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IMMUNOLOGY. Sherko A Omer MB ChB, MSc., PhD. THE COMPLEMENT SYSTEM. Complement system includes more than 30 soluble and cell-bound proteins . The biological activities of this system affect both innate and acquired immunity .
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IMMUNOLOGY Sherko A Omer MB ChB, MSc., PhD
THE COMPLEMENT SYSTEM Complement system includes more than 30 soluble and cell-bound proteins. The biological activities of this system affect both innate and acquired immunity. They are proteins or glycoproteins synthesized mainly by hepatocytes, although significant amounts are also produced by blood monocytes, tissue macrophages, and epithelial cells of the gastrointestinal and genitourinary tracts.
THE COMPLEMENT SYSTEM These 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, which removes an inhibitory fragment and exposes the active site. The complement-reaction sequence starts with an enzyme cascade.
THE COMPLEMENT SYSTEM Nomenclature Classical pathway components are labelled with a C and a number (e.g., C1, C3). Alternative pathway components are lettered (e.g., B, P, D). Some components are called factors (e.g., factor B, factor D). Activated components or complexes have a bar over them to indicate activation (e.g., C4b2a).
THE COMPLEMENT SYSTEM Nomenclature Cleavage fragments are designated with a small letter after the component (e.g., C3a and C3b are fragments of C3). Inactive C3b is designated iC3b. Polypeptide cell membrane receptors for C3 are abbreviated CR1, CR2, CR3, and CR4.
THE COMPLEMENT SYSTEM Complement activation can occur by three different mechanisms: The classical pathway The alternative pathway The lectin pathway
THE CLASSICAL PATHWAY Classical pathway is initiated by Formation of soluble antigen-antibody complexes (immune complexes) or with the binding of antibody to antigen on a suitable target, such as a bacterial cell. IgM and certain subclasses of IgG (human IgG1, IgG2, and IgG3) can activate the classical complement pathway.
Structure of the C1 macromolecular complex. (a) Diagram of C1qr2s2 complex. A C1q molecule consists of 18 polypeptide chains arranged into six triplets, each of which contains one A, one B, and one C chain. Each C1r and C1s monomer contains a catalytic domain with enzymatic activity and an interaction domain that facilitates binding with C1q or with each other. (b) Electron micrograph of C1q molecule
THE ALTERNATIVE PATHWAY The alternative pathway is initiated by pathogens and particles of microbial origin (lipopolysaccharides from gram-negative bacteria, teichoic acid from gram-positive cell walls, fungal and yeast cell walls, some viruses and parasites) Non pathogen materials (cobra venom factor, nephritic factor, heterologous erythrocytes and pure carbohydrates) and aggregated IgA.
THE LECTIN PATHWAY The lectin pathway is activated by the binding of mannose-binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms including certain Salmonella,Listeria, and Neisseria strains, as well as Cryptococcus neoformans and Candida albicans. MBL is an acute phase protein produced in inflammatory responses. Its function in the complement pathway is similar to that of C1q.
THE LECTIN PATHWAY The lectin pathway, like the alternative pathway, does not depend on antibody for its activation. However, the mechanism is more like that of the classical pathway, because after initiation, it proceeds, through the action of C4 and C2, to produce a C5 convertase, this mechanism also called the MB Lectin pathway or mannan-binding lectin pathway.
REGULATION OF COMPLEMENT SYSTEM The classical pathway is regulated by C1 inhibitor (C1 Inh), a serine esterase inhibitor that causes C1r2s2 to dissociate from C1q preventing further activation of C4 or C2. C1Ihb absence leads to a condition called hereditaryangioedema. Factor J is a cationic glycoprotein that also inhibits C1 activity. C4-binding protein (C4bBP) disassembles the C4b2a complex, allowing factor I to inactivate C4b.
REGULATION OF COMPLEMENT SYSTEM Factor H or decay- accelerating factor (DAF) compete with factor B for binding to C3b (e.g., to produce C3bH), decreasing the half-life of the C3bBb complex and causing dissociation of the complex into C3b and Bb. Factor I acts on C3bH to degrade C3b.
REGULATION OF COMPLEMENT SYSTEM The MAC is regulated by S protein which binds soluble C5b67 and prevents its insertion into cell membrane. Membrane bound factors such as homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL) bind to C5b678 on autologous cells, blocking binding of C9. Anaphylatoxin inactivator are soluble factors that inactivates anaphylatoxin activity of C3a, C4a, and C5a by carboxypeptidase N removal of C-terminal Arg.
