2. Fill free to ask questions during my lectures. In addition, I would also be happy to answer any questions after class. My office is in Room 441 BSB. My email address is boacklrj@musc.edu and my phone number is 792-2550. Robert J. Boackle, Ph.D. Please be sure to study the chapter on Complement

3. Complement A series of Blood Serum components which exist in a NON-ACTIVE state

4. The Primary Functions of the Complement System 1) Quickly Neutralize anything that activates complement by permanently coating the activator 2) Enhance the Phagocytosis of that complement- coated substance 3) Directly damage that complement-coated membrane (e.g., if the activator is a susceptible microbe) The Primary Functions of the Complement System

5. Complement Components are produced Primarily by “HEPATOCYTES” MACROPHAGES and many different cell types also produce complement components

6. Fast Acting (2 min) Potent Tightly Controlled Cascading/Amplifying Sequence COMPLEMENT

7. 1) FRAGMENTATION 2) CONFORMATIONAL CHANGE 3) ACTIVATION

8. Activation by Immune Complexes The Classical Pathway (Most Powerful Pathway)

9. C1 C4 C2 C3 C5 C6 C7 C8 C9 Membrane damage (Amplification Loop - Alternative Pathway) C3b, Factor B, Factor D Membrane Attack Complex C4b, C3b, iC3b, C3dg, C3d Immune Adherence Enhanced Phagocytosis C3a, C5a Anaphylotoxins Chemotactic Factors ( ) Membrane Damage & Potential for Cell Lysis Classical Pathway Activation by Immune Complexes Lectin Pathway weakest pathway MBL MASP1 + MASP2

10. C1 C4 C2 C3 C5 C6 C7 C8 C9 C4 C2 C3 C5 C6 C7 C8 C9 C4 C2 C3 C5 C6 C7 C8 C9 Cascading - Amplifying Sequence At many steps, especially at C1 and C3 C3 C5 C6 C7 C8 C9 C3 C5 C6 C7 C8 C9 (Amplification Loop) Control at these two steps is important.

11. C1q Stem region C1q Globular Heads C1q is a subcomponent of the first complement component C1

12. (Native C1) The first Complement Component, C1 = Macromolecular C1qr 2 s 2 C1r 2 and C1s 2 C1q

13. C1 C1q Globular Heads bind to the exposed Fc region of IgM, IgG3 or IgG1 Immune Complexes, but not to IgA nor IgG4

14. C1 Structure, after C1 activation C1r = Catalytic Domain Activated C1s

15. CH2 CH3 Movement within the Hinge Region IgG3 or IgG1

16. Movement within the Hinge Region CH2 CH3 IgG3 or IgG1 ANTIBODIES C1q globular heads bind CH2 gamma, but only if the Fab regions have bound antigen and moved so as to expose the CH2 gamma Foreign (Antigenic) cell/viral surface Antigenic determinant Antigenic determinant

17. CH2 CH3 C1 IgG3 or IgG1 Antigenic Surface At least two or more IgG antibodies are required but only one IgM (5 Fc regions) At least two adjacent IgG

18. CH2 CH3 IgG3 or IgG1 Antigenic Surface At least two adjacent IgG C1q conformational changes are induced after binding to adjacent IgG antibodies on the antigenic surface; Followed by C1r 2 then C1s 2 activation

19. IgG3 or IgG1 Antigenic Surface At least two adjacently deposited IgG antibodies are needed to bind C1, but the more deposited IgG on the antigenic surface the better the interaction with C1 becomes. This requirement for sufficiently deposited levels of antibody insures that complement is not inadvertently activated. However, pathogens that either mutate their immunogenic epitopes or slough their epitopes defeat or circumvent the proper action of the classical complement pathway.

20. FOREIGN SURFACE Active Host Enzymes now on the Antigen CH 2 Flexibility of C1q C1r 2 - C1s 2 C1q

21. FOREIGN SURFACE C1S 2 C4 Activated

22. FOREIGN SURFACE C1S 2 C4b Short-lived Active Binding Site

23. FOREIGN SURFACE C1S 2 C4b Covalent Binding Virus Neutralization

24. C4b C4 Binding Protein C4b that did not bind to the antigen is quickly degraded If C4b Remains Unbound FACTOR I (protease) Note: Bordetella pertussis accumulates serum C4 Binding Protein, resulting in Factor I-mediated inactivation of bound C4b on its surface

25. Antigenic Surface C1s C4b C1q C1r 2 C4a 2 C4b C4

26. Antigenic Surface C1q C1r 2 2 C4b C2 C2b C2a C4b C2a C4b C2a C2b C1s C4b2a is a C3 Convertase enzyme complex

27. C2a C4b ANTIGEN C4b2a is a C3 convertase enzyme C1s is no longer needed. C1 Inhibitor travels between the now loosened C1q stems and Irreversibly binds and inactivates C1r and C1s

28. C2 C4b Native C3 GLY- CYS - GLY- GLU - GLU -THR SC O

29. C4b ANTIGEN GLY- CYS - GLY- GLU - GLU -THR H ANTIGEN C3b COS Both C4b and C3b are covalently bound to the antigen via the short-lived active binding site.

