Effector cells : Lymphocytes that can mediate the removal of pathogens or antigens from the body. Inflammatory response : One of the major responses to injury, ( and constitutes a process targeted at bringing the injured area back to its normal state.)
Complement refers, historically, to fresh serum capable of lysing antibody (Ab)-coated cells . This activity is destroyed (inactivated) by heating serum at 56 C for 30 minutes. The lytic activity of complement is decreased in certain diseases, e.g. SLE, serum sickness, chronic infections, complement deficiencies, etc.
Opsonization – the coating of a particle, such as bacterium, with antibody and/or a complement component (an opsonin that leads to enhanced phagocytosis by phagocytic cells. C3b – primary opsonin, also C4b and iC3b C3b coats immune complexes or antigen which are quickly taken up and destroyed by phagocytic cells (neutrophils, macrophages) Bound by complement receptors (CR1, CR3, CR4) on phagocytes Production of Anaphylatoxins: Substances such as C5a, C3a, and C4a capable of releasing histamine from mast cells and basophils. They bind to complement receptors on mast cells and basophils and induce degranulation histamine release which lead to increased smooth muscle contraction and increased vascular permeability Which leads to influx of phagocytic cells (macrophages and neutrophils) and antibody which enhance the response to the pathogens. Chemotactic for neutrophils – neutrophils circulating in the blood migrate to the site of inflammation, and destroy the foreign material Induce smooth muscle contraction Lysis of Cells: This is done by the formation of MAC on the surface of the cell, which is the final stage in all three pathways.
Enhancing B Cell Responses to Antigens: Complement components enhance antibody removal in several ways: Interactions of the complement fragment C3d and CR2 with a microbial antigen enhance the B cell response to the antigen B cell processing of T-dependent antigens is more rapid when the antigen is bound to C3d than when it is not – binding enhances uptake and processing of antigen Complement plays a part in the induction of memory responses: C3d covalently bound to antigen in immune complexes is taken up by germinal center dendritic cells that express CR2. This provides a long-lasting deposit of antigen.
Deposition of C3b on large antigen-antibody complexes disrupt the bonds that hold the complex together. Complex breaks up into smaller pieces that can be cleared by macrphages Deposition allows binding to erythrocytes (RBC) which express the CR1 receptor on their surface – they clear the complexes from the circulation through the spleen via phagocytotic macrophages that destroy them.
Necrotic cell -- Necrotic tissue is dead tissue. Subcellular membranes – midocondria and endoplasmic reticulum activate classical and alternative pathways and are cleared in the same manner. Complement removes dead or dying cells from the tissues and helps in maintaining homeostasis .
Complement binding: leads to opsonization of the virus and lysis of the virion. Interferes with the virus’s ability to interact with the membrane of its target cells and thus blocks viral entry into the cell.
The test system : a sample ( patient serum , if you are searching for antibody) and specific antigen (for example, virus antigen). Step #1 – incubate serum + test antigen; if Antibodies to the test antigen are present, immune complexes will form, if not only free antigen is present Complement Step #2 Add complement: if Antibody/Antigen complexes are present, complement will bind to them and be used up = ‘fixed’; if not present then free complement remains. The indicator system : sheep red cells and hemolysin (= antibody which binds sheep red cells. Step #3 Add the indicator: anti-red blood cell antibodies equal hemolysin bind to the rbc. If free complement is available, it will bind the antibody-coated cells and lyse them Success of the assay depends on having exactly the right amount of complement present; test and indicator sera are heated to inactivate any complement present in them, if not hen the red blood cell survive. Assay can be made quantitative by measuring amount of Hb released, by spectrophotometry.
