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L4 Complement System.pdf

  1. The Complement System By Dr. B.P. Nayak
  2. Innate Immune Response • Inflammatory response enhances phagocytosis through acute phase proteins • Mannose-binding lectin (MBL) • Binds to bacterial surface with particular spatial arrangement of mannose or fucose • C-reactive protein (CRP) • Binds to phosphorylcholine on bacterial surface • Complement • Set of proteins which bind to bacterial surface • Inflammatory response • Accumulation of fluid and cells at infection site (swelling, redness, heat and pain)
  3. Adaptive Immune Response • Creates millions of different B and T cells for specific antibody-mediated and cell-mediated immunity • Antibody-Mediated Immunity (AMI) • Involves B lymphocytes, plasma cells and antibodies • Humoral immunity • Name derives from antibodies found in body fluids (humors - old medical term) • Cell-Mediated Immunity (CMI) • Involves T lymphocytes, antigen-presenting cells and MHC (major histocompatibility complex) molecules • Cellular immunity
  4. Antibody binds antigens on the surface of target cells Fc receptors on NK cells recognize bound antibody Cross-linking of Fc receptors signals the NK cell to kill the target cell Target cell dies by apoptosis and membrane damage ADCC Around 1960-70, it was found that, ADCC is possible without the help of NKs Then, HOW ?
  5. The complement system  Complements act as a bridge between innate and adaptive immunity.  It not only initiates a robust innate immunity (innate detection and elimination of pathogenic infections), it can effectively modulate the adaptive immune responses through regulation of interplay between B andT cells
  6. The complement system  Complement refers to a complex set of 14 distinct serum proteins (nine components) + 12 surface proteins that are involved in three separate pathways of activation.  Components numbered in order of discovery.  Sequence of activation is not in numerical order.  Components circulate in inactive precursor form, develop activity upon activation.  Complement proteins designated by “C” followed by numbers and letters.
  7. General Properties
  8. The complement system • A defensive system consisting of over 30 proteins produced by the liver and found in circulating blood serum. • Complement kills microbes in three different ways 1. Opsonization 2. Inflammation 3. Cytolysis
  9. •The complement works as a cascade system. • Cascade is when one reaction triggers another reaction which trigger others and so on. • These types of systems can grow exponentially very fast. A Cascade system
  10. • Complement proteins are often designated by an uppercase letter C and are inactive until they are split into products. • Example: C1 • When the products are split they become active. The active products are usually designated with a lower case a or b. • Example: C1a and C1b Cascade activation
  11. • The complement pathway can be activated by either of three different pathways. THREE Pathways
  12. The Classical Pathway
  13. The building of a C3 activation complex
  14. C3 Activation complex
  15. C3b • Many C3b molecules are produced by the C3 activation complex. • The C3b bind to and coat the surface of the bacteria. • C3b is an opsonin • Opsonins are molecules that bind both to bacteria and phagocytes • Opsonization increases phagocytosis by 1,000 fold. Opsonins
  16. C3a C3a increases the inflammatory response by binding to mast cells and causing them to release histamine C3a
  17. Building the C5 activation complex
  18. • Eventually, enough C3b is cleaved that the surface of the bacteria begins to become saturated with it. • C2b and C4b which make up the C3 activation complex has a slight affinity forC3b and C3b binds to them • When C3b binds to C2b and C4b it forms a new complex referred to as the C5 activation complex Building the C5 activation complex
  19. The C5 activation complex • The C5 activation complex (C2b, C4b, C3b) activates C5 proteins by cleaving them into C5a and C5b • Many C5b proteins are produced by the C5activation complex. • These C5b begin to coat the surface of the bacteria. C5 activation complex
  20. C5a •C5a disperses away from the bacteria. • Binds to mast cells and increases inflammation. • Most powerful chemotactic factor known for leukocytes
  21. Building the Membrane Attack complex
  22. Building the Membrane Attack complex • C5b on the surface of bacteria binds to C6 • The binding of C6 to C5b activates C6 so that it can bind to C7 • C7 binds to C8 which in turn binds to many C9’s • Together these proteins form a circular complex called the Membrane attack complex (MAC)
  23. Membrane Attack complex • The MAC causes Cytolysis. • The circular membrane attack complex acts as a channel in which cytoplasm can rush out of and water rushes in. • The cells inner integrity is compromised and it dies
  24. Overview
  25. The alternative pathway  Cleavage of C3 and activation of the remainder of the complement cascade occurs independently of antibody.  Triggers for the alternative pathway include  Bacterial cell walls  Bacterial lipopolysaccharide  Fungal cell walls  Virally infected cells  Rabbit erythrocytes
  26.  Molecules of C3 undergo cleavage at continuous low level in normal plasma.  At least 4 serum proteins (factor B, factor D, properdin (P), and initiating factor (IF) function in this pathway.  C3b attaches to appropriate site (activating surface) which is actually a protective surface  Action by the 4 serum proteins on C3b proceeds to the C3 activator stage without participation of C1, C4 or C2.  Activation sequence: C3, C5, C6, C7, C8, C9. The alternative pathway The alternative pathway
