Complement proteins are normally present. Activation of mast cells, resulting in the release of histamine, which dilates capillaries and causes local redness during inflammation and allergic reactions; this function is associated with fragments C5a, C3a, Ba,

Without regulatory mechanisms acting at many stages, the complement system would be ineffective; unlimited consumption of its components could lead to severe, potentially fatal damage to the cells and tissues of the body. At the first stage, the C1 inhibitor blocks the enzymatic activity of Clr and Cls and, consequently, the cleavage of C4 and C2. Activated C2 lasts only a short time, and its relative instability limits the lifetime of C42 and C423. The C3-activating enzyme of the alternative pathway, C3bBb, also has a short half-life, although the binding of properdin to the enzyme complex prolongs the lifetime of the complex.

IN serum there is an inactivator of anaphylatoxins - an enzyme that cleaves off the N-terminal arginine from C4a, C3a and C5a and thereby sharply reduces their biological activity. Factor I inactivates C4b and C3b, factor H accelerates the inactivation of C3b by factor I, and a similar factor, C4-binding protein (C4-bp), accelerates the cleavage of C4b by factor I. Three constitutional proteins of cell membranes - PK1, a membrane cofactor protein and a factor that accelerates decay (FUR) - destroy C3- and C5-convertase complexes that form on these membranes.

Other components of cell membranes- associated proteins (among which CD59 is the most studied) - can bind C8 or C8 and C9, which prevents the incorporation of the membrane attack complex (C5b6789). Some blood serum proteins (among which protein S and clusterin are the most studied) block the attachment of the C5b67 complex to the cell membrane, its binding of C8 or C9 (i.e., the formation of a full-fledged membrane attack complex) or otherwise prevent the formation and incorporation of this complex.

The protective role of complement

Neutralization viruses C1 and C4 are enhanced by antibodies and increase even more when C3b is fixed, which is formed along the classical or alternative pathway. Thus, complement is of particular importance in the early stages of a viral infection, when the number of antibodies is still low. Antibodies and complement also limit the infectivity of at least some viruses by forming the typical complement "holes" visible on electron microscopy. The interaction of Clq with its receptor opsonizes the target, i.e. facilitates its phagocytosis.

C4a, C3a and C5a are fixed by mast cells, which begin to secrete histamine and other mediators, leading to vasodilation and edema and hyperemia characteristic of inflammation. Under the influence of C5a, monocytes secrete TNF and IL-1, which enhance the inflammatory response. C5a is the main chemotactic factor for neutrophils, monocytes and eosinophils capable of phagocytizing microorganisms opsonized by C3b or its cleavage product iC3b. Further inactivation of cell-bound C3b, leading to the appearance of C3d, deprives it of its opsonizing activity, but its ability to bind to B-lymphocytes is retained. C3b fixation on the target cell facilitates its lysis by NK cells or macrophages.

C3b binding with insoluble immune complexes solubilizes them, since C3b, apparently, destroys the lattice structure of the antigen-antibody complex. At the same time, it becomes possible for this complex to interact with the C3b receptor (PK1) on erythrocytes, which transfer the complex to the liver or spleen, where it is absorbed by macrophages. This phenomenon partly explains the development of serum sickness (immune complex disease) in individuals with C1, C4, C2, or C3 deficiency.

Complement system

Membrane attack complex causing cell lysis.

Complement system- a complex of complex proteins that are constantly present in the blood. This is a cascade system of proteolytic enzymes, designed for the humoral protection of the body from the action of foreign agents, it is involved in the implementation of the body's immune response. It is an important component of both innate and acquired immunity.

History of the concept

At the end of the 19th century, it was found that blood serum contains a certain “factor” with bactericidal properties. In 1896, a young Belgian scientist Jules Bordet, who worked at the Pasteur Institute in Paris, showed that there are two different substances in the serum, the combined action of which leads to the lysis of bacteria: a thermostable factor and a thermolabile (losing its properties when serum is heated) factor. The thermostable factor, as it turned out, could only act against certain microorganisms, while the thermolabile factor had nonspecific antibacterial activity. The thermolabile factor was later named complement. The term "complement" was coined by Paul Ehrlich in the late 1890s. Ehrlich was the author of the humoral theory of immunity and introduced many terms into immunology, which later became generally accepted. According to his theory, cells responsible for immune responses have receptors on their surface that serve to recognize antigens. We now call these receptors "antibodies" (the basis of the variable receptor of lymphocytes is an IgD class antibody attached to the membrane, less often IgM. Antibodies of other classes in the absence of the corresponding antigen are not attached to cells). The receptors bind to a specific antigen, as well as to the heat-labile antibacterial component of the blood serum. Ehrlich called the thermolabile factor "complement" because this component of the blood "serves as a complement" to the cells of the immune system.

Ehrlich believed that there are many complements, each of which binds to its own receptor, just as a receptor binds to a specific antigen. In contrast, Bordet argued that there is only one type of "complement". At the beginning of the 20th century, the dispute was resolved in favor of Bordet; it turned out that complement can be activated with the participation of specific antibodies or independently, in a non-specific way.

General view

Components of the complement system

Complement is a protein system that includes about 20 interacting components: C1 (a complex of three proteins), C2, C3, ..., C9, factor B, factor D and a number of regulatory proteins. All these components are soluble proteins with a mol. weighing from 24,000 to 400,000, circulating in the blood and tissue fluid. Complement proteins are synthesized mainly in the liver and make up approximately 5% of the total globulin fraction of blood plasma. Most are inactive until activated either by an immune response (involving antibodies) or directly by an invading microorganism (see below). One of the possible results of complement activation is the sequential association of the so-called late components (C5, C6, C7, C8 and C9) into a large protein complex that causes cell lysis (lytic, or membrane attack complex). Aggregation of late components occurs as a result of a series of successive proteolytic activation reactions involving early components (C1, C2, C3, C4, factor B and factor D). Most of these early components are proenzymes activated sequentially by proteolysis. When any of these proenzymes is specifically cleaved, it becomes the active proteolytic enzyme and cleaves the next proenzyme, and so on. Because many of the activated components bind tightly to membranes, most of these events occur on cell surfaces. The central component of this proteolytic cascade is C3. Its activation by cleavage is the main reaction of the entire complement activation chain. C3 can be activated in two main ways - classical and alternative. In both cases, C3 is cleaved by an enzyme complex called C3 convertase. Two different pathways lead to the formation of different C3 convertases, however, both of them are formed as a result of spontaneous association of two complement components activated earlier in the chain of the proteolytic cascade. C3 convertase cleaves C3 into two fragments, the larger of which (C3b) binds to the target cell membrane next to C3 convertase; as a result, an even larger enzyme complex with an altered specificity, C5-convertase, is formed. Then the C5 convertase cleaves C5 and thereby initiates the spontaneous assembly of the lytic complex from the late components - from C5 to C9. Since each activated enzyme cleaves many molecules of the next proenzyme, the activation cascade of early components acts as an enhancer: each molecule activated at the beginning of the entire chain leads to the formation of many lytic complexes.

