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1Adapted from the Presentation of Medical School - Duluth The Complement SystemAdapted from the Presentation ofJean F. Regal, Ph.D.Medical School - DuluthLike the clotting system, the complement system sits around waiting for something to happen and then it jumps into action.Circulating proteins with membrane bound regulators.
2Learning ObjectivesExplain the importance of the complement system in host defense and inflammation and the clinical consequences of complement deficiencies.Describe the biochemistry of activation of the three different pathways including the initiators, sequence of reactions, important enzymes, and fragments.List the proteins which control the complement system and where they act.Describe the biological responses mediated by the different complement receptors.Describe the biological effects of complement activation.
3Complement: Location of Complement Proteins Complement is not a single protein but a complex of proteins that are found constitutively in the plasma.Complement proteins are present in secretions, such as bronchial fluids, where they protect portals of entry.Complement proteins are present in interstitial fluids where they protect against agents that penetrate the protective barriers (skin, mucosal membranes, etc.).
4Production of Complement Proteins The molecular weights of complement proteins range widely from kDa.Complement proteins are synthesizedPrimarily by liver hepatocytes and by tissue macrophages,Secondarily by epithelial cells, fibroblasts and monocytes.Concentration ranges in plasma:1 or 2 ug/ml – Mannose-Binding Lectin and Factor D300 ug/ml – C41200 ug/ml – C3Albumin concentration is ~40 mg/ml with total serum protein being ~60 mg/ml
5Roles of Complement Complement proteins are activated on demand. Complement proteins are activated in a cascade.In these ways, complement proteins are similar to clotting proteins.Complement proteins are non-specific proteins that play roles both in the innate immune system and in the adaptive immune system.Destroy bacteriaDestroy fungiDestroy viruses
6Importance of Complement The complement system is so important to our defense against microorganisms that there are several pathways by which the complement system can be activated.Classical pathwayAlternative pathwayMannose-binding lectin pathway (aka, lectin pathway)
7Nomenclature of Complement Proteins Complement proteins in the common portions of the Classical PathwayDenoted with the letter “C” followed by a number and are named C1 through C9.Proteins in the Mannose-Binding Lectin Pathway areMannan-binding lectin (MBL)MBL-associated serine protease-1 (MASP-1)MBL-associated serine protease-2 (MASP-2)Proteins in the Alternative Pathway that lead to the common portions of the classical complement pathwayDenoted as factors (Factor B and Factor D).
8Function of the Complement System The complement system acts as an auxiliary system in immunity, both on its own and in conjunction with humoral immunity.In its role in innate immunity, it is a primitive surveillance and defense system for microbes, independent of T cells and antibodies.In its role in adaptive immunity, it is a major effector system for humoral immunity.Lowest organisms have some aspects of the complement system.Primitive vertebrates like the lamprey and hagfish.Invertebrates such as the horseshoe crab and insects have alternative pathway.
9Specific Functions of the Complement System Chemotactic AgentActivator ofInflammationComplement also augments stimulation of B cells through complement receptor 2 (CR2/CD21) to increase the humoral immune response.
