1. Introduction
• Complement – A series of serum and
membrane expressed proteins involved
in the effector role of the immune
response to pathogens
• Made up of approximately 30 circulating
and membrane-bound proteins.
• Synthesized in the liver and by cells
involved in the inflammatory response.
3. Key role in defense against many
foreign invaders.
• Most important functions are:
• Production of opsonins
• Production of anaphylatoxins
• Direct killing of organisms
• Enhancing antigen-specific immune
response
• Maintaining homeostasis
4. Complement Activation
Pathways
• Involves a “cascade” of successive
components.
• Enhances a small initiating signal.
• Components are cleaved into
activated fragments.
• Fragments induce intense
inflammatory responses to eliminate
infectious agents.
5. Pathways of the complement
activation
• Distinct recognition events for each
pathway
– Classical - C-reactive protein
– Lectin – Mannose-binding lectin (MBL)
– Alternative – serum factors B, D, and P
6. The Classical Pathway
• Antigen-Antibody complexes are main
activators of this pathway.
• Activated by the formation of soluble Ag-
Ab complexes or binding of Ab (IgM or
IgG) to Ag on a target cell.
• C-reactive protein binds to the surface of
many bacteria and are also activators.
14. Activation Effectiveness
• IgM is more effective at activating
complement than IgG
• C1q binds to the CH2 domain of Ig and
requires at least two adjacent Fc regions
• Activation of the Thiol-Ester bond and
covalent attachment to antigen
15. The Lectin Pathway
• Antibody-independent pathway
• Activated by mannose-binding lectin to
mannose residues on foreign surface
• Binding activates MASP-1 and MASP-2 that
cleave and activate C4 and C2
• Cleaved C4 and C2 generate C3 convertase
• Converges with the classical pathway at
activation of C3
17. The Alternative Pathway
• Does not require Ag-Ab complex
formation
• Initiated by foreign cell surface proteins
• Produces active C3 and C5 convertase
• Active C3 is generated spontaneously
• Host cells regulate the progression
21. Activation of C3
• Cleavage of C3 is a critical step in all three
pathways.
• C3 convertases split C3 into two fragments:
C3a---smaller, fluid-phase
anaphylatoxin
C3b---larger, continues the
sequential activation of
successive components
22. Activities of activated C3
• C3a promotes inflammation
• C3b fixation to surfaces leads to
opsonization
• C3b fixation leads to immune complex
clearance
• Generation of the C5 Convertase activity
23. Activation of C5
• Cleavage of C5 produces two fragments:
C5a---released into the fluid phase,
potent anaphylatoxin
C5b---binds to the cell surface,
nucleus for binding the terminal
complement components
24. The Terminal Sequence
• Terminal components of the complement
cascade:
C5b, C6, C7, C8, and C9
• Components are common to all pathways
• Bind to each other and form a MAC
• Results in cell lysis
30. Regulation Of Complement
Activity
• Regulators may:
– dissociate the convertase
– cleave the complement component that is
left on the cell surface
– Act as a cofactor for this cleavage
32. Biological Activities
Of Complement
• Production of Opsonins
• Production of Anaphylatoxins
• Lysis of Cells
• Enhancing B Cell Response to Antigens
• Controlling the Formation and Clearance of Immune
Complexes
• Removing Dead and Dying Cells
• Responses to Viruses
42. Detection of complementary
deficiency by ELISA
• Complement concentration in plasma reflects
activity of the immune system
– Activated complement components are unstable and
extensive formation of immune complexes may
deplete complement faster than it can be replaced by
the liver.
• Complement Fixation Assay
– Detects immune complexes
43. The Assay Contains Three
Components and Three Steps
Components:
1. The test system
• Incubate serum + test antigen
1. Complement
• Add complement
1. The indicator system
• Add the indicator system
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.)