The main stages of activation of the complement system.

Classical and alternative ways of activation of the complement system.

The complement system works as a biochemical cascade of reactions. Complement is activated by three biochemical pathways: the classical, alternative, and lectin pathways. All three activation pathways produce different variants of C3 convertase (a protein that cleaves C3). classic way(it was discovered first, but evolutionarily new) requires antibodies to activate (specific immune response, adaptive immunity), while alternative And lectin pathways can be activated by antigens without the presence of antibodies (nonspecific immune response, innate immunity). The result of complement activation in all three cases is the same: C3 convertase hydrolyzes C3, creating C3a and C3b and causing a cascade of further hydrolysis of complement system elements and activation events. In the classical pathway, activation of C3 convertase requires the formation of the C4bC2a complex. This complex is formed upon cleavage of C2 and C4 by the C1 complex. The C1 complex, in turn, must bind to class M or G immunoglobulins for activation. C3b binds to the surface of pathogens, which leads to a greater “interest” of phagocytes in C3b-associated cells (opsonization). C5a is an important chemoattractant that helps attract new immune cells to the area of ​​complement activation. Both C3a and C5a have anaphylotoxic activity, directly causing degranulation of mast cells (as a result, release of inflammatory mediators). C5b starts the formation of membrane attack complexes (MACs) consisting of C5b, C6, C7, C8 and polymeric C9. MAC is the cytolytic end product of complement activation. MAC forms a transmembrane channel that causes osmotic lysis of the target cell. Macrophages engulf pathogens labeled by the complement system.

biological functions

Now there are the following functions:

1. opsonizing function. Immediately following the activation of the complement system, opsonizing components are formed that cover pathogens or immune complexes, attracting phagocytes. The presence of the C3b receptor on the surface of phagocytic cells enhances their attachment to opsonized bacteria and activates the absorption process. This tighter attachment of C3b-bound cells or immune complexes to phagocytic cells has been termed immune attachment phenomenon.
2. Solubilization (i.e. dissolution) of immune complexes (C3b molecule). With complement deficiency, immunocomplex pathology (SLE-like conditions) develops. [SLE = systemic lupus erythematosus]
3. Participation in inflammatory reactions. Activation of the complement system leads to the release of biologically active substances (histamine, serotonin, bradykinin) from tissue basophils (mast cells) and basophilic blood granulocytes, which stimulate the inflammatory response (inflammatory mediators). Biologically active components that are formed during splitting C3 And C5, lead to the release of vasoactive amines, such as histamine, from tissue basophils (mast cells) and blood basophilic granulocytes. In turn, this is accompanied by relaxation of smooth muscles and contraction of capillary endothelial cells, and increased vascular permeability. Fragment C5a and other complement activation products promote chemotaxis, aggregation and degranulation of neutrophils and the formation of oxygen free radicals. Administration of C5a to animals resulted in arterial hypotension, pulmonary vasoconstriction, and increased vascular permeability due to endothelial damage.
   Functions of C3a:
   • act as a chemotactic factor, causing the migration of neutrophils towards the place of its release;
   • induce attachment of neutrophils to the vascular endothelium and to each other;
   • activate neutrophils, causing them to develop a respiratory burst and degranulation;
   • stimulate the production of leukotrienes by neutrophils.
4. Cytotoxic or lytic function. In the final stage of activation of the complement system, a membrane attack complex (MAC) is formed from the late complement components, which attacks the membrane of a bacterial or any other cell and destroys it.

Activation of the complement system

classic way

The classical path is triggered by the activation of the complex C1(it includes one C1q molecule and one C1r and C1s each). The C1 complex binds via C1q to class M and G immunoglobulins associated with antigens. Hexameric C1q is shaped like a bouquet of unopened tulips, the “buds” of which can bind to the antibody site. A single IgM molecule is sufficient to initiate this pathway, activation by IgG molecules is less efficient and requires more IgG molecules.

С1q binds directly to the surface of the pathogen, this leads to conformational changes in the C1q molecule, and causes the activation of two molecules of C1r serine proteases. They cleave C1s (also a serine protease). The C1 complex then binds to C4 and C2 and then cleaves them to form C2a and C4b. C4b and C2a bind to each other on the surface of the pathogen to form the classical pathway C3 convertase, C4b2a. The appearance of C3 convertase leads to the splitting of C3 into C3a and C3b. C3b forms, together with C2a and C4b, the C5 convertase of the classical pathway. C5 is cleaved into C5a and C5b. C5b remains on the membrane and connects to the C4b2a3b complex. Then C6, C7, C8 and C9 are connected, which polymerizes and a tubule appears inside the membrane. Thus, the osmotic balance is disturbed and, as a result of turgor, the bacterium bursts. The classical way is more accurate, since any foreign cell is destroyed in this way.

Alternative path

An alternative pathway is triggered by hydrolysis of C3 directly on the surface of the pathogen. Factors B and D are involved in the alternative pathway. With their help, the enzyme C3bBb is formed. Protein P stabilizes it and ensures its long-term functioning. Further, PC3bBb activates C3, as a result, C5-convertase is formed and the formation of a membrane attack complex is triggered. Further activation of the terminal complement components occurs in the same way as in the classical pathway of complement activation. In the liquid in the C3bBb complex, B is replaced by the H factor and, under the influence of a deactivating compound (H), turns into C3bi. When microbes enter the body, the C3bBb complex begins to accumulate on the membrane. It connects to C5, which splits into C5a and C5b. C5b remains on the membrane. Then C6, C7, C8 and C9 are connected. After C9 is combined with C8, C9 is polymerized (up to 18 molecules are crosslinked with each other) and a tube is formed that penetrates the bacterial membrane, water is pumped in and the bacterium bursts.

