Complement System PDF

6/27/2024 Complement Chapter 7 Preamble ▪ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam ▪ Each student is responsible for READING the TEXTBOOK for details to answer...

6/27/2024 Complement Chapter 7 Preamble ▪ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam ▪ Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES ▪ Unit Objectives are your study guide (not this PowerPoint) ▪ Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/27/2024 Chapter Overview Pathways of the complement system System controls Complement receptors and their biological roles Biological manifestations of complement activation Complement and disease states Complement therapeutics Complement abnormalities and their laboratory detection Introduction to Complement ▪ Complement is a series of more than 50 soluble and cell- bound proteins that interact to enhance host defense mechanisms against foreign cells. ▪ Most plasma complement proteins are synthesized in the liver. – Exceptions: ▪ C1 components (produced by intestinal epithelial cells) ▪ Factor D (made in adipose tissue) 2  6/27/2024 Introduction to Complement (continued) White blood cells are Most of these proteins additional sources of are zymogens that are early complement converted to active components: enzymes in a very C1, C2, C3, C4 precise order. ▪ Cause lysis of foreign cells ▪ Act as opsonins – Specific receptors are present on Functions of phagocytic cells Complement – Enhance clearance of immune complexes Components ▪ Increase vascular permeability ▪ Recruit monocytes and neutrophils to the area of antigen concentration ▪ Trigger secretion of immunoregulatory molecules that amplify the immune response 3  6/27/2024 Complement System Activation Three pathways: More than one End product: lysis Classical pathway pathway can be of the invading Lectin pathway activated cell Alternative pathway simultaneously Complement Pathways 4  6/27/2024 The Classical Pathway Has three stages: Recognition Activation Membrane Attack complex ▪ Antibody dependent pathway contains: ▪ 9 proteins ▪ Antibody directed mechanism Activated by IgM, IgG1, IgG2, and IgG3 IgM: Most efficient due to multiple binding sites The Classical Pathway Divided into three main stages – each dependent on certain reactants 1. Recognition unit – involves C1 2. Activation unit – involves C4, C2, C3 3. Membrane attack complex (MAC) involves C5–C9 5  6/27/2024 Substances That Initiate the Classical Pathway Antibodies (IgG or C-reactive protein IgM) bound to bound to ligand antigen Certain viruses* Mycoplasmas* Certain gram- Some protozoa* negative bacteria* The Classical Pathway Begins With C1 ▪ C1 – First complement component to bind – Consists of three subunits: ▪ C1q ▪ C1r ▪ C1s – At least two of the globular heads of C1q must be bound to antibody to initiate the pathway. – Stabilized by calcium 6  6/27/2024 Initiation of the Classical Pathway by Antibodies ▪ Clq “recognizes” the Fc region of two adjacent antibody molecules. The Classical Pathway: Recognition Unit The zymogens C1r and C1s are converted into active enzymes as binding of C1q occurs. Autoactivation of C1r results from a change that takes place as C1q is bound. When activated, C1r cleaves a thioester bond on C1s, which activates it. C1s has a limited specificity, with its only substrates being C4 and C2. When Cls is activated, the recognition stage ends 7  6/27/2024 The Classical Pathway: The Activation Unit C1s cleaves C3b binds to C3 C4 to create C4b2a to convertase C4b and form C5 (C4b2a) is cleaves C2 to convertase formed. create C2a. (C4b2a3b). The Classical Pathway: Membrane Attack Complex C5 convertase splits C5 into C5a and C5b. C5b attaches to the cell membrane to initiate formation of the MAC (C5b6789). 8  6/27/2024 The Lectin Pathway ▪ Activated by carbohydrates on microbial cell walls ▪ Serves as a defense mechanism in infancy in between loss of maternal antibody and development of full antibody response ▪ Involves three classes of recognition molecules (lectins, ficolins, collectins) ▪ Mannan-binding lectin (MBL) – An acute-phase protein – Binds to mannose or related sugars on microbes to initiate pathway The Lectin Pathway (continued) ▪ Complex associates with MBL-associated serine proteases (MASP-1, MASP-2, MASP-3). – Complex acts like C1qrs. – MASP-2 cleaves C4 and C2. ▪ Remaining steps are identical to the classical pathway. 