30. C2a C3b may also attach covalently to C4b on C4b2a C3b As soon as C4b and C3b are Covalently bound to the antigenic surface C3a C3a does not bind to antigen, it is released Antigenic Surface w V w V C4b Regions on C4b and C3b become “Exposed.” Host Phagocytes attach to these regions on the deposited complement

31. Antigenic Surface C4b C2a C3b w C4b C2a C3b w w w Immune Adherence & Enhanced Phagocytosis of the complement-coated antigen PMN or any Phagocyte One of the “major” functions of complement is to enhance Phagocytosis

32. Each activated complement component has at least one inhibitor or inhibitory mechanism Complement Regulation The powerful fast-acting complement system must be controlled at each step or disease ensues due to non-productive depletion of complement.

33. iC3b C4b C2a C3b C4b C2a C3b Antigenic Surface C3c C3d C3b Release of C1 C1 Inhibitor Inactivation and removal of C1 occurs as C4b and C3b deposit on the Fab (CH1) or on the antigenic determinants and disrupt the immune complexes, which in turn cause a loosening of the C1qr 2 s 2 complex and allows entrance of C1-Inhibitor. C3b Dissolution of Immune Complexes Release of Ab C1-Inhibitor binds and then removes the inactivated C1r and C1s. However, when low levels of deposited IgG are present, the entire C1qr 2 s 2 complex is removed by the action of C1-Inhibitor. Trace amounts of heparin bind to C1q and facilitate the C1-inhibitor mediated inactivation of activated C1r 2 and C1s 2 and the removal of C1qr 2 s 2 thereby allowing a more efficient maintenance/usage of the (residual) complement components.

34. Deficiency of Early Complement Components (C1r, C1s, C4 or C2) Insufficient complement activation on the antigen- No loosening of the Immune Complexes SLE and/or Glomerulonephritis- Inability to clear immune complexes faster than they are forming Possible consequences

35. C1q C1r C1s Activated C1, when not bound to immune complexes is rapidly inhibited by C1- Inhibitor Complement Regulation Prevents Auto- activation of Native C1 by Activated C1

36. C1q C1r C1s Activated C1, when not bound to immune complexes is rapidly inhibited by C1- Inhibitor Complement Regulation In the absence of sufficient control by C1-Inhibitor, activated C1r in one (released) activated C1 will activate C1r in other Native C1 (C1 auto-activation) resulting in escalating C1 auto- activation, C4 and C2 consumption and continual depletion of C1-Inhibitor.

37. C1 Inhibitor Deficiency Genetic: 1. Silent Gene - 25% of Normal C1 INH 2. Dysfunctional Gene Product In Both Cases C1 Inhibitor Function is down and C4 levels are lower than normal Acquired: Lymphoproliferative Disorders

38. Angioneurotic edema (Angioedema) NO Control over unbound activated C1 resulting in total depletion of C4 and C2 and most importantly temporary depletion of C1-inhibitor, resulting in lowered control over Kallikrein and subsequent Bradykinin formation. Plasma Prekallikrein circulates complexed with high molecular weight kininogen. (Kallikrein activity is controlled by C1 INHIBITOR), Uncontrolled Activated Kallikrein cleaves kininogen to release Bradykinin.

39. In Breast Cancer Elevated Ca ++ levels are often observed that may slightly disrupt C1qr 2 s 2, making it more difficult to activate complement on an antibody-coated cancer cell surface Poor specific antibody responses (to cancer cells) Elevated levels of complement inhibitory molecules on the surface of the cancer cells Small levels of complement deposition actually cause surviving cancer cells to become more resistant to apoptosis. Complement and Breast Cancer

40. COMPLEMENT (C3b & C4b) Neutralizes Endotoxin Neutralizes Viruses Virus no longer binds to the target host cell properly Changes the nature of the substance..no longer endotoxin

41. C2a C4b C3b ANTIGEN C5 C5a C5a is a “very strong Chemotatic Factor” C3b C5 joins the complex after one or two C3b molecules bind. C5a C5b

