Complement system
- 1. Santosh Yadav M.Sc. Clinical Microbiology Dept. of Microbiology Institute of Medicine Tribhuvan Univarsity Teaching Hospital, Nepal COMPLEMENT SYSTEM
- 2. Santosh HISTORY Research on complement began in the 1890s, when Jules Bordet at the Institut Pasteur in Paris showed that sheep antiserum to the bacterium Vibrio cholerae caused lysis of the bacteria and that heating the antiserum destroyed its bacteriolytic activity. He named those substances as Alexins. Paul Ehrlich coined the term complement.
- 3. Santosh It is named “complement system” because it was first identified as a heat-labile component of serum that “complemented or augment” antibodies in the killing of bacteria. Consists of serum and cell surface proteins involved in defense against pathogens and tissue damage mediated by antibodies The Complement system is the major effector of cellular and humoral branch of immune system. Plays major role in both innate and adaptive immunity. INTRODUCTION
- 4. Santosh Complement system represents a group of about 30 proteins which augment or complement the immune response. Most of these proteins are found in serum or on cell surfaces. Synthesized in liver as inative precursors and are activated by proteolysis durig their interaction in a sequential manner. Also produced by blood monocytes, tissue macrophages and epithelial cells of he gastrointestinal and genitourinary tract.
- 5. Santosh General properties Present in serum of all animals but its concentration is maximum in serum of guinea pig. Complement of one species are able to react with antibodies of other species but not to the same extent. C- proteins constitute about 5% of normal serum protein . Are glycoproteins. Are synthesized rapidly in inflammatory responses – hence are called acute phase proteins. Heat labile and lost activity at 56⁰ C for 30 mins and inactivated. Immunoglobulins are not inactivated at this temperature. Binds with Fc potion of immunoglobulns .
- 6. Santosh Three main effects of complement are: 1. Lysis of cells (bacteria, allografts, tumor cells) 2. Generation of mediators of inflammation 3. Opsonization – enhancement of phagocytosis
- 7. Santosh Over 30 serum and cell surface proteins: Complement components- - Components are designated by numbers (E.g. ; C1 – C9) or latters (E.g. : Factor D) - (in serum inactive, activated sequentially as a cascade) Complement receptors (cell surface, recognize activated components) Regulatory proteins of complement (both in serum and cell surface, inhibit activated components) system
- 8. Santosh Complement proteins: are proenzymes - activation by cleavage. Example: C4 Exception: C2: C2a = large fragment C2b = small fragment a = smaller fragment. -Diffusion b= larger fragment. -remains bound to microbe C4a C4b
- 9. Santosh Complement Pathway Three pathway of complement activation 1.Classical pathway:- Is antibody dependent pathway and triggered by formation of soluble antigen-antibody complex or by binding of the antibody to the antigen present on the target cell surface. 2.Alternative pathway:- Is antibody independent pathway stimulated by antigen directly eg. Bacterial cell surface components. 3.Lectin Pathway:- Also antibody independent but resembles classical pathway.
- 10. Santosh Stages of complement Activation Three main stages in the activation of complement by any pathway are Formation of C3 convertage Formation C5 convertage Formation of membrane attack complec(MAC) • The initiation and formation of C3 convertage are different in classical and alternative pathway . These then follow the parralel route to merge at C5 convertage stage and finally generate the MAC by a common route.
- 11. Santosh Sequential activation of complement components occurs via one of three pathways: the classic pathway, the lectin pathway, and the alternative pathway . Of these pathways, the lectin and the alternative pathways are more important the first time we are infected by a microorganism because the antibody required to trigger the classic pathway is not present. The lectin pathway and the alternative pathway are, therefore, participants in the innate arm of the immune system.
- 12. Santosh All three pathways lead to the production of C3b, the central molecule of the complement cascade. The presence of C3b on the surface of a microbe marks it as foreign and targets it for destruction. C3b has two important functions: (1) It combines with other complement components to generate C5 convertase, the enzyme that leads to the production of the membrane attack complex and (2) it opsonizes bacteria because phagocytes have receptors for C3b on their surface.