The alternative pathway differs from the classical one in the following way: activation of the complement system does not require the formation of immune complexes; it occurs without the participation of the first complement components - C1, C2, C4. It also differs in that it works immediately after the appearance of antigens - its activators can be bacterial polysaccharides and lipopolysaccharides (they are mitogens), viral particles, tumor cells.

Lectin (mannose) pathway of activation of the complement system

The lectin pathway is homologous to the classical pathway of activation of the complement system. It uses the mannose-binding lectin (MBL), a protein similar to the classical C1q activation pathway, that binds to mannose residues and other sugars on the membrane, allowing for the recognition of a variety of pathogens. MBL is a serum protein belonging to the group of collectin proteins, which is synthesized mainly in the liver and can activate the complement cascade by directly binding to the surface of the pathogen.

In blood serum, MBL forms a complex with MASP-I and MASP-II (Mannan-binding lectin Associated Serine Protease, MBL-binding serine proteases). MASP-I and MASP-II are very similar to C1r and C1s of the classical activation pathway and may have a common evolutionary ancestor. When several MBL active sites bind to specifically oriented mannose residues on the pathogen's phospholipid bilayer, MASP-I and MASP-II are activated and cleave the C4 protein into C4a and C4b, and the C2 protein into C2a and C2b. C4b and C2a then combine on the surface of the pathogen to form C3 convertase, and C4a and C2b act as chemoattractants for cells of the immune system.

Regulation of the complement system

The complement system can be very dangerous to host tissues, so its activation must be well regulated. Most of the components are active only as part of the complex, while their active forms can exist for a very short time. If during this time they do not meet with the next component of the complex, then the active forms lose their connection with the complex and become inactive. If the concentration of any of the components is below the threshold (critical), then the work of the complement system will not lead to physiological consequences. The complement system is regulated by special proteins that are found in blood plasma in even higher concentrations than the complement system proteins themselves. The same proteins are present on the membranes of the body's own cells, protecting them from attack by the proteins of the complement system.

Regulatory mechanisms mainly operate at three points.

1. C1. Inhibitor C1 controls the classical and lectin activation pathways. It acts in two ways: it limits the action of C4 and C2 by binding to C1r and C1s proteases and similarly turns off the lectin pathway by removing MASP enzymes from the MBP complex.
2. C3 convertase. The lifetime of C3-convertase is reduced by decay accelerating factors. Some of them are found on the surface of their own cells (for example, DAF and CR1). They act on C3 convertases in both the classical and alternative pathways of activation. DAF accelerates the breakdown of the alternative pathway C3 convertase. CR1 (C3b/C4b receptor) is located mainly on the surface of erythrocytes and is responsible for the removal of opsonized immune complexes from blood plasma. Other regulatory proteins are produced by the liver and are dissolved in the blood plasma in an inactive state. Factor I is a serine protease that cleaves C3b and C4b. C4-binding protein (C4BP) cleaves C4 and helps factor I cleave C4b. Factor H binds to glycosaminoglycans that are present on self cells but not on pathogen cells. This protein is a factor I cofactor and also inhibits C3bBb activity.
3. C9. CD59 and Homologous Limiting Factor inhibit C9 polymerization during the formation of the membrane attack complex, preventing it from forming.

The role of the complement system in disease

The complement system plays a large role in many immune-related diseases.

CORRESPONDENCE ACADEMY OF POSTGRADUATE EDUCATION

CORRESPONDENCE ACADEMY OF POSTGRADUATE EDUCATION

K. P. Kashkin, L. N. Dmitrieva

PROTEINS OF THE COMPLEMENT SYSTEM: PROPERTIES AND BIOLOGICAL ACTIVITY (Lecture)

Department of Immunology, Russian Medical Academy of Postgraduate Education, Ministry of Health of the Russian Federation, Moscow

Protection of the body from foreign agents is carried out with the participation of many so-called non-antigen-specific cellular and humoral immunity factors. The latter are represented by various blood proteins and peptides. also present in other body fluids. Humoral antigen-specific immunity factors either themselves have antimicrobial properties or are able to activate other humoral and cellular mechanisms of the body's immune defense.

In 1894 V. I. Isaev and R. Pfeiffer showed that fresh blood serum of immunized animals has bacteriolytic properties. Later, this antimicrobial serum factor was named alexin (Greek alexo - protect, reflect), or complement, and characterized as a thermolabile factor that ensures the lysis of microbes in the immune serum, as well as the lysis of erythrocytes sensitized by antibodies.

According to modern ideas, complement is a system of serum proteins that can be activated as a result of the interaction of some of the initial components of the system with antigen-antibody complexes or with other molecules that activate the system.

Proteins of the complement system are represented by 13 blood plasma glycoproteins. The system is regulated by seven blood plasma proteins and a multitude of proteins and receptors associated with cell membranes.

In the literature, the complement system is denoted by the Latin letter C", while individual components are additionally given Arabic numerals (Cl, C2, C3, etc.) or capital letters (factors: B, D): complement subunits, as well as cleavage or activation products of proteins systems - additionally in small Latin letters (for example: Clq, C3a, C3b, etc.);

activated forms of complement components can be indicated by a stroke from above (Cl, C3, B, etc.). The numbering of components C" corresponds to the chronology of their discovery and does not always coincide with the sequence of involvement of the components in the activation reaction of the complement system.

Activation of the complement system occurs as a result of the interaction of some proteins of the complement system circulating in the blood with activating agents. Such an interaction changes the conformational structure of the molecules of the corresponding complement components, so that protein molecules open up regions that can interact with subsequent components of the system, fix them, and sometimes split them.

This "cascade" type of activation is characteristic of both the complement system and many other blood protein systems. When the complement system is activated, plasma-soluble native complement proteins are “consumed” and fixed on various insoluble carriers (aggregates of molecules, cell surfaces, etc.).

Classic pathway of complement system activation

Two main complement activation pathways are known - the classical, discovered first, and the alternative, established later. The classical pathway differs from the alternative one in that the activation of the system is initiated by the Clq subcomponent of complement as a result of the interaction of Clq with the Fc fragment of conformationally altered IgG and IgM blood. Conformational changes in Fc fragments in IgG and IgM occur when these blood immunoglobulins interact with antigens, as well as artificially as a result of thermal (63°C, 10 min) or chemical (diazobenzidine) treatment of immunoglobulins.

