FUN2: 11:00-12:00Scribe: Ashley Holladay

Thursday, October 23, 2008Proof: Teresa Kilborn

Dr. BarnumImmunologyPage1 of 6

Ab- Antibody, Ag- Antigen, TNF- Tumor Necrosis Factor, IL- Interlukin

The Complement System

  1. Introduction [S1 & S2]:
  2. The complement system has been mentioned before. It is a fairly complex mechanism of the innate immune system with a lot of different proteins and functions and there are different pathways and specific mechanisms of activation. This is just going to touch on the basic points of how the system works-what activates complement pathways and biological functions in terms of host defense.
  3. Don’t have to worry about specifics or how to draw out pathways or how things derived. This is just an overview.
  4. Videos used later to help you understand- animated versions of how complement system activated- helps you see where proteins come from and touches on biological activity.
  5. There should be copies of these in the Learning Resource Center set up in sections with questions if you have trouble.
  6. Basic definition of complement- group of sequentially reacting proteins, which upon activation, mediate anumber of biological reactions important to host defense.
  7. Sequential pathway- cascade, ex. Coagulation cascade. There are several different pathways. We will talk about the classical and the alternative pathway.
  8. Upon activation- normally complement found in your blood, tissue, body fluids is in an inactive state. That is very important because the complement system is a very potent mediator of innate immune response and can kill eukaryotic as well as bacterial, viral and parasitic cells.

Ex. If you put a kidney into someone that is not a match- the complement system and Ab will turn the kidney into a blob of useless tissue within minutes.

