1. ALLAH does not want from you to be the BEST ALLAH wants from you to do your BEST ALLAH will take care of the REST

2. Complement Hazem M. Abu-Eisha Hazem M. Abu-Eisha Ph.D, MD Assistant Professor of Immunology Pathology Department King Saud University King Khaled University Hospital E mail : hmt149@yahoo.com

3. Outlines 1. 1. General considerations 2. 2. Complement Activation Pathways 3. 3. Consequences of Complement Activation ( Functions ) 4. 4. Regulations of Complement Activity 5. 5. Complement Deficiency 6. 6. Measurement of complement activity

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5. Story of Complement started in 1890s, in Institute Pasteur in Paris when Jules Bordet found that sheep antiserum to bacterium Vibrio Cholerae cause lysis of this bacteria Heating of this antiserum destroy its bacteriolytic activity & this ability was restored by adding fresh serum that doesn't contain Abs against this bacterium Explanation of this bacteriolytic activity is : 1. 1. Sheep antiserum contain substances { Specific antibacterial Abs } survive heating process 2. 2. Heat sensitive component responsible for the lytic activity

6. General Considerations   A complex group of normally found inactive plasma proteins that interact with each other in a cascading fashion to protect against infectious agents   Historically, the term complement (C) was used to refer to a heat labile serum component that was able to lyse bacteria

7.  Proteins of complement are mainly synthesized by liver hepatocytes. Also, it can be formed by monocytes & macrophages  Complement components are designated by numbers ( C1 - C9 ) or by letters ( Factor B )  Many components are proenzymes ( i.e. : Inactive until proteolytic cleavage occurs )  Activated components of complement are over-lined ( e.g. C1qrs )

8.  When complement component was enzymatically cleaved, it is divided into two fragments ( a & b ) The letter “a” refers to the smaller fragment which initiates localized inflammatory response and the letter “b” refers to the larger fragment which binds to the target ( e.g. C3a and C3b ) The letter “a” refers to the smaller fragment which initiates localized inflammatory response and the letter “b” refers to the larger fragment which binds to the target ( e.g. C3a and C3b ) The exception is for C2 in which the larger fragment is given letter “a” and the smaller fragment is given the letter “b” The exception is for C2 in which the larger fragment is given letter “a” and the smaller fragment is given the letter “b”

9. Complement proteins *C1 ( qrs ), C2, C3, C4, C5, C6, C7, C8 & C9 * Factors B, D, H, I & Properdin ( P ) * Mannose - Binding Lectin (MBL ), Mannose- associated serine proteases ( MASP -1 & MASP - 2 ) * C1 inhibitor ( C1INH ), C4b - binding protein ( C4 - BP ) & Decay accelerating factor ( DAF ) * Complement receptors ( CR1, CR2, CR3 & CR4 ) Serine Proteases ARE : Proteolytic enzymes that cleave the next components in the cascade [ C1r, C1s, C2b & Factors D & B ]

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12. 1. Classical Pathway Activation of classical pathway is antibody - dependent Activation of classical pathway is antibody - dependent IgM & IgG ( IgG3, IgG1 & IgG2 ) are the most important two isotypes in activating this pathway IgM & IgG ( IgG3, IgG1 & IgG2 ) are the most important two isotypes in activating this pathway Sequence : Sequence : 1. C1 is firstly activated & cleaved ( C1q, C1r & C1s ) 2. C1s activates C2 & C4 3. C2a & C4b activate C3 ( C3 convertase ) 4. Finally, C4b2a3b is formed ( C5 convertase )

13. 2. Alternative Pathway  Antibody- independent pathway  It is activated when C3 binds to the surface of a microorganism  C3b cleaves factors B & D with release of Ba & Bb fractions  Together with C3b, factor Bb forms C3bBb complex ( C3 convertase )  Factor P ( Properdin ) stabilizes C3bBb complex as it binds with it  C3bBb binds other C3b fragments producing C3bBb3b ( C5 convertase )

14. 3. Lectin Pathway Lectin pathway is antibody - independent It is closely similar to classical pathway Lectins are proteins that can bind carbohydrate ( CHO ) and normally found in serum The pathway is activated by binding of Mannose - Binding Lectin ( MBL ) to mannose residues on glycoprotein or CHO on the surface of microorganisms forming a complex ( MBL - serine protease 1 & 2 ) greatly similar to C1 MBL- associated serine protease ( MASP ) acts on C4 & C2 to generate C3 convertase

