* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download receptors - EuroBiotech Project
Survey
Document related concepts
Hygiene hypothesis wikipedia , lookup
Complement system wikipedia , lookup
Immune system wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Molecular mimicry wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adaptive immune system wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Transcript
European Biotechnology project no 134310-LLP-1-2007-1-ITERASMUS-ENW Work package: Application of Immunology in Biotechnology E. Walajtys-Rode, Rzeszow University of Technology With the support of the Lifelong Learning Programme of the European Union EUROBIOTECH – European Biotechnology Agr. n° 2007-2566 / 001-001 project n° 134310-LLP-1-2007-1-IT-ERASMUS-ENW This project has been funded with support from the European Commission. This publication reflects the views only of the author, and the Commission cannot be held responsible for any use which may be made of the information contained therein. Leader partner: Prof. Dr Sc. Elzbieta Walajtys -Rode, Rzeszow University of Technology, PL Participants: Prof. Brigitte Schmitz, Reinische Friedrich Wilhelms University of Bonn, RFN Prof. Olli Vainio, University of Oulu, SU Dr Maria Czygier, Rzeszow University of Technology, PL Dr Maryta Sztukowska, Rzeszow University of Technology, PL Dr Magdalena Dabrowska, Nencki Institute of Experimental Biology, Polish Academy of Sc. PL E. Walajtys-Rode, Rzeszow University of Technology Application of immunology in biotechnology course for Bachelor of Biotechnology degree Lectures for students of Technical Universities Prerequisite requirements: Basic courses of: •Molecular cell biology with genetics, •Biochemistry, •Molecular biology, E. Walajtys-Rode, Rzeszow University of Technology INTRODUCTION The purpose and rationale of biotechnology-oriented immunology course The modern immunology is an interdisciplinary science comprising and combining most recent developments in the fields of biology, biochemistry, molecular biology, genetics and genetical engineering The molecular and cellular mechanisms of immune response create basis for many immunological techniques applied in biotechnology, basic sciences, medicine and industry E. Walajtys-Rode, Rzeszow University of Technology Levels of todays immunology investigations: Whole body Intercellular Intracellular • Clinical immunology: • Tolerance and acquired immunodeficiences, • Hypersensitivity and autoimmunity, • Transplantation and tumor immunology. • Cellular immunology: • Proliferation, differentiation lymphocytes (Th1/Th2 subtype), • Effector cell function: cytotoxic lymphocytes, antibody production, cytokine production • Cell biology: • Signal transduction pathways • Antigen processing and presentation • Antigen receptors and accesory molecules E. Walajtys-Rode, Rzeszow University of Technology Levels of todays immunology investigations: Gene and genome Gene and genome • Genetics and molecular biology: • Lymphocyte maturation and expression of antigen receptor genes • Congenital immunodeficiences • Genetic engineering: • Gene targeting and expression • Transgenic organisms E. Walajtys-Rode, Rzeszow University of Technology Short story of long experiences and investigations creating todays immunology • Immunity comes from Latin immunitas and means excluded, • Historically immunity means protection from the infectious disease, • The concept of immunity was recognized by ancient Chinese who protect children against smallpox by the inhalation of the powdered skin lessions from recovering patients. • The term of immunity was mentioned by Thucydides in Athens at V century BC in connection with resistance to „plague” (epidemy) E. Walajtys-Rode, Rzeszow University of Technology •First controlled manipulation of the immunological response belongs to Edward Jenner, who in 1798 year created concept of vaccination by protecting people from smallpox by giving them cowpox pustule material •Great progress in the field of vaccination was made by Ludwig Pasteur, ( 1880) who investigated and created vacine against cholera in chicken and rabies vaccine for human E. Walajtys-Rode, Rzeszow University of Technology Recognition of general properties and functions of immune response • The humoral immunity was described by Emil von Behring and Shibasaburo Kitasato in 1980, whereas antibody recognition was due to sudies by Karl Landsteiner and Paul Ehrlich in 1900 who developed theory for the specificity of antigenantibody reaction E. Walajtys-Rode, Rzeszow University of Technology Recognition of general properties and functions of immune response The cellular immunity started with discovery of phagocytes by Elie Metchnikoff in 1893, developed by Almroth Wright in 1900 and established by George Mackaness in 1950 who prooved that resistance to intracellular bacterium can be transferred with cells. E. Walajtys-Rode, Rzeszow University of Technology • Further progress in immunology was correlated with the development and advances in many fields including recombinant DNA methodology and genetic engineering creating transgenic and knock-out animalas, cell cultures, immunochemistry, x-ray crystalography, monoclonal antibody production and many others which