9  6/27/2024 The Alternative Pathway Acts mainly as an amplification loop for classical or lectin pathways Can be activated directly by bacterial or fungal cell walls, viruses, tumor cells, or some parasites Involves C3, C5 – C9, Factors B, D, and P Steps of the Alternative Pathway 10  6/27/2024 Alternate Pathway ▪ Acts as a natural defense system ▪ Need substances named FACTORS in the alternative pathway ▪ First named Properdin System, thought to be a protein that caused binding – now it is known to stabilize the complex ▪ C1, C2, C4 are NOT utilized in this system ▪ C3 is the key in both pathways Animation of Complement 11  6/27/2024 Convergence of the Three Complement Pathways System Controls in Plasma SERUM MOLECULAR CONCEN- FUNCTION INVOLVED PROTEIN WEIGHT (KD) TRATION PATHWAYS (MG/DL) C1-INH 105 240 Dissociates C1r and C1s from Classical C1q Lectin Factor I 88 35 Cleaves C3b and C4b Classical Lectin Factor H 150 300–450 Cofactor with I to inactivate C3b; Alternative prevents binding of B to C3b C4BP 520 250 Acts as a cofactor with I to Classical inactivate C4b Lectin S protein 84 500 Prevents attachment of C5b67 Classical (vitronectin) complex to cell membranes Lectin Alternative 12  6/27/2024 System Controls in the Alternative Pathway Cellular Complement Controls RECEPTOR LIGAND CELL TYPE FUNCTION DAF (CD55) C3b, C4b RBCs, neutrophils, platelets, Dissociates C2b or Bb monocytes, endothelial cells, from binding sites, thus fibroblasts, T cells, B cells, preventing formation of C3 epithelial cells convertase MIRL (CD59) C8 RBCs, neutrophils, platelets, Prevents insertion of C9 monocytes, endothelial cells, into cell membrane epithelial cells MCP (CD46) C3b, C4b Neutrophils, monocytes, Cofactor for factor I macrophages, platelets, T cells, B cleavage of C3b and C4b cells, endothelial cells 13  6/27/2024 Biological Roles of Complement Receptors RECEPTOR LIGAND CELL TYPE FUNCTION CR1 (CD35) C3b, iC3b, C4b RBCs, neutrophils, monocytes, Cofactor for factor I; macrophages, eosinophils, B mediates transport of and T cells, follicular dendritic immune complexes cells CR2 (CD21) C3dg, C3d, iC3b B cells, follicular dendritic cells, B-cell co-receptor for epithelial cells antigen with CD19 CR3 iC3b, C3d, C3b Monocytes, macrophages, Adhesion and in-creased (CD11b/CD18) neutrophils, NK cells activity of phagocytic cells CR4 iC3b, C3b Monocytes, macrophages, Adhesion and increased (CD11c/CD18) neutrophils, NK cells, activated activity of phagocytic T and B cells, dendritic cells cells Biological Manifestations of Complement Amplifies inflammatory response Anaphylatoxins (C5a, C3a) increase vascular permeability Chemotaxins (C5a) attract phagocytic cells to a specific area Opsonins (C3b, C4b, iC3b, C3dg) coat damaged or foreign cells to enhance phagocytosis. 14  6/27/2024 Biological Manifestations of Complement (continued) Role in uptake and presentation Facilitates B-cell Role in neural of antigens in activation development adaptive immune response Complement and Disease States Complement can be harmful if: It is activated systemically on a large scale, as in gram- negative septicemia. It is activated by tissue necrosis and results in obstruction of the blood supply, as in myocardial infarction. Lysis of RBCs occurs, as in cold agglutinin disease or autoimmune hemolytic anemia. 15  6/27/2024 DEFICIENT ASSOCIATED DISEASE COMPONENT C1 (q, r, or s) Lupus-like syndrome; recurrent infections C2 Lupus-like syndrome; recurrent infections; atherosclerosis C3 Severe recurrent infections; glomerulonephritis Complement C4 Lupus-like syndrome C5–C8 Neisseria infections Deficiencies C9 No known disease association C1-INH Hereditary angioedema DAF Paroxysmal nocturnal hemoglobinuria MIRL Paroxysmal nocturnal hemoglobinuria Factor H or factor I Recurrent pyogenic infections MBL Pneumococcal diseases, sepsis, Neisseria infections Properdin Neisseria infections MASP-2 Pneumococcal diseases Other Complement Abnormalities Atypical Caused by mutations in genes coding for Factor Hemolytic Uremic Rare kidney disorder that results in acute renal H, MCP, Factor I, Factor B, Syndrome failure in children. C3 or thrombomodulin; or to autoantibodies against (aHUS) Factor H. Inflammation of the renal glomeruli that may be due C3 to autoantibodies that glomerulopathy cause C’ dysregulation (C3 nephritic factors; C3NeFs). 