In 1899 Belgian Jules Bordet discovered that serum contained two components, a heat stable one that had the activity of agglutination and precipitation, later identified by Paul Erhlich as antibodies, and a heat liable one that was responsible for bacterial lysis. However, it wasn’t until the 1950’s that the first system of heat liable serum proteins whose function served to “complement” the antibacterial action of antibodies was elucidated. Even earlier, though poorly understood, the role for the heat liable serum proteins was first noted in early attempts in blood transfusions and these observations has helped us understand how complement works.
Opsonins – molecules that enhance phagocytosis by coating with C3b fragments.
Anaphylatoxins – peptides that activate local and systemic inflammatory responses.
Homeostasis – maintenance of the body’s internal stability, via the removal of immune complexes and dead or dying cells.
Cascade – one component starts enzymatic function that starts the activation of the next component in the sequence. Enhancement is due to one active enzyme molecule able to split (cleave) multiple substrate molecules.
Activated fragments are designated by lower-case letters, (lower-case a denotes smaller fragment, b denotes larger fragments). Potency of theses responses may damage the host or make them become susceptible to infectious diseases (ex. - Each pathway has specific events and components that are recognized upon initiation of each pathway. Same components are used in the later stages of each pathway.
Ag-Ab complexes are major effector of the humoral adaptive immune response.
Classical pathway: C-reactive proteins: Found in serum; produced by hepatocytes as part of the acute phase response that is expressed on the surface of many bacteria, activates classical pathway
. Inflammation induced by bacterial infection, necrosis of tissue, trauma, or malignant tumors may cause an increase in the serum concentration within 48 hours of the inducing condition.
C1 is a complex that is made from the combination of 3 different proteins:
The first component of the classical pathway is C1q, an 18-polypeptide chain component of C1, with two molecules each of C1s and C1r
When C1 binds to the Ab in an antigen-antibody complex it initiates the classical pathway and becomes enzymatically active and is referred to as C1s esterase
Activation of C1 occurs when the globular head regions of the subunit C1q bind to the Fc regions of either the IgM or two closely spaced IgG molecules bound to the antigen
Here is an actual picture of the C1qrs complex with distinct polar head regions and the stalk section.
Classical pathway –as we can see from this diagram, the globular head regions of the C1q subunits bind to C1q-specific receptors on the Fc region of the IgM or two closely spaced IgG molecules, (as depicted in this illustration).
C1s esterase cleaves the next component, C4, into two fragments—(C4a =small piece, remains in fluid and C4b which covalently binds to the surface of the bacterium.)
C4b then binds C2 which is then cleaved by C1s
This diagram shows that several proteins regulate the classical pathway C3 convertase by binding to C4b and displacing C2a from the complex:
C4BP (C4 binding proteins) which is a serum protein.
DAF; CD55 – decay-accelerating factor which are widely distributed cell-surface molecules
MCP; CD46 – membrane cofactor protein
CR1; CD35 – complement receptor 1
Complement receptors (CRs) – are cell surface proteins on various cells that recognize and bind complement proteins that have bound pathogens or other antigens. CRs on phagocytes allow them to identify pathogens coated with complement proteins for uptake and destruction.
Factor 1 – serum protein that cleaves C4b on the cell surface after C2a has been displaced.
Cofactors for factor 1-mediated cleavage (CR1, MCP, and C4BP), factor 1 cleavage of C4b must have one or more of these molecules present.
IgM is the first immunoglobulin to appear on the surface of B cells and the first to be secreted following B cell stimulation with antigen.
Ag-Ab complex induces conformational change in Ab exposing binding sites for C1 (ex. IgM exposes 3 binding sites for C1q
Pentameric IgM molecules bind to antigens on bacterial surface and adopt ‘stable’ form
IgG molecules binds to Antigens on bacterial surface also.
C1q binds to one IgM molecule
C1q binds to at least two IgG molecules
IgM more stable due to the use of just one molecule as apposed to IgG which needs two molecules before binding can take place.
Cysteine & glutamic acid form thiol-ester bond
The lectin pathway is initiated by terminal carbohydrate residue on the surface of bacteria.
The generation of C3 convertase is like the classical pathway
Mannose-binding lectin (MBL) pathway is structurally the same as C1q in the classical pathway.