allowed to explain the immune sysstem and its function on the molecular level. • The combined effords of the investigators provide concepts of the modern immunology and brougt to many of them Nobel prise (>30 from the beginning of 20 century ) E. Walajtys-Rode, Rzeszow University of Technology PART I: BASIC MOLECULAR AND CELLULAR MECHANISMS OF IMMUNE SYSTEM This part is devoted to a description of the components of immune system: cells and tissues , their anatomic organization and structure-function relationships. General properties and fundamental principles of immune responses are also introduced. E. Walajtys-Rode, Rzeszow University of Technology The physiological function of the immune system is defence of the organism against pathogens which depends on the recognition of forigne and self molecules Realization of such a purpose requires coordinate action of all compounds of immune system which create the innate and adaptive immune response: Humoral Adaptive response Cytotoxic Immune system Innate response E. Walajtys-Rode, Rzeszow University of Technology Constitutive compounds Organs, tissues, lymphatic vessels, cells and cytokins comprise immune system which elecit immune responses Bone marrow and thymus peripheral organs , tissues and lymphatic vessels Cells of the immune system and cytokines E. Walajtys-Rode, Rzeszow University of Technology Cells and tissues of the immune system act in a coordinate and collective manner (network) Primary lymphatic organs: bone marrow and thymus Secondary lymphatic organs: spleen, lymph nodes, BALT, GALT Cytokines Cells of the immune system localized in organs and tissues Cells of the immune system circulating with lymph and blood E. Walajtys-Rode, Rzeszow University of Technology Cells of the immune system (in adult life) all come from common precursor: self-renewing bone marrow stem cells which develop into two progenitor lines: lymphoid and myeloid which differentiate into various types of cells Selfrenewing stem cell Pluripotent stem cell Myeloid progenitor Lymphoid progenitor E. Walajtys-Rode, Rzeszow University of Technology Bone marrow blood tissue immature dendritic cell megakaryocyte mature dendritic cell( lymph nodes) platelets macrophage monocyte myeloid progenitor erythroblast neutrophil basophil mast cell eosinophil Myeloid cell lineage E. Walajtys-Rode, Rzeszow University erythrocyte of Technology Cells derived from the myeloid progenitor: mononuclear phagocytes 1. Mononuclear phagocytes are devoted to host defense by phagocytosis. They originate from bone marrow, circulate in the blood , mautre and become activated in a various tissues, • Monocytes are incompletly differentiated cells circulating in the blood , • After entering tissues monocytes mature to macrophages assuming different morphologic forms named after specific locations. Macrophages are found in all organs , connective tissues, and submucosal layers and belong to one of the three types of profesional phagocytes. May remain in the tissue for extended periods and can undergo cell division. E. Walajtys-Rode, Rzeszow University of Technology Cells derived from the myeloid progenitor: mononuclear phagocytes • Related to lineage of mononucler phagocytes are myeloid dendritic cells: immature cells are located in epithelia of the skin (Langerhands cells) and respiratory and gastroinestinal systems, during migration to the lymph nodes dendritic cells mature and become proffesional antigen-presenting cells (APC). They are both phagocytic and macropinocytic E. Walajtys-Rode, Rzeszow University of Technology Maturation of mononuclear phagocytes Bone marrow Stem cell blood tissues monocyte macrophage Microglia (CNS) Alveolar macrophages (lungs) Kupfer cells (liver) Ostoclasts (bones) Epithelioid cells (skin) Immature dendritic cells Mature dendritic cells E. Walajtys-Rode, Rzeszow University of Technology Lymph nodes Other cells of myeloid lineage 2. Polymorphonuclear leucocytes or granulocytes are primarily secretory cells releasing contents of their granules upon activation Short living cells produced during immune responses, migrating from blood to sites of infection or inflammation ; – Neutrophils: third phagocytic cell of immune system, activates of bactericidal mechanisms. Contains granules comprising degradative enzymes (lysozyme, collagenase and elastase) and lysosomes called azurophilic granules . Neutrophils are produced at 1011 per day and survive for 6 hours, – Eosinophils: involved in killing of extracellular parasites.. – Basophils: function not well recognized (they are not precursors of mast cells) E. Walajtys-Rode, Rzeszow University of Technology Other cells of myeloid lineage 3. Mast cells: tissue cells releasing the granule contents and producing many mediators of inflammatory and allergic reactions 4. Megakaryocytes: precursors of platelets involved in blood coagulation 5. Erythroblasts: precursors of erythrocytes E. Walajtys-Rode, Rzeszow University of Technology Lymphoid cell lineage Bone marrow Thymus Blood and lymph nodes (peripheral tissues) Effector cells T cell B cell lymphoid progenitor NK cell E. Walajtys-Rode, Rzeszow dendritic cellUniversity of Technology activated T cell plasma cell (also in bone marrow) activated NK cell Cells derived from