2THE COMPLEMENT SYSTEMThe complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens.Approximately 30 components:activating moleculesregulator factorscomplement receptorsmembrane proteins wich inhibit the lysis of host cellsMost of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogen). Activation is induced by proteolitic cleavage.
3AMPLIFICATION OF THE COMPLEMENT CASCADE limitedproteolysisinactive precursorsenzymeactivating surfaceActivating surface needed!
4ACTIVATION OF THE COMPLEMENT SYSTEM COMPLEMENT ACTIVATIONRECRUITMENT OF INFLAMMATORY CELLSOPSONIZATION OF PATHOGENSDIRECT KILLING OF PATHOGENSClearence of Immune complexesFACILITATING PHAGOCYTOSIS
6THE C1 COMPLEX Collagen „legs” Gobular „heads” C1 is always present in serum but it can operate on an activating surface in normal caseLow affinity binding to the Fc region of antibody conformational change activation Multiple interaction with immune complexes
7In the innate immune response, the classical pathway of complement activation is initiated by the binding of the C1q component of C1 to the acute-phase protein C-reactive protein, which is a pentamer. In the adaptive immune response, the classical pathway is initiated by pentameric IgM. This is not a coincidence. In all likelihood the preexisting predilection of C1q for binding to pentameric C-reactive protein selected for the evolution of pentameric antibody molecules.
9Different isotypes of antibodies activate the complement system differently The IgM and IgG3 isotypes are the most effective at activating the complement cascade.
10The classical pathway: Fixation of complement, generation of C3b by the classical C3 convertaseWhen soluble pentameric IgM in the 'planar' conformation establishes multipoint binding to antigens on a pathogen surface, it adopts the 'staple' conformation and exposes its binding sites for the C1q component of C1. Activated C1 then cleaves C2 and C4, and the C2a and C4b fragments form the classical C3 convertase on the pathogen surface. Conversion of C3 to C3b leads to the attachment of C3b to the pathogen surface and the recruitment of effector functions (i.e. opsonisation). C3a recruits phagocytic cells to the site of infection.
11ACTIVATION VIA THE MANNAN-BINDING LECTIN PATHWAY
12GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES Eukariotic cellsProkariotic cellsMannoseGalactoseGlucoseamineMannoseNeuraminic acid(sialic acid)
13MANNAN-BINDING LEKTIN ACTIVATES THE COMPLEMENT SYSTEM MBL belongs to the collectinsMASP = MBL associated serin protease
15C3b can derive from classical or the lectin pathway too Alternative pathway is instantly inactivated on eukariotic cell surfaces (in the presence of sialic acid molecules)In the plasma close to a microbial surface the thioester bond of C3 spontaneously hydrolyzes at low frequency. This activates the C3, which then binds factor B. Cleavage of B by the serine protease factor D produces a soluble C3 convertase, called iC3Bb, which then activates C3 molecules by cleavage into C3b and C3a. In this complex the Bb fragment of factor B provides the protease activity to cleave C3, and the C3b fragment of C3 locates the enzyme to the pathogen's surface.
16THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM Complement fixationStrong covalent bindingC3 proteis have one of the highest levels in the serum: 1.2 mg / ml( molecules/ml)
17C5-CONVERTASEC3-convertase + C3b=C5-convertase(C4bC2bC3b)Figure 2.12 Complement component C5 is cleaved by C5 convertase to give a soluble active C5b fragment.The C5 convertase of the alternative pathway consists of two molecules of C3b and one of Bb (C3b2Bb). C5 binds to the C3b component of the convertase and is cleaved into fragments C5a and C5b, of which C5b initiates the assembly of the terminal complement components to form the membrane-attack complex.The classical and alternative C3-convertase is different in structure but common in function
18MEMBRANE ATTACK COMPLEX (MAC) live and deadbacteriaMAC in the cell membranePore formation osmotic lysis of pathogens
28Upper panel: the soluble protein properdin (factor P) binds to C3bBb and extends its lifetime on the microbial surface. Middle panel: factor H binds to C3b and changes its conformation to one that is susceptible to cleavage by factor I. The product of this cleavage is the iC3b fragment of C3, which remains attached to the pathogen surface but cannot form a C3 convertase. Lower panel: when C3bBb is formed on a human cell surface it is rapidly disrupted by the action of one of two membrane proteins: decayaccelerating factor (DAF) or membrane cofactor protein (MCP). In combination, these regulatory proteins ensure that much complement is fixed to pathogen surfaces and little is fixed to human cell surfaces.