Activated by the terminal mannose residue of proteins and polysaccharides located on the surface of bacteria.
MASP = (mannose associated serine protease)
MASP-1 cleaves C4 and C2 sequentially
Forms the classical pathway C3 convertase C4b2a
The alternative pathway initiator is spontaneously activated C3, which is continually generated at a low rate and is always circulating in the blood system.
Since it does not require Ag-Ab complex formation, the activation of this pathway is an effector arm of the innate immune response ( which is the Ag-specific mechanism involved in the early phase of the response to foreign invaders – such as: phagocytocic cells, cytokines.
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Initiators of the alternative pathway: pathogens and particles of microbial origin such as
Lipopolysaccharides (aka endotoxins) from gram-negative bacteria; Teichoic acid from gram-positive cell walls
parasites (trypanosomes); Fungal and yeast cell walls (zymosan)
Some tumor cell (Raji); Some viruses and viruses-infected cells
Pure carbohydrates expressed on cell surfaces.
Nonpathogens
Human IgG, IgA, and IgE in complexes
Rabbit and Guinea pig IgG in complexes
Pure carbohydrates (agarose, inulin)
Cobra venom factor
Heterologous erythrocytes (rabbit, mouse, chicken)
In the alternative pathway:
C3b binds foreign surface antigen on bacteria
Can bind host cell as well – role of sialic acid
Factor B binds C3b and exposes site cleaved by Factor D (Bb)
Cleavage generates C3bBb binding protein (C3 convertase) that is bound by properdin (factor P – a positive regulator that stabilizes C3b bind protein.)
C3bBb – hydrolyzes C3 into C3a and C3b to amplify response (amplification loop) – done in a short time
During the activation of the alternative pathway both C3a and C5a are generated; both are anaphylatoxins (anafhilatoxins) and cause degranulation of mast cells.
Factor H is a soluble protein that blocks formation of C3 convertase by binding C3b; cofactor of C3b by factor 1
Convertases…an enzyme component in the complement cascade that “converts” the inactive form of the next component in the pathway into an active form by cleaving it. C3 is cleaved into fragments C3a (released into the fluid phase) and C3b (deposits on the cell surface) C5 is cleaved into C5a (released into fluid phase) and C5b (deposits on the cell surface).
Anaphylatoxin—substances such as C3a, C5a capable of releasing histamine from mast cells and basophils.
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Histamine---a vasoactive amine stored in mast cell granules that is released by Ag binding to IgE molecules on mast cells, causing dilation of local blood vessels and smooth muscle contraction and produces some of the symptoms of immediate hypersensitivity reactions. C3b binding to either the classical or alternative pathway C3 convertases allows the next component, C5, to bind and be cleaved.
The C3 convertases with bound C3b are referred to as C5 convertases
The terminal sequence of complement activation involves C5b, C6, C7 C8 and C9. These components interact to form a structure known as the MAC (membrane attack complex). A large transmembrane channel is created that disrupts the membrane and enables ions and small molecules to diffuse freely.
C5 is cleaved by the C5 convertase of either the Classical or Alternative Pathways. The C5 is cleaved into C5a and C5b. The C5a fragment diffuses away, and the C5b fragment binds to the surface of the target cell. This component is very labile and is inactivated within two minutes unless C6 binds and stabilizes its activity.
(MAC) Formation Steps:
1st step in MAC formation---C6 binding to C5b on a cell surface.
2nd C7 then binds to C5b and C6 and undergoes a hydrophilic-amphiphilic structural transition. Transition is required to bind phospholipid and inserts into the target cell outer membrane.
3rd C8 binding to C5bC6C7 induces a hydrophilic-amphiphilic structural transition creating a small pore that can lyse red blood cells (not nucleated cells). C5b-C8 acts as a receptor on the cell membrane.
Additional C9 molecules interact with the C9 molecule in the complex to form polymerized C9 (poly-C9)
C5 is cleaved by the C5 convertase of either the Classical or Alternative Pathways. The C5 is cleaved into C5a and C5b. The C5a fragment diffuses away, and the C5b fragment binds to the surface of the target cell. This component is very labile and is inactivated within two minutes unless C6 binds and stabilizes its activity.
