Innate immunity. Monika Raulf. Lecture Tasks of the immune system. Body protection against damaging influences

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1 Innate immunity Monika Raulf Lecture Tasks of the immune system Body protection against damaging influences Deployment of a strong defense 2

2 The line of defence Skin Mucosa Sweat Digestive fluids Lysozyme Lactoferrin Cilia Cough, Sneezing, Vomiting, Diarrohea 3 Innate immunity natural/mechanistic barriers Skin, mucus moves by cilia (air- or fluid-flow along the epithelia; normal gut flora) chemical barriers proteolytic enzymes in body fluids digestive enzymes (in stomach ph ), lysozyme (enzyme able to cleave peptidoglycan of bacterial cell walls) sweat (ph ) low ph-value two Key player -cell types Monocytes/macrophages + granulocytes Endozytose Complement system 4

3 Immune defense Problem: How can the host differ between the large amount of different pathogens? Answer: Development of multiple receptors, which detect conserved motifs of pathogens, that do not occur on Eukaryotes. 5 Pattern recognition receptors (PRR) 4 main groups (classified on the basis of their cellular localization and their function) - free receptors in the serum (e.g. MBL) - membrane-bound phagocytic receptors (e.g. Dectin-1, Scavenger receptors) - membrane-bound signaling receptors (e.g. TLR* 1 ) - cytoplasmic signaling receptors (e.g. NODR* 2 ) * 1 lead to an activation of the pro-inflammatory pathway * 2 NOD = Nucleotide-binding Oligomerization Domain 6

4 Comparison of the characteristics of recognition molecules of the innate and adaptive immune systems Receptor characteristics Innate immunity Adaptive immunity Specificity inherited in the genome yes no Expressed by all cells of a particular type (e.g. macrophages) yes no Triggers immediate response yes no Recognizes broad classes of pathogens yes no Interacts with a range of molecular structures of a given type yes no Encoded in multiple gene segments no yes Requires gene rearrangement no yes Clonal distribution no yes Able to discriminate between even closely related molecular structures no yes 7 Pathogen-associated molecular patterns Pathogen-associated molecular patterns (PAMPs) Endotoxin (lipopolysaccharide) Peptidoglycan bacterial lipoprotein 1 3 -D-glucan Lipoteichoic acid Flagellin HSPs dsrna Cpg DNA are recognized by pattern recognition receptors (PRRs) 8

5 What is endotoxin? 9 Structure of the lipopolysaccharides (LPS, endotoxins) Variability Hex Hex Hep p p Kdo Kdo p GlcN n Hex Hex Hex external Hep Hep Kdo p internal GlcN p O-antigen (repeat 40 units) Nucleus region Lipid A 10

6 Interaction of the organism with endotoxin Route of exposure Contact with blood Oral Inhalative 11 Interaction of LPS with cells gram-negative bacteria cell membrane body barrier (e.g. respiratory tract) scd14 macrophages LBP LPS LBP CD14 NFkB LBP = Lipopolysaccharides, LPS LBP = LPS-binding protein (Acute-Phase-Protein) 12

7 Signal transduction way in detail LPS MD 2 IL-1β IL-6 LBP CD 14 TLR 4 IL-8 TNF-α MyD88 Cytoplasma TRAF 6 IRAK Translation Iк K P Iк B RNA NFк B Transcription nucleus DNA 13 Release of pro inflammatory cytokines gram-negative bacteria cell membrane Body barrier (e.g. respiratory tract) scd14 macrophage LPS LBP LBP CD14 blockade NFkB IL-6 TNF- BPI BPI LBP CD14 activation IL-8 IL-1 LBP BPI = Lipopolysaccharides, LPS CD18 = LPS-binding protein (Acute-Phase-Protein) = Bactericidal-Permeability-Increasing-Protein neutrophils O 2 14