the lymphoid progenitor • Lymphocytes. There are three major classes of lymphocytes: 1. B lymphocytes or B cells mature in bone marrow and produce antibodies after activation 2. T lymphocytes or T cells precursors arise in the bone marrow and then migrate and mature in the thymus. T lymphocytes consist of two main types: •Helper T lymphocytes (Th) after activation effect and stimulate other cells such as B lymphocytes or macrophages •Cytotoxic T lymphocytes (CTL) after activation kill cells infected with intracellular pathogen Lymphocytes B and T are the only cells capable of very specific recognition of antigen determinants due E. Walajtys-Rode, Rzeszow University to the presence of unique variants of antigen receptors of Technology Cells derived from the lymphoid progenitor 3. NK cells are the third lineage of the lymphoid cells. This cells lack of the antigen-specific receptor, but instead they have other receptors recognizing foreign antigens (PRP receptors) and are the part of innate immune defence • Lymphoid dendritic cells: the minor subpopulation of dendritic cells arising from lymphoid progenitor E. Walajtys-Rode, Rzeszow University of Technology Lymphoid organs and tissues, the site of immune cell development and maturation • Generative or primary lymphoid organs where B and T lymphocytes express their antigen receptors and attain functional maturity: – Bone marrow : the site of hematopoiesis ( the process of generation of all blood cells ) and site of B cell maturation, – Thymus: the primary organ of mammals in which T cells mature and reach a stage of functional competence. Cells leaving primary limphatic organs are naive – without contact with forigne antigens E. Walajtys-Rode, Rzeszow University of Technology Lymphoid organs and tissues, the site of immune cell development and maturation •Peripheral or secondary lymphoid organs where the lymphocyte response to foreign antgens is initiated and developed consist: –Lymphatic sysstem (lymph nodes and lymph vessels) –Spleen, –Cutaneous immune system, –Mucosal immune system, –Aggregates of lymphocytes found in in connective tissue of many organs. E. Walajtys-Rode, Rzeszow University of Technology Structure and function of the primary lymphoid organs During fetal development the hematopoiesis occurs in yolk sac and para-aortic mesenchyme and later in the liver and spleen Bone marrow In adults hematopoiesis occurs in bone marrow of flat bones (sternum, vertebrae, iliac bones and ribs). Under emergency conditions the liver and spleen can be involved in hematopoiesis. • The bone marrow has vascular and extravascular compartment. The vascular compartment is supplied by artery which branches into smaller opening into sinuses from which blood is carried via vein into general circulation E. Walajtys-Rode, Rzeszow University of Technology • Bone marrow •The extravascular compartment consists of a stroma of a fibrous connective tissue , stromal cells (fibroblasts, fat cells, numerous plasma cells) and pluripotent stem cells (1 per 104 stromal cells). •The epithelial cells of the capillary sinuses contain narrow openings that permit the mature red and white blood cells to enter sinus space and be transferred into circulation. E. Walajtys-Rode, Rzeszow University of Technology Structure and function of primary lymphoid organs: thymus T lymphocytes mature in the thymus, derived from ectoderm lobular organ located in the anterior mediastinum, covered by connective tissue capsule and lobulated by septa originating from capsule, • Thymus continues to grow after birth, then undergoes involution by which the volume of active tissue is reduced, • Each lobule consists of outer cortex and inner medulla both containing lymphocytes (thymocytes) of different density. Thymus contains also: macrophages , epithelio-reticular cells supporting the framework, dendritic cells (also lymphoid lineage), E. Walajtys-Rode, Rzeszow University of Technology Structure and function of primary lymphoid organs: thymus • Thymus has rich vascular and lymphatic veasculature. Trough the former immature T lymphocytes enter thymic cortex . After maturation lymphocytes enter medulla and exit the thymus through efferent lymphatic vessels • Physical cellular blood-thymus barrier prevent the contact of immature lymphocytes with blood born antigens E. Walajtys-Rode, Rzeszow University of Technology Structure and function of peripheral lymphoid organs: lypmhatic system • Principial function of lymphatic system is collection of the antigens from their entry sites an delivery to the lymph nodes by the lymph vessels • Lymphatic system present in higher vertebrates is vital to the development of defence mechanisms and is structurally and functionally related to the blood vascular system • Lymphatic system is open-enden and linear without central driving force, whereas blood vascular system is closed and blood is pumped by the heart E. Walajtys-Rode, Rzeszow University of Technology Structure and function of peripheral lymphoid organs: lypmhatic system • Lymph vessels are present in almost all tissues except brain, cornea and retina, epidermis and cartilage • Lymph is an interstitial fluid made of plasma exudate from blood capillaries which enter the lymphatic capillaries in peripheral