Once C7 binds, the complex undergoes a hydrophilic-amphiphilic transition that exposes hydrophobic regions, which allow the complex to be inserted into the phospholipid bilayer.-- (If the reaction occurs on an immune complex or other noncellular surface, the complex cannot be anchored and it is released causing "innocent bystander lysis". In a number of diseases in which immune complexes are produced, tissue damage results from such innocent-bystander lysis.)–
Binding of C8 induces a structural change in C8 exposing a hydrophobic region, which interacts with the plasma membrane. A small pore is formed.
The final step in the sequence is the polymerization of C9 ( a perforin-like molecule). As many as 10-16 C9s can be polymerized by a single C5bC6C7C8 complex.
Once bound, C9 undergoes a hydrophilic-amphiphilic transition, so that it too can be inserted into the plasma membrane. This result in a hole formation. Even nucleated cells cannot maintain osmotic stability and are lysed by an influx of water and loss of electrolytes.
Tubular in form
Ions and small molecules diffuse without regulation
.
Once bound, C9 undergoes a hydrophilic-amphiphilic transition, a transmembrane channel is formed by the poly-C9, which disturbs the osmotic equilibrium of the cell. Ions pass through the channel, and water enters the cell. Cell swells, membrane becomes permeable to macromolecules, which then escape from the cell. Result is cell lysis.
Here depicts the bursting or lyses of a cell membrane
Example:
C1 inhibitor ( a soluble protein) causes C1r’s and C1s’s to dissociate from C1q in the classical pathway.
C4b-binding protein ( a soluble protein) blocks formation of C3 convertase by binding C4b; cofactor for cleavage of C4b by factor 1 in the classical and lectin pathways.
Factor H (soluble protein) blocks formation of C3 convertase by binding C3b; cofactor for cleavage of C3b by factor 1
Our own cells are protected from spontaneously activated complement pathways because they possess other proteins that inactivate the complement cascade.
C1-inhbitor binds to C1r, C1s and removes it from C1qrs complex resulting in inactivation
Some microorganisms have evolved a defense strategy that involves the presence of their own cascade inhibitors. But most do not have this defense and the protein complex that pierces the microorganism, called perforin, creates a large hole in the membrane of the pathogen. This loss of membrane integrity destroys the pathogen’s ability to control the concentration of salts within itself, thus killing it.
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.
Some viruses also bind to complement receptors and thus gain entry into cells.
Coating of the Epstein-Barr virus -- Epstein-Barr virus, frequently referred to as EBV, is a member of the herpes virus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age have been infected. Infants become susceptible to EBV as soon as maternal antibody protection (present at birth) disappears. Many children become infected with EBV, and these infections usually cause no symptoms or are indistinguishable from the other mild, brief illnesses of childhood. In the United States and in other developed countries, many persons are not infected with EBV in their childhood years. When infection with EBV occurs during adolescence or young adulthood, it causes infectious mononucleosis 35% to 50% of the time.
Both inherited and acquired disorders involving the complement system are associated with increased susceptibility to infections. Deficiencies in any defense mechanism of the host can lead to severe microbial infections; these are of clinical relevance. Although patients with these deficiencies are very uncommon, their identification and detailed study has proved enormously worthwhile.
These patients suffer with recurrent bacterial infections from organisms that are normally susceptible to opsonisation or lysis by complement.
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Complement components, regulators, or receptors deficiencies may result in an increase susceptibility to infection with pus-forming bacteria (pyogenic) or the development of inflammatory conditions.