REGULATION OF COMPLEMENT SYSTEM MCP: membrane cofactor protein
BIOLOGIC CONSEQUENCES OF COMPLEMENT ACTIVATION Cell lysis is achieved through MAC, The MAC formed by complement activation can lyse gram-negative bacteria, parasites, viruses, erythrocytes, and nucleated cells. Cleavage products of complement components mediate inflammation. C3a, C4a and C5a have anaphylatoxin activity. Anaphylatoxins, bind to receptors on mast cells and blood basophilsand induce degranulation, with release of histamine and other pharmacologically active mediators.
BIOLOGIC CONSEQUENCES OF COMPLEMENT ACTIVATION The anaphylatoxins also induce smooth muscle contraction and increased vascular permeability. C3a, C5a, and C5b67 each can induce monocytes and neutrophils to adhere to vascular endothelial cells, extravasate through the endothelial lining of the capillary, and migrate toward the site of complement activation in the tissues.C5a is most potent in mediating these processes.
BIOLOGIC CONSEQUENCES OF COMPLEMENT ACTIVATION C3b is the major opsonin of the complement system, although C4b and iC3b also have opsonizing activity. The amplification that occurs with C3 activation result in a coating of C3b on immune complexes and particulate antigens. Phagocytic cells, as well as some other cells, express complement receptors (CR1, CR3, and CR4) that bind C3b, C4b, or iC3b.
CLEARING IMMUNE COMPLEXES FROM CIRCULATION The coating of soluble immune complexes with C3b is thought to facilitate their binding to CR1 on erythrocytes. Erythrocytes play an important role in binding C3b-coated immune complexes and carrying these complexes to the liver and spleen. In these organs, immune complexes are stripped from the red blood cells and are phagocytosed, thereby preventing their deposition in tissues.
COMPLEMENT ASSAYS Complement protein levels assayed by: Nephelometry Agar gel diffusion Radial immunodiffusion ELISA. Functional assays include hemolytic assaysto measure functional activity of specific components of either pathways.
COMPLEMENT ASSAYS The total hemolytic complement assay (CH50) measures the ability of the classical pathway and the MAC to lyse sheep RBC to which antibodies has been attached. The alternative pathway CH50 measuresthe ability of the alternative pathway and the MAC to lyse rabbit RBC.
ANTIGEN-ANTIBODY INTERACTION A bimolecular association similar to an enzyme-substrate interaction. It does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an antibody and an antigen involves various noncovalent interactions between the antigenic determinant (epitope), of the antigen and the paratope region of the antibody, (VH/VL) domain, particularly the hypervariable regions, or complementarity-determining regions (CDRs).
ANTIGEN-ANTIBODY INTERACTION The antigen antibody complex is not bounded firmly and may dissociate spontaneously Binding is affected by environmental factors like pH in which binding is weaker in pH <4 or >10, increased salt concentration leads to weaker binding. Temperatures of 50-55 C cause stronger binding.
ANTIGEN-ANTIBODY INTERACTION The noncovalent binding is critically dependent on the distance (d) between the interacting groups. As force is proportional to 1/d2 for electrostatic force and 1/d7 for Vander Waals force, so accordingly there must be a high degree of fitness between antigen and antibody (complementary binding) in order to these forces come to work.
ANTIGEN-ANTIBODY INTERACTION Affinity
ANTIGEN-ANTIBODY INTERACTION The combined strength of the noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope. Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. In some biological reactions high affinity is superior to low affinity like in haemagglutination, haemolysis, complement fixation and enzyme inactivation.
ANTIGEN-ANTIBODY INTERACTION Experimentally antigen antibody complexes containing low affinity antibody persist longer in circulation and localized in glomerular basement membranes, this may lead to impairment of renal function. In contrast high affinity antigen-antibody complexes are readily removed from circulation and tend to localize in mesangium of kidney and have little effect on kidney’s function.
ANTIGEN-ANTIBODY INTERACTION The strength with which a multivalent antibody binds a multivalent antigen, avidity, is affected by affinity and valency. Multivalent means that the molecule has more than one binding sites. A simple IgG molecules is multivalent as it has two antigen binding sites while an antigen may be monovalent (e.g. in hapten) or multivalent. When an antigen binds an antibody with more than two binding sites the avidity become grater than the sum of individual binding sites (individual affinities).
ANTIGEN-ANTIBODY INTERACTION Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Cross-reactivity occurs if two different antigens share an identical or very similar epitope. In the latter case, the antibody’s affinity for the cross-reacting epitope is usually less than that for the original epitope.
ANTIGEN-ANTIBODY INTERACTION Cross-reactivity usually characterised by less avidity than specific reaction which occur between antibody and the original antigen.
ANTIGEN-ANTIBODY INTERACTION A number of viruses and bacteria have antigenic determinants identical or similar to normal host-cell components. In some cases, these microbial antigens have been shown to elicit antibody that cross-reacts with the host-cell components, resulting in a tissue-damaging autoimmune reaction.