Depending on the role played by individual complement components in the process of activation and ensuring the function of the system, complement proteins can be divided into several blocks: recognizing (Cl), activating the system (C2, C4, C3) and attacking cell membranes (C5, C6 , C7, C8, C9). The properties of the proteins included in these blocks are summarized in Table. I. Activation of the complement system in the classical way begins with the Clq subcomponent of complement, conformational changes in molecules of which "trigger" this process (Fig. 1). Clq is a serum glycoprotein built from 18 polypeptide chains of three types: A, B and C. Chains A, B and C from the N-terminus of the chains are brought together to form six globular heads. The A, B, and C chains themselves are held together by disulfide bonds, forming six collagen-like triple helices. The C-terminals of the polypeptide chains of all six Clq helices are held together. The shape of the Clq molecule resembles a mollusk with six tentacles (Fig. 2). Like collagen, Clq contains large amounts of glycine, hydroxyproline, and hydroxylysine. About 8% of the Clq mass is made up of carbohydrates, among which glycosylgalactosyl residues predominate. Clq does not have enzymatic activity, but with the help of its six collagen-like three-helix filaments - "tentacles" - it interacts both with complexes of C1r and Cls subcomponents of complement circulating in the blood (sections of filaments between the globular heads and the central part of the Clq molecule), and with Fc regions of conformationally changed IgG and IgM molecules (globular heads at the free ends of six Clq strands). Isolated from the blood, the Clr component of complement is a dimer (C1Gs), dissociating into two monomeric C1g molecules at pH 5.0. Each C1r monomer is represented by a polypeptide chain of 688 amino acid residues. The polypeptide chain of the monomer forms one domain at the end of the molecule. During dimerization, the contact binding site of the monomers is located between these domains so that the C1r3 dimer has an asymmetric "X" shape. Activated C1r2 is a serine protease and in the construction of active

Rice. 1. The classical way of activation of the complement system.

a - complement components in the aqueous phase; b- complement components, immobilized on cell membranes; Ag - antigens on the cell membrane;at- antibodies to corresponding antigens of the IgM and IgG classes; POPPY. - membrane attack complex.

The term "complement" was first proposed by Borclet as a result of the observation that in order to realize a number of immunological effects (hemolysis, bactericidal activity), along with antibodies, a serum factor is required, which is destroyed when heated to +56°C. Over 70 years of studying complement, it has been established that it is a complex system of 11 serum proteins, the activity of which is regulated by at least the same number of factors. Complement is a system of cascade-acting highly efficient proteases that are sequentially activated by the cleavage or addition of peptide fragments and ultimately leads to bacteriolysis or cytolysis. In terms of complexity, the complement system is comparable to the blood coagulation system, with which it is connected, as well as with the kinin system, by functional links. In phylogenesis, the complement system appeared before the immune system. Ontogenetically, this manifests itself in the fact that already a 6-week-old fetus is able to synthesize individual components of the system, and from the 10th week, hemolytic activity of the synthesized factors can be detected, although normal concentrations of all C-components are determined only during the first year after birth. Of the total amount of whey proteins, the complement system accounts for about 10%. It is the basis of the body's defenses. Functional defects in the complement system can lead to severe recurrent infections and pathological conditions due to immune complexes. There is a direct functional relationship between the complement system and the phagocytic system, since direct or antibody-mediated binding of complement components to bacteria is a necessary condition for phagocytosis (opsonization of microorganisms). Complement is the dominant humoral component of the inflammatory response, since its products are chemotaxins and anaphylactoxins that have a pronounced effect on phagocytes, metabolism, and the blood coagulation system. Thus, complement is considered to be an important element of the resistance system, as well as an effective component of humoral immunity. In addition, the complement system includes important factors in the regulation of the immune response.

Synthesis and metabolism of C-factors. The formation of C-factors occurs mainly in the liver, bone marrow and spleen. A special position is occupied by C1, which is apparently synthesized in the epithelium of the small intestine. Macrophages play a decisive role in the synthesis of complement components, reflecting the close phylogenetic relationship between these two systems. The continuous use of C-factors in the body and the high level of their catabolism determine the need for their continuous synthesis, and the rate of synthesis is relatively high. For C3, for example, 0.5-1.0 mg of protein per 1 kg of body weight is synthesized every hour. Both activation and inhibition, as well as consumption and synthesis, are in a labile equilibrium. At the same time, the serum concentrations of individual factors, on the one hand, and the content of fragments and cleavage products, on the other hand, make it possible to assess the state and level of activation of the entire system.

C-factors usually consist of several polypeptide chains. C3, C4 and C5 are synthesized in the form of a single polypeptide chain, as a result of proteolytic cleavage of which either C3 and C5, or only C4 are formed. The C1 and C8 polypeptide chains are synthesized separately. Glucosylation occurs immediately before secretion and is a necessary prerequisite for this process.

A decrease in the synthesis of complement components is observed in severe liver disease, uremia and the use of high concentrations of corticosteroids, affecting mainly C3, C4 and C5. A reduced serum C3 concentration is also determined in chronic immunocomplex pathology due to the activation of an alternative pathway with an increased consumption of this component. At the same time, a decrease in the synthesis of this component may occur, which indicates the existence of a negative feedback on the regulation of its synthesis through C3d.

Mechanisms of activation of the complement system. Activation after the initial stage can develop in several directions:

Classical pathway of complement activation, starting at C1;

Alternative complement activation pathway starting at C3;

Specific activation of complement with the formation of various cleavage products.

I. The classical way of activation of the complement system. The classical pathway of complement activation is an immunologically mediated process initiated by antibodies. Immunological specificity is provided by the interaction of antibodies with antigens of bacteria, viruses and cells. The antigen-antibody reaction is associated with a change in the configuration of the immunoglobulin, which leads to the formation of a binding site for Clq on the Fc fragment near the hinge region. Immunoglobulins can bind to C1. C1 activation occurs exclusively between two Fc fragments. Therefore, the activation cascade can be induced even by a single IgM molecule. In the case of IgG antibodies, the proximity of two antibody molecules is necessary, which imposes severe restrictions on the density of antigen epitopes. In this regard, IgM is a much more effective initiator of cytolysis and immune opsonization than IgG. Quantitatively, this estimate corresponds to a value of 800:1. The process of complement activation itself can be divided into certain stages:
1- recognition of immune complexes and formation of C1;
2 - formation of C3-convertase and C5-convertase;
3 - formation of a thermostable complex C5b, 6.7;
4 - membrane perforation.