16  6/27/2024 Laboratory Detection of Complement Abnormalities Immunologic assays of Techniques involve: individual components Measurement of Automated nephelometry components as antigens or immunoturbidimetry in serum Measurement of Radial immunodiffusion functional activity of (RID) complement pathways Laboratory Detection of Complement Function Classical pathway assays Hemolytic titration (CH50) assay CH50 test for lysis of liposomes ELISA to detect C9 epitopes exposed after binding in MAC Alternative pathway assays AH50 ELISA to detect C3bBbP or C3bP Test systems assessing all three pathways Measurement of complement activation products (e.g., C5a, soluble C5b-9) 17  6/27/2024 CH50 Test Complement Fixation Test 1. Complement can be used as a reagent in Complement Fixation Tests 2. Technique used for the detection of a. Viral antibodies b. Fungal antibodies c. Rickettsial antibodies 3. Two step process a. Test system with antigen and antibody – one of which is known b. An indicator system consisting of sheep RBCs coated with a hemolysin which will lyse in the presence of complement 18  6/27/2024 Complement Fixation Test 1. Destroy any complement present in patient serum by heating serum at 56°C for 30 minutes 2. Dilutions of serum are combined with know antigen and a measured amount of Guinea Pig complement 3. If patient antibody is present it will bind with the reagent antigen and complement will be bound. (Test can also be used with reagent antibody to detect presence of antigen in sample) 4. Add sheep RBC’s that are coated with a hemolysin (will lyse if any complement is present) 5. Incubate and centrifuge tubes; Read for hemolysis ▪ Hemolysis present – No Patient antibody – Test is negative ▪ Hemolysis absent – Patient antibody bound to complement – Test is Positive 6. Results are expressed as highest dilution showing NO hemolysis  Patient 1 – Positive, 1:10 1:10 1:20 1:40 1:80 Patient 1  Patient 2 – Positive, 1:40 Patient 2 Positive versus Negative 19  6/27/2024 Interpretation of Laboratory Findings IMPAIRED FUNCTION CLASSICAL PATHWAY LECTIN PATHWAY ALTERNATIVE OR DEFICIENCY PATHWAY C1q, C1r, C1s Low Normal Normal C4, C2 Low Low Normal MBL, MASP2 Normal Low Normal B, D, P Normal Normal Low C3, C5, C6, C7, C8, Low Low Low C9 C1-INH Low Low Normal Factors H and I Low Low Low Improperly handled Low Low Low sera Complement Therapeutics Target key points in the complement pathways to treat disease. Recombinant C1-INH is used to treat HAE. Monoclonal antibody to C5 (eculizumab) is used to block formation of C5a and the MAC Used to treat aHUS and PNH Leads to decreases in soluble C5b-9 levels, CH50, and AH50 20  6/27/2024 Summary The complement system is a series of more than 50 soluble and cell-bound proteins that interact with the innate and adaptive immune systems to enhance host defenses against infection. Complement activities include lysis of foreign or damaged cells, opsonization, increase in vascular permeability, and attraction of monocytes and macrophages to areas where needed. Summary (continued_1) The classical complement pathway is triggered by IgG or IgM binding to the surface of pathogens. Three distinct units are involved Recognition unit, consisting of C1qrs in the classical pathway: Activation unit, consisting of C2, C4, and C3 MAC, consisting of C5, C6, C7, C8, and C9 21  6/27/2024 Summary (continued_2) The lectin pathway is activated by carbohydrates present in microbial cell walls. Molecules distinct to the lectin pathway include mannose-binding lectin (MBL), MASP-1, MASP-2, and MASP-3. The alternative pathway is triggered by bacterial and fungal cell walls, yeast, viruses, tumor cells, and certain parasites. Summary (continued_3) Plasma protein regulators allow for control of the complement system. Soluble regulators include C1-INH, C4BP, factor H, factor I, and S protein. Cell-bound regulators include CR1, MCP, and DAF. Specific complement receptors on host cells enhance phagocytosis and stimulate other accessory cells. 22  6/27/2024 Summary (continued_4) Complement proteins play a major role in recognition and presentation of antigens, activation of B cells, and maintenance of immunologic memory. Anaphylatoxins (C5a, C3a) increase vascular permeability. Chemotaxins (C5a) attract phagocytic cells to a specific area. Opsonins (C3b, C4b, iC3b, C3dg) coat damaged or foreign cells to enhance phagocytosis. Summary (continued_5) Complement deficiencies can place an individual at risk for certain infections. Complement abnormalities that affect the kidneys are aHUS and C3 glomerulopathy. Therapeutic agents can replace specific deficient complement proteins or block complement proteins involved in disease pathology. 23  6/27/2024 Summary (continued_6) Laboratory assays to quantitate individual complement components include nephelometry, immunoturbidimetry, and radial immunodiffusion. The hemolytic titration or CH50 assay is a functional assay for the classical pathway. It measures lysis of antibody-coated sheep RBCs. The AH50 assay measures the activity of the alternative pathway. Postamble ▪ READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. ▪ USE THE UNIT OBJECTIVES AS A STUDY GUIDE ▪ All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 24