Complement Cascade

● Jules Bordet
○ Belgian immunologist and microbiologist
○ Nobel Prize in Physiology or Medicine 1919 because of his work in complement
○ Bordetella was named after him
● Complement System
○ a collection of circulating and membrane-associated proteins that are important
in defense against microbes
○ these proteins are mostly synthesized in the liver
■ Proteins: C1-C9
■ C1 is synthesized by intestinal epithelial cells, monocytes,
macrophages, polymorphonuclear cells, T-cells
■ Factor C - synthesized in the adipose tissue
■ The enzyme activation is sequential hence the name complement
cascade
○ Properties of Complement
■ Complement is a complex of 9 major components that interact with each
other in the classical, alternative and lectin pathway
■ Classical - part of the adaptive humoral immunity
■ Alternative and lectin are part of the natural and innate immunity
■ Complement plays a role in the cytolytic destruction of cellular antigens
by specific antibodies
■
■ Complement activity in antigen-antibody reaction is destroyed by heating
sera to 56oC for 30 minutes
■ Complement is present in all mammalian sera and most lower animals. It
does not increase with immunization
■ Complement from one specie will usually react with antibodies of another
specie from the same taxonomic order
■ Complement may bind to all antigen-antibody complexes
■ Immune complex - antigen and antibody combination
■ Complement can be activated by nonserologic reactions
■ may be activated without an immune complex - seen in lectin
pathway
○ Nomenclature
■ C=Complement
■ fragment a = fragment b, attached through peptide bonds
■
■ The first protein is not always the smaller one like in the case of
C2; a is the bigger fragment, b is active in complement
■ If there is a horizontal line above the name, it is activated
■
■ lowercase i means inactivated
■ C3 convertase - a combination of C4bC2b, activated
● Classic Pathway
■ C4 comes first because they were named based on order of isolation, not
activity
 ■ Anaphylatoxin - substances that can activate mast cells and basophils
causing it to release its granular contents, leading to hypersensitivity
reactions
■ Antibody Isotopes - refers to the different types of antibodies, there's IgM
and IgG in the classic complement pathway
■
■ IgM is generally known as a pentomer
○ Classic Complement Pathway
■ C1 can only be activated if there are at least 2 FC portions of IgG that are
adjacent to each other and it only requires 1 IgM
● Activation of complement system happens on surfaces e.g. bacterium surface
● It is initiated by antibodies
● Alternative Pathway
○ a.k.a Properdin Pathway
○
○ -there are additional factors (B,D) and properdin-
○ Properdin - serves as the stabilizer for the complex formed in the alternative
pathway
○ C3 activation - marks the beginning of the alternative pathway
■ Tick-over -
■ C3 is not stable in plasma therefore water hydrolyses the thioester bond,
and spontaneously activates C3 which produces C3i
■
■ Exposure to other cells (bacterial cell walls, fungal cell walls, tumor cell
lines, parasites especially trypanosome) also activates C3; activated also
in the presence of IgA
■ B - factor B attaches to C3i and it is stabilized by magnesium
■ D - factor D would cleave factor B, segmenting it into two, Ba, and Bb.
■ Ba - released into the cell
■ Bb - stays attached to C3i
■ C3 convertase activity - activates more C3, binds more factor B. It is
cyclical.
■ Properdin attaches to the Bb after factor D cleaves it
■ C3bBbP - called this when properdin is attached to the complex,
■ C5 Convertase Formation
■ C3bBb3b - C5 convertase
■ C5b will attach to C6 and C7, attach to the cell surface with C7
inserted into the membrane, and then C8 will attach to the
complex, which would activate the formation of C9 and C9 will
form the transmembrane 4 which will cause water to get into the
cell and lyse it
● Lectin Pathway
○
○ Begins when in the presence of the carbohydrate, mannose. Lectin binds to
mannose
○ Mannose - present on the surface of microbes
○ Mannose binding lectin - activates proteins on the classical pathway but is part of
the innate immunity
○
○ Biologic Activities
■
 ■ Inflammation
■ Phagocytosis - C3B is an opsonin
■ Elimination of Immune Complexes
■ Osmotic lysis - the purpose of complement activation
■ How Would a Mast Cell Recognize C3B?