29Regulatory proteins on human cells protect them from complement-mediated attack
30CD59 prevents assembly of terminal complement components into a membrane pore
31Regulation of complement system Factor Ia-2macroglC1InhDAFCR1MCPC4bpLECTIN PATHWAYCD59HRFS-proteinC-pept.ase NProperdinpositive feedbackThe opened available thioeter bound is active only for millisecondsAnafilatoxins are regulated by carboxipeptidases (Carboxipeptidase N releases c-term arginine: des-Arg)Decay accelerating factorsDAF, CR1, C4bpProperdin-like activity: autoantibody: nephritic factorAnaphilatoxin inactivator: Carboxipeptidase NAlpha-2 macroglobulin: inhibitor of MBLS-protein=vitronectinCR1=CD35MCP=CD46DAF=CD55HRF=Homolog restriction factorMIRL=protectin=CD59Factor IFact-HCR1MCPDAFmembrane proteinsoluble molecule
32MAJOR REGULATING FACTORS OF COMPLEMENT SYSTEM C1Inh: C1-inhibitor (serine-protease inhibitor)Factor I: inhibits both C3 convertases in the presence of co-factors (C4bp – classical pw., factor H – alternative pw., MCP – both)DAF(CD55): Decay Accelerating FactorMCP: Membrane Cofactor ProteinMIRL(CD59): Membrane Inhibitor of Reactive LysisProperdin: stabilize convertases of alternative pathway
33Deficiencies of complement system – cascade molecules Not the lysis of cells is the most important function of the complement system
35One of the major function of C1 INHIBITOR C1q binds to IgM onbacterial surfaceC1q binds to at least two IgGmolecules on bacterial surfaceBinding of C1q to Ig activates C1r, which cleavesand activates the serine protease C1sC1INH dissociates C1r and C1s from the active C1 complex
36HEREDITARY ANGIONEUROTIC EDEMA (HANE) (HEREDITARY C1INH DEFECT) 17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting)appendectomia normal appendixsimilar symptoms occured repeatedly earlier in his life with watery diarrheafamily history of prior illnessimmunologist’s suspicion: hereditary angioneurotic edemalevel of C1INH: 16% of the normal meandaily doses of Winstrol (steroid) – marked diminution in the frequency andseverity of symptomsintravenous purified C1INH became avaible by the timeMain symptoms:swellings of skin, guts, respiratory tractsserious acute abdominal pain, vomitinglarynx swelling – suffocation, may cause deathTreatment:iv C1INH, FFP, steroidkallikrein and bradykinin receptor antagonistsFFP= Fresh Frozen PlasmaChild with symptoms of HANE
37Pathogenesis of hereditary angioneurotic edema Inhibition by C1INH in many stepsactivation of XII factorbradykinin and C2-kinin:enhance the permeability ofpostcapillar venulesby contraction of endothelholes in the venule wallsedema formationC1 is always active withoutactivating surface becauseplasmine is always activeactivation ofkallikreinactivation ofproactivatorcleveage of kininogento generate bradykinin,vasoactive peptidecleveage of C2a togenerate C2-kinin,vasoactive peptidecleveage ofplasminogen to generate plasmincleveage of C2 togenerate C2aactivation of C1
38Q&AHANE1. Activation of complement system results in the release of histamine and chemokines, which normally produce pain, heat and itching. Why is the edema fluid in HANE free of cellular components, and why does the swelling not itch?Histamine release on complement activation and recruiting of leukocytes is caused by C3a and C5a, both generated by the C3/C5 convertases. In HANE C1 constantly activate C2 and C4 in the plasma but C4b is rapidly inactivated because it does not bind to activating surface; for that reason, and because the concentrations of C2 and C4 are relatively low, no C3/C5 convertase is formed.Edema is caused by C2-kinin and bradykinin.2. Which complement component levels will be decreased? Why?C2 and C4, because of the continous cleavage by activated C1.