- 13. Santosh • C-activation: alteration of C proteins such that they interact with the next component • C-fixation: utilization of C by Ag-Ab complexes • C-inactivation: denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity • Convertase/esterase: altered C-protein which acts as a proteolytic enzyme for another C-component Some Definitions
- 14. Santosh Classical pathway Part of acquired immunity. In the classic pathway, antigen–antibody complexes activate C1 to form a protease, which cleaves C2 and C4 to form a C4bC2a complex, C2a and C4b split off. The C4bC2a is C3 convertase, which cleaves C3 molecules into two fragments, C3a and C3b. C3b forms a complex with C4b,2b, producing a new enzyme, C5 convertase (C4b2a3b), which cleaves C5 to form C5a and C5b C5b binds to C6 and C7 to form a complex that interacts with C8 and C9 to produce the membrane attack complex (C5b,6,7,8,9), which causes cytolysis. Note that the "b" fragment continues in the main pathway, whereas the "a" fragment is split off and has other activities except C2a which binds and C2b which
- 15. Santosh Only IgM and IgG fix complement. One molecule of IgM can activate complement; however, activation by IgG requires two cross-linked IgG molecules. Of the IgGs, only IgG1, IgG2, and IgG3 subclasses fix complement; IgG4 does not. C1 is bound to a site located in the Fc region of the heavy chain. C1 is composed of three proteins, C1q, C1r, and C1s. C1q is an aggregate of 18 polypeptides that binds to the Fc portion of IgG and IgM. It is multivalent and can cross-link several immunoglobulin molecules. C1s is a proenzyme that is cleaved to form an active
- 16. Santosh
- 17. Santosh C4b-2a-3b functions as the classical C5 convertase:
- 18. Santosh Classical Pathway Generation of C3-convertase
- 19. Santosh Classical Pathway Generation of C3- convertase C4b C4b2a is C3 convertase
- 20. Santosh Classical Pathway Generation of C5-convertase C4b C3b C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway
- 21. Santosh Lytic pathway assembly of the lytic complex b C6 C7
- 22. Santosh Lytic pathway: insertion of lytic complex into cell membrane b C6 C7 C 9 C 9 C 9 C 9C 9 C 9C 9 C 9 C 9
- 23. Santosh
- 24. Santosh Classic Pathway Components of the Classical Pathway Native component Active component(s) Function(s) C1(q,r,s) C1q Binds to antibody that has bound antigen, activates C1r. C1r Cleaves C1s to activate protease function. C1s Cleaves C2 and C4. C2 C2a Unknown. C2b Active enzyme of classical pathway; cleaves C3 and C5. C3 C3a Mediates inflammation; anaphylatoxin. C3b Binds C5 for cleavage by C2b. Binds cell surfaces for opsonization and activation of alternate pathway. C4 C4a Mediates inflammation. C4b Binds C2 for cleavage by C1s. Binds cell surfaces for opsonization.
- 25. Santosh Classic Pathway Components of the Membrane-Attack Complex Native component Active component(s) Function(s) C5 C5a Mediates inflammation; anaphylatoxin, chemotaxin. C5b Initiates assembly of the membrane-attack complex (MAC). C6 C6 Binds C5b, forms acceptor for C7. C7 C7 Binds C5b6, inserts into membrane, forms acceptor for C8. C8 C8 Binds C5b67, initiates C9 polymerization. C9 C9n Polymerizes around C5b678 to form channel that causes cell lysis.
- 26. Santosh Alternative pathway Ab independnt pathway. In the alternative pathway, many unrelated cell surface substances, e.g., bacterial lipopolysaccharides (endotoxin), fungal cell walls, and viral envelopes, can initiate the process by binding C3 and factor B. This complex is cleaved by a protease, factor D, to produce C3bBb. This acts as a C3 convertase to generate more C3b. Alternative pathways are more important the first time we are infected by a microorganism because the
- 27. Santosh • Usually activated by products of micro-organisms like endotoxin • Other activators include: 1. Complexes containing IgA 2. Some virus-infected cells (e.g. EBV) 3. Many gram negative and gram positive organisms 4. Parasites – Trypanosomes, Leishmania 5. Erythrocytes 6. Carbohydrates (agarose)
- 28. Santosh
- 29. Santosh Alternative Pathway Components of the Alternate Pathway Native component Active component(s) Function(s) C3 C3a Mediates inflammation; anaphylatoxin. C3b Binds cell surfaces for opsonization and activation of alternate pathway. Factor B B Binds membrane bound C3b. Cleaved by Factor D. Ba Unknown. Bb Cleaved form stabilized by P produces C3 convertase. Factor D D Cleaves Factor B when bound to C3b. Properdin P Binds and stabilizes membrane bound C3bBb.
