Complement & its biological role.

THE COMPLEMENT SYSTEM & ITS BIOLOGICAL ROLE

A Presentation By

Isaac U.M.Associate Professor & HOD, Dept. of

Microbiology, College of Medicine, International Medical &

Technological University, Dar-Es-Salaam, Tanzania

Introduction

• The complement system is an alarm and a weapon against infection, especially bacterial infection.

• The complement system is activated directly by bacteria and bacterial products (alternate or properdin pathway), by lectin binding to sugars on the bacterial cell surface (mannose-binding protein), or by complexes of antibody and antigen (classical pathway).

• Activation by either pathway initiates a cascade of proteolytic events that produce chemotactic factors to attract phagocytic and inflammatory cells to the site, increase vascular permeability to allow access to the site of infection, bind to the agent to promote their phagocytosis (opsonization) and elimination, and directly kill the infecting agent.

• The three activation pathways of complement coalesce at a common junction point, the activation of the C3 component.

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Figure 2.7. Schematic overview of the complement cascade. There are three pathways of complement activation: the classical pathway, which is triggered by antibody or by direct binding of complement component C1q to the pathogen surface; the MB-lectin pathway, which is triggered by mannan-binding lectin, a normal serum constituent that binds some encapsulated bacteria; and the alternative pathway, which is triggered directly on pathogen surfaces. All of these pathways generate a crucial enzymatic activity that, in turn, generates the effector molecules of complement. The three main consequences of complement activation are opsonization of pathogens, the recruitment of inflammatory cells, and direct killing of pathogens.

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History

• Research on complement began in the 1890s, when Jules Bordet at the Institut Pasteur in Paris showed that sheep antiserum to the bacterium Vibrio cholerae caused lysis of the bacteria and that heating the antiserum destroyed its bacteriolytic activity.

• Surprisingly, the ability to lyse the bacteria was restored to the heated serum by adding fresh serum that contained no antibodies directed against the bacterium and that was unable to kill the bacterium by itself.

• Bordet correctlt reasoned that bacteriolytic activity requires two different substances: first, the specific antibacterial antibodies, which survive the heating process, and a second, heat-sensitive component responsible for the lytic activity.

• Bordet devised a simple test for the lytic activity, the easily detected lysis of antibody coated red blood cells, called hemolysis.

• Paul Ehrlich in Berlin independently carried out similar experiments and coined the term complement, defining it as “the activity of blood serum that completes the action of antibody.”

History

• In ensuing years, researchers discovered that the lytic action of complement was the result of a complex group of proteins.

• It was further shown that the results of complement activation range far beyond the originally observed antibody-mediated cell lysis and that complement plays a key role in both innate and adaptive immunity.

Jules Bordet (1870 – 1961)

Paul Ehrlich (1854 – 1915)

The Multiple Activities of the Complement System

Serum complement proteins and membrane-bound complement receptors partake in a number of immune activities: lysis of foreign cells by antibody-dependent or antibody-independent pathways; opsonization or uptake of particulate antigens including bacteria, by phagocytosis; activation of inflammatory responses; and clearance of circulating immune complexes by cells in the liver and spleen. Soluble complement proteins are schematically indicated by a triangle and receptors by a semicircle; no attempt is made to differentiate among individual components of the complement system here.

The Components of Complement

• The soluble proteins and glycoproteins that constitute the complement system are synthesized mainly by liver hepatocytes, although significant amounts are also produced by blood monocytes, tissue macrophages, and endothelial cells of the gastrointestinal and genitourinary tracts.

• Complement components constitute 5% (by weight) of the serum globulin fraction.

• Most circulate in the serum in functionally inactive forms as proenzymes or zymogens, which are inactive until proteolytic cleavage removes an inhibitory fragment and exposes the active site of the molecule.

• The complement reaction starts with an enzyme cascade.• Complement components are designated by numerals (C1-C9), by letter

symbols (e.g. factor D), or by trivial names (e.g., homologous restriction factor).

• Peptide fragments formed by activation of a component are denoted by small letters.

The Components of Complement

• In most cases, the smaller fragment resulting from cleavage of a component is designated “a” and the larger fragment designated “b” (e.g. C3a, C3b; note that C2 is an exception: C2a is the larger cleavage fragment).

• The larger fragments bind to the target near the site of activation, and the smaller fragments diffuse from the site and can initiate localized inflammatory responses by binding to specific receptors.