10Biochemistry of the Complement System Activation of the complement systemThe classical pathwayThe mannose-binding lectin pathwayThe alternative pathwayControl of complement activationActivation/Inactivation of C4b and C3bComplement receptors
142 IgG/1 IgM C1q C1q C1r C1r C1s C1s C4 C4b Activation of C1C1 is present in plasma as an inactive C1qr2s2 complexBinding of two arms of the complex to immunoglobulin (2 IgG or 1 pentameric IgM) causes conformational change in C1q. This initiates a cascade of events.C1q conformational change C1r conformational changeC1r conformational change C1r active enzymeC1r active enzyme C1s enzymatic cleavageC1s enzymatic cleavage C1s active enzymeC1s active enzyme C4 cleavageThis result of this cascade is often referred to as the C1 esterase cleavage of C4.Cleavage of C4 is controlled by the C1 inhibitor (C1INH)The absence or mutation of C1 inhibitor leads to hereditary angioedema (swelling of the face and respiratory airways, as well as abdominal cramps).2 IgG/1 IgM C1q C1q C1r C1r C1s C1s C4 C4bCirculating IgM is not in the right conformation for C1q binding, but antigen bound IgM is.A single molecule of IgM on a foreign RBC can activate the classical pathway, whereas some 1000 molecules of IgG on a foreign RBC are required to make sure that 2 IgG molecules are close enough to each other to initiate C1q binding. (within nm of each other).Italics = conformational changeColor = enzyme activity
16Activation of C4 C1 esterase cleaves C4. C4a can act a chemoattractant C4b has a thioester region which forms covalent bonds with molecules on the target surface.C4b can act as an opsonin and interacts with complement receptors (CR1).
17Activation of C2C2 interacts with C4b and is cleaved by C1s, forming a C4b2a complex on the surface.C4b2a is the classical pathway’s C3 convertase.Thus, C4b2a is an enzyme that cleaves C3 to C3a and C3b.Note: There is some disagreement among scientists about the nomenclature for the cleavage products for C2. For example, some scientists identify the C3 convertase as the C4b2b complex.Generally, it is the larger fragment that has enzymatic activity. In the case of C2 cleavage products, C2a is the smallest and contributes to the enzymatic activity of the C4b2a complex.
18C3 activationC4b2a cleaves C3, activating a labile thioester bond on C3b.This thioester can bind COVALENTLY to free hydroxyl or amino groups, resulting in C3b covalently binding to target surfaces.C3b bound to a surface acts as an opsonin.Key points for the classical pathwayActivation occurs in conjunction with specific antibodyC3b and C4b covalently bind to target via thioester bondsBecause there is a series of enzymatic cleavage events, there is tremendous amplification of the signal as the signal progresses down the series.
19Review of Activation of the Classical Pathway The sequence of complement protein activation in the classical pathway is 1>4>2>3>5>6>7>8>9Note that 4b gets “before (b 4)” its expected place.The classical pathway is triggered by antigen binding to (crosslinking) two IgG molecules or two subunit parts of one IgM molecule.The cascade of proteolytic steps in the classical pathway are performed by serine esterases.C4b and C3b bind covalently to surfaces via thioester bonds.
20Sequential Enzymatic Cleavage Events in Complement Activation
22Activation through C5Involves proteolytic cleavage steps, liberating smaller fragments from C2 through C5. The smaller fragments are soluble and can have biologic effects. The larger fragments remain bound in a complex required for the next activation step.By convention,Smaller fragments are denoted by the letter ‘a’ (e.g., C3a, C5a)Larger fragments by ‘b’ (e.g., C3b, C5b)Notable exception is C2 (C2a is the larger, active fragment).Complexes with enzymatic activity are often denoted by a line over the top of the numbers or letters, as in(C4b2a)
24MBL PathwayActivation of the MBL Pathway is primarily mediated by a protein constituent in the plasma called mannan-binding lectin (also called the mannose-binding lectin or MBL).Activation of the MBL Pathway does not require specific antibody for activation.Activation of the MBL Pathway occurs by a C1-independent mechanism.Activation of the MBL pathway occurs when MBL binds to specific sugar residues like N-acetyl glucosamine or mannose that are present in the cell wall polysaccharides of microorganisms such as Salmonella, Listeria, Neisseria, Candida, etc.MBL, which resembles C1q, interacts with MASP-1 and MASP-2 by a mechanism similar to C1q interaction with C1r and C1s, resulting in the formation of the classical pathway C3 convertase (C4b2a).Lectin is something that recognizes specific sequences of sugar residues.Mannose type receptor on phagocytes recognizes N acetyl glucosamine, mannose, glucose, fucose.Pathogen associated molecular patterns (on pathogen) interact with pattern recognition receptors.