■ through receptors
■
■ EBV -Epstein-barr virus
● Controls, Diseases, & Tests
○ Biologic Activities
■ Osmotic Lysis
■ Inflammatory response
■ anaphylatoxins;
■ C5a - most potent, induces blood vessels to dilate, also a
chemotaxin, stimulates the motility of WBC and respiratory
burst
■ C4a - least potent, induces the release of histamine
■ C3a
■ Opsonization and Phagocytosis
■ C3b - another opsonin but has a role mainly in b cell activation
■ Elimination of immune complexes
■ if C3b is attached to the antigen, B-cell activation and humoral
responses occur
■ B cell activation*
■ Harmful consequences of complement activation
■ large scale activation in gram negative septicemia
■ activated by tissue necrosis such as myocardial infarction
■ lysis of red blood cells occur
○ Importance of Complement Controls
■ Limit inflammation
■ avoid excessive activation
■ prevent bystander lysis of host cells
■ host cells are not a target cell but is near the site of complement
activation, it could be destroyed if C3b attaches to it
○ Regulators of Complement
■ Fluid-phase Regulators
■ C1 inhibitor (C1 INH)
■ Factor H
■ C4-binding protein
■ S protein (vitronectin)
■ Clusterin
■ Factor I
■ Membrane-bound regulators
■ CR1
■ Decay accelerating factor (DAF/CD55)
■ MCP (CD46) - membrane cofactor protein
■ CD 59 (protectin)
■ Homologous Restriction Factor
○ Complement Controls
 ■ brought about by (Ag-Ab complex) C1 INH (gives rise to C4 and C2),
prevents C1 from binding to antibodies; binds to C1, dislodges the entire
complex from the Ig
■ C1 INH - produced by the liver
■ Inhibition of C3 convertase
■ 2 types:
■ C4b2A (classical complement pathway) - inhibits the
production of C3 and C5 convertase
■ C3bBb (alternative pathway)
■ Factor 1 &
■ factor H (P) - soluble protein that binds to C3b and
prevents factor b from binding to C3b; cofactor of
complement receptor 1; principle regulator of the
alternative pathway
■ C4BP (P) - binds fluid phased C4b; takes the place of C2a
and destroys the C3 convertase or make it inactive
■ CR1 (M) - binds to C3b (higher affinity) and C4b; exposes
it for degradation by factor I; found in the peripheral blood
cells (WBCs, follicular dendritic cells, T lymphocytes, B
lymphocytes); receptors on platelets and RBCs (CR1 binds
to it, taking it to the spleen or liver, removing the immune
complex; does not destroy platelet or RBCs)
■ Immune adherence - the ability of the cells to bind
complement coated particles
■ MCP (M) - a.k.a CD46
■ expressed in almost every cell except RBCs; most
efficient cofactor of factor I (also destroys C4b)
■ DAF (M) - decay accelerating factor; found in PB cells, endothelial
cells, fibroblasts, epithelial cells of GI mucosa, renal tubules
■ capable of dissociating both C3 convertases of classical
and alternative pathways
■ deficiency causes PNH - paroxysmal nocturnal
hemoglobinuria
■
○ Regulation of MAC
■ same for all pathways
■ C4b2a3b - C5 convertase of the classical complement pathway
■ C3bBb3b - C5 convertase of the alternative pathway
■
■ Protein S (vitronectin) - binds to the C5bC6C7 complex, and inhibits C9
polymerization (hence, no pore formation)
■ MIRL - membrane inhibitor of reactive lysis, aka protectin, HRF20
■ found in all circulating cells, podocytes, sperm, endothelial cells,
epithelial cells, some cells in the CNS
■ binds in the beta chain of C8 and C9, inhibits MAC formation on
the host cell
■ Homologous Restriction Factor (HRF) - expressed on RBCs, platelets, B-
lymphocytes, T-lymphocytes, neutrophils, and monocytes
■ binds to C8, C9 is not formed
■ Clusterin - SP40
 ■ prevents the insertion of C5b6,7, complex protein; inhibits
production of C8, C9
○ Inactivation In Vitro
■ Heat
■ 56oC for 30 mins - C1,C2,C4 (inhibition of classic complement
pathway)
■ 50oC for 20 mins - factor B (inhibition of alternative pathway)
■ Use of Anticoagulants
■ EDTA - binds to calcium and magnesium
■ calcium is needed in the C1 complex
■ magnesium is need in the C3 convertase of all 3 pathways
■ Heparin
■ Prolonged Storage - causes deterioration
■
■ Complement receptors are found on cells not to help in the complement
control but for the cell to recognize the complement fragment
■ CR1 - important in immune adherence; enhances phagocytosis
■ CR2 - helps in activating B-lymphocytes; also an EBV receptor
■ CR3 - recognizes IC3b (formed in the alternative pathways); present in
phagocytes (not in dendritic cells) and NK cells; enhances phagocytosis
■ CR4 - recognizes IC3B and CR3; increases phagocytic activity
■ DAF, protects all cells where it is found
■ MIRL - a ligand; for inhibition of membrane attack complex
○ Mechanisms to Evade Complement
■ Bacterial capsule - prevents C3b from attaching to the cell membrane so
it will not be recognized by the phagocytes
■ Synthesis of proteins that
■ prevents C3 convertase formation through the recruitment of
Factor H; some microbes express sialic acid inhibiting the
alternative pathway
■ mimic human complement regulatory proteins
■ C1qBP by E.coli
■ Staph. complement inhibitor (SCIN)
■ binds C3
■ glycoprotein C-1 in HSV
■ properdin - stabilizes the C3bBb complex
■
■ C2 and Factor B
■ C3 - most severe
■ Genetic Mapping - used to know if there are complement deficiencies
■ 4th column - incidence rate
■ Test for Complement
■ Quantitative Assay -amount/ level or complement protein in
the blood
■ radial immunodiffusion - fused with antibodies;
produces precipitate, precipitin ring; done by
comparing with standard
■ nephelometry
■ ELISA
■ flow cytometry
 ■ advantage: highly specific, simpler to perform,
require fewer specialized agents, cheaper, less
time consuming
■ disadvantage: no info on complete complement
activity
■ Functional Assay -
■ CH50 assay - CH (complement hemolysis) titration
assay; measures the amount of complement
activity in patient serum
■ AH50 assay - for alternative pathway
■
■ ELISA
■ Rabbit RBC - indicator of alternative pathway
activation
■ hemolysis - end point of both AH50 and CH504
■ Assays for Classical Pathway
■ CH50 - measures the amount of px serum required
to lyse 50% of a standardized concentration of ab
sensitized sheep erythrocytes
■ Variations
■ lysis of liposomes that release enyme when
lysed
■ measurement of radial hemolysis in agarose
plates containing sensitized rabbit RBC
■ ELISA
■
■ What specimen is used for complement testing?
■ serum, collected in a tube without any serum
separator/clot activator
■ should be tested within 1-2 hours
■ Causes for decreased complement proteins
■ decreased production
■ increased consumption
■ poor specimen handling
■ inadequate refrigeration (lower concentration)
■ multiple freeze-thaws (lower concentration)
■ prolonged storage (lower concentration)
■ -control serum must always be included-