2. SZEMINRIUM

3RD SEMINARINNATE IMMUNITY: ANTIVIRAL STATE,KILLER CELLS, THE COMPLEMENT SYSTEMConventional DCPlasmacytoid DC58737109NLRIL-1IL-12/23IL-10IFNIFN124616RLRDANGER SIGNALS ARE TRANSLATED TO CYTOKINE SECRETION THROUGH VARIOUS MOLECULAR SENSORS IN DC SUBTYPESTLR1 bacterial lipoprotein (together with TLR2)TLR2 bacterial lipoprotein, peptidoglycane, lipoteicholic acid (heteromer with TLR1 and TLR6)TLR3 viral dsRNATLR4 bacterial LPSTLR5 bacterial flagellinTLR6 bacterial lipoprotein (together with TLR2)TLR7 viral ssRNATLR8 viral ssRNATLR9 unmethylated CpG DNATLR10 modified viral nucleotidesNLRs microbial products, DAMPsRLRs viral dsRNARLRTHE TYPE I INTERFERON RESPONSE: ANTIVIRAL STATE

plasmacytoid dendritic cellsPlasmacytoid dendritic cells (pDCs) produce 1000x more type I interferon than other cells (Natural Interferon Producing Cells NIPC)After viral infection they are accumulated at the T cell zone of the lymph nodes3VIRUS-INDUCED TYPE I INTERFERON PRODUCTIONparacrineautocrineInfected cellsubtypesIFN-IFN-IFN responseIRF-3IRF-7VirusIFN-IFN-NFBAP-1Type I IFN receptorIFN responseIRF: interferon regulatory factorIRF-3INTERFERON EFFECTOR PATHWAYSinduction of the antiviral state1. Mx GTPase pathwayblock viral transcription

2. 2',5'-oligoadenylate-synthetase (OAS)-directed Ribonuclease L pathwaydegrade viral RNA

3. Protein kinase R (PKR) pathway (Ser/Thr kinase, dsRNA-dependent)inhibit translation

4. ISG15 ubiquitin-like pathwaymodify protein function

CONTROL ALL STEPS OF VIRAL REPLICATIONTRIFTANKIKKTBK1IRF-3TRIFTRAMTLR3TLR4MyD88IRF-5TLR7TLR8TLR9IFN-, IFN- RIG-1Stimulation of Ig-productionin B-cellsType I interferon receptorIRF-7

Increased citotoxicity and proliferation of NK-cellsActivation of - and T-cellsIncreased antigen presentationin myeloid dendritic cellsIRAK-1TRAF-6IRF-7MULTIPLE EFFECTS OF TYPE I INTERFERONSEFFECTOR MECHANISMS OF INNATE IMMUNITY

KILLER CELLSPHAGOCYTIC CELLSCOMPLEMENT SYSTEMNK CELLS

Similar functions to cytotoxic T cells but:

larger than lymphocytesno rearranged antigen-specific receptorscontain large cytoplasmic granulesrespond fast, circulate in a partly activated state

KIRKARKIRKARRECOGNITION AND KILLING BY NK CELLSContents of lytic granules:Perforin: forming pores in the target cell membrane lysisGranzyme: inducing apoptosis in the target cell9Lysis of infected cellKinetics of the activity of the complement system and NK cells in virus infectionIFNIL-12

Complement systemNK-cellsdaysRelative level/activityNK-CELLSVirus-infectedcellPRRRECOGNITIONACTIVATIONRECOGNITION OF ALTERED HOST CELLSNATURAL KILLER CELL ACTIVATION EFFECTOR MECHANISMS OF INNATE IMMUNITY

KILLER CELLSPHAGOCYTIC CELLSCOMPLEMENT SYSTEMTHE 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 which inhibit the lysis of host cells

Most of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogen). Activation is induced by proteolytic cleavage.

