Complement Activation 2024 PDF

Complement Activation Istanbul University, Istanbul Medical Faculty Division of Virology and Fundemental Immunology Learning Objectives Complement Components Complement Activation Pathways Regulation of Complement Biological Consequences Complement Deficiences Complement It is a ter...

Complement Activation Istanbul University, Istanbul Medical Faculty Division of Virology and Fundemental Immunology Learning Objectives Complement Components Complement Activation Pathways Regulation of Complement Biological Consequences Complement Deficiences Complement It is a term that refers to heat-sensitive factors that kill pathogens through immune cytolysis in serum and are complementary to the defense system. It contains at least 50 different proteins that exist as pro-enzyme (zymogens) and become active upon stimulation. Most complement components are synthesized by hepatocytes in the liver,but some are also produced by monocytes,macrophages,fibroblasts and epithelial cells of the gastrointestinal and genitourinary tracts. Complement components constitute approximately 15 % of the globulin protein fraction in plasma. Functions of complement 1.Chemotaxis of phagocytic cells (C3a,C5a) 2.Bacterial opsonization (C3b) 3.Bacteriolysis 4.Adhesion 5.Removal of immune complexes 6.Immune regulation 7.Viral neutralization Activation of complement components C5 to C9 forms the membrane attack complex. History Research on complement began in the 1890s when Jules Bordet showed that sheep antiserum to the bacterium Vibrio cholerae caused bacterial lysis (membrane destruction). Lytic activity destroyed when heated at 56 C for 30 min Surprisingly, the ability to lyse the bacteria was restored to the heated serum by adding fresh serum that contained no antibacterial antibodies. Bordet reasoned that bacteriolysis required two different substances: 1. The heat-stable, cholera specific antibodies that bound to the bacterial surface, 2. Heat-labile (sensitive) component responsible for the lytic activity. Jules Bordet (1870-1961) Institute Pasteur Jules Bordet won the Nobel Prize in 1919 (Complement-mediated bacteriolysis) History The famous immunologist Paul Ehrlich, working independently in Berlin, conducted similar experiments and coined the term complement, defining it as; “the activity of blood serum that completes the action of antibody.” General features Complement is a term that refers to heat labile factors in the serum that cause immune cytolysis. As a group, "complement" comprises at least 50 distinct proteins that effect multiple biological functions. Most complement components are synthesized in the liver by hepatocytes, although some are also produced monocytes, macrophages, fibroblasts, and epithelial cells of the gastrointestinal and genitourinary tracts. Complement components constitute approximately 10-15% of the globulin protein fraction in plasma. The Role of Complement in the Immune system A major role for this system is the recognition and destruction of pathogens based on recognition of pathogen-associated molecular patterns (PAMPs), rather than on Ab specificity. Many components are proenzymes (zymogens), which are functionally inactive until proteolytic cleavage, which removes an inhibitory fragment and exposes the active site. Reaction starts with an enzyme cascade.  Complement Complement Complement Regulators Receptors Component C1 C2 C3 C4 C5 C6 C7 C8 C9 Functional Categories of Complement Proteins  Structure of the C1 macromolecular complex In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q and two molecules each of the serine proteases C1r and C1s, held together in a Ca - stabilized complex (C1qr s ). The C1q molecule itself is composed of 18 polypeptide chains that associate to form six collagen-like triple helical arms, the tips of which bind the C 2 domain of the antigen-bound antibody molecule. Complements C2, C3, C4, C5 consist of two fragments 1- The fragment with enzymatic activity Inaktive 2- The fragment with a biological function The fragments are activated by cleavage by proteolytic enzymes.  