28Alternative PathwayPhylogenetically the oldest of the C3 activating pathways.Does not require specific antibody/antigen binding for activation.Can be triggered by a low level of spontaneous lysis of C3 by water to C3i that functions in a manner similar to C3b.Can be amplified by C3b binding to foreign surface structures (LPS) or by additional cleavage by bacterial proteases.Things deficient in sialic acid are good activators.An ‘activating surface’ is in large part one without adequate regulatory protein function to control alternative pathway activation, or one that is not favorable to control of the alternative pathway by factor H.
29Some Initiators or Activators of the Alternative Pathway of Complement Activation Many Gram negative and Gram positive bacteriaLPS from Gram negative bacteriaTeichoic acid from Gram positive cell wallsFungal and yeast cell walls (zymosan)Some viruses and virus infected cellsSome tumor cellsSome parasitesHuman IgA, IgG and IgE in complexesAnionic polymers (dextran sulfate)Pure carbohydrates (agarose, inulin)Targets bearing surface clusters of both charge and neutral sugar.Alternative pathway is always autoactivating and an activating surface lacks the regulators to keep it under control.
30Activators Lectin Pathway Classical Pathway Alternative Pathway MBL MASP-1, MASP-2C1qC3 + H2OFactor BFactor DC1r2 C1s2C4C2C3C3 convertase (C4b2a)C3b (Opsonin)C3aC5aC5 convertase (C4b2a3b)C5b C6 C7 C8 C9Membrane Attack ComplexC5Terminal lytic PathwayAntigen AntibodyComplexes (IgG/IgM)Polysaccharides onMicrobes; Also IgAForeign Surfaces (LPS);Spontaneous (Nucleophiles)Activators(Anaphylatoxins)A nucleophile is a molecule that can donate electrons. For example, Cl-, hydrogen peroxide, carboxylate anions, amines, nitrates, or NH3 (ammonia)
32Formation of the Alternative Pathway C3 Convertase (C3bBb) C3 tickover - spontaneous conformational change of a few C3 molecules, leading to water hydrolyzing the thiolester bond of C3 to form C3 H20 or C3i.C3i is then deposited in a random and non-specific manner on the surfaces of host cells and pathogenic organisms alike.On the normal host cell, bound C3i can inactivated by binding to Factor I and Factor H.On the pathogenic organism, bound C3i can be further activated by binding to Factor B to form C3iB which is then cleaved by Factor D to form C3iBb (C3 convertase).Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb)Surfaces rich in carbohydrate and deficient in sialic acid tend to be the best activators.
33Activation and Inactivation of C3b C3 = Complement C3FB = Factor BFD = Factor DFI = Factor I (in conjuction with Factor H,inactivates soluble C3b and C4b whendeposited on the surface of a normal cell)FH = Factor H (cofactor of Factor I in mediatingcleavage of C3b to its inactive form C3biaka C3iC3 tickover is about 1% of the total C3 per hour.C3bBb is stabilized by properdinStablized byproperdinTarget Cell MembraneNormal Cell Membrane
34Amplification of C3 Cleavage by Membrane-Bound C3bBb Amplification – 1 C1 molecule 6 C4b2a 1200 C3bProperdin stabilizes C3bBb
35Activation of C5 and the Terminal Complement Pathway C5 is cleaved by either the Classical Pathway C5 convertase (C4b2aC3b) or by the Alternative Pathway C5 convertase (C3bBbC3b) into 2 fragments: C5a and C5b.Cleavage of C5 is the last enzymatic stepC5b binds to a target and then interacts with C6, C7, C8 and C9 to form the Membrane Attack Complex in the lipid membrane.The Membrane Attack Complex is a transmembrane channel that allows passage of ions, compromises of the semi-permeable membrane, and causes lysis of the cell.