42. C3b C5b C5a C3a PMN PMNPMN TISSUE INFECTION Concentration Gradient of C5a and C3a

43. C3a & C5a PMN C5a C3a PMN Complement Coated Antigen

44. UP-REGULATION -- Higher Expression of Complement Receptors on PMN PMN C3a & C5a

45. TISSUE INFECTION Infected Tissue ARTHUS REACTION PMNPMN PMN Complement Coated Antigen

46. C3a and C5a bind to Mast Cells and Basophiles Mast Cell Then Mast Cells and Basophiles release Histamine and Heparin C3a C5a

47. Mast Cell Histamine Release C3a C5a

48. Histamine

49. Edema

50. C5-9 Classical Pathway C1 C4 C2 C3 C5 C6 C7 C8 C9 “Membrane damage” Lectin Pathway Alternative Pathway (Amplification Loop) Membrane Attack Complex (MAC)

51. Polymerized C9 C7 C8 C5b C6 C5b C6 C7 Cell Membrane Transmembrane Channel C8 enters the membrane, then the polymerized C9 causes the lesion. Then the cell swells. Membrane Attack Complex, C5-C9

52. MAC cell surface lesions

54. Bacteria (Certain E. coli) SHED PILI eliminate MAC

55. Protective Mechanisms on Host Cell Membranes Host Cells Must Protect Themselves from Inadvertent Complement Attack

56. Complement Receptor 1 CR1 (CD35) Decay Accelerating Factor DAF (CD55) Membrane Cofactor Protein MCP (CD46) Protectin (CD59) Protective Mechanisms on Host Cell Membranes Over-expressed on host cells that are located in inflamed areas or on Cancer Cells Xenograph Organ Transplant Research Goal: Genetically program foreign cells (animal organs) to express these “human” complement regulatory substances and defeat destruction by Ab and the Classical Complement Pathway

57. Protective Mechanisms on Host Cell Membranes How do these normal cell surface proteins protect host cells in areas of complement-mediated inflammation from inadvertent complement attack? As an example, we will discuss the mechanism for Membrane Cofactor Protein MCP (CD46) and Complement Receptor One (CR1) (the textbook chapter provides more complete information)

58. HOST CELL Factor I (a serum protease) Inactivated iC3b Complement Cascade is stopped MCP If C3b inadvertently deposits on a host cell. Then, CR1 and or MCP as a part of that host cell membrane will serve as cofactors for serum Factor I MCP

59. Alternative Complement Pathway in the absence of Classical Pathway activation 1) Represents a first line of defense before substantial levels of Ab are produced 2) Is always ready to be activated by a susceptible surface Always remember that the Amplification Loop of the classical pathway has the same molecular steps as the Alternative Pathway

60. The Alternative Pathway Non - specific (does not require antibody) Less Efficient (requires more of the activator) Mg ++ Dependent By definition does not require C1, C4 or C2 although the Classical Pathway will activate it!

61. C1 C4 C2 C3 C5 C6 C7 C8 C9 Membrane damage C3a, C5a Anaphylotoxins Chemotactic Factors C4b, C3b, iC3b, C3dg, C3d Immune Adherence Enhanced Phagocytosis Alternative Pathway C3b, Factor B and Factor D “Must start with a Bound C3b That has not been inactivated” C3b C3 Amplification Loop or

62. Thus, serum FACTOR B will bind to any Deposited C3b that has not been Inactivated C3b Factor B PRO-ENZYME now ready to become activated Activator / Antigen

63. C3b Factor B b Activator / Antigen Factor D activated

64. C3bBb activates more C3 “Amplification of C3b deposition” C3b Factor Bb (Activated) Native C3 Antigen

65. Activated Factor B when bound to deposited C3b will now activate more native C3 C3b Factor B b Enzyme NATIVE C3 C3a Amplification Loop Antigen Note that Factor Bb has enzymatic functions almost like C2a

66. C3b Factor Bb C3b C3 ANTIGEN More C3b is Deposited C3 Amplification Loop C3bBb enzyme

67. C3b Factor B C3b C3 ANTIGEN FACTOR B More Factor B binds so more C3 can be activated

68. Factor C3bBb activates C3 and C5 C3b FACTOR B ( similar to C2 ) MORE Activator / Antigen MORE

69. C3b Factor B Adheres to deposited C3b, if C3b is bound to an antigen. If the surface to which C3b binds has no built-in protection against complement (like host cells do) e.g. no CR1, MCP or DAF that surface will activate the Alternative Pathway (the C3b-Amplification Loop). This includes most microbes and artificial substances, for example “Cellophane filters used in leukophoresis”