- 30. Santosh Lectin Pathway Also known as the MBL Pathway In the lectin pathway, mannan-binding lectin (MBL) (also known as mannose-binding protein) binds to the surface of microbes bearing mannan (a polymer of the sugar, mannose). Binding causes activation of MASP (MBP- associated serine proteases) cleave C2 and C4 and activate the classic pathway. Note that this process bypasses the antibody-requiring step and so is protective early in infection before antibody is formed.
- 32. Santosh
- 33. Santosh
- 34. Santosh Membrane attack complex Cleavage of C5 into C5a and C5b. C5 (structurally homologous to C3 and C4, lacks internal thioester bond ) C5b initiates formation of MAC (complex of C5b, C6, C7, C8 and multiple C9 molecules ) binds to C6, and C7 , recruits C8 and complex penetrates more deeply into the membrane. C9, a pore-forming molecule with homology to perforin. The complex of C5b678 forms a nidus for C9 binding and polymerization Penetrates membrane bilayers to form pores Disrupt the osmotic barrier, leading to swelling and lysis of susceptible cells Abbas et.al.Cellular&Molecular immunology 6th edition
- 35. Santosh
- 36. Santosh Biologic Effects of complement: 1. Opsonization • C3b & C1q; enhance phagocytosis 2. Chemotaxis • C5a and C5,6,7 complex attract neutrophils • C5a – enhance adhesiveness of neutrophils to the endothelium 3. Anaphylatoxin (C3a, C4a, C5a) • Cause degranulation of mast cells • Bind directly to smooth muscles of bronchioles bronchospasm
- 37. Santosh C3b is an opsonin Opsonins are molecules that bind both to bacteria and phagocytes Opsonization increases phagocytosis by 1,000 fold.
- 38. Santosh 4. Cytolysis (MAC) • Disrupt the membrane & the entry of water and electrolytes into the cell 5. Enhancement of antibody production • Binding of C3b to its receptors on the surface of activated B cells enhanced antibody production
- 39. Santosh Regulation of Complement System 1. C1 inhibitor • Important regulator of classic pathway • A serine protease inhibitor (serpin) • Irreversibly binds to and inactivates C1r and C1s, as well as MASP in lectin pathway 2. Factor H • Regulate alternative pathway • Reduce amount of C5 convertase available • With both cofactor activity for the factor I- mediated C3b cleavage, and decay accelerating activity against C3bBb (C3 convertase)
- 40. Santosh 3. Properdin • Protects C3b and stabilizes C3 convertase 4. Factor I • Cleaves cell-bound or fluid phase C3b and C4b inactivates C3b and C4b 5. Decay accelerating factor (DAF) • Glycoprotein on surface of human cells • Prevents assembly of C3bBb or accelerates disassembly of preformed convertase no formation of MAC • Acts on both classical and alternative
- 41. Santosh 6. C4b-binding protein (C4BP) • Inhibits the action of C4b in classical pathway • Splits C4 convertase and is a cofactor for factor I 7. Complement Receptor 1 (CR-1) • Co-factor for factor I, together with CD46 8. Protectin (CD59) and Vitronectin (S protein) • Inhibits formation of MAC by binding C5b678 • Present on “self” cells to prevent complement from damaging them
- 42. Santosh Clinical Aspects of complement 1. Deficiency of C5-C8 & Mannan-binding lectin • Predispose to severe Neisseria bacteremia 2. Deficiency of C3 • Severe, recurrent pyogenic sinus & resp. tract infections 3. Deficiency of C1 esterase inhibitor • Angioedema inc. capillary permeability and edema 4. Deficiency of DAF • Increased complement-mediated hemolysis paroxysmal nocturnal hemoglobinuria
- 43. Santosh 5. Transfusion mismatches • Activation of complement generate large amounts of anaphylatoxins & MAC red cell hemolysis 6. Autoimmune diseases • Immune complexes bind complement low complement levels + activate inflammation tissue damage 7. Severe liver disease • Deficient complement proteins predispose to infection with pyogenic bacteria
- 44. Santosh 8. Factor I deficiency • Low levels of C3 in plasma due to unregulated activation of alternative pathway recurrent bacterial infections in children • Mutations in factor I gene implicated in development of Hemolytic Uremic Syndrome
- 45. Santosh THANK U