In it's simplest form the test is used to detect a patient serum antibody, so an ANTIGEN that is recognized by that antibody is the first reagent shown. If the antibody is present in the patient's serum it binds to the antigen, and the complement reagent is completely consumed in the reaction. (The test can also be used to look for antigen in the serum by modifying the reagents used). The complement fixation assay indicator system uses sheep red blood cells (SRBC) and anti-SRBC antibody. If the antibody specific for the antigen in the assay is present in the patient's serum, then complement is completely consumed in the reaction and there is none left to bind to the SRBC/anti-SRBC complexes. A Test Positive For Ab = NO HEMOLYSIS
If there is NO ANTIBODY PRESENT in the patient's serum the antigen is not bound, and the complement reagent does not have immune complexes with which to react. Complement is still present in the indicator reaction and binds strongly to the SRBC/anti-SRBC complexes. This causes the SRBCs to burst in a process called hemolysis. A Test Negative For Ab = LOTS OF HEMOLYSIS
Introduction <ul><li>Complement – A series of serum proteins involved in the effector role of the immune response to pathogens </li></ul><ul><li>Made up of approximately 30 circulating and membrane-bound proteins. </li></ul><ul><li>Synthesized in the liver and by cells involved in the inflammatory response. </li></ul>
Complement: History <ul><li>Discovered in 1894 by Bordet </li></ul><ul><li>It represents lytic activity of fresh serum </li></ul><ul><li>Its lytic activity destroyed when heated at 56C for 30 min </li></ul>
Complement functions <ul><li>Host benefit: </li></ul><ul><ul><li>opsonization to enhance phagocytosis </li></ul></ul><ul><ul><li>phagocyte attraction and activation </li></ul></ul><ul><ul><li>lysis of bacteria and infected cells </li></ul></ul><ul><ul><li>regulation of antibody responses </li></ul></ul><ul><ul><li>clearance of immune complexes </li></ul></ul><ul><ul><li>clearance of apoptotic cells </li></ul></ul><ul><ul><li>Host detriment: </li></ul></ul><ul><ul><li>Inflammation, anaphylaxis </li></ul></ul>
Proteins of the complement system (nomenclature) <ul><li>C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9 </li></ul><ul><li>factors B, D, H and I, properdin (P) </li></ul><ul><li>mannose binding lectin (MBL), MBL associated serine proteases (MASP-1 MASP-2) </li></ul><ul><li>C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF), Complement receptor 1 (CR1), protein-S (vitronectin) </li></ul>
Definitions <ul><li>C-activation : alteration of C proteins such that they interact with the next component </li></ul><ul><li>C-fixation : utilization of C by Ag-Ab complexes </li></ul><ul><li>C-inactivation : denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity </li></ul><ul><li>Convertase/esterase : altered C-protein which acts as a proteolytic enzyme for another C-component </li></ul>
Activation product of complement proteins (nomenclature) When enzymatically cleaved, the larger moiety , binds to the activation complex or membrane and the smaller peptide is released in the microenvironment Letter “b” is usually added to the larger, membrane-binding, peptide and “a” to the smaller peptide ( e.g. , C3b/C3a, C4b/C4a, C5b/C5a), EXCEPT C2 (the larger, membrane-binding moiety is C2a; the smaller one is C2b) Activated component are usually over-lined: e.g. C1qrs
Biological Activities of Classical Pathway Components Opsonin C4b Anaphylotoxin C4a Opsonin Activation of phagocytic cells C3b Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead to anaphylaxis C3a Prokinin; cleaved by plasmin to yield kinin, which results in edema C2b Biological Activity Component
Biological properties of C-activation products Product Biological Effects Regulation anaphylactic as C3, but much more potent; attracts & activates PMN causes neutrophil aggregation, stimulation of oxidative metabolism and leukotriene release C5a ( chemotactic factor ) carboxy-peptidase-B (C3-INA) C5b67 protein-S chemotaxis, attaches to other membranes
Control of Classical Pathway Components C4 binding protein (C4-BP) and Factor I; C4-BP facilitates degradation of C4b by Factor I; C4-BP also prevents the association of C2a with C4b thus blocking formation of C3 convertase C4b C3a-INH C4a Factors H and I; Factor H facilitates the degradation of C3b by Factor I C3b C3a-inactivator C3a C1-inhibitor (C1-INH); dissociates C1r and C1s from C1q All Regulation Component
Complement Deficiencies and Disease Classical Pathway Opsonization of immune complexes help keep them soluble, deficiency results in increased precipitation in tissues and inflammation Predisposition to SLE C1, C2, C4 Overproduction of C2b (prokinin) Hereditary Angioedema C1INH Mechanism Disease Pathway Component
Complement Deficiencies and Disease Lectin Pathway Inability to initiate lectin pathway Susceptibility to bacterial infections in infants or immunosuppressed MBL Mechanism Disease Pathway Component
Complement Deficiencies and Disease Alternative Pathway Inability to attack the outer membrane of Gram-negative bacteria Susceptibility to Gram-negative infections C5, C6, C7 C8, or C9 Lack of opsonization and inability to utilize the membrane attack pathway Susceptibility to bacterial infections C3 Lack of sufficient opsonization of bacteria Susceptibility to pyogenic (pus-forming) bacterial infections Factors B or D Mechanism Disease Pathway/Component
Complement Deficiencies and Disease Alternative Pathway cont. Uncontrolled activation of C3 via alternative pathway resulting in depletion of C3 C3 deficiency and susceptibility to bacterial infections Factors H or I Lack of opsonization of bacteria Susceptibility meningococcal meningitis Properdin (X-linked) Mechanism Disease Pathway Component
The Assay Contains Three Components and Three Steps <ul><li>Components: </li></ul><ul><li>The test system </li></ul><ul><ul><ul><li>Incubate serum + test antigen </li></ul></ul></ul><ul><li>Complement </li></ul><ul><ul><ul><li>Add complement </li></ul></ul></ul><ul><li>The indicator system </li></ul><ul><ul><ul><li>Add the indicator system </li></ul></ul></ul>