36Activation of C5C5 is cleaved into 2 fragments (C5a and C5b) by eitherThe Alternative Pathway C5 convertase (C3bBbC3b) orThe Classical Pathway C5 convertase (C4b2aC3b).Cleavage of C5 is the last enzymatic step.
40Assembly of C9 ChannelIf C9 molecules are bound to the C5bC6C7C8 complex, they form the Membrane Attack Complex that can punch a hole in the lipid membrane.Since the Membrane Attack Complex is a transmembrane channel that allows passage of ions, it will compromise the semi-permeability of the membrane and result in lysis of the cell.
41Notes on C9 AssemblyIf the interaction with C5b through C9 occurs in proximity to a membrane, then the MAC assembly occurs in that membrane and lysis is the end result.Alternatively, C5b-9 can bind to S protein in the fluid phase. In this case, lysis does not occur.
42Summary of Pathways of Activation Three Primary Pathways of Activation with different start signalsClassical – antigen antibodyMannose binding lectin - mannoseAlternative – LPS, carbohydrates, etcProteolytic cleavages of complement components operate through C5Non-proteolytic events for assembly of C6789 membrane attack complex
43Summary of Names You Need to Know Classical Pathway:C1q, C1r, C1s, C4, C2Mannose Binding lectin pathway:MBL (mannose binding lectin)MASP-1 (MBL-associated serine protease)MASP-2Alternative Pathway:Factor BFactor DProperdinCommon to all pathways:C3Terminal Lytic pathway:C5, C6, C7, C8, C9
44ControlWhat stops the activation? Or Why don’t we lyse all of our own cells?
45Things That Limit Complement Activation Short half life of the enzymes formedProperties of non-activator surfacesInhibitorsFluid phase inhibitorsSo active fragments don’t go too farMembrane bound inhibitorsOn our own membranesSo C3b and C4b don’t attach or don’t lead to lysis of our own cells
46Activation and Inactivation of C3b C3 = Complement C3FB = Factor BFD = Factor DFI = Factor I ( inconjuction with Factor H,inactivates soluble C3b and C4b whendeposited on the surface of a normal cell)FH = Factor H (cofactor of Factor I in mediatingcleavage of C3b to its inactive form C3biaka C3iC3 tickover is about 1% of the total C3 per hour.C3bBb is stabilized by properdinStablized byproperdinTarget Cell MembraneNormal Cell Membrane
49What If You Lack Control? Deficiencies of complement control proteins can lead to uncontrolled activation of the complement systemConsequences of activation – lysis, etcConsumption (exhaustion) of the complement components leading to the consequences of secondary complement deficiency (immune-complex disease and infections)
50C1 Inhibitor Deficiency Roles of the C1 inhibitorInhibits C1 esteraseAlso inhibits kallikrein, plasmin, Factor XIa and Factor XIIaDeficiency in C1 inhibitor leads to recurrent episodes of localized edema in skin, GI tract, or larynxResults in HAE (hereditary angioedema)Prevalence: 2-10 per 100,000First case of homozygous C1 inhibitor deficiency reported in 2006 (JACI 118:1330, 2006)Homozygous patients had undetectable C1q, reduced C1s, circulating active C1r, and a C1Inh mostly in its cleaved inactive form.The most common HAE treatment is attenuated androgens, which increase the C1INH gene transcription levels.C1INH is a serpin plasma serine protease inhibitor.Also inhibits MASP
51Hereditary angioedema Figure 3-6. Hereditary angioneurotic edema (HAE). A and B, Angioedema of the hands. C, Five-year-old girl with a moderate attack of angioedema. D, Small bowel barium radiograph during an acute episode of pain in the proband. Two segments of jejunum have mural edema: "thumbprinting" (thick arrows) and spiculation (thin arrows). This condition is one of several that are caused by deficiencies and dysfunction on complement regulator proteins. HAE is an autosomal dominant condition caused by a deficiency or nonfunction of C1 esterase inhibitor . Patients with this abnormality have recurrent episodes of swelling of the soft tissues, primarily of the face, extremities, larynx, and intestinal mucosa. Symptoms usually begin in adolescence, and attacks are commonly triggered by emotional stress and trauma (see panels A-D). Without the C1 esterase inhibitor, there is continued activation of the classic complement pathway and dysregulation of the activity of clotting, kinin, and fibrinolysis pathways. The causative factor leading to the angioedema may be C3a, C4a, and C5a; the anaphylotoxins; or C2 kinin from kinin activity. C4 levels are decreased in patients with HAE. Aminocaproic acid is used in the treatment of patients with acute attacks and for prevention before a medical procedure such as surgery. For long-term therapy, androgens such as danazol have been shown to stimulate C1 esterase inhibitor production. (Panels A and B from Arreaza et al. , with permission; panel C from Weinstock et al. , with permission; panel D from Nielsen et al. ; with permission.)