15. Membrane Attack Complex (MAC / C5b-9) C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathways cleaves C5 into C5a and C5b C5b rapidly associates with C6 and C7 and inserts into the membrane. The C5b67 complex is referred to as the Membrane Attack Complex (MAC) Subsequently C8 binds, followed by binding of several molecules of C9 The C9 molecules form a pore in the membrane through which the cellular contents leaks out and lysis occurs

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19. Functions of Complement Biological Consequences of Complement Activation o o Lysis of bacterial cells & viruses o o Antigen opsonization that facilitate phagocytosis o o Viral neutralization o o Initiation of immune responses as : Inflammation o o Solubilization & clearance of immune complexes

20. Complement has a central role in inflammation causing chemotaxis of phagocytes, activation of mast cells and phagocytes, opsonization and lysis of pathogens, and clearance of immune complexes.

21. 1. Cell Lysis Cells susceptible to complement mediated - lysis are : 1. 1. Viruses 2. 2. Gram negative bacteria But some gram negative bacteria & most of gram positive bacteria are generally resistant to complement mediated - lysis WHAT ARE THE EVADING MECHANISMS ? 1. 1. Long polysaccharide side chain in the cell wall that prevents insertion of MAC into the bacterial membrane 2. 2. Capsulated bacteria prevents interaction between C3b deposited in the membrane & CR1 on phagocytic cells 3. 3. Some bacteria have elastase which inactivates C3a & C5a

22. C3b is the major & potent opsonin of complement system Activation of C3 Coating of C3b on immune complexes & different antigens Phagocytic cells ( Neutrophils, monocytes & macrophages ) express complement receptors ( CR1, CR3 & CR4 ) can bind C3b that will enhance phagocytosis 2. Antigen Opsonization

23. 3. Viral Neutralization Mechanisms of Viral neutralization : 1. 1. Binding of Ab & or complement to the viral particles forms a thick protein coat which neutralizes viral infectivity by blocking attachment to the host cells ( C4 ) 2. 2. Binding of viral particles to cells possessing Fc or CR1 ( Phagocytic cells ) thus enhancing phagocytosis & intracellular destruction of ingested viral particle 3. 3. Lysing enveloped viruses that leads to fragmentation of the envelope & disruption of the nucleocapsid

24. 4. Inflammatory Response Smaller fragments resulting from complement cleavage, which can bind to receptors on basophiles & mast cells degranulations with release of pharmacologically active mediators : Smaller fragments resulting from complement cleavage, C3a, C4a & C5a called ANAPHYLATOXINS which can bind to receptors on basophiles & mast cells degranulations with release of pharmacologically active mediators : 1. Smooth muscle contraction 2. Increased vascular permeability So complement activation influx of fluids that carries antibody & phagocytic cells to the site of antigen entry So complement activation influx of fluids that carries antibody & phagocytic cells to the site of antigen entry C3a, C5a & C5b67 are the most important chemotactic factors with C5a is the most potent in mediating this process C3a, C5a & C5b67 are the most important chemotactic factors with C5a is the most potent in mediating this process

26. 5. Solubilization of Immune Complexes This function is evident in patients with SLE Complement deficiency ( C4 ) leads to SLE as it interfere with effective solubilization & clearance of immune complexes which in turn leads to their persistence TISSUE DAMAGE ( Type III hypersensitivity reaction )

27. RBCs express CR1. Coating the immune complexes with C3b helps in binding to CR1 on RBCs. These immune complexes are carried to liver & spleen where they are separated from RBCs to be phagocytosed & prevented from their deposition in tissues

28. Are you tired like this ? Have a rest !!!!!!!

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30. Regulation of the Complement System Complement can be activated spontaneously through the alternative pathway It must be controlled by regulatory proteins to prevent complement mediated damage of healthy autologous cells

31. C3 is unstable compound & without inhibition, spontaneous break down occur with production of very reactive C3b fragment C3b can react to two common chemical functional groups ; Amino & Hydroxyl groups Unless C3b is neutralized by water ; many organisms contain these two functional groups where C3b attach to the pathogen and is not broken down

32. Regulatory Mechanisms 1. 1. Serum proteins enzymatically attack complement components so inactivate them 2. 2. Serum proteins bind to & inhibit complement component 3. 3. Regulatory proteins in cell membranes