70. C3b FACTOR H In addition, Factor H is the primary Inhibitor of unbound C3b When C3b fails to bind to antigen, Factor H “quickly” binds to the C3b, this attracts the Factor I protease, then C3b is rapidly inactivated by Factor I to form iC3b FACTOR I serum protease i

71. Pathogens have mechanisms to circumvent the immune system. Streptococcus pyogenes Neisseria gonorrhoeae Candida albicans Each of these pathogens express molecules that attract and accumulate host serum Factor H onto its microbial surface that promote a rapid serum Factor I mediated cleavage of any deposited C3b to form iC3b. [Inactivated C3b (iC3b) can not participate in the amplification loop.] As a result, less C3b is deposited on these organisms.

72. If C3b becomes inadvertently bound to a host cell or becomes bound to an antibody-coated cancer cell Host Cell Surface Co-Factor either CR1 or MCP (CD46) C3b Factor I Host Cell has evaded the attack no amplification loop i

73. PMNs can endocytose small amounts of C3b or iC3b or C3d that inadvertently deposit on their surfaces

74. Complement Receptor 1 CR1 (CD35) Decay Accelerating Factor DAF (CD55) Membrane Cofactor Protein MCP (CD46) CD46 just happens to be a receptor for measles virus (MV) MV infection may cause host immune suppression, secondary to signaling events through CD46 on dendritic cells and macrophages. Protectin (CD59). Host Cell Protective Mechanisms

75. Decay Accelerating Factor DAF (CD55) Blocks the interaction of C4b and C3b with the subsequent complement components Protectin (CD59) Blocks the function of C8 and C9 Two additional the host cell surface Protective Mechanisms phosphatidylinositol “anchored” to the host cell

76. Paroxysmal Nocturnal Hemoglobinuria (PNH) An acquired disorder of phosphatidylinositol "anchors" on selected hemopoietic stem cell lines and their particular erythrocyte progeny. The patients develop anemia associated with the intermittent passage of dark urine. The hemoglobinuria is due to an increased susceptibility of the abnormal population of innocent bystander erythrocytes to complement- mediated lysis, when complement is activated. The deficiency of the phosphatidylinositol anchoring system is reflected by deficiencies of DAF (CD55) and Protectin (CD59). Type I PNH red cells have normal levels or slightly lowered levels of these two proteins and usually show normal resistance to complement-mediated hemolysis. Type II PNH erythrocyte populations lack DAF and have intermediate sensitivity to hemolysis. Type III PNH erythrocyte populations lack both proteins and are very sensitive to hemolysis.

77. SSSS S S  chain C3  chain C3d C3g Tissue protease or plasmin S S C3dg C3c Factor I C3c goes into the fluid phase SSSS S S iC3b Factor I with co-factor H or CR1 or MCP SSSS S S C3b C3 Convertase C4bC2a or C3bBb C3a SSSS Covalently bound to the antigen The speed of the C3b breakdown (catabolism) depends on the nature of the substance onto which C3b covalently binds

78. Phagocytes have receptors for all of the covalently-bound C3 fragments

79. COMPLEMENT RECEPTOR EXPRESSED ON BINDING SPECIFICITY ERYTHROCYTES PMN MONOCYTES / MACROPHAGES CR1 universal GLOMERULAR PODOCYTES C3b C4b CR4 PMN MONOCYTES / MACROPHAGES CR3 PMN MONOCYTES / MACROPHAGES iC3b C3d C3dg

80. COMPLEMENT RECEPTOR EXPRESSED ON BINDING SPECIFICITY CR2 B cells iC3b C3dg C3d (EB virus receptor) Complement deposition (C3b and C3d) on Antigens amplifies the stimulation of B Cells (that have CR2 and CR1, in addition to their specific Ab-receptors for the antigen) and enhances the subsequent Antibody production by a 1000 fold. Dendritic Cells

81. Role of Complement Receptor 1 (CR1) on Erythrocytes

82. Complement Coated Immune Complexes are picked-up from the serum by Erythrocytes and Delivered to Phagocytes. Complement Coated ANTIGEN ANTIBODY COMPLEXANTIGEN ANTIBODY COMPLEX Erythrocyte CR1

83. Complement Coated Immune Complexes are picked up from the serum by Erythrocytes and Delivered to Phagocytes. CR1 CR3 CR4 Phagocyte Complement Coated ANTIGEN ANTIBODY COMPLEXANTIGEN ANTIBODY COMPLEX Erythrocyte CR1