Membrane Attack Complex (MAK) C5b, C6, C7, C8, C9 form a complex (MAK). This complex has a tubular form and forms 70-100 A° pores on the pathogen surface. The Major Pathways of Complement Activation There are three major pathways by which the complement cascade can be initiated: 1. the classical pathway, 2. the lectin pathway, and 3. the alternative pathway. Although the initiating event of each of the three pathways of complement activation is different, they all converge in the generation of an enzyme complex that cleaves the C3 molecule. Enzymes that cleave C3 into two fragments, C3a and C3b, are referred to as C3 convertases. The Complement Pathways The classical and lectin pathways use the dimer C4b2a for their C3 convertase activity, while the alternative pathway uses C3bBb. However, the final result of both C3 convertase activities is the same: a dramatic increase in the concentration of C3b, a critically important, multifunctional complement protein. There is a second set of convertase enzymes generated in the early stages of complement activation. The C5 convertases are formed by the addition of a C3b component to each of the two C3 convertases. C5 convertases cleave C5 into C5a, an inflammatory mediator, or anaphylatoxin, and C5b, which is the initiating factor of the membrane attack complex. Inititation of Clasical Pathway The classical pathway of complement activation begins with the formation of antigen-antibody complexes. These complexes may be soluble, or they may be formed when an antibody binds to antigenic determinants, or epitopes, situated on viral, fungal, parasitic, or bacterial cell membranes. Only complexes formed by antigens with antibodies of the IgM, IgG 1, IgG2 and IgG3 antibodies are capable of activating the classical complement pathway. The formation of an antigen-antibody complex induces conformational changes in the nonantigen-binding (Fc) portion of the antibody molecule. This conformational change exposes a binding site on the antibody for the C1 component of complement. Intermediates in the Classical Pathway of Complement Activation up to the Formation of the C5 Convertase C1q binds to Ag-bound Ab Binding of C1q to Fc induces a conformational change in C1r C1r converts to an active serine protease enzyme, C1r, which cleaves C1s to a similar active enzyme, C1s. C1s has two substrates, C4 and C2 ↓ C1s hydrolyzes C4 into C4a and C4b, and hydrolyze C2 into C2b and C2a ↓ C4b and C2a form a C4b2a complex, also called C3 convertase, referring to its role in converting the C3 into an active form. C4b2a (C3 convertase) hydrolyzes C3 into C3b and C3a ↓ C3b binds to C4b2a and form C4b2a3b (C5 convertase) ↓ C4b2a3b cleaves C5 into C5b and C5a opsonization inflammatory inflammatory responses responses © The Lectin Pathway is Initiated when Soluble Proteins Recognize Microbial Antigens The lectin pathway of complement activation, like the classical pathway, proceeds through the activation of a C3 convertase composed of C4b and C2a. However, instead of relying on antibodies to recognize the microbial threat and to initiate the complement activation process, this pathway uses lectins—proteins that recognize particular carbohydrate components—as its specific receptor molecules. Because it does not rely on antibodies from the adaptive immune system, the lectin pathway is considered to be an arm of innate, rather than adaptive, immunity.  Initiation of the lectin pathway relies on lectin receptor recognition of microbial cell surface carbohydrates Mannose-binding lectin (MBL), the first lectin demonstrated to be capable of initiating complement activation, binds close-knit arrays of mannose (sugar) residues that are found on the surfaces of microbes such as; Salmonella, Listeria, and Neisseria bacteria, Cryptococcus neoformans and Candida albicans fungi, The membranes of some viruses such as HIV1 and respiratory syncytial virus (RSV).  The Lectin Pathway - In the blood, mannose-binding lectin (MBL) is associated with MBL- associated serine proteases (MASP) proteins. Three MASP proteins—MASP-1, MASP-2, and MASP-3—have been identified, but most studies of MASP function point to the MASP-2 protein as being the most important player in the next step of the MBL pathway. - MASP-2 is structurally related to the serine protease, and when MBL binds to a microbial surface, associated MASP-2 molecules cleave both C2 and C4, giving rise to the C4b2a C3 convertase that we encountered in our discussion of the classical pathway. The Alternative Pathway Initiation of the alternative pathway, like the lectin pathway, is independent of antibody-antigen interactions. Therefore, this pathway is also considered to be part of the innate immune system. However, unlike the lectin pathway, the alternative pathway uses a different set of C3 and C5 