Membrane perforation. Each formed C5b, 6,7 complex, regardless of membrane binding or S-protein shielding, binds to 1 C8 molecule and 3 C9 molecules. The free C5b-C9 complex acts hemolytically, whereas the complex with the S-protein does not have this effect. Two membrane-associated C5b-C9 complexes form a ring pair in the membrane, which leads to a sharp change in the osmotic pressure in the cell. If erythrocytes are highly sensitive to the formation of such a membrane defect, then nucleated cells are capable of repairing defects of this type and have a certain resistance to complement attack. In this regard, the determining factor in the interaction of complement with the membrane is the total number of Clg molecules bound to the cell, which depends on the number and class of antibodies bound to the cell. Among bacteria, there are species that are resistant to the action of complement. In this case, the effect of opsonization of microorganisms with subsequent phagocytosis is decisive. Lysozyme plays a role in the complement attack of Gram-negative bacteria. Some features of complement activation follow from general patterns and are determined by the initial activation of C1 by soluble or precipitated immune complexes. The reaction proceeds identically up to the formation of the C5b complex, 6,7, which leads to the production of chemotactic factors and anaphylatoxins. Similar processes occur with intravenous administration of aggregated IgG. Clinical manifestations in this case can vary from serum sickness to anaphylactic shock. The combination of Fc fragments with adhesive components C5b, 6,7 in the composition of soluble immune complexes can lead to their deposition on endothelial cells and association with blood cells, causing a number of systemic lesions. Such immunocomplex mechanisms create the basis for type III allergic reactions, a cascade of complement activation reactions, an avalanche-like involvement of complement components in the reaction with an increase in the number of pharmacologically active fragments.

Alternative pathway for complement activation. With an alternative pathway of complement activation, factors C1, C4, C2 do not participate in the reactions. Activation begins when C3 is cleaved into C3a and C3b fragments. The further course of the process is identical to the classical path.

Pillemer first described the Mg+ dependent "properdin system" in which C3 was activated by zymosan (a polysaccharide) without the participation of antibodies. Other insoluble polysaccharides can also act as activators (inulin, high molecular weight dextran), in addition, bacterial endotoxins, aggregated IgG4, IgA and IgE, immune complexes with F fragments, proteases (plasmin, trypsin), cobra venom factor, C3b can serve as activators. . In the alternative activation pathway, two C3 convertases act. C3Bb has little activity and appears when C3 interacts with B, D and properdin. C3Bb removes a small amount of C3b, which leads to the formation of a highly active C3b convertase, the result of which is C3b. There is a positive feedback that greatly enhances the reaction. The suppression of such spontaneous enhancement is carried out by C3b-INA, which inhibits C3b formed in a soluble form. Cobra venom factor is a functional and structural analogue of C3b, but is not inhibited by C3b-INA. Endotoxins and polysaccharides activate properdin and thus create conditions for the binding and stabilization of C3b, which is inhibited by C3b-INA only in the free state. The defining step in the alternative activation pathway is the formation of C3b, which is transferred to the activated surface. The process begins with the binding of C3b to B, this step being dependent on the presence of Mg2+. C3bB is activated by D to the C3b Bb complex. Properdin binds C3b and thus stabilizes the spontaneously dissociating Bb complex. A specific inhibitor of the alternative pathway is B1H. It competes with factor B for the C3b bond, displacing it from the C3bB complex and making C3b available for C3b-INA action. The cytolytic activity of the alternative pathway is completely determined by the properties of the shell of microorganisms and the cell membrane. Glycoproteins and glycolipids containing terminal residues of sialic acid render the membrane resistant to the action of complement activated via the alternative pathway, while treatment with neuraminidase abolishes this resistance and makes the cells highly sensitive. Sialic acids play an important role in the resistance of microorganisms. Most bacterial species do not contain sialic acids in their shell, but many pathogenic species do. Antibodies can change surface properties and thus increase the sensitivity of targets to complement. An important step in surface activation is the binding of properdin, resulting in a high-affinity receptor for C3b and, at the same time, a stable C3Bb complex is formed. In this regard, there are two types of alternative pathway activators: 1) properdin-dependent activators (polysaccharides, endotoxins, antibodies); 2) property-independent activators (cobra venom factor, proteases).

The C5-convertase of the alternative pathway of activation results from the binding of C3b to the C3Bb complex as part of the amplification mechanism, and the subsequent course of the process corresponds to the classical pathway of activation.

Alternative complement activation is an important component of the system of nonspecific resistance to bacteria, viruses and unicellular microorganisms. The transition from nonspecific protection to antibody-mediated reactions occurs smoothly, or both processes proceed in parallel. As a pathogenetic link, alternative complement activation is involved in many diseases. Examples are:
- membranoproliferative nephritis with hypocomplementemia;
- acute glomerulonephritis after streptococcal infection;
- nephritis with SLE;
- disease of pigeon breeders;
- fungal infections;
- septicemia with shock caused by endotoxins;
- nocturnal paroxysmal hemoglobinuria;
- partial lipodystrophy.

An alternative pathway is also observed in some cases of complement activation along the classical pathway. With nephritis, the C3NeF factor is detected, which is a complex of autoantibodies with C3bBb, resistant to the action of p1H and functioning as a C3 convertase. Endotoxins due to lipid A are effective activators of not only the alternative pathway of complement activation, but also the coagulation system, as well as the kinin system. Activation of factor XII plays a decisive role in this.

Non-specific complement activation. Non-specific complement activation can be carried out by proteases (trypsin, plasmin, kallikrein, lysosomal proteases and bacterial enzymes) at each step from C1 to C5. The initial activated factor is much more effective than the inducing protease, and when activated in the liquid phase, activation can begin in several processes at once. Anaphylatoxins arise, which, in addition to the hemolytic effect, give a complete picture of shock in acute pancreatitis and severe infections. Nonspecific activation is one of the components of acute inflammation.