  1. Requires activation- It needs to be activated only when you need it to be. There are specific activators that turn that system on.
  1. Complement Nomenclature [S3]
  2. There is difficult nomenclature with complement system just like every other part of immunology as well.
  3. “C”- 11 proteins that start with letter C. C1-C9
  4. There are 11 because C1 is actually a complex of C1Q, C1R, C1S.
  5. These proteins (esp. C1-C4)- are part of the classical pathway of complement.
  6. C5-C9 are part of terminal or lytic pathway. These are involved in formation of membrane attack complex (MAC)- common to all the complement activation pathways that we will talk about. It is the part of the complement system that leads to the lysis of invading pathogens or possibly your own tissues if it has been misdirected.
  7. There are a lot of proteins in the system called factors. Many of these (Factor B, FD) are important components in the alternative pathway.
  8. In literature, you may find proteins with an overbar- that designates that it is an active component.
  9. Lower case letters-proteolytic fragment of parent molecule (“a” is usually the smaller of the 2 fragments)
  10. Ex. C3 cleaved to C3a and C3b
  11. C2 is exception- C2a is the larger fragment and C2b is the smaller. (Sometimes different in different textbooks.)
  12. “R”- Many of the biological functions of the complement system are receptor mediated (C5a, C3a and others derived from complement proteins when the system has been activated.
  13. Proteins of the complement system [S4]
  14. Important thing to know for this slide is that a fair portion of the complement system is serum soluble- found in blood and other tissues, and of those most of them are involved in activation (of alternative, classical or MNP/Ficolin Pathway)
  15. About half of the proteins in the system are involved in regulation
  16. Out of the ~40 proteins for the complement system- about half involved in regulating protein activation
  17. This means it is very important to control this system, or else you will have problems
  18. Individuals who are deficient in some of these proteins have very well described clinical syndromes.
  19. Some regulatory molecules are membrane bound (CR1, CD59). They are designed to protect your own tissues from complement mediated lysis. They are found in all cells in your body, and all receptors are membrane bound.
  20. [S5] There are a few families of proteins in the complement system. Since many of the proteins are cleaved to get fragments involved in biological function, many of the proteins used are enzymes- serine proteases(trypsin, chymotrypsin). Many of the enzymes in the alternative and classical pathways (C1s, C1r, C2, FB, FD)- serine proteases
  21. There are a number of molecules in the collectin family (Ex. C1)
  22. They have a collagen stalk and multiple globular head domains that allow them to interact with multiple different proteins so that they can bridge gaps. They are critical in activation events. (Ex. C1q and MNP)
  23. Proteins involved in lysis of cells share common domains found in C9 and perforin
  24. Perforin- found in cytotoxic T-cells- what they use to punch holes into cells they are trying to kill (virally or bacterially infected)
  25. Almost all the complement regulatory proteins and most of the receptors have a Short Consensus Repeat Domain (SCR)- This is a short 60-70 amino acid domain that is common to all of these proteins. It can be up to 30-40 of these molecules in a role that makes up the entire composition of the protein. This protein motif is found in other proteins of the immune system as well.
  26. True complement proteins- C3, C4, C5- Most of the biological activities of the complement system are derived from cleavage fragments from these proteins.
  27. Complement Biosynthetic Sites [S6]
  28. Complement is made primarily in the liver. About 90% of the complement you find in your blood and tissues comes from the liver.
  29. The other 10% come from lots of different cell types.
  30. But the liver constitutively is pushing out a lot of complement protein. Other types are not making very much unless they are signaled to do so. (Cells like monocytes, macrophages, fibroblasts, endothelial cell, astrocytes and neurons)
  31. When there is an infection, trauma or injury where proinflammatory cytokines are being liberated- it will turn those cells on for the production of complement. You would get complement being produced in cells all over the body.
  32. QUESTION: Hepatocytes make 90% of complement and the other cells listed make 10%? ANSWER: No, nobody knows where the other 10% comes from. It is probably a combination of different cell types. Cells like astrocytes and neurons are not going to contribute very much to the blood because they are behind the blood/brain barrier. It is probably cells like fibroblasts, endothelial cells, and macrophages.
  33. Complement can be me made throughout the body- important part of the innate immune system that is ready to go anytime you get an infection anywhere in the body.
  34. Complement Pathways [S7]
  35. What is on the slides is as complicated of a pathway as we will be responsible for.
  36. We will focus on the classical, alternative and more recently described mannose binding protein/ficolin pathway. MBP/ficolin feed into classical anyway. The end result is the same. There are just a couple of proteins that are involved in starting off that pathway
  37. All of these pathways, once they become activated, serve to generate multi-molecular protein complexes called convertases.
  38. There are convertases that cleave C3 or C5. There are different convertases for the alternative and the classical pathway but the functional outcome is the same. When you cleave C3 by the classical or the alternative pathway, the cleaveage sites and the fragments are the same, it is just different routes to get to the same end.