Classical pathway: SLE (systemic lupus erythematose deficiencies –impaired ability to process and clears immune complexes. Although homozygous C1 and C4 deficiencies are quite rare, reported individuals with these deficiencies also suffer principally from rheumatologic diseases
C3 deficiencies: Membranoproliferative glomerulonephritis is an inflammation of the capillary loops in the glomeruli of the kidney and is associated with circulating immune complexes. Increased cell number and a thickening of capillary walls. C3 participates in numerous effector mechanisms responsible for inflammation and host defense. Consequently it is not surprising that C3 deficiency leads to multiple, severe derangements including immune complex disease, impaired immune responses, impaired chemotaxis, reduced opsonophagocytosis and abnormal serum bactericidal activity. As with classical component deficiencies, these individuals are at increased risk for rheumatologic disease, 79% demonstrating a lupus-like syndrome.
Alternative pathway deficiencies: Of components with functions strictly related to the alternative pathway, inherited defects have been described for properdin and for factor D. Individuals with properdin deficiency exhibit a marked propensity for infections with encapsulated organisms, especially N.meningitidis. Patients with properdin deficiency do not appear to have the same propensity for rheumatologic diseases as do individuals with either classical component or C3 deficiency states.
Hereditary angioneurotic edema:
A genetic form of angioedema. (Angioedema is also referred to as Quinke's disease.) Persons with it are born lacking an inhibitor protein (called C1 esterase inhibitor) that normally prevents activation of a cascade of proteins leading to the swelling of angioedema. Patients can develop recurrent attacks of swollen tissues, pain in the abdomen, and swelling of the voice box (larynx) which can compromise breathing. The diagnosis is suspected with a history of recurrent angioedema. It is confirmed by finding abnormally low levels of C1 esterase inhibitor in the blood. Treatment options include antihistamines and male steroids (androgens) that can also prevent the recurrent attacks. Also called hereditary angioedema.
Autoimmune disease -- Autoimmunity
is the failure of an organism to recognise its own constituent parts (down to the sub-molecular levels) as "Self", which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease, the prominent examples being Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and Rheumatoid Arthritis (RA).
Paroxysmal nocturnal hemogloblinuria (PHN)
-- A rare, acquired disorder of red blood cells in which an abnormal cell surface molecule leads to premature destruction (hemolysis) of the cells. This destruction is intermittent (paroxysmal).
The exact cause is unknown. It is thought to be a disorder of the stem cell (the precursor of blood cells) in which a sensitivity to complement (a substance produced by the immune system) occurs in the cell membrane. The disease can affect people of any age. Red blood cell, white blood cell and platelet counts may be low. Urine may be intermittently red or brown, signifying the breakdown of red blood cells with release of hemoglobin into the circulation and then the urine. Blood clots may form in some people.
Prevention There is no known way to prevent this disorder.
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Symptoms intermittent dark urine, abdominal pain, back pain, headache
shortness of breath, easy bruising or bleeding in the skin
Signs and tests, CBC may show low white blood cell count, red blood cell count, and platelets, sucrose hemolysis test is positive, Ham's (acid hemolysin) test is positive.
urinalysis shows hemosiderin and hemoglobin.
This disease may also alter the results of the following tests:
hemoglobin; serum
Detection by ELISA:
To be useful in diagnostic work, screening procedures for the detection of complement deficiencies, should be simple and rapid, and should clearly distinguish between defects within the functional units of the complement system, i.e. the classical activation pathway (C1, C2, C4), the alternative activation pathway (C3, factor B, factor D, properdin), and the terminal sequence (C5-C9). Furthermore the assays should not be influenced by rheumatoid factors, and the reagents should be commercially available. The ELISA procedure appears to meet these requirements.
Serum complement is measured by immunoassay of individual proteins or by the ability of complement to destroy antibody-coated erythrocytes. The specific components depleted can reveal which pathway has been activated: C2 and C4 are used by the classical pathway, not the alternative pathway; C3 is depleted by both.
Complement Fixation Assay:
Can be used to detect either antigen or antibody – I will describe its use to detect antiviral antibody. This assay is based on competition for a limited amount of complement by two different antigen-antibody complexes in succession.
The first contains viral antigen, plus antibodies to it that may be present.
The second contains sheep red blood cells plus antibody that binds to them.
detects immune – the more antibody viral antigen is present, the more immune complexes will form, the more complement will bind to these complexes, and less complement will be left over to bind to the antibody-coated red cells.
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