tissues and through larger lymphatic vessels and thoracic duct flows back into the blood vascular system for recirculation • Lymph carries immune cells including lymphocytes and antigen-presenting cells as well as nutritions (dietetary fat and fat-soluble vitamins A,D,E and K from digestive system) and soluble and particulate E. Walajtys-Rode, Rzeszow University antigens . of Technology Function of the lymphatic system Lymph is collected in peripheral tissues by lymphatic capillaries together with soluble and particulate antigens Afferent lymphatics carry lymph into lymph nodes where it percolates through the nodal stroma. Antigens are extracted by APC (including B cells) and presented to T cells. Efferent lymphtics may become afferent for another node. Lymph carrying lymphocytes is collected in larger lymphatic vessels and into thoracic duct and emptied into superior E. Walajtys-Rode, Rzeszow University vena cava , returning to the blood stream of Technology Lypmhatic system : lymph nodes and lymphatic vessels • Lymph nodes are the sites where the adaptative immune responses are initiated and are the most organized of the lymphatic organs. Are localized along lymphatic vessels, • Lymph nodes are enveloped by the fibrous capsule, the entry of afferent lymphatic vessels which empty the lymph into marginal sinus • Lymph passes the node into medullary sinus and leaves the node through the efferent lymphatic vesels in the hilum, E. Walajtys-Rode, Rzeszow University of Technology The distribution of lymphoid tissues in the body tonsils adenoids cervical nodes thymus axillary nodes Intercostal nodes appendix Peyer’s patch spleen inguinal nodes bone marrow E. Walajtys-Rode, Rzeszow University of Technology Lypmhatic system : lymph nodes and lymphatic vessels • Interior architecture of lymphatic node is made of outer cortex and inner paracortex beneth which is medulla consisting of medullary cords that lead to to the medullary sinus and collagen fibers on which lymphocytes are localized • Cortex contains bulk of lymphatic tissue diffused and organized into primary and secondary follicles (germinal centers). Paracortex containis mostly T lymphocytes, dendritic cells and macrophages • Medulla consists medullary cords populated by macrophages and plasma cells and medullary sinus. E. Walajtys-Rode, Rzeszow University of Technology The lymph node organization primary lymphoid follicle germinal center marginal sinus Secondary lymphoid follicle cortex paracortical area medullary cords afferent lymphatic vessel efferent lymphatic vessel medullary sinus E. Walajtys-Rode, Rzeszow University ofvein Technology artery The different classes of lymphocytes undergo Sequestration in the specific areas of the lymph node: Primary follicles Secondary follicles, germinal centers Paracortex • Mature, naive B cells • Proliferating B cells, • Producing antigens B cells • Memory B cells • T cells E. Walajtys-Rode, Rzeszow University of Technology Lymph and blood flow through lymph nodes carrying antigens and naive and activated cells 1. Lymph flow: • afferent lymphatic vessels bring lymph to the node from periphery towards the hilus where it drains via efferent lymphatic vessels 2. Blood flow: • Blood enters via arteriole at the hilus and through capillaries into the outer cortex and leaves via vein at the hilus. • Lymphocytes carried in the blood enter the node and return to the circulation via the lymphatic vessels. E. Walajtys-Rode, Rzeszow University of Technology The lymphatic system cervical nodes subclavian trunk thoracic duct axillary nodes vessels from stomach vessels from large intestine cisterna chyli inguinal nodes vessels from small intestine E. Walajtys-Rode, Rzeszow University of Technology Structure and function of peripheral lymphoid organs: spleen • The spleen is part of the circulatory system since it is a storage and processing station for erythrocytes. It contains also a very large population of lymphocytes and is a major site of immune responses to blood-borne antigens, • The spleen is surrounded by the collagenous capsule from which arise fibrous trabeculae subdividing the interior volume of the organ, • Interior of spleen comprises the aggregated masses of cels: the „red pulp” contains mostly erythrocytes, macrophages, dendritic and plasma cells, splenic cords Walajtys-Rode, Rzeszow University and blood sinuses E.between them, of Technology Structure and function of peripheral lymphoid organs: spleen • The „white pulp” comprises the lymphocyte population in the form of periarteriolar lymphocyte sheath (PALS) containing mainly T lymphocytes. To the PALS are attached lymphoid follicles, some of them contains germinal centers, which contains predominantly B cells. These structures are surrounded by perifollicular zone comprising lymphocytes and macrophages • Antigens and lymphocytes enter spleen through the splenic arteria into vascular sinusoids and leave organ through splenic vein. E. Walajtys-Rode, Rzeszow University of Technology Function of the spleen Blood carrying lymphocytes and antigens enter spleen through splenic artery, which divides into progressively smaller branches. The arterioles end in vascular sinusoids scattered with erythrocytes, macrophages, dendritic and plasma cells (red pulp). The spleen is the major site for clearing the blood of microbes by red pulp macrophages Through the small arterioles antigens are delivered to T lymphocytes (PALS) and B lymphocytes (lymphoid follicles and germinal centers ) Anatomical organization of spleen allowes for very efficient interaction and activation of B and T lymphocytes and antigen presenting cells E. Walajtys-Rode, Rzeszow University of Technology Cutaneous immune system • Skin creates the largest physical barrier between external environment and organisms and is a site of many immune responses to foreign antigens, • This purpose is realized by the specific cell populations present within epidermis and dermis. • Immature dendritic cells (Langerhans cells) form almost continuous meshwork in epidermis and capture antigens entering through the skin. After stimulation by cytokines Langehrans cells migrate into the dermis and to the lymph nodes via lymphatic vessels E. Walajtys-Rode, Rzeszow University of Technology Cutaneous immune system •Many intraepidermal T cells express antigen receptor, an uncommon type, not clearly definied yet. •Dermal T lymphocytes (both CD4+ and CD8+ activated and memory cells) are located mostly around venules and lymphatic vessels together with macrophages E. Walajtys-Rode, Rzeszow University of Technology Cellular components of the cutaneous immune system Epidermis Dermis • Keratinocytes (produce cytokines) • Langerhans cells (immature dendritic cells) • Intraepidermal lymphocytes (mostly CD8+, also T cells) • Lymphocytes predominantly perivascular (CD4+ and CD8+) • Macrophages perivascular and scattered E. Walajtys-Rode, Rzeszow University of Technology Mucosal immune system Mucosal epithelia are barriers between the internal and external environments and create first line of defence against forigne antigens due to specialized cell populations of different locations and distinct phenotypic and functional characteristic E. Walajtys-Rode, Rzeszow University of Technology Mucosal immune system Gut associated lymphoid tissue: In the mucosa of the gastrointestinal tract lymphocytes are located : within epithelial layer ( mostly CD8+, also T cells expressing antigen receptor) scattered throughout the lamina propria, In the lamina propria as aggregates in the small intestines (Peyer’s patches). Some epithelial cells of Peyer’s patches are specialized M cells (multifenestrated cells), which are pinocytic and transport antigens from the intestinal lumen into subepithelial tissues. Similar structures are found in appendix, tonsils and adenoids E. Walajtys-Rode, Rzeszow University of Technology Organized lymphoid tissue of the mucosal immune system comprises mucosal follicles reach in B cells and often containing germinal centers and interfollicular regions which are T cell regions. The same basic architecture share other mucosal systems: • Bronchial associated lymphoid tissue (BALT) • Mucosal-associated lymphoid tissue (MALT) gut lumen epithelium M cells gut wall T cell area follicle B cell area germinal center E. Walajtys-Rode, Rzeszow University of Technology Lymphocytes circulate between blood and lymph • Effective immune response to foreign antigens requiers lymphocyte recirculation via the blood stream and lymphatics among peripheral lymphoid tissues and peripheral sites of antigen entry and inflammatory reaction. • Recruitment of lymphocytes into specific tissue location (lymphocyte homing) depends on adhesive interactions : – between lymphocytes and specialized vascular endothelil cells (high endotelial venules(HEV), the site of lymphocyte extravasation into tissue – between lymphocytes and extravascular connective E. Walajtys-Rode, Rzeszow University tissue components of Technology Lymphocytes circulate between blood and lymph • The process of lymphocyte recirculation is due to adhesion molecules responsible for interactions between lymphocytes and vascular endothelial cells: the molecules on lymphocytes are homing receptors the different ligands for homing receptors on endothelial cells are adressins E. Walajtys-Rode, Rzeszow University of Technology vein thoracic duct efferent lymphatic Superior vena cava artery arteriole aorta heart venule lymph node lymph venous blood afferent lymphatic arterial blood ativated and memory T cell naive T cells peripheral tissue Pathwys of T lymphocyte circulation E. Walajtys-Rode, Rzeszow University of Technology Innate and adaptive immunity Defence against microbes and other forigne antigens is mediated by the early reactions of innate immunity and the later responses of adaptive immunity The various components of the mammalian immune system developed during phylogeny and become increasingly specialized with evolution E. Walajtys-Rode, Rzeszow University of Technology Evolution of the immune system All organisms have mechanisms defending host organism against forigne invaders Bacteria protect themself from parasites (plasmids) and pathogens (bacteriphages) with restriction nucleases and against competing bacteria with atimicrobial peptides Innate immunity: Invertebrates respond to microbes and forigne antigens by phagocytosis with specialized cells and by antimicrobial peptides and also by the ancestral three-compound complement system