• The complement fragments interact with one another to form functional complexes.

• Those complexes that have enzymatic activity are designated by bar over the number or symbol (e.g. C4b2a, C3bBb).

Figure 12-8 The classical, lectin, and alternate complement pathways. Despite different activators, the goal of these pathways is cleavage of C3 and C5 to provide chemoattractants and anaphylotoxins (C3a, C5a), an opsonin (C3b) that adheres to membranes, and to initiate the membrane attack complex to kill cells. MASP, MBP-

associated serine protease; MBP, mannose-binding protein. (Redrawn from Rosenthal KS, Tan JS: Rapid review microbiology and immunology, St. Louis, 2002, Mosby.)

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© 2005 Elsevier

Complement Pathways

Classical Pathway

• The classical complement cascade is initiated by binding to the Fc portion of antibody that is bound to cell surface antigens, or in an immune complex with soluble antigens.

• Aggregation of antibody (IgG or IgM, not IgA or IgE) changes the structure of the heavy chain to allow binding to complement.

• The first complement component, designated C1, consists of a complex of three separate proteins designated C1q, C1r, and C1s.

• One molecule each of C1q and C1s with two molecules of C1r comprises the C1 complex or recognition unit.

• C1q facilitates binding of the recognition unit to cell surface antigen-antibody complexes.

• Activation of the classical complement cascade requires linkage of C1q to two IgG antibodies through their Fc regions.

• In contrast, one pentameric IgM molecule attached to a cell surface may interact with C1q to initiate the classical pathway.

• Binding of C1q activates C1r (referred to now as C1r*) and in turn C1s (C1s*).

• C1s* then cleaves C4 to C4a and C4b, and C2 to C2a and C2b.

Classical Pathway

• The ability of a single recognition unit to split numerous C2 and C4 molecules represents an amplification mechanism in the complement cascade.

• The union of C4b and C2a produces C4b2a, which is known as C3 convertase. This complex binds to the cell membrane and cleaves C3 into C3a and C3b fragments.

• The C3b protein has a unique thioester bond that will covalently attach C3b to a cell surface or be hydrolyzed.

• The C3 convertase amplifies the response by splitting many C3 molecules.

• The interaction of C3b with C4b2a bound to the cell membrane produces C4b3b2a, which is termed C5 convertase.

• This activation unit splits C5 into C5a and C5b fragments and represents yet another amplification step.

Figure 12-9 Cell lysis by complement. Activation of C5 initiates the molecular construction of an oil-well-like membrane attack complex (MAC).


© 2005 Elsevier

Classical PathwayFigure 2.14. 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. Intact C4 consists of an α, a β, and a γ chain with a shielded thioester bond on the α chain. This is exposed when the α chain is cleaved by C1s to produce C4b. The thioester bond (marked by an arrow in the third panel) is rapidly hydrolyzed (that is, cleaved by water), inactivating C4b unless it reacts with hydroxyl or amino groups to form a covalent linkage with molecules on the pathogen surface. The homologous protein C3 has an identical reactive thioester bond that is also exposed on the C3b fragment when C3 is cleaved by C2b. The covalent attachment of C3b and C4b enables these molecules to act as opsonins and is important in confining complement activation to the pathogen surface.



Alternate Pathway

• The alternate pathway is activated directly by bacterial cell surfaces and their components (e.g., endotoxin, microbial polysaccharides), as well as other factors.

• This pathway can be activated before the establishment of an immune response to the infecting bacteria because it does not depend on antibody and does not involve the early complement components (C1, C2, and C4).

• The initial activation of the alternate pathway is mediated by properdin factor B binding to C3b and then with properdin factor D, which splits factor B in the complex to yield the Bb active fragment that remains linked to C3b (activation unit).

• The C3b sticks to the cell surface and anchors the complex.• Inactive Ba is split from this complex, leading to cleavage and activation

of many C3 molecules (amplification). • The complement cascade continues in a manner analogous to the

classical pathway.

Lectin Pathway

• The lectin pathway is also a bacterial and fungal defense mechanism.• Mannose-binding protein (previously known as RaRF) is a large

serum protein that binds to nonreduced mannose, fucose, and glucosamine on bacterial and other cell surfaces.

• Mannose-binding protein resembles and replaces the C1q component and on binding to bacterial surfaces, activates the cleavage of mannose-binding protein-associated serine protease.