convertases. The alternative pathway C3 convertase, is made up of one molecule of the C3b protein fragment and one molecular fragment unique to the alternative pathway, Bb (C3bBb). A second C3b is then added to make the alternative pathway C5 convertase, C3bBbC3b. Recent investigations have revealed that the alternative pathway can be initiated in three distinct ways. The first mode of initiation to be discovered, the “tickover” pathway, uses the four serum components; C3, factor B, factor D, and Second mode of activation for the alternative pathway have also been identified: one is initiated by properdin, and the other by proteases such as thrombin and kallikrein (It is shown invitro).  The Alternative Tickover Pathway The term tickover refers to the fact that C3 is constantly being made and spontaneously inactivated. The alternative tickover pathway is initiated when C3, which is at high concentrations in serum, undergoes spontaneous hydrolysis at its internal thioester bond, yielding the molecule C3(H O). The conformation of C3(H O) is different from that of the C3 parent protein. C3(H O) accounts for approximately 0.5% of plasma C3. In the presence of serum Mg , C3(H O) binds another serum protein, factor B. When bound to C3(H O), factor B becomes susceptible to cleavage by a serum protease, factor D and releasing a smaller Ba subunit that diffuses away, leaving a catalytically active Bb subunit bound to C3(H O). The C3(H O)Bb complex is referred to as the fluid-phase C3 convertase because it remains in the blood plasma and is not bound to any cells. Membrane-bound C3 convertase The C3(H O)Bb complex is not very stable in a healthy host and it is rapidly degraded, hence it needs other pathways. The fluid-phase convertase cleaves many molecules of C3 into C3a and C3b. If there is an infection present, some of the newly formed C3b molecules bind nearby microbial surfaces via their thioester linkages. It turns out that factor B is capable of binding not only to C3(H O) but also to the C3b fragment as well. In the presence of a microbial infection, factor B binds the newly attached C3b molecules on the microbial cell surface, and becomes susceptible to cleavage by factor D, with the generation of C3bBb complexes. Finally there is membrane-bound C3bBb C3 convertase that amplifies fluid-phase and results in microbial destruction. Initiation of the alternative pathway by specific, noncovalent binding of properdin to the target membrane. Properdin (factor P) binds to components of microbial membranes, and stabilizes the binding of C3bBb complexes of the alternative complement pathway. The difference between this and the tickover pathway is that properdin binds first and initiates complement deposition on the membrane. The Three Complement Pathways Converge at the Membrane-attack Complex  Formation of Membrane-attack Complex When C5b binds to the serum protein C6, the resulting complex interacts reversibly with the cell membrane via both ionic and hydrophobic bonds. Binding of C7 to C5bC6 induces a conformational change in C7 that exposes hydrophobic regions on its surface capable of inserting into the interior of the microbial membrane  Formation of Membrane-attack Complex C8 is made up of two peptide chains: C8β and C8αγ. Binding of C8β to the C5b67 complex induces a conformational change in the C8αγ dimer such that the hydrophobic domain of C8αγ can insert into the interior of the phospholipid membrane. The C5b678 complex is capable of creating a small membrane pore, 10 Å in diameter. The final step is the binding and polymerization of C9 to the C5b678 complex. As many as 10 to 19 molecules of C9 can be bound and polymerized by a single C5b678 complex. During polymerization, the C9 molecules undergo a conformational transition, so that they, too, can insert into the membrane. The completed membrane-attack complex (MAC) has a tubular form and functional pore size of 70 – 100 Å  Formation of Membrane-attack Complex Photomicrograph of poly-C9 complex formed by in-vitro Relative locations of the members of polymerization of C9 and complement-induced lesions on the membrane attack complex: the membrane of a red blood cell. These lesions result from C5b, C6, C7, C8, and C9. formation of membrane attack Complement Receptors Connect Complement Tagged Pathogens to Effector Cells Complement Receptors Connect Complement Tagged Pathogens to Effector Cells  The Regulation of Complement Activity C1 INH