E. Walajtys-Rode, Rzeszow University of Technology Innate immunity: Fruit fly Drosophila melanogaster have cell surface receptors (Toll receptors) activating expression of antimicrobial peptides found also in vertebrates, which suggests presence of the common signalling pathway for innate immune response . Mammals have more than 10 Toll-like receptors activating different signalling pathways of innate immune response E. Walajtys-Rode, Rzeszow University of Technology Adaptive immunity: • Jawed fishes and higher vertebrates developed antibody molecules Fishes have only IgM type, amphibians , reptiles and birds have 3 classes of immunoglobulins and 7-8 antibody classes are found in mammals • Jawed fishes and higher vertebrates have also both major histocompatibility complexes (MHC I and II class) • Lymphocytes of general characteristics are present in all vertebrates and become specialized into T i B class in amphibians, birds and mammals • The lymph nodes are found in some birds and in mammals E. Walajtys-Rode, Rzeszow University of Technology Specificity of innate and adaptive immunity: both systems recognize self from nonself Innate immunity Adaptive immunity • Recognize „molecular patterns” of different types of microbes • Receptors encoded in the germline, nonclonally distributed • Recognize structural details of microbial and nonmicrobial antigens • Receptors encoded by gene segments rearrangement during development (somatic recombinations), clonally distributed E. Walajtys-Rode, Rzeszow University of Technology Specificity and function of innate immunity • The principal feature of innate immunity is constitutive presence of all components and mechanisms and lack of memory, • The innate immunity , phylogenetically oldest mechanisms, provide the first line of defence against infection by the common pathogen organisms and can elimnate the microbes. • The cells of innate immune system play a crucial part in the initiation and subsequent direction of the adaptative immunity mechanisms and cooperate in elimination of microbes by phagocytosis and complement system E. Walajtys-Rode, Rzeszow University of Technology Components of innate immune system • Epidermal and mucosal surface barriers consist epithelil cell layers, produced by epithelial cells peptides –defensins and cryptocidins (acting as antibiotics ) and mucus entrapping bacteria • Efector cells of innate imunity: neutrophils, mononuclear phagocytes and natural killer (NK) cells recognize, attack and destroy microbes that entered into circulation or tissue. E. Walajtys-Rode, Rzeszow University of Technology Components of innate immune system •Effector circulating proteins: complement system, mannose-binding lectin (collectin), C-reactive protein (pentraxin) and other mediators of inflammation, coagulation factors •Cytokines and chemokines - protein molecules produced by activated cells which coordinate immune response Same components of innate immune system are functioning all the time (surface barriers), other , normally inactive ,become rapidly activated in the presence of microbes (phagocytosis, complement E. Walajtys-Rode, Rzeszow University system). of Technology The major family of phagocytes: macrophages and neutrophils play a key role in innate immunity. They can recognize, ingest and destroy pathogens without the aid of an adaptive immunity • Both macrophages and neutrophils have surface receptors of phagocytosis recognizing molecules displayed by pathogens: Ligation of phagocytic receptors induce internalization of the pathogen in phagosome, and fusion with lysosomes to generate phagolysosome in which pathogen undergos destruction by lysosomal enzymes. E. Walajtys-Rode, Rzeszow University of Technology Phagocytes after activation produce a variety of toxic products that help kill engulfed organisms 1. Reactive oxygen intermediates(ROI) toxic to the cells: Superoxide anion radical O 2, hydrogen peroxide H2O2, singlet oxygen 1O2 , hydroxyl radical OH , hypohalite OCl produced as a result of activation of NADPH oxidase by microbial antigens 2. Reactive nitrogen intermediates (RNI) toxic to the cells: Nitric oxide free radical NO , nitrosonium ion NO+ , nitroxyl anion NO, nitrite NO2 , nitrogen dioxide NO2, Excessive formation of nitric oxide is due to activation of inducible isoenzyme of nitric oxide synthase (iNOS) during host injury and inflammatory processes E. Walajtys-Rode, Rzeszow University of Technology Phagocytes after activation produce a variety of toxic products that help kill engulfed organisms Superoxide anion radical and nitric oxide react to give peroxynitrite ONOO a potent factor capable of protein nitration and oxidation and inducer of cell death. 