as an opsonin or reinitiate the pathway to form another molecule of C3b,Bb convertase.

Thus, the alternative pathway activates through an amplification loop that can proceed on the surface of a pathogen, but not on a host cell. This same amplification loop enables the alternative pathway to contribute to complement activation initially triggered through the classical or MB-lectin pathways Fig. The alternative pathway of complement activation can amplify the classical or the MB-lectin pathway by forming an alternative C3 convertase and depositing more C3b molecules on the pathogen.

C3b deposited by the classical or MB-lectin pathways can bind more The C3 convertases resulting from activation of the classical and MB-lectin pathways C4b,2b and from the alternative pathway C3b , Bb are apparently distinct. However, understanding of the complement system is simplified somewhat by recognition of the close evolutionary relationships between the different complement proteins.

Thus the complement zymogens, factor B and C2, are closely related proteins encoded by homologous genes located in tandem in the major histocompatibility complex MHC on human chromosome 6. Furthermore, their respective binding partners, C3 and C4, both contain thioester bonds that provide the means of covalently attaching the C3 convertases to a pathogen surface.

Only one component of the alternative pathway appears entirely unrelated to its functional equivalents in the classical and MB-lectin pathways; this is the initiating serine protease, factor D. Factor D can also be singled out as the only activating protease of the complement system to circulate as an active enzyme rather than a zymogen. This is both necessary for the initiation of the alternative pathway through spontaneous C3 cleavage, and safe for the host because factor D has no other substrate than factor B when bound to C3b.

This means that factor D only finds its substrate at a very low level in plasma, and at pathogen surfaces where the alternative pathway of complement activation is allowed to proceed. Comparison of the different pathways of complement activation illustrates the general principle that most of the immune effector mechanisms that can be activated in a nonclonal fashion as part of the early nonadaptive host response against infection have been harnessed during evolution to be used as effector mechanisms of adaptive immunity.

It is almost certain that the adaptive response evolved by adding specific recognition to the original nonadaptive system. This is illustrated particularly clearly in the complement system, because here the components are defined, and the functional homologues can be seen to be evolutionarily related Fig.

There is a close relationship between the factors of the alternative, MB-lectin, and classical pathways of complement activation. Most of the factors are either identical or the products of genes that have duplicated and then diverged in sequence. The more The formation of C3 convertases is the point at which the three pathways of complement activation converge, because both the classical pathway and MB-lectin pathway convertases C4b,2b, and the alternative pathway convertase C3b , Bb have the same activity, and they initiate the same subsequent events.

They both cleave C3 to C3b and C3a. C3b binds covalently through its thioester bond to adjacent molecules on the pathogen surface; otherwise it is inactivated by hydrolysis.

C3 is the most abundant complement protein in plasma, occurring at a concentration of 1. Thus, the main effect of complement activation is to deposit large quantities of C3b on the surface of the infecting pathogen, where it forms a covalently bonded coat that, as we will see, can signal the ultimate destruction of the pathogen by phagocytes.

The next step in the cascade is the generation of the C5 convertases. In the classical and the MB-lectin pathways, a C5 convertase is formed by the binding of C3b to C4b,2b to yield C4b,2b,3b. By the same token, the C5 convertase of the alternative pathway is formed by the binding of C3b to the C3 convertase to form C3b 2 , Bb. C5 is captured by these C5 convertase complexes through binding to an acceptor site on C3b, and is then rendered susceptible to cleavage by the serine protease activity of C2b or Bb.

This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, as C5 can be cleaved only when it binds to C3b that is part of the C5 convertase complex. Thus, complement activation by both the alternative, MB-lectin and classical pathways leads to the binding of large numbers of C3b molecules on the surface of the pathogen, the generation of a more limited number of C5b molecules, and the release of C3a and C5a Fig.

Complement component C5 is cleaved when captured by a C3b molecule that is part of a C5 convertase complex. As shown in the top panel, C5 convertases are formed when C3b binds either the classical or MB-lectin pathway C3 convertase C4b,2b to form C4b,2b,3b, more The most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytic cells.

This occurs by the specific recognition of bound complement components by complement receptors CRs on phagocytes. These complement receptors bind pathogens opsonized with complement components: opsonization of pathogens is a major function of C3b and its proteolytic derivatives.

C4b also acts as an opsonin but has a relatively minor role, largely because so much more C3b than C4b is generated. The five known types of receptor for bound complement components are listed, with their functions and distributions, in Fig. The best-characterized is the C3b receptor CR1 CD35 , which is expressed on both macrophages and polymorphonuclear leukocytes. Binding of C3b to CR1 cannot by itself stimulate phagocytosis, but it can lead to phagocytosis in the presence of other immune mediators that activate macrophages.