Mechanisms of regulation of the complement activation system

I. Inhibitory mechanisms. Each step of the complement activation cascade is in equilibrium with an inactivated state. Pronounced pharmacological effects of activation products require regulation at various levels.

The limiting factor in the classical pathway activation system is C2, which is present in the lowest concentration.

Another limiting group of factors is the need for interaction of Clq with two Fc fragments of antibodies and the possibility of access to the resulting binding sites for activators and reaction substrates (C2a, C4b, C3b, etc. up to C9). The instability of C2a, C4b, C5b and Bb in the liquid phase prevents the unlimited development of the reaction and determines the concentration of the process on the activated surface. Described specific inhibitors for Clr, Cls, C4b, C2, C3b, C6, C5b-6-7, Bb, C3a and C5a.

II. Incentive mechanisms. The most important mechanism for enhancing complement activation is positive feedback, as a result of which the appearance of C3b leads to a significant acceleration of the formation of this activation product. Activated properdin stabilizes Bb. The effect of pathological autoantibodies is realized in a similar way.

Biological effects of the complement system

I. Cytolysis and bactericidal. Cytolysis and bactericidal activity can be induced as follows:
- immune cytolysis due to IgM and IgG antibodies;
- CRP (C-reactive protein) - connection with subsequent complement activation;
- direct activation of properdin through an alternative pathway of activation by cells and bacteria;
- side effects in the reaction of immune complexes;
- involvement of activated phagocytes.

II. Formation of anaphylatoxins. The term "anaphylatoxin" was first introduced by Friedberger. In this case, we meant the C3a fragment and the C5a fragment, which bind to the corresponding cell membrane receptors and have similar pharmacological effects:
- release of histamine and other mediators from mast cells and basophils (C5a is more effective than C3a);
- contraction of smooth muscles and effects on microcirculation (C3a is more effective than C5a);
- activation of phagocytes and secretion of lysosomal enzymes (the efficiency of C3a and C5a is comparable).

Neutralization of viruses. The complement system is an important factor in natural resistance against viral infection. Some RNA-containing oncogenic viruses are capable of directly binding Clq. Classical complement activation in this case leads to lysis of the infectious agent. Some other viruses interact with complement via CPB. In addition, complement is able to inactivate the virus located in the soluble immune complex, which leads to its opsonization and phagocytosis.

The antiviral action of complement is due to the following processes:
- lysis of the virus due to fragments from C1 to C9;
- aggregation of the virus due to immune conglutinins;
- opsonization and phagocytosis;
- blockade of viral ligands for the corresponding cell membrane receptors;
- blockade of virus penetration into the cell.

By itself, complement is not able to inactivate the cell affected by the virus.

Destruction of immune complexes. The appearance of immune complexes containing antibodies of the IgG and IgM classes is associated with constant complement activation. Activated complement components bind to components of immune complexes, including both antibodies and antigens, thereby preventing the formation of large aggregates due to steric effects. Since complement activation is associated with the appearance of protease activity, partial loosening and splitting of the formed aggregates occurs. Removal of decay products from the bloodstream is carried out due to opsonization with the help of immunophagocytosis and immunoendocytosis, and therefore the accessibility to binding to cell receptors associated with C3b complexes plays an important role. The immune complexes deposited in the tissues are also removed by phagocytosis, and plasmin and lysosomal enzymes play a significant role in this process.

Complement, blood coagulation and the kinin system. Complement, the blood coagulation system and the kinin system are closely related functionally. We are talking about a complex set of mechanisms, the activation of each of which leads to the activation of the entire complex. This is clearly seen in the endotoxin-induced Sanarelli-Schwartzmann reaction and conditions caused by immune complexes. Kallikrein, plasmin, and thrombin activate C1 and cleave C3, C5, and factor B. Factor XIIA can also activate C1, with C1 being cleaved first by plasmin, and then the cleavage products are used by kallikrein and factor XIIA. Platelet activation is carried out through the interaction of C3, factor B, properdin, fibrinogen and thrombin. Activated macrophages and phagocytes are important sources of tissue proteases and thromboplastin in all types of inflammation. Activation of all three systems occurs through the activation of factor XII (Hageman factor). On the other hand, C1 = 1NH inhibits both kallikrein and factor XIIA. Protease inhibitors - antitrypsin, macroglobulin and antichymotrypsin - have the same effect. As a result, a system with complex dynamics is formed, which can not only perform protective functions, but also participate in pathological processes.

Complement and T cell-mediated immune responses. The complement system has a regulatory effect on both the T-system and B-lymphocytes, with C3 fragments, factor B and B1H acting as the main mediators. Membrane-associated factors and complement components C5, C6, C7, C8 and C9 were detected on cytotoxic lymphocytes (CTLs). On the other hand, the study of CTL target cells using an electron microscope showed that structures similar to pores formed under the action of complement system factors on the membrane are determined in the area of ​​intercellular contact.

Diagnostic value of the complement system. Assessment of the complement system aims to address the following practical questions:
- Are activated components of the complement system involved in the pathogenesis of the disease?
Are there any defects in the complement system?

To answer these questions, the total complement activity is first determined using ram erythrocytes and inactivated antiserum. As a source of complement, the test serum is used in serial dilutions and the titer corresponding to 50% hemolysis is determined. The results are expressed in units of CH50. Rabbit erythrocytes can directly activate an alternative pathway of complement activation, in which case the activity of the serum under study is measured in units of AP 50. With acute and progressive complement consumption, as well as its defects, a decrease in complement activity is observed. To detect a defect in a certain factor, sera are used that do not contain the studied factor, which are added to the test sample. Immunochemical determination of individual components of the complement system (rocket electrophoresis and radial immunodiffusion) is also used, but this approach cannot replace functional tests, since functionally inactive abnormal proteins and inactive cleavage products can lead to erroneous determinations. All test samples should be stored at -70 °C until use. The study of complement consumption can be carried out using radioimmunoassay and enzyme immunoassay methods for determining the cleavage products of C3, C4 and B. Of particular importance is quantitative RIA for determining the concentration of C5a, which is an indicator of anaphylactic reactions. When identifying primary and secondary complement defects, it is recommended to use the following research program:
- determination of CH50, and possibly AP50 for screening;
- quantitative determination of C4 and C3 to clarify the role of the classical and alternative activation pathways;
- detailed analysis of Clq, C5, P and other factors.