3. Antimicrobial peptides: defensins and cationic peptides 4. Enzymes: lysozyme disrupting proteoglicans cel wall Gram-positive bacteria, acid hydrolases –digest bacteria 5. Competitors binding nutritiens essential for bacteria: lactoferrin (binds Fe 2+), vitamin B12 –binding proteins. E. Walajtys-Rode, Rzeszow University of Technology Extracellular and intracellular microbicidal mechanisms of neutrophils and macrophages Extracellular microbes NADPH oxidase O2 Phagosome with ingested microbes ROIs NO arginine ROIs oxidase lysosome with enzymes phagolysosome with ingested microbes E. Walajtys-Rode, Rzeszow University of Technology iNOS Pathogen recognition and tissue damage initiate inflammatory response Infalammation plays the essential role in defence against infection by two general mechanisms: • providing physical barrier against spreading of the microbes via the blood stream by initiation of microcoagulation process • accumulation effector cells at the site of infection E. Walajtys-Rode, Rzeszow University of Technology Inflammation is accompanied by pain, redness , heat and swelling at the site of infection due to release of variety of inflammatory mediators acting on different cell types, •Under influence of the inflammatory mediators endothelial cells lining the blood vessels become separated leading to an exit of fluid from the blood and express adhesion molecules promoting binding and migration of circulating leucocytes into tissues in the process called extravasation E. Walajtys-Rode, Rzeszow University of Technology Mediators released by macrophages after recognition of pathogens involved in innate response 1. Cytokines: major cytokines of inflammatory reaction: Interleukin-1, Interleukin-6 and Tumor necrosis factor - (TNF-), Interleukin-12: activator of NK cells, 2. Chemokines: Interleukine-8: chemoattraktant for neutrophils, 3. Lipid mediators of inflammation produced enzymatically by the degradation of plasma membrane phospholipids: prostaglandins, leukotrienes, thromboxanes, platelet-activating factor (PAF) E. Walajtys-Rode, Rzeszow University of Technology Inflammatory mediators released by macrophages after recognition of pathogens during innate response Cytokines:IL-1, IL-6 TNF-, IL-12 Chemokines: IL-8 Lipid mediators: prostaglandins, leukotrienes, tromboxanes, PAF INFLAMMATION E. Walajtys-Rode, Rzeszow University of Technology Infection stimulates macrophages to initiate inflammatory response Activated macrophage cytokines Expression of adhesion molecules Adhesion of circulating leucocytes to the endothelial cells chemokines Blood vessel Blood clotting in the microvessels Extravasation of leukocytes E. Walajtys-Rode, Rzeszow University of Technology Cytokines released by phagocytes (IL-1, IL-6 and TNF-α ) activate the systemic response that contribute to host defence by: 1. Acting on hypothalamus (endogenous pyrogens) and on muscle and fat cells altering energy mobilization to increase the body temperature. The decrease bacterial and viral replication 2. Stimulating synthesis plasma: fever of acute phase proteins by liver and release into C-rective protein – pentraxin, comprising five identical subunit which bind to phosphocholine portion of certain bacterial and fungal cell-wall lipopolisaccharides and activate the complement system Mannose binding lectin and pulmonary surfactant proteins SP-A and SP-D promoting phagocytosis of pathogens 3. Inducing leucocytosis (an increase in circulating neutrophils) and migration (maturation) of dendritic cells from peripheral tissues to lymph nodes E. Walajtys-Rode, Rzeszow University of Technology The phagocyte cytokines have a wide spectrum of biological activities coordinating the body response to infection IL-1/IL-6/TNF-α Liver Bone marrow endothelium Hypothalamus Acute phase proteins: C-reactive protein Mannose – binding lectin Neutrophil mobilization Activation of complement Phagocytosis Increased body temperature Fat, muscle Protein and energy mobilization Decreased viral and bacterial replication. Increased antigen processing E. Walajtys-Rode, Rzeszow University of Technology Dendritic cells TNF-α stimulates migration to lymph nodes and maturation Initiation of adaptative immune response Complement system consists of many proteins distributed through body fluids and tissues that interact with pathogens to mark them for destruction by phagocytes • Complement components are activated by induced proteolytic enzyme cascade • There are three distinct pathways of the complement activation depending on different molecules of pathogen surfaces but as a result the same set of effector molecules are generated for protection of the organism against infection: E. Walajtys-Rode, Rzeszow University of Technology 1. Activated complement components bind to pathogens, opsonizing them for receptordependent phagocytosis 2. Activated complement components act as chemoattractants for phagocytes and activate them 3. Terminal components of complement system act directly on bacteria by creating pores in the bacterial membrane E. Walajtys-Rode, Rzeszow University of Technology Three pathways of complement activation CLASSICAL PATHWAY MB-LECTIN PATHWAY ALTERNATIVE PATHWAY Antigen-antibody complexes Lectin binding to pathogen surface Pathogen surface molecules COMPLEMENT ACTIVATION Opsonization and phagocytosis of pathogens Recruitment of cells to the site of infection E. Walajtys-Rode, Rzeszow University of Technology Direct killing of pathogens Complement system consists a few functional classes of proteins • Binding to pathogen surfaces ant antigen-antibody complexes (C1q) • Binding to mannose compound of bacterial cell-wall (MBL) • Enzymes activating components of complement (serine proteases, convertases) • Membrane binding proteins (C4b, C3b) • Mediators of inflammation-peptides (C5a, C4a, C3a) • Peptides