52Deficiency in Decay Accelerating Factor (CD55) & CD59 DAF deficiency causes increased susceptibility of erythrocytes to membrane attack complex-mediated lysisSee as complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH)DAF deficiency is due to a defect in a post-translational modification of the peptide anchors that bind the proteins to the cell membraneRecent studies suggest that DAF deficiency can be treated with an antibody to C5 reduces hemolysis
54Review: What does complement do? Lyses cells (MAC)Inflammatory mediators (C3a, C5a)OpsonizationSolubilization and clearance of immune complexesAugmentation of humoral immunity
55Review: What does complement do? Lyses cells (MAC)Inflammatory mediators (C3a, C5a)OpsonizationSolubilization and clearance of immune complexesAugmentation of humoral immunity
56Anaphylatoxins C3a C3a receptor Response C3a and C5a can mimic the symptoms of inflammation and anaphylaxisChemotaxis, smooth muscle contraction, increased vascular permeability, degranulation of mast cells, etc.Distinct receptors on many cell types
58Review: What does complement do? Lyses cells (MAC)Inflammatory mediators (C3a, C5a)OpsonizationSolubilization and clearance of immune complexesAugmentation of humoral immunity
59Things C4b and C3b can do Complement Activation C4b and/or C3b on surfacesParticipate in continuedpathway activation leadingto MACDegraded to fragmentsInteract withCR2 and CR3Interact with CR1LysisOpsonizationClearance of ICAugmentation of humoralimmunityOpsonizationClearance of IC
60CR1 (CD35) Major ligands C3b, C4b Monocytes, macrophages, PMN, Eosinophil, RBC, B and T cellsTransport of immune complexes by RBCPromotes immune adherence (binding of opsonized microbes to primate RBCs)Promotes phagocytosis in cooperation with Fc receptorsBlocks formation of C3 convertase
62Review: What does complement do? Lyses cells (MAC)Inflammatory mediators (C3a, C5a)OpsonizationSolubilization and clearance of immune complexesAugmentation of humoral immunity
63C3 fragment interaction with Complement Receptors Bacteria or ICClearance of Immune ComplexAugments humoralimmunity
64Immune Complex Disease High incidence of Immune Complex disease in individuals who are deficient in C1, C4, C2 or C3Immune complexes are not solubilized and clearedComplement can also play a significant role in tissue damage in Immune Complex diseases such as SLE (systemic lupus erythematosus)Excess immune complexes cause pathological complement activation inflammation, tissue damage
65Immune Complex Solubilization And Transport Complement prevents formation of insoluble immune complexes (solubilization).Deposition of insoluble aggregates in the tissues can cause damage and immune complex disease.Binding of C3b to the antigen antibody complex interferes with lattice formation, limits its growth, prevents precipitation of the antigen antibody complexes and keeps them soluble.