In the acute phase of inflammation, with tumors and during the postoperative period, complement activity is increased.

Complement in diseases of the immune system. The complement system plays an important role in allergic diseases type II (cytotoxic antibodies) and type III (immunocomplex pathology, Arthus phenomenon). The role of complement is confirmed by the following data:
- pronounced complement consumption (CH50 is reduced, activity and concentration of factors are below normal);
- the appearance of degradation products of components in the serum (C4a, fragments C3, C5a);
- determined by immunohistochemical analysis of specific antibodies (anti-C3, anti-C4, etc.) complement deposits in tissues;
- production of cytotoxic antibodies;
- evidence of chronically increased complement consumption.

Typical examples are the following diseases:
- acute viral infections (especially often the effects of immune complexes are manifested in rubella, measles, hepatitis B, infection with the ECHO virus);
- acute bacterial infections (activation of complement by immune complexes in streptococcal infections, for example, in scarlet fever; activation of the alternative pathway in infection with gram-negative microorganisms or endotoxin);
- glomerulonephritis;
- autoimmune hemolytic anemia;
immune thrombocytopenia;
- systemic lupus erythematosus;
- antibody-induced graft rejection;
- rheumatoid arthritis;
- serum sickness;
- cryoglobulinemia, amyloidosis, plasmacytoma.

In all these diseases, complement assessment is not entirely informative, nor is it in a wide range of chronic diseases. However, the study of this system allows us to draw a conclusion about the individual dynamics of the course of the disease. Complement testing is mandatory if there is a history of frequent bacterial infections due to the possibility of genetically determined anomalies. This is also true for SLE, which is often associated with birth defects of the complement system.

26.1. General concept
Complement is a complex protein complex in the blood serum.
A. The complement system consists of 30 proteins (components, or fractions, of the complement system).
B. The complement system is activated due to a cascade process: the product of the previous reaction acts as a catalyst for the subsequent reaction. Moreover, when the fraction of the component is activated, in the first five components, its splitting occurs. The products of this cleavage are referred to as the active fractions of the complement system.
1. The largest of the fragments (denoted by the letter b) formed during the cleavage of the inactive fraction remains on the cell surface - complement activation always occurs on the surface of the microbial cell, but not on its own eukaryotic cells. This fragment acquires the properties of an enzyme and the ability to act on the subsequent component, activating it.
2. The smaller fragment (denoted by the letter a) is soluble and "leaves" in the liquid phase, i.e. into the blood serum.
B. Fractions of the complement system are designated differently.
1. Nine - discovered first - proteins of the complement system are designated by the letter C (from the English word complement) with the corresponding number.
2. The remaining fractions of the complement system are designated by other Latin letters or their combinations.
D. The value of complement for the macroorganism is large and varied (for more details, see section 26.6).
1. Part of the active fractions of the complement system are proteases.
2. Some - bind to the antigen-antibody complex (immune complex).
3. Others - activate mast cells and associated vascular inflammatory responses.
4. And, finally, part of the complement fractions perforates the membranes of bacterial cells.

26.2. Complement activation pathways
There are three complement activation pathways: classical, lectin, and alternative.
A. The classical pathway of complement activation is the main one. Participation in this pathway of complement activation is the main function of antibodies.

Figure 26.2-2. Diagram of the classical complement activation pathway

1. Complement activation in the classical way is triggered by the immune complex: a complex of antigen with immunoglobulin (class G - the first three subclasses - or M). The place of the antibody can be "taken" by C-reactive protein - such a complex also activates complement along the classical pathway.
2. The classical way of complement activation is carried out as follows (Fig. 26.2-1).
A. First, the C1 fraction is activated: it is collected from three subfractions (C1q, C1r, C1s) and converted into the C1-esterase enzyme (C1qrs).
b. C1-esterase cleaves the C4 fraction.
V. The active C4b fraction covalently binds to the surface of microbial cells (but not to the macroorganism's own eukaryotic cells) and here attaches the C2 fraction to itself.
d. The C2 fraction in complex with the C4b fraction is cleaved by C1-esterase to form the active C2b fraction.
e. Active fractions C4b and C2b into one complex - C4bC2b - with enzymatic activity. This is the so-called classical pathway C3 convertase.
e. C3 convertase cleaves the C3 fraction, producing large amounts of the active C3b fraction.
and. The active fraction of C3b joins the C4bC2b complex and converts it into C5-convertase (C4bC2bC3b).
h. C5 convertase cleaves the C5 fraction.
And. The resulting active fraction C5b adds fraction C6.
j. The C5bC6 complex attaches the C7 fraction.
l. The C5bC6C7 complex is incorporated into the phospholipid bilayer of the microbial cell membrane.
m. Protein C8 joins this complex.
n. Being together with the whole complex in the phospholipid bilayer of the microbial cell membrane, the C8 protein catalyzes the polymerization of 10-16 molecules of the C9 protein. This polymer forms a non-falling pore with a diameter of about 10 nm in the membrane of a microbial cell (Figure 26.2-2), which leads to the lysis of the microbe (since many such pores form on its surface - the “activity” of one unit of C3-convertase leads to the appearance of about 1000 pore). The C5bC6C7C8C9 complex resulting from complement activation is called the membrane attack complex (MAC).

B. The lectin pathway of complement activation is triggered by a complex of a normal blood serum protein, the mannan-binding lectin (MBL), with carbohydrates of the surface structures of microbial cells (with mannose residues). The MSL-associated serine protease activated as a result of this process acts similarly to the C1-esterase of the classical pathway, along which, in fact, further events develop, ending with the formation of MAC (Fig. 26.2-3).
C. The alternative pathway of complement activation (Fig. 26.2-4) begins with the covalent binding of the active C3b fraction - which is always present in the blood serum as a result of the spontaneous cleavage of the C3 fraction that constantly occurs here - with the surface molecules of not all, but some microorganisms.