  39. A lot of the functions are redundant. It is just different pathways.
  40. [S8] You will end up cleaving C3 to C3a and C3b
  41. The C5 convertase will give you C5a and C5b.
  42. The C5b portion will combine with C6, C7, C8, and C9 to form the membrane attack complex. At this point there is no proteolytic cleaveage- just self association of the molecules
  43. There are multiple pathways with different activators that all lead to cleavage of C3 and C5 to give you the main proteolytic cleavage fragments that are involved in host defense functions.
  44. C3a and C5a are both very potent chemoattractant molecules. When you activate complement near the site of infection and liberate these molecules, they serve as a mechanism to attract macrophages and neutrophils to traffic through the tissue and into the site where the infection has occurred.
  45. C5a is probably one of the most potent chemoattractant molecules known
  46. C3a and C5a are both involved in inflammation and both can almost be considered a form of a cytokine because they mediate a lot of the same functions that the proinflammatory cytokines mediate in the acute phase response (TNF, IL1 and IL6). They induce the production of adhesion molecules and other cytokines and more complement.
  47. C3b important for opsonization
  48. Opsonization= tagging. Opsonizing something is to tag it or make it palatable for phagocytic cell. It marks it for destruction by a phagocytic cell. An Ab can do the same thing. An Ab to a given bacteria is another way of tagging the bacteria so phagocytic cells know that it is a foreign invader and they should get rid of it.
  49. C3b can also neutralize invading pathogens (especially when alternative pathway activated because can generate a lot of C3b)
  50. Difference in opsonizing with a complement or with an Ab is that complement opsonization is covalent. Covalent attachment of C3b or C4b to an invading pathogen tags it permanently for removal by phagocytic cells.
  51. If you get a lot of C3b all over the surface of an invading pathogen, it can’t interact with host tissues and cause an infection.
  52. QUESTION: What is covalent? ANSWER: Binding of C3b or C4b to an invading pathogen to permanently mark.
  53. C5b- part of the membrane attack complex that can kill invading pathogens (lytic).
  54. Complement Activation [S9]
  55. The classical pathway is activated by Ag-Ab complexes.
  56. Discriminatory complements for this pathway- best activators- IgM and IgG
  57. Have to have at least 1 molecule of IgM in order to activate classical pathway. Structure of the IgM molecule is pentomeric. There are 5 Fc regions.
  58. Have to have at least 2 IgG molecules to activate and they have to be in close proximity.
  59. MBP Pathway-invading pathogens that have mannose or n-acetylglucosamine in their membrane will activate that pathway.
  60. Alternative pathway is activated best by lipopolysaccharides (LPS) which is a membrane component of different bacteria.
  61. These are just a couple of examples of what activate different pathways, but these are all we will have to know.
  62. [S10] To activate classical pathway, you have to have a recognition molecule to bridge the gap between an Ag-Ab complex where a bacteria, for example, has an Ab bound to it- activate complement system.
  63. Molecule that recognizes that complex is C1. C1 is made up of C1q (collectin molecule), and 2 each of C1r and C1s (serine proteases that help kick off the activation of the classical pathway).
  64. 1st Video- Classical Pathway [S11]
  65. Classical pathway activation- this happens on the surface of the invading pathogen
  66. 2 IgG molecules in order to activate in close enough proximity that the C1 complex can bridge the gap.
  67. C1 protein kicks off activation by binding to the Fc region of the Ab through the globular domains of IgG. Activation involving IgM is so easy because you have 5 Fc regions all in close proximity all the time.
  68. Confirmational changes occur in the C1q portion of the molecule that cause autoactivation of C1r and C1s. Once this complex is fully active, the classical pathway is kicked off.
  69. The complex cleaves C4 protein to C4a and C4b. C4a floats off with no known function, and C4b covalently attaches to the surface of the invading pathogen through an ester or an amide linkage.
  70. Then C1 cleaves C2 into C2a and C2b
  71. Bimolecular complex of C4b and C2a is the C3 convertase of the classical pathway.
  72. C3 is cleaved by the C3 convertase into C3a and C3b. Some of the C3b that is generated can bind to the C3 convertase- then you have trimolecular complex of C4b, C2a, and C3b.
  73. That new complex can now cleave C5 to C5a and C5b. (adding the new fragment changes the specificity)
  74. C5a is very potent chemoattractant. They attract phagocytic cells and activate mast cells and basophils and neutrophils and macrophages and bind to the receptors on their surface. That aids in the phagocytic cells activation.
  75. The C5b fragment binds nonspecifically to the surface of the invading pathogen (and initiates the terminal part of the pathway) and C6,C7, C8 and C9 will self associate with C5b without proteolytic cleavages. When C8 attaches you can get leaky membranes.
  76. C5b, C6, C7, C8 and C9- Membrane attack complex.
  77. Then there is the association of multiple C9s (up to 18 can self associate and make a pore)
  78. The pore allows for lysis because of the movement of water and free molecules.
  79. QUESTION: What is the most important thing to get from the video? ANSWER: The biological functions. Certain activators that activate certain pathways and there are certain biological functions that occur from these proteolytic cleavage fragment. NOT questions like what is the next fragment generated. Focus on biological functions.
  80. QUESTION: Do we need to know things like C5b initiates terminal pathway? ANSWER: Showing how pathways are activated and where they come from. NOT going to ask specific steps in the pathway.