66Immune complex transport The complement system is a major mechanism for removal of immune complexes (transport).Immune complexes coated with C3b bind to CR1. More than 85% of the CR1 in the circulation is on the RBC.CR1 receptors on the erythrocyte are responsible for the transport of immune complexes to the reticuloendothelial system for clearance (macrophages in spleen, etc). The immune complex coated with C3b is transferred from the RBC CR1 receptor to the macrophage CR1 receptor. The immune complex is then internalized and degraded.
68Review: What does complement do? Lyses cells (MAC)Inflammatory mediators (C3a, C5a)OpsonizationSolubilization and clearance of immune complexesAugmentation of humoral immunity
69CR2 (CD21) Major ligands C3d, C3dg, iC3b B cells, activated T cells, epithelial cellsCR2 forms an additional signal with antibody to augment stimulation of the B cell to increase the humoral immune response (CR2/CD19/CD81).CR2 has high affinity for an envelope protein of Epstein Barr virus, allowing the virus to enter the B cell.
71Complement Deficiencies Deficiencies of the various complement components often present as infectionsPyogenic infections and infections with encapsulated bacteria (classical and alternative)Opsonization and phagocytosis are a primary host defense.Neisseria infections (C3, alternative pathway and terminal lytic pathway)Immune complex or autoimmune diseaseClassical pathway or C3 deficienciesMBL deficiency results in serious pyogenic infections as well
72Complement Deficiencies and Associated Diseases Recurrent infection by pyogenic (pus forming) bacteria such as steptococci and staphylococci. These organisms are gram positive and generally resistant to the lytic effects of the MAC. Nevertheless, the early complement components ordinarily prevent recurrent infection by mediating a localized inflammatory response and opsonizing the bacteria.Wikipedia, 2011A pyogenic infection is an infection that is characterized by severe local inflammation, usually with pus formation, generally caused by one of the pyogenic bacteria.Some common disease processes caused by pyogenic infections are impetigo, osteomyelitis, septic arthritis, and necrotizing fasciitis. Pyogenic bacteriaA great many species of bacteria may be pyogenic. The most commonly found include:Staphylococcus aureusStaphylococcus epidermidisStreptococcus pyogenesEscherichia coli (Bacillus coli communis)Streptococcus pneumoniae (Fraenkel's pneumococcus)Klebsiella pneumoniae (Friedländer's bacillus)Salmonella typhi (Bacillus typhosus)Pseudomonas aeruginosaNeisseria gonorrhoeaeActinomycesBurkholderia mallei (Glanders bacillus)Mycobacterium tuberculosis (tubercle bacillus)From Wikipedia, the free encyclopediaJump to: navigation, searchSEM micrograph of an encapsulated bacterium (Pneumococcus).Polysaccharide encapsulated bacteria, frequently referred to simply as encapsulated bacteria and less precisely called encapsulated organisms, are a group of bacteria that have an outer covering, a bacterial capsule, made of polysaccharide.Contents[hide]1 Examples of encapsulated bacteria2 Role in disease2.1 Asplenia2.2 Children3 See also4 References Examples of encapsulated bacteriaHaemophilus influenzae type b (Hib)Streptococcus pneumoniae (pneumococcus)Neisseria meningitides (meningococcus)Group B streptococcus (GBS)Klebsiella pneumoniaeSalmonella typhi
75Factor H is One Fluid Phase Inhibitor of C3 Convertase Factor H is a fluid phase inhibitor of C3 convertase. If it sees C3bBb floating around, it binds and dissociates the Bb, thus inactivating the C3bBb.‘Decay acceleration of the convertase’
76Factor H Can Inactivate C3bBb on the Surface of a Normal Cell If Factor H sees C3bBb on a membrane with sialic acid (like our membranes), it will bind to the sialic acid residue and C3b, displacing Bb from the convertase and inactivating C3bBb. Factor I than can degrade the C3b, with Factor H as a cofactor.An activator surface (such as bacteria) does not have sialic acid and therefore Factor H cannot bind and displace the Bb. In this case, the Factor H does not inhibit the C3 convertase activity.