inhibits both the C1r2s2 serine proteases of the classical pathway and the MASP-2 protease of the lectin pathway, inhibiting further activation of C4 and C2 and formation of the C3 convertase. Decay-Accelerating Factor promotes decay of C3 convertases. Factor I is a soluble, constitutively active serine protease that cleaves C3b and C4b into inactive fragments only when it is associated on host cell membranes with the necessary cofactors. CD59 (Protectin) Inhibits the MAC Attack  Biological Effects of Complement Component 1. Cell lysis The membrane-attack complex can lyse a broad spectrum of cells: G(-) bacteria Parasites Erythrocyte nucleated cells (tumor cells) Additional viral neutralization Because the activation of alternative and lectin pathways is Ab- independent, these pathways serve as important innate immune defenses against infectious microorganisms.  Biological Effects of Complement Component 2. Opsonization Phagocytosis is mediated by many different complement receptors on the surface of macrophages and neutrophils, including CR1, SIGN-R1, and C1qRp. Phagocytes, using their Fc receptors, also bind to antigens opsonized by antibody binding. C3b is the major opsonin of the complement system although C4b and iC3b also have opsonizing activity  Biological Effects of Complement Component 3. Inflammation Various peptides generated during activation of complement play a decisive role in the development of an effective inflammatory response. C3a, C5a (called anaphylatoxin) bind to complement receptors on mast cells and basophils and induce degranulation with release of histamine and other mediators. The anaphylatoxins also induce smooth-muscle contraction, increased vascular permeability, extravasation, and chemoattraction.  Biological Effects of Complement Component 5. Clearance of immune complexes Antigen-antibody complexes generated during the course of an immune response are opsonized by C3b and removed from the circulation after recognition by CR1 receptors on erythrocytes.  Complement Deficiencies and Disease Classical Pathway Pathway Component Disease Mechanism C1INH Hereditary Overproduction of C2b Angioedema (prokinin) C1, C2, C4 Predisposition Opsonization of immune to SLE complexes help keep them soluble, deficiency results in increased precipitation in tissues and inflammation 47  Complement Deficiencies and Disease Lectin Pathway Pathway Component Disease Mechanism MBL Susceptibility to Inability to initiate bacterial infections lectin pathway in infants or immunosuppressed 48  Complement Deficiencies and Disease Alternative Pathway Pathway/Component Disease Mechanism Factors B or D Susceptibility Lack of sufficient to pyogenic opsonization of bacteria (pus-forming) bacterial infections C3 Susceptibility Lack of opsonization and to bacterial inability to utilize the infections membrane attack pathway C5, C6, C7 C8, or Susceptibility Inability to attack the outer C9 to Gram- membrane of Gram- negative negative bacteria infections 49  Complement Deficiencies and Disease Alternative Pathway cont. Pathway Component Disease Mechanism Properdin (X-linked) Susceptibility Lack of opsonization of meningococcal bacteria meningitis Factors H or I C3 deficiency Uncontrolled activation of and C3 via alternative susceptibility to pathway resulting in bacterial depletion of C3 infections 50 All three activation pathways of complement converge at a common point which is the activation of the C3 component  Classic pathway summary It is the first recorded complement mechanism. The most important factor that enables the activation of the classical complement reaction is the antigen-antibody complex. Classical pathway is activated by antigen (bacteria)antibody complex. Complement activation begins with the binding of the immunoglobulin Fc part to the C1 component. When C3 convertase occurs C3 turns into C3a and C3b active forms. Lectin pathway Activated macrophages stimulate hepatocytes through the cytokines they secrete and secrete a lectin called mannose-binding protein. Mannose-containing protein (MCP) is a factor that activates the classical complement pathway without antigen antibody complexes. MCP,can bind to gram negative gram positive bacteria,cryptococci,candida and many viruses. Alternative pathway summary Pathogen membranes such as gram negative microorganisms are activated by bacterial endotoxins and complex polysaccharides. Begins with C3 activation. C1,C2 and C4 do not take part.