  1. 2nd Video- Alternative Pathway [S12]
  2. Alternative Pathway- trickier in some ways. Mostly as it relates to activation of this pathway.
  3. The complement system is inactive unless it is activated- basically a lie.
  4. In the alternative pathway, there is a continuously occurring event in tissues and blood called C3 tickover- continuous cleavage of C3 to C3a and C3b in your tissues. This is important for activation of the alternative pathway.
  5. QUESTION: Is it constitutive? ANSWER: Yes, it happens all the time but at a very low rate.
  6. Important thing to know- in the absence of an invading pathogen, the proteolytic fragments derived from C3 are going to be degraded and there won’t be continued activation of the alternative pathway. If there is no invading pathogen- alternative pathway is shut down.
  7. In the presence of an invading pathogen, C3b combines to that pathogen and serves as an initiation of the alternative pathway.
  8. In this video- see how C3 tickover works and leads to the generation of C3 and C5 convertases in the alternative pathway.
  9. QUESTION: Does invading pathogen bind C3a or C3b? ANSWER: C3b. The only thing that binds C3a is the C3a receptor.
  10. Tickover of the C3 molecule requires hydrolysis by water to give you C3-H20.
  11. C3-H20 combines Factor B (important enzyme in alternative pathway) FB is a substrate for Factor D.
  12. FD will cleave FB into Ba and Bb and leave you with C3-H20-Bb bimolecular complex.
  13. C3-H20-Bb is the C3 initiation convertase of the alternative pathway. Enzymatic activity resides in the Bb portion of the molecule, and that will cleave C3 molecules into C3a and C3b.
  14. In the presence of invading pathogen, C3b can covalently bind to the surface of the pathogen and that is important for kicking off the alternative pathway.
  15. C3b has a binding site for FB just like the initiation convertase.
  16. FD will cleave FB to Ba and Bb portions.
  17. This bimolecular complex produced- C3b-Bb- Alternative pathway C3 convertase and works like classical.
  18. C3b-Bb will cleave C3 to C3a and C3b. Every time this happens you generate a new C3b molecule which can start the formation of another convertase.
  19. Additional C3b molecules can bind nearby the site of the initial convertase and very quickly you can generate lots of C3 convertases and some of the C3b can bind to C3 convertase and make trimolecular complex of 2 C3b and Bb which is the C5 convertase of alternative pathway.
  20. The fragments derived from cleavage of C3 and C5 in the alternative pathway are identical to that of the classical with the exact same functions.
  21. You get the same biological activities derived from both pathways.
  22. Then you get the formation of the membrane attack complex as in the classical pathway.
  23. Key thing to remember for alternative pathway- you always have turnover of C3 at a very low rate in your tissues. C3 tickover is mediated by C3 initiation convertase which is a composition of C3-H20 bound by FB. FD cleaves FB to Ba and Bb fragments. Complex of C3-H20-Bb is the initiation convertase of the alternative pathway. This complex will cleave C3 into C3a and C3b. And in the presence of invading pathogen, C3b binds covalently to the surface of the pathogen and starts the formation of C3 convertase.
  24. When you activate the alternative pathway, you can very quickly cleave thousands of C3 molecules and coat the surface of the invading pathogen with C3b which will continue the cycle.
  25. QUESTION: How do C3 and C3b molecules differentiate between self and non-self? ANSWER: They don’t. That’s why you have regulatory proteins on your tissues. They see that complement fragments bound to surface are cleaved and nonfunctional on your own tissues. (CD59, DAF, CDP)
  26. QUESTION: What is it that makes C3 bind to water? ANSWER: Crosstalk between the pathways. Has to do with a thioester bond and covalent linkage with amine bond or ester linkage.
  27. Biological functions [S13]
  28. C3a and C5a- potent chemotactic molecules. If you activate enough complement, you can almost get an anaphylaxis type situation-like with Type 1 hypersensitivity where you get a lot of release of mast cell components.
  29. These molecules can cause degranulation of mast cells, basophils, and eosinophils. That releases a lot of histamine and basoactive amines that are present in these cells. So, at sites of infection where complement gets activated, there is tissue mast cells all over your body. You get the release of these different basoactive molecules.
  30. This is important because it leads to increased vascular permeability and edema at the site of infection. This is important becauseit allows bloodborne components of the immune system (Ab, other proteins and phagocytic cells) to get to the site of infection and help clear it up.
  31. These cells are almost cytokine like because the can induce the production of other cytokines, increase expression of adhesion molecules, acute phase proteins- all helps ramp up the acute phase response to eliminate an invading pathogen.
  32. When these molecules bind to phagocytic cells like macrophages and neutrophils they can help augment the respiratory burst that generates superoxides molecules that are toxic to invading pathogens.
  33. [S14] C5b starts the formation of the membrane attack complex.
  1. Intermediates leading to the formation of the MAC might be involved in signal transduction events particularly in self tissues to help them recognize that they are being attacked by complement and to help them protect against complement mediated damage.
  1. [S15] C3b very important in opsonization of Ag-Ab complexes and it can also bind covalently to bacteria or viruses in the absence of Ab
  2. It is very important in clearing those complexes and neutralizing pathogens so they don’t have a chance to interact with host tissues and set up infection.
  3. It can also help break up immune complexes.