39Q&AHANE3. Would you expect the alternative pathway components to be low, normal orelevated?C1 plays no part in the alternative pathway. This pathway is not affected.4. What about the levels of the terminal components?The unregulated activation of the early components does not lead to the formationof the C3/C5 convertase, so the terminal components are not abnormally activated.5. Despite the complement deficiency in patients with HANE, they are notunduly susceptible to infection. Why not?The alternative pathway of complement activation is intact and these arecompensated for by the potent amplification step from the alternative pathway.6. How might you decide the background of the laryngeal edema(HANO or anaphylactic reaction)?If the laryngeal edema is anaphylactic, it will respond to epinephrine.If it is due to HANO, it will not, C1INH needed.
40PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) Acquired clonal mutation of PIG-A gene in myeloid progenitors – no GPI-enchored proteins in the cell membrane of affected cells (rbc, plt, wbc)CD59 and CD55 complement regulatory proteins are GPI-enchored proteinsNo CD59 and/or CD55 PNH patients are highly susceptible to complement-mediated lysisThe lysis of red blood cells leads to high levels of hemoglobins in the blood that appears in the urine (hemoglobinuria)Elevated levels of TF derived from complement-damaged leukocytes cause thrombosesPIG-A = Phosphatidylinositol N-acetylglucosaminyltransferase subunit AGPI= glycosylphosphatidylinositolParoxysmal = sudden attacksNocturnal = occuring at night
41Change in the colour of urine samples taken from PNH patient during the day
42Paroxysmal nocturnal hemoglobinuria (PNH) symptoms and therapy Haemolytic anaemia andassociated symptomsHaemoglobin and its products in the urineThrombosis: in brain veins, mesentheric veins, vv. hepaticae (Budd-Chiari-syndrome)Transformation to acut myelogenous leukemia (AML), aplastic anaemia, myelodisplastic syndrome (MDS)Specific th.: eculizumab (Soliris - anti-C5 monoclonal antibody)Curative th.: bone marrow transplantationAlternative th.: steroids (general immunosuppression)Anticoagulants: sc. heparin p.o. kumarinIron replacementTransfusion (filtered-irradiated blood)Frequency: 1-2/million/year. Mean survival: 10 years.Over 10% of aplastic anemia patients and 1% to 3% of MDS patients develop PNH (secunder form). DAF and MIRL proteins are absent from the cell surfaces flow cytometry is diagnostic.In 75%-of patients hemolysis occures regardless of time of day (not only at night, ‘nocturn’), following the activation of the complement system or decrease of the pH of the blood.Transfusion can provoke hemolysis, this is why it has to be done with blood products free of plasma and white blood cells.
43AbbreviationsC1Inh: inhibitor of C1 and MBL (serin protease inhibitor – multiple effects)α2-macroglobulin: inhibitor of MBLC4bp: C4 binding protein - inhibitor of the classical C3 convertaseFactor H: inhibitor of the alternative C3 convertaseFactor I: cleaves C4b and C3bProperdin: stabilizes the convertases of the alternative pathwayDAF (CD55): Decay Accelerating Factor (of C3 convertases)MCP (CD46): Membrane Cofactor Protein, cleavage of C3 convertases with factor ICR1: complement receptor 1, inhibitor of C3 convertasesCD59 (MIRL): Membrane Inhibitor of Reactive Lysis – inhibits binding of C9 to C8HRF: Homologous Restriction Factor (inhibits binding of C8 and C9)Ebből és a következőből 1 ábrát kellene csinálni