AMPLIFICATION OF THE COMPLEMENT CASCADEinactive precursorslimitedproteolysisactivating surfaceenzymeActivating surface needed!ACTIVATION OF THE COMPLEMENT SYSTEM

Clearence of Immune complexes14THE CLASSICAL PATHWAY

C1 is always present in serum but it requires an activating surface for activationLow affinity binding to the Fc region of antibody conformational change activation Collagen legsGobular headsTHE C1 COMPLEXACTIVATION OF THE C1 COMPLEX

THE CLASSICAL PATHWAY: FIXATION OF COMPLEMENT, GENERATION OF C3b BY THE CLASSICAL C3 CONVERTASE18When 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.THE MANNAN-BINDING LECTIN PATHWAYEukariotic cellsglucoseaminemannose (polymer = mannan)galactoseneuraminic acid(sialic acid)GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIESProkariotic cellsMANNOSE-BINDING LECTIN (MBL) PATHWAYMBL: part of the collectin family

similar structure to C1 complex, MASP-1,2 ~ C1r,s

binds mannose and similar sugar molecules on the surface of bacteria, fungi, protozoa and viruses conformational change cleavage of C2 and C4 molecules

MASP = MBL-associated serin protease

ACTIVATION OF THE MBL COMPLEXThe literature sometimes refers to the bigger fragment of C2 as C2a but it is more consequent if we use b for the bigger fragments of all complement molecules22THE ALTERNATIVE PATHWAY

C3b can derive from classical or the lectin pathway tooAlternative pathway is instantly inactivated on eukaryotic 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.24

THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM (3 900 000 000 000 000 molecules/ml)Strong covalent bindingComplement fixationC3 proteis have one of the highest levels in the serum: 1.2 mg / ml25C5-CONVERTASE

C3 convertase + C3b = C5 convertase(C4bC2bC3b)The classical and alternative C3 convertase is different in structure but common in functionFigure 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. 26

MACs in the cell membraneMEMBRANE ATTACK COMPLEX (MAC = C5b-C9n)

Pore formation osmotic lysis of pathogens27COMPLEMENT ACTIVATIONSUMMARYAntigen-antibodycomplexMannose Pathogen surfaceC1q, C1r, C1sSerin proteaseC4, C2MBLMASP-1/MASP-2

Serin proteaseC4, C2C3B, DCOMPLEMENT SYSTEMCLASSICAL PATHWAYMB-LECTIN PATHWAYALTERNATIVE PATHWAYC3 CONVERTASEC4a*C3a, C5aInflammatory peptide mediatorsPhagocyte recruitmentC3bOpsonizationBinding to phagocyte CRImmune complex removalTerminal C5b C9MACPathogen/cell lysisTHE ANAPHYLATOXINS: C3a, C4a, C5a

OPSONIZATION COMPLEMENT-MEDIATED PHAGOCYTOSIS

MOVIES: ACTIVATION VIA THE CLASSICAL PATHWAY

REGULATION OF THE COMPLEMENT SYSTEM

33DEFICIENCIES OF COMPLEMENT COMPONENTS AND REGULATORSDeficient complement proteinEffects of deficiencyC1, C2, C4C3Immune-complex diseases (similar to SLE), susceptibility to pyogenic infectionsMAC, alternative pathway componentsSusceptibility to Neisserial infectionsC1INHHereditary angioneurotic edema (HANE)DAF (CD55), MIRL (CD59)

Paroxysmal nocturnal hemoglobinuria (PNH)HEREDITARY ANGIONEUROTIC EDEMA (HANE)(HEREDITARY C1INH DEFECT)Main symptoms: swellings of skin, guts, respiratory tracts serious acute abdominal pain, vomiting larynx swelling suffocation, may cause death

Treatment: iv C1INH, FFP, steroid kallikrein and bradykinin receptor antagonists

Children with symptoms of HANE

bradykinin and C2-kinin: enhance the permeability of postcapillar venules edema

C1 is always active without activating surface because plasmin is always activeInhibition by C1INH in many steps

FFP= Fresh Frozen PlasmaAcquired 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 proteins

No CD59 and/or CD55 PNH patients are highly susceptible to complement-mediated lysis

The 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 thromboses

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)

PIG-A = Phosphatidylinositol N-acetylglucosaminyltransferase subunit AGPI= glycosylphosphatidylinositolParoxysmal = sudden attacksNocturnal = occuring at nightChange in the colour of urine samples taken from PNH patient during the day

Only minority of patients have this symptom. 37PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)SYMPTOMS Haemolytic anaemia and associated symptoms

Haemoglobin and its products in the urine

Thrombosis:brain veins,mesentheric veins,hepatic veins (Budd-Chiari-syndrome)

May transform to leukemia or other bone marrow diseases

THERAPYSpecific th.: eculizumab (Soliris) = anti-C5 monoclonal antibody

Curative th.: bone marrow transplantation

Alternative th.: steroids (general immunosuppression)

Anticoagulants: s.c. heparin p.o. kumarin

Iron replacement

Transfusion (filtered-irradiated blood)