8 The receptor Toll was identified in Drosophila 15 TLRs are members of the IL-1R superfamily and are membrane spanning molecules found on the cell surface and in endosomes Cytokines Pathogen-associated molecules Endogenous damage signals IL-1RII Cell-membrane TIR IL-1-family TIR TLR 1-11 IL-1R, IL-18R, T1/ST2, IL-RAcP, IL-18RAcP Signal transduction Cytokines 16

9 Toll-like-Receptors LPS (Lipid A) Lipid protein Peptidoglycan TLR4 TLR2 TIR NF- B nucleus NF- B I B IL-8 17 Lipid Mediators Microbe Chemokine N-formylmethionyl Peptide LPS CD14 Detection of the microbes, mediators Seven -helical transmembranereceptors Toll-like receptor Mannose receptor Cell response increased integrin activity; cytosceletal changes Cytokine production, reactive oxygen intermediate (ROIs) Phagocytes of microbe into phagosome Appropriate results Migration into tissues Killing of microbes Killing of microbes 18

10 DAMPs = Damage-associated molecular patterns (molecules that are responsible for endogenous adjuvant activity) PAMPs = Pathogen-associated molecular patterns; exogenous molecules which share a number of different recognizable biochemical features Alarmins = endogenous molecules which induce tissue and cell damage 19 from Yang D. et al. : Alarmins link neutrophils and dendritic cells. Trends Immunol 2009; 30:

11 Rock KL et al. Innate and adaptive immune responses to cell death. Immunol Rev 2011; 243: Innate immunity relevant cells 22

12 1. First Cellular Frontline Phagocytes 2. Cellular Defence line Macrophages (in tissue (mononuclear)) (development) Monocytes (in blood) long-lasting at activation cytokines* and other mediators are released, which allure among others neutrophile granulocytes Neutrophile granulocytes = PMN=polymorphonuclear neutrophil leucocytes (just in blood, not in tissue) short-living *important for local inflammatory response and mediation of the induced, not adapted reaction 23 Cellular response 1) Phagocytic cells: Neutrophile granulocytes (60-70%) bacteria 2) NK-cells (natural killer cells) Polymorph neutrophils Monocytes ( macrophages) (5%) Eosinophilic granulocytes (1.5%) parasites 24

13 Phagocytosis, activation of anti bacterial mechanisms Killing of parasites (by antibodies coated) 25 Like mast cells, to find in blood Release of granule, which contain e.g. histamine and other active substances 26

14 Phagocytosis and activation of anti bacterial mechanisms Antigen presentation Found in tissue and blood; characteristic markers: CD1a, CD11c, MHC II DC take up proteins by pinocytosis and present fragments of these to T-cells Antigen admission in the periphery Antigen presentation in the lymph node 27 Phagocytosis Microbe coated with antibodies which bind to Fc receptors on the sell surface The macrophage wall encased the microbe Fusion of the membranes and generation of a vesicle = phagosom Lysomes fused with the phagosom: generation of a phagolysosom 28

15 Opsonisation I Seasoning the victim 29 A bacterial infection triggers an inflammatory response Microbes activate macrophages releasing cytokines and chemokines Vascular dilatation and enhanced permeability of the vascular wall caused redness, swelling and heating Inflammatory cells invaded the tissue and released inflammatory mediators, which provoked pain Fluid from: Immunologie, Janeway et al. 30

16 Inflammatory response Induced by detection of pathogens and at tissue damages Three important functions of the inflammation in the fight against infection: Reinforcement of the first line of defence (macrophages) by further effector-molecules and -cells Construction of a physical barrier, which prevents the distribution Encouragement of the cure of the damaged tissue Marker of the inflammation: pain, redness, heat, swelling The release are the cytokines 31 Cytokines MW ~ 25 kda released of different cells bind on specific receptors Special class Three structural main families haematopoietic family, e.g. IL-6 TNF family Chemokine family Chemokine = chemo attractors, e.g. IL-8 32

17 Effect of the cytokines bloodstream Effect autocrine paracrine endocrine hormone molecules target cell 33 From the cytokine to the biological function Cytokine Receptor Intracellular signal kascade Redundant function TNF IL-1 fever IL-6 Cell nucleus Pleiotropic effect Gene activation Biological function IL-1 fever cytokine production proliferation 34