  27. • C3 contains in unstable thioester bond. • This unstable bond makesC3 subject to slow spontaneous hydrolysis to C3b and C3a • The C3b is able to bind to foreign surface antigens. Initiation: The alternative pathway Mammalian cells contain sialic acid which inactivates C3b
  28. Factor B • C3b on the surface of a foreign cells binds to another plasma protein called factor B
  29. Factor D • The binding of C3b to factor B allows a protein enzyme called Factor D to cleave Factor B to Ba and Bb. • Factor Bb remains bound to C3b while Ba and Factor D disperse away. Factor D
  30. The C3 activation complex • Properdin, also called factor P, binds to the C3bBb complex to stabilize it. • C3bBbP make up the C3 activation complex for the alternative pathway
  31. The C3 activation Complex • The C3 activation complex causes the production of more C3b. • This allows the initial steps of this pathway to be repeated and amplified • 2X106 molecules can be generated in 5 minutes The C3 activation complex
  32. C5 activation complex • When an additional C3b binds to the C3 activation complex it converts it into a C5 activation complex. • The C5 activation complex cleaves C5 into C5a and C5b. • C5b begins the production of the MAC. C5 activation complex
  33. Overview
  34. Lectin Pathway • Activation of the lectin pathway begins when mannose-binding protein (MBP) binds to the mannose groups of microbial carbohydrates. • Two more lectin pathway proteins called MASP1 and MASP2 (equivalent to C1r and C1s of the classical pathway) bind to the MBP. • This forms an enzyme similar to C1 of the classical complement pathway that is able to cleave C4 and C2 to form C4bC2b, the C3 convertase capable of enzymatically splitting hundreds of molecules of C3 into C3a and C3b
  35. LECTIN PATHWAY  The beneficial results are the same as in the classical complement pathway: Trigger inflammation (C5a>C3a>C4a); Chemotactically attract phagocytes to the infection site (C5a); Promote the attachment of antigens to phagocytes via enhanced attachment or opsonization (C3b>C4b); Serves as a second signal for the activation of naive B-lymphocytes (C3d); Cause lysis of gram-negative bacteria and human cells displaying foreign epitopes (MAC).  And remove harmful immune complexes from the body (C3b>C4b). Lectin Pathway
  36. Regulation: Complement Cascade  Modulating mechanisms are necessary to regulate complement activation and control production of biologically active split products  First type of control is extreme lability of activated complement If activated complement does not combine within milliseconds the activity is lost or decreased. Active fragments rapidly cleared from the body.  Second type of control involves specific control proteins C1 inhibitor blocks activity of C1r and C1s. Factor H and I, activators in the presence of certain cofactors inactivates C3b and C4b. A number of proteins act to control membrane attack unit
  37. Regulation: Complement Cascade
  38. Complement Deficiencies  Major Pathway components  Hereditary deficiency of any component except C9 results in increased susceptibility to infection and delayed clearance of complexes.  Deficiency of MBL important during infant transition to making their own antibody, associated with pneumonia, sepsis and meningococcal disease.  C3 deficiency most serious, key mediator in ALL pathways. Prone to serious life threatening infections and immune complex disease.  Deficiency of terminal components causes increased susceptibility to Neisseria infections.
  39. Complement Deficiencies •Regulatory Factor components • PNH – RBCs deficient in DAF, results in hemolysis due to “bystander” affect. • Hereditary angiodema results in excess cleavage of C4 and C2, • keeps classical pathway going • Results in increased vascular permeability causing edema. • Usually spontaneously subsides but can be life threatening – oropharynx • Inhibitors of factors H and I produces constant turnover of C3 to depletion – recurrent bacterial infections occur. Complement Deficiencies
  40. Tests for Complement To test the classical pathway • Hemolytic titration (CH50) assay, • Lytic activity, • ELISA To test the alternative pathway • AH50 • ELISA test. Other tests • QuantitativeTests • Test all three pathways using test strips coated with reagents specific for each pathway. • Use complement fixation test to detect viral, fungal and rickettsial antibodies, hemolysis of RBCs is a positive result. Tests for Complements
  41. CH50 • Hemolytic titration (CH50) assay • Serial dilutions of the sample are made and incubated with antibody‐sensitized sheep erythrocytes. • The results are reported as the reciprocal of the dilution at which 50% hemolysis is seen (CH50). CH50
  42. AH50 • A similar assay, using rabbit (or guinea pig) erythrocytes, which specifically activate the alternative pathway by virtue of their decreased sialic acid content. • Additionally, activation of the classical pathway is further blocked by chelation of calcium by EGTA. • The results are again reported as the reciprocal dilution at which 50% hemolysis is seen (AH50). • A second method to quantitate classical pathway activation is to measure the neoantigen formed when the MAC complex (C7, 8, 9) is generated. AH50
  43. • Quantification of the majority of individual complement components can be performed through standard immunochemical assays, including immunodiffusion, nephelometry, and turbidometry. • The most commonly used methods for quantification of C3 and C4 measures intact proteins as well as the major soluble fragments formed during activation. • The presence of cell‐bound regulatory proteins such as CD55, CD59, and CD35 (CR1) can be assessed using flow cytometry. Quantitative Tests