1. Further events develop as follows.
A. C3b binds factor B (which is structurally and functionally homologous to factor C2) to form the C3bB complex.
b. When bound to C3b, factor B acts as a substrate for factor D (serum serine protease), which cleaves it to form the active C3bBb complex. This complex has enzymatic activity, is structurally and functionally homologous to the classical pathway C3 convertase (C4bC2b) and is called the alternative pathway C3 convertase.
V. Alternate pathway C3 convertase itself is unstable. In order for the alternative pathway of complement activation to continue successfully, this enzyme is stabilized by factor P (properdin).
d. What happens next is similar to the classical pathway of complement activation.
1. A lot of C3b is produced and the C3bBbC3b complex is formed, which is a C5-convertase.
2. Activation of C5 gives rise to the formation of a membrane attack complex (see sections 26.2.A.2.i - 26.2.A.2.n).
2. The main functional difference of the alternative pathway of complement activation, in comparison with the classical one, is the rapid response to the pathogen: since it does not take time for the accumulation of specific antibodies and the formation of immune complexes.
D. It is important to understand that both the classical and alternative pathways of complement activation operate in parallel, amplifying (i.e. enhancing) each other. In other words, the complement is activated not “either by the classical or by the alternative”, but by “both by the classical and by the alternative” pathways of activation. This, with the addition of the lectin activation pathway, is a single process (see Fig. 26.2-5), the different components of which may simply manifest themselves to different degrees.

26.3. Anaphyllotoxins
The active complement fractions C3a and C5a are called anaphylotoxins, as they are involved, among other things, in an allergic reaction called anaphylaxis (see below). The strongest anaphylotoxin is C5a.
A. Anaphylotoxins act on different cells and tissues of the macroorganism.
1. Their action on mast cells causes degranulation of the latter.
2. Anaphylotoxins also act on smooth muscles, causing them to contract.
3. They also act on the vessel wall: they cause activation of the endothelium and increase its permeability, which creates conditions for extravasation of fluid and blood cells from the vascular bed during the development of an inflammatory reaction.
B. In addition, anaphylotoxins are immunomodulators, i.e. they act as regulators of the immune response.
1. C3a acts as an immunosuppressor (i.e. suppresses the immune response).
2. C5a is an immunostimulant (i.e. enhances the immune response).

26.4. Receptors for complement components
Complement fractions can affect the cells of the macroorganism only if the latter have corresponding receptors.
A. Phagocytes have a receptor for C3b. This receptor causes a greater activity of phagocytes in relation to opsonized microbes (namely, to those of them, on the surface of which there is a C3b fraction).
B. Erythrocytes have specific receptors for C3b and C4b fractions. With these receptors, erythrocytes bind the corresponding complement fractions in the composition of circulating immune complexes (CIC) and transport these complexes to macrophages of the spleen and liver, which destroy them, thereby carrying out clearance (i.e., purification) of the blood from the CIC.
C. Receptors for the C5a fraction are localized on mast cells, through which this anaphylatoxin activates these cells and causes their degranulation.
D. Macrophages have the same receptor, due to which the C5a fraction also activates these cells.

26.5. Regulation of the complement system
Normally, in the absence of a pathogen in the internal environment of the macroorganism, the level of spontaneous activity of the complement system is low. The cascade mechanism of complement activation is “launched” by activators, and the regulation of its work by the “feedback” type is triggered by inhibitors, without which each activation episode would end in complete exhaustion of the entire system.
A. The activators of the complement system are molecular complexes located on the surface of the microorganism and triggering the process of complement activation in one way or another. They have already been mentioned above (see section 26.2).
1. Two complexes act as activators of the classical pathway of complement activation.
A. Immune complex (antigen-antibody complex).
b. Complex of antigen with C-reactive protein.
2. The activator of the lectin pathway of complement activation is the complex of a normal blood serum protein - mannan-binding lectin (MBL) - with carbohydrates of the surface structures of microbial cells (namely, with mannose residues).
3. Two complexes act as activators of the alternative pathway of complement activation.
A. The complex (as a result of covalent binding) of the active fraction C3b - which is always present in the blood serum as a result of the spontaneous cleavage of the C3 fraction constantly occurring here - with the surface molecules of not all, but some microorganisms.
b. Immunoglobulins of classes A and E aggregated on the surface of the microbe.
B. Inhibitors of the complement system are localized in the blood serum or on the cell membrane.
1. Five proteins are localized in the blood serum - inhibitors of the complement system.
A. C1 inhibitor (C1inh) inactivates the active fraction of C1qrs (i.e. C1 esterase).
b. C4 binding protein (C4BP) makes factor C4b available for degradation by factor I.
V. Factor H - makes factor C3b available for degradation by factor I.
d. Factor I cleaves C3b (in complex with factor H) and C4b (in complex with C4BP).
e. Protein S binds to the C5bC6C7 complex and prevents further formation of the membrane attack complex.
2. Mammalian (and, accordingly, human) cells contain three proteins - inhibitors of the complement system.
A. DAF (decay-accelerating factor) inactivates C4bC2b (as it binds to C4b instead of C2).
b. MCP (membrane proteolysis cofactor) makes factor C3b available for degradation by factor I.
V. Protectin (also referred to as the CD59 molecule) inactivates the proteins of the membrane attack complex (prevents C-mediated lysis of its own cells)

26.6. Functions of the complement system
The complement system plays a very important role in host defense against pathogens.
A. The complement system is involved in the inactivation of microorganisms, incl. mediates the action of antibodies on microbes.
B. Active fractions of the complement system activate phagocytosis.
C. Active fractions of the complement system take part in the formation of the inflammatory response.

26.7. Determination of the activity of the complement system
To determine the activity of complement in modern immunological laboratories, the hemolysis reaction and enzyme immunoassay (ELISA) are used, which replaced the Mancini radial immunodiffusion reaction.
A. The hemolysis test is used to determine the complement titer and to measure the overall activity of the complement system.
1. Complement titer is defined as the maximum dilution of blood serum that causes lysis of sheep erythrocytes loaded with anti-erythrocyte antibodies (the so-called heme system).
2. The total activity of the complement system is understood as the amount of complement that ensures the lysis of 50% of the erythrocytes of the heme system (denoted as CH50).
B. ELISA is used to determine the serum concentration of individual components of the complement system (C1q, C1s, C2, C3, C4, C5, C6, C7, C8, C9, properdin, factor B, C1 inhibitor). Previously, the concentration of the most functionally important fractions of the complement system (more often C3 and C4) was determined using the Mancini immunodiffusion reaction, but in modern laboratories equipped with ELISA analyzers, enzyme immunoassay is used for this purpose, which significantly expanded the possibilities for assessing the functional state of a patient its complement system.