For example, the small complement fragment C5a can activate macrophages to ingest bacteria bound to their CR1 receptors Fig. C5a binds to another receptor expressed by macrophages, the C5a receptor , which has seven membrane-spanning domains.

Receptors of this type couple with intracellular guanine-nucleotide-binding proteins called G proteins , and the C5a receptor signals in this way. Proteins associated with the extracellular matrix, such as fibronectin, can also contribute to phagocyte activation; these are encountered when phagocytes are recruited to connective tissue and activated there.

C3a, which has inflammatory activities similar to those of C5a, although it is a less potent chemoattractant, binds to its own specific receptor, the C3a receptor, which is homologous in structure to the C5a receptor.

Distribution and function of receptors for complement proteins on the surfaces of cells. There are several different receptors specific for different bound complement components and their fragments. CR1 and CR3 are especially important in inducing phagocytosis more The anaphylotoxin C5a can enhance phagocytosis of opsonized microorganisms. Activation of complement, either by the alternative or the MB-lectin pathway, leads to the deposition of C3b on the surface of the microorganism left panel.

C3b can be bound more Like several other key components of complement, C3b is subject to regulatory mechanisms and can be cleaved into derivatives that cannot form an active convertase.

One of the inactive derivatives of C3b, known as iC3b see Section acts as an opsonin in its own right when bound by the complement receptors CR2 or CR3. A second breakdown product of C3b, called C3dg , binds only to CR2. CR2 is found on B cells as part of a co-receptor complex that can augment the signal received through the antigen -specific immunoglobulin receptor. Thus a B cell whose antigen receptor is specific for a given pathogen will receive a strongly augmented signal on binding this pathogen if it is also coated with C3dg.

The activation of complement can therefore contribute to producing a strong antibody response see Chapters 6 and 9. This example of how an innate humoral immune response can contribute to activating adaptive humoral immunity parallels the contribution made by the innate cellular response of macrophages and dendritic cells to the initiation of a T-cell response, which we will discuss later in this chapter.

The central role of opsonization by C3b and its inactive fragments in the destruction of extracellular pathogens can be seen in the effects of various complement deficiency diseases. Whereas individuals deficient in any of the late components of complement are relatively unaffected, individuals deficient in C3 or in molecules that catalyze C3b deposition show increased susceptibility to infection by a wide range of extracellular bacteria , as we will see in Chapter The small complement fragments C3a, C4a, and C5a act on specific receptors see Fig.

When produced in large amounts or injected systemically, they induce a generalized circulatory collapse, producing a shocklike syndrome similar to that seen in a systemic allergic reaction involving IgE antibodies see Chapter Such a reaction is termed anaphylactic shock and these small fragments of complement are therefore often referred to as anaphylotoxins. Of the three, C5a is the most stable and has the highest specific biological activity.

All three induce smooth muscle contraction and increase vascular permeability, but C5a and C3a also act on the endothelial cells lining blood vessels to induce adhesion molecules. The changes induced by C5a and C3a recruit antibody , complement, and phagocytic cells to the site of an infection Fig. Local inflammatory responses can be induced by small complement fragments, especially C5a. The small complement fragments are differentially active: C5a is more active than C3a, which is more active than C4a.

They cause local inflammatory responses by more C5a also acts directly on neutrophils and monocytes to increase their adherence to vessel walls, their migration toward sites of antigen deposition, and their ability to ingest particles, as well as increasing the expression of CR1 and CR3 on the surfaces of these cells.

In this way C5a and, to a smaller extent, C3a and C4a, act in concert with other complement components to hasten the destruction of pathogens by phagocytes. C5a and C3a signal through transmembrane receptors that activate G proteins ; thus the action of C5a in attracting neutrophils and monocytes is analogous to that of chemokines, which also act via G proteins to control cell migration.

One of the important effects of complement activation is the assembly of the terminal components of complement Fig. The reactions leading to the formation of this complex are shown schematically in Fig. The end result is a pore in the lipid bilayer membrane that destroys membrane integrity. This is thought to kill the pathogen by destroying the proton gradient across the pathogen cell membrane.

The terminal complement components assemble to form the membrane-attack complex. Assembly of the membrane-attack complex generates a pore in the lipid bilayer membrane. The sequence of steps and their approximate appearance are shown here in schematic form.

C5b triggers the assembly of a complex of one molecule each of C6, C7, and more The first step in the formation of the membrane-attack complex is the cleavage of C5 by a C5 convertase to release C5b see Fig. In the next stages, shown in Fig. First, one molecule of C5b binds one molecule of C6, and the C5b,6 complex then binds one molecule of C7.