• Mannose-binding protein-associated serine protease cleaves the C4 and C2 components to produce the C3 convertase, the junction point of the complement cascade.


Figure 2.13. 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 microscope (lower panels) has been described as looking like ‘a bunch of tulips.' Associated with this complex are two serine proteases, MBL-associated serine protease (MASP)-1 and -2. The structural disposition of MASP proteins in the complex is not yet determined. On binding of MBL to bacterial surfaces, these serine proteases become activated and can then activate the complement system by cleaving and activating C4 and C2. Photograph courtesy of K.B.M. Reid.


Biological Activities of Complement Components

• Cleavage of the C3 and C5 components produces important factors that enhance clearance of the infectious agent by promoting access to the infection site and by attracting the cells that mediate protective inflammatory reactions.

• C3b is an opsonin that promotes clearance of bacteria by binding directly to the cell membrane to make the cell more attractive to phagocytic cells such as neutrophils and macrophages, which have receptors for C3b.

• C3b can be cleaved further to generate C3d, which is an activator of B lymphocytes.

• Complement fragments C3a and C5a serve as powerful anaphylatoxins that stimulate mast cells to release histamine, which enhances vascular permeability and smooth muscle contraction.

• C3a and C5a also act as attractants (chemotactic factors) for neutrophils and macrophages. These cells also express receptors for C3b, are phagocytic, and promote inflammatory reactions.

Membrane Attack Complex• The terminal stage of the classical pathway involves creation of the

membrane attack complex, which is also called the lytic unit.• The five terminal complement proteins (C5 through C9) associate into a

membrane attack complex on target cell membranes to mediate injury.• Initiation of membrane attack complex assembly begins with C5

cleavage into C5a and C5b fragments. • A (C5b,6,7,8)1(C9)n complex forms and drills a hole in the membrane,

leading to the hypotonic lysis of cells. • The C9 component is similar to perforin, which is produced by cytolytic

T cells and natural killer cells.

Regulation of Complement Activation

• Humans have several mechanisms for preventing generation of the C3 convertase to protect against inappropriate complement activation.

• These include C1 inhibitor, C4 binding protein, Factor H, Factor I, and the cell surface proteins, which are decay-accelerating factor (DAF) and membrane cofactor protein.

• In addition, CD59 (protectin) prevents formation of the membrane attack complex.

• Most infectious agents lack these protective mechanisms and remain susceptible to complement.

• A genetic deficiency in these protection systems can result in disease.

Hereditary Complement Deficiencies & Microbial Infection

• Inherited deficiencies of C1q, C1r, C1s, C4, and C2 components are associated with defects in activation of the classic complement pathway that lead to greater susceptibility to pyogenic (pus-producing) staphylococcal and streptococcal infections.

• These bacteria escape detection by γδ T cells. • A deficiency of C3 leads to a defect in activation of both the classic and

the alternative pathways, which also results in a higher incidence of pyogenic infections.

• Defects of the properdin factors impair activation of the alternative pathway, which also results in an increased susceptibility to pyogenic infections.

• Finally, deficiencies of C5 through C9 are associated with defective cell killing, which raises the susceptibility to disseminated neisserial infections.

The Complement Fixation

• The assay contains three components and three Steps Components:

1. The test system.• Incubate serum + test antigen.

2. Complement.• Add complement.

3. The indicator system.• Add the indicator system.

Complement Fixation Test (Positive test)

Complement Fixation Test(Negative test)

Summary

• The complement system comprises a group of serum proteins, many of which exist in inactive forms.

• Complement activation occurs by the classical, alternative, or lectin pathways, each of which is initiated differently.

• The three pathways converge in a common sequence of events that leads to generation of a molecular complex that causes cell lysis.

• The classical pathway is initiated by antibody binding to a cell target; reactions of IgM and certain IgG subclasses activate this pathway.

• Activation of the alternative and lectin pathways is antibody-independent. These pathways are initiated by reaction of complement proteins with surface molecules of microorganisms.

• In addition to its key role in cell lysis, the complement system mediates opsonization of bacteria, activation of inflammation, and clearence of immune complexes.

• Interactions of complement proteins and protein fragments with receptors on cells of the immune system control both innate and adaptive immune responses.

Summary

• Because of its ability to damage the host organism, the complement system requires complex passive and active regulatory mechanisms.

• Clinical consequences of inherited complement deficiencies range from increases in susceptibility to infection to tissue damage caused by immune complexes.

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