33. ComponentRegulatory Component Function C1C1- Inh ( 2 )Dissociates C1r and C1s from C1q C3aC3a inactivator ( 1 )Inactivates C3a C3bFactors H ( 2 ) and I ( 1 )Factor H facilitates the degradation of C3b by Factor I MAC CD59 ( MAC inhibitor ) ( 3 )Prevents formation of MAC C4bC4b - binding protein (C4b-BP) ( 2 ) and Factor I ( 1 ) C4b-BP facilitates degradation of C4b by Factor I. C4b-BP also prevents association of C2a with C4b thus blocking the formation of C3 convertase C3 convertase Decay accelerating factor ( DAF ) ( 3 ) Accelerates decay & prevent assembly of C3 convertase C5b67S protein ( Vitronectin ) ( 3 ) Binds soluble C5b67 & prevent its insertion into cell membrane

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35. Complement Deficiency Deficiency of one of the regulatory components can lead to a significant disease Deficiency of one of the regulatory components can lead to a significant disease Example : Example : Deficiency of C1 inhibitor ( C1Inh ) Hereditary Angioedema There is activation of Classical Pathway It may be fatal if not treated & controlled ; as if it occurs in Larynx that end with fatal swelling & oedema which can obstruct the airway

37. Deficiency or dysfunction of CD59 can leads to Increased susceptibility of erythrocytes ( RBCs ) to lysis ( Low levels of autologous complement that is much lower than normally required ) in a diseases called Paroxysmal Nocturnal Haemoglobinuria ( PNH ) The upper diagram ( 1 ) : Assembly of MAC in absence of the regulator CD59. C9 binds C5b-8, with further recruitment of C9 molecules, which in turn forms MAC The upper diagram ( 1 ) : Assembly of MAC in absence of the regulator CD59. C9 binds C5b-8, with further recruitment of C9 molecules, which in turn forms MAC In the lower diagram ( 2 ) : CD59 binds the C5b-8 complex and prevents insertion of C9, which is essential for the initiation of MAC pore formation. In the lower diagram ( 2 ) : CD59 binds the C5b-8 complex and prevents insertion of C9, which is essential for the initiation of MAC pore formation. 1 1 2

38. C3 is essential for all complement pathway ( Classic, Alternative & Lectin ) Patients with C3 deficiency usually suffer from severe bacterial infections, reflecting the central role of C3 in activating C5 that ends with MAC formation

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42. Measurement of Complement Activity Complement Fixation Test ( CFT ) depends on formation of Ag/Ab complex that based on consumption of complement CFT can be used to identify one of them if the other is known ( Usually AB ) Mainly used in viral infections

43. First step : o Ag & Ab are mixed o Known amount of complement is added o o If Ag/Ab complexes are not formed, no complement will be fixed o o If Ag/Ab complexes are formed, complement will be fixed

44. Second step : o o Add an indicator system { Sensitized red blood cells } Indicator system is used where Standard amount of RBCs that have been pre-coated with anti-erythrocyte antibodies is added

45. RESULT o o If Ag/Ab complexes are not formed, complement will be fixed by the indicator system, All RBCs will be lysed & the test is NEGATIVE o o If Ag/Ab complexes are formed, complement will be fixed by these complexes & some of the complement will be consumed by these complexes so : Not all RBCs of the indicator system will be lysed & the test is POSITIVE

46. Simply, measuring the amount RBCs lysis by measuring the release of haemoglobin into the medium : Indirectly measure the amount of AG / AB complexes in the tested sample

47. Complement Haemolytic Assay ( CH50 ) o o Functional evaluation of Classical pathway with assessment of MAC o o CH50 measures complement required to obtain 50% haemolysis of sheep RBCs under standard conditions o o Haemolysis is measured by amount of haemoglobin released from lysed RBCs

48. 1. Classical pathway activity is measured using antibody-sensitized sheep erythrocytes in a buffer containing both Ca 2+ and Mg 2+ ions 2. Standards and samples diluted in the specific buffer are incubated with the relevant target cell and the amount of hemolysis measured 3. The results are converted mathematically to standardized hemolytic units (CH50)

49. Measurement of Complement Components Measurement of Complement components especially : C3 & C4   ELISA   Single radioimmunodiffusion   Nephlerometry Mainly in Immunodeficiency diseases & autoimmune disorders ( SLE )

50. TEXT BOOK FOR ADVANCED READING Kuby Immunology 4 th edition Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne ISBN 0 - 7167 - 3331 - 5

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