84. RELEASE OF COMPLEMENT COATED IMMUNE COMPLEXES TO PHAGOCYTE CR1 interaction weakens Erythrocyte CR1 CR3 CR4 Phagocyte ANTIGEN ANTIBODY COMPLEXANTIGEN ANTIBODY COMPLEX Complement Coated

85. RELEASE OF COMPLEMENT COATED IMMUNE COMPLEXES TO PHAGOCYTE CR1 interaction weakens Erythrocyte CR1 CR3 CR4 Phagocyte ANTIGEN ANTIBODY COMPLEXANTIGEN ANTIBODY COMPLEX Complement Coated

86. Deficiency of 1) Early Complement Components (C1r, C1s, C4 or C2) 2) CR1 (on erythrocytes) SLE and/or Glomerulonephritis- Inability to clear immune complexes Possible consequences

87. HIV-1 In addition to remaining dormant inside the DNA of host cells (such as CD4 positive cells), and continually changing its immunodominant epitopes, HIV-1 virions (when released into the serum from an infected host cells) are protected from the complement system in several ways:

88. HIV-1 1) Acquires DAF (CD55) upon leaving the host cell 2)Attracts Factor H from serum 3)Sheds many of its trimeric gp160 spikes In addition to continually changing its immunodominant epitopes, these above properties allow a percentage of HIV-1 particles to escape neutralization by antibody and complement. A low level, non-neutralizing C3b-iC3b-C3d deposition may actually help HIV-1 to be more persistent in lymph nodes.

89. HIV-1 Virion Structure ~ 100 nm diameter 20 faced icosahedron with 12 vertices 3 envelope spikes on each face and one at each vertex = 72 spikes –Based on underlying structure Actual mature HIV-1 virion has only 7-15 spikes due to shedding limits IgG aggregation humanvaccine.duke.edu, 1/12/06 Y Y

90. PANCREATITIS CARDIAC INFARCTION ORGAN FAILURE SEVERE BURNS Sudden Release of Proteases

91. C5a Induced Granulocyte Aggregation Blockage of Capillaries by Granulocytes + Superoxide Stimulation Massive Complement Activation SHOCK

92. RESPIRATORY DISTRESS SYNDROME EXTENDED DAMAGE IN CARDIAC INFARCTION RETINAL DAMAGE - TEMPORARY BLINDNESS C5a Induced Granulocyte Aggregation

93. ACTIVATION OF CLASSICAL AND ALTERNATIVE PATHWAYS BY BIOMATERIALS Hemodialysis (Cellophane Filters) Oxygenators (Silicone Polymers) Filtration Leukopheresis (Nylon Fibers) Contrast Media (X-Ray Examination) No CR1, no MBP, so No Way To make C3b inactive

94. The Lectin Complement Pathway Very Similar to C1qr 2 s 2 and C1-Mediated Activation Like the Alternative Pathway, this is another primitive back-up system to activate complement (and save your life), before substantial levels of specific IgG antibodies are produced.

95. MBL and Ficolins are carbohydrate binding Lectins in human serum that look like C1q, all three have a umbrella- like appearance.

96. However, rather than C1q, the Lectin Complement Pathway begins with the binding of host serum glycoproteins termed: 1) Mannan Binding Lectin (MBL) or by 2) Ficolins (FCNs) Normally, both of these lectins are at a relatively low concentrations in human serum.

97. C1qr 2 s 2 MBL- MASP1, MASP2 & MASP3 FCN- MASP1, MASP2 & MASP3 Pro-enzymes Lectin Pathway (ancient & weakest Pathway) 1) Human Mannan Binding Lectin (MBL) or 2) Human Ficolins (FCNs) These are Human Serum Lectins, which remain associated with serum serine proteases termed MASPs (rather than C1r and C1s). Upon binding to microbial structures, such as Mannan, Lipoteichoic Acid, or Peptioglycan, the MASP (pro-enzymes) proteases within the Lectin-MASP complexes become activated and in turn activate C4, C2 and C3. Mannan is a capsular substance of pathogenic fungi and yeasts (e.g., Cryptococcus neoformans and Candida albicans).

98. C4 C2 C3 C5 C6 C7 C8 C9 C1qr 2 s 2 MBL-MASP1, MASP2 & MASP3 FCN-MASP1, MASP2 & MASP3 All three “lectins” remain associated with their respective serum pro-enzymes. After the lectins bind to their respective targeted substance, their associated activated enzymes are capable of activating early complement components.