This reaction leads to a conformational change in the constituent molecules, with the exposure of a hydrophobic site on C7, which inserts into the lipid bilayer. Similar hydrophobic sites are exposed on the later components C8 and C9 when they are bound to the complex, allowing these proteins also to insert into the lipid bilayer.

The membrane-attack complex, shown schematically and by electron microscopy in Fig. The disruption of the lipid bilayer leads to the loss of cellular homeostasis, the disruption of the proton gradient across the membrane, the penetration of enzymes such as lysozyme into the cell, and the eventual destruction of the pathogen. Although the effect of the membrane-attack complex is very dramatic, particularly in experimental demonstrations in which antibodies against red blood cell membranes are used to trigger the complement cascade, the significance of these components in host defense seems to be quite limited.

To date, deficiencies in complement components C5 —C9 have been associated with susceptibility only to Neisseria species, the bacteria that cause the sexually transmitted disease gonorrhea and a common form of bacterial meningitis. Thus, the opsonizing and inflammatory actions of the earlier components of the complement cascade are clearly most important for host defense against infection. Formation of the membrane-attack complex seems to be important only for the killing of a few pathogens, although, as we will see in Chapter 13, it might have a major role in immunopathology.

Given the destructive effects of complement , and the way in which its activation is rapidly amplified through a triggered-enzyme cascade, it is not surprising that there are several mechanisms to prevent its uncontrolled activation. As we have seen, the effector molecules of complement are generated through the sequential activation of zymogens, which are present in plasma in an inactive form.

The activation of these zymogens usually occurs on a pathogen surface, and the activated complement fragments produced in the ensuing cascade of reactions usually bind nearby or are rapidly inactivated by hydrolysis. These two features of complement activation act as safeguards against uncontrolled activation. Even so, all complement components are activated spontaneously at a low rate in plasma, and activated complement components will sometimes bind proteins on host cells.

The potentially damaging consequences are prevented by a series of complement control proteins, summarized in Fig. As we saw in discussing the alternative pathway of complement activation see Section many of these control proteins specifically protect host cells while allowing complement activation to proceed on pathogen surfaces.

The complement control proteins therefore allow complement to distinguish self from nonself. The reactions that regulate the complement cascade are shown in Fig.

Interestingly, the amount of anti-erythrocyte antibodies in the 19 sera samples correlated with the number of immune complexes present. Our data suggest that high levels of anti-erythrocyte antibody will often be associated with high levels of immune complexes. We speculate that underlying immune complex mediated chronic inflammation broadly disrupts homeostasis, an important regulatory function of the complement system [ 32 ], and could potentially contribute to increased severity of AIHA illness for SLE patients.

Based on the association of anti-erythrocyte antibody levels and high immune complex levels, our data suggest that a classical pathway complement inhibitor that would also block immune complex-initiated complement activation might be advantageous for treating AIHA in the setting of SLE.

A molecule such as PA-dPEG24 that can also bind hemoglobin and block hemoglobin-mediated peroxidase activity [ 33 ], which is felt to be the predominant mechanism of free hemoglobin-mediated acute kidney injury [34], could potentially add clinical benefit for treating AIHA in the setting of SLE. Future testing of this hypothesis in an AIHA animal model is warranted.

National Center for Biotechnology Information , U. Author manuscript; available in PMC Jul 6. Pamela S. Hair , 1 Daniel W.

Krishna , 3 and Kenji M. Cunnion 1, 4, 5. Daniel W. Neel K. Kenji M. Author information Copyright and License information Disclaimer. Corresponding author: Kenji M. Cunnion, M. Copyright notice. The publisher's final edited version of this article is available at Lupus. See other articles in PMC that cite the published article. Abstract Introduction. Introduction Autoimmune hemolytic anemia AIHA is a disease with an estimated prevalence of , individuals per year [ 1 ].

Clinical Data All clinical data were maintained by Dr. Reagents Commercially obtained serum was acquired from a 25 y. Table 1: Demographics and classification criteria of the SLE study patients. Characteristics Value No. Open in a separate window. Erythrocyte opsonins The erythrocytes were further processed to recover bound opsonins.

Figure 1. Figure 2. Figure 3. Figure 4. Erythrocyte-initiated complement activation and SLE disease In order to further evaluate associations between erythrocyte-initiated complement activation in the sera samples and different aspects of SLE, we dichotomized the complement activation markers. Table 2: Medications and severity of illness. Table 3. Plasma autoantibodies and complement levels. References 1. Zanella A, Barcellini W.

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