18 Pro-inflammatory cytokines have a broad spectrum of functions from: Immunologie, Janeway et al

19 Effect of cytokines I IL-1 = Fever, T-cell- and macrophage-activation IL-2 = Increase of T-cells IL-3 = Haematopoiesis (Basophile) IL-4 = Th2-cell activation, B-cell activation, IgE-switch IL-5 = Growth and differentiation of eosinophils 37 Effect of cytokines II IL-6 = Acute phase proteins, increase of T- and B-cells IL-9 = Mast cell activation IL-10 = Inhibition of macrophages and T-cells IL-12 = Differentiation of Th1, Activation of NK-cells IL-13 = B-cell activation, Inhibition of macrophages 38

20 Effect of cytokines III TNF- = Local inflammation IFN- = Macrophage-activation, MHCexpression high, antiviral IFN- / = antiviral, MHC I high, Th1 IL-8 = Chemotaxis of neutrophils and T- cells MCP = Chemotaxis of monocytes MIP-1 = Chemotaxis of monocytes, T-cells and eosinophils 39 Chemokine = small chemoattract proteins that stimulate the migration and activation of cells They induce a directed chemotaxis at adequate reactive cells Abbreviation: CCL and CXCL 40

21 Chemotaxis: Comes to help Steps of an inflammatory reaction: Chemotactic substances (e.g. C5a, LTB 4, IL-8) are released or activated and diffuse from the site of infection to the surrounding tissue and the capillaries Pavementation: Adhesion of the phagocytes to the endothelia (epithelia) of the blood vessels Diapedesis: Resolving of the basement membrane by the phagocytes and pass through the vascular wall Chemotaxis: Wandering against the concentration gradient 41 Chemokine are a family of proteins with similar structures, binding on chemokine receptors, which belong on the other hand to the big family of the G-protein-coupled receptors all chemokine own similar amino acid sequences and their receptors are all integral membrane proteins with seven membrane traverses Helices. Classification in two large groups: binds C-C Chemokine CCR (1-9) on e.g. MCP-1 (spec. monocytes) bind C-X-C Chemokine CXCR (1-5) on e.g. IL-8 (spec. neutrophils) IL-8 and MCP-1 possess similar, but complementary functions IL-8 leads PMN to leave the blood circulation and to immigrate in the surrounding tissue MCP-1 however, affects monocytes and influences their wandering of the vessels and therewith their development to tissue macrophages also fmlp (bacterial peptide) = Chemokine for neutrophils (C=Cystein) 42

22 Unspecific (innate) immune response genetically fixed (innate) not directed toward specific pathogens but against pathogen associated molecular patterns PAMPs reduces invasion of pathogens into the tissue reacts immediately, slows down distribution of pathogen until specific immunity has evolved e.g. : humoral: cellular: protease, complement, Interferons macrophages, granulocytes humoral = solubilized in blood (lat. umor = Liquid) 43 Important immune responses innate adaptive Phagocytosis Antigenprocessing Antigen-presentation in association with MHC II Receptor-detection of antigens in association with MHC II Release of cytokines as IFN- and IL-2 T Penetration of infective agents (antigens) Skin, mucosal membrane Macrophage B helper cell Release of cytokines and chemotactic factors as IFN-, IL-1 and IL-6, TNF, etc.; Neopterin Lymphocyte activation T- and B-lymphocytes T Lymphocyteproliferation B T T B B Production and release of antibodies T-cell-mediated immunity (Immunglobulines M, G, A and E) 44

23 Pathogens IL-4 Detection TLRs APC Intake IL-4 Antigen presentation Co-stimulatory molecules naive T-cell Th2 IL-4, IL-5, IL-10, IL-13 Effectorcytokines IL-2, IFN Th1 Cytokines IFNg Innate immune system Adaptive Immune system 45 Which cells are involved in the antigen presentation? 46

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