forming pore: membrane attack proteins (MAC: C5b, C6, C7, C8, C9) E. Walajtys-Rode, Rzeszow University of Technology Assamble of the membrane attack complex generates pore in the lipid bilayer membrane ( 10 nm) C7 C8 C6 C9 C5b67 complex C5b Lipid bilayer Pathogen E. Walajtys-Rode, Rzeszow University of Technology 10-16 molecules of C9 bind to form a pore in the membrane Main components and effector actions of complement system CLASSICAL PATHWAY MB-LECTIN PATHWAY First complement protein C1q binds to pathogen surfaces through pathogenbinding protein ( Cprotein)and antigen/ antibody complexes and initiates activation of enzymes C1r and C1s Mannose binding lectins bind mannose on pathogens and initiates activation of enzymes MASP-1 and MASP-2 ALTERNATIVE PATHWAY Many pathogen surfaces initiates directly spontaneous hydrolysis of C3 generating C3 convertase C3 convertase C3a, C5a Peptide mediators of inflammation C3b C5b, C6, C7, C8, C9 Opsonization of Membrane attack complex pathogens Lysis pathogens and cells Binds to complement E. Walajtys-Rode, Rzeszow University receptors of Technology Complement system is involved in innate as well as adaptative immune response Pathogens marked by opsonization with complement are recognized by cells with surface receptors for complement compounds and stimulate different cell functions: • Stimulation of phagocytosis by macrophages, monocytes, polymorphonuclear leucocytes, dendritic cells, B cells, erythrocytes (transport of immune complexes) CR1, CR 3 and CR4 • Part of antigen receptor complex of B cell (CR2) • Receptors for C3a and C5a activate endothelial cells and mast cells E. Walajtys-Rode, Rzeszow University of Technology Recognition of the self from nonself by the cells of innate immune system is due to the molecular pattern recognizing receptors • Activated macrophages and neutrophils destroy pathogens through phagocytosis and/or by initiation of the inflammatory response Both patways are dependent on the binding specific pathogen molecules to the various surface receptors: Soluble receptors : • mannose binding lectin (MBL) initiating lectindependend pathway of complement activation, • collectins opsonizing bacteria E. Walajtys-Rode, Rzeszow University of Technology Cell-surface receptors Fagocytic receptors: • Macrophage mannose receptor: cell bound lectins with several carbohydrate recognition domains rcognizing sugars (mannose, fucose) present on the surface many bacteria and some viruses • Scavanger receptors recognize anionic polymers and acetylated low-density lipoproteins. Some of these receptors recognize structures of normal host cells depleted of sialic acid (eg. senescent red blood cell • Receptors for opsonins: Receptors FcγR1 for Fc region of IgG antibodies coating microbes Receptors for components of the complement system opsonizing E.bacteria ( especially C3) Walajtys-Rode, Rzeszow University of Technology Receptors resposible for migration cells into tissue (receptors dependent on protein G): • chemotactic receptors: receptors recognizing N-formylated bacterial (also mitochondrial) peptides, chemokines (IL-8) and lipid mediators of inflammation (PAF, prostaglandin E) Receptors for cytokins produced by cells of the innate and adaptive response following activation by various effectors • Receptors for cytokines of inflammatory reaction (TNF-α, IL-6 and IL-1) • Receptors for cytokines produced by lymphocytes (interferon- INF- expressed during innate immune response by lymphocytes NK and by lymphocytes T during cell-mediated adaptive reactions E. Walajtys-Rode, Rzeszow University of Technology Signalling Toll-like (TLR) receptors: First Toll receptors were described in the fruit fly where they trigger (in adult fly) formation of antimicrobial peptides in response to infection. Homologous proteins in mammals are named Tolllike receptors. So far 10 genes are indentified in mice and humans encoding TLR recognizing „molecular pattern” – a particular molecular structures that are components of pathogenic microorganisms but are not present in vertebrates E. Walajtys-Rode, Rzeszow University of Technology Toll-like receptors are founded on innate response vertebrate cells and also in plant defence mechanisms against viruses and other pathogens. This signalling pathway is probably present in all multicellular organisms E. Walajtys-Rode, Rzeszow University of Technology Molecular pattern of microorganisms comprises repeating structural motifs of surface molecules and nucleic acids • bacterial DNA contains unmethylated repeats of CpG dinucleotide • viruses express double – strainded dsRNA as a part of life cycle • Gram –negative bacteria have lipoplisaccharide (LPS) as cell-wall component • bacterial flagella are composed of specific protein (flagellin) • Gram-positive bacteria express peptidoglycans as a cell wall component E. Walajtys-Rode, Rzeszow University of Technology Molecular partterns recognized by Toll-like receptors (acc. to Beutler et al.2005) Toll-like receptor Ligand TLR-1 dimer Peptidoglicans Lipoproteins Lipoarabinomannan (mycobacteria) (glycosylphosphatidylinositol (T.cruzi) Zymosan (yeast) TLR-2/TLR-6 dimer TLR-3 dsRNA TLR-4 dimer Plus CD14 LPS (Gram-negative bacteria) TLR-5 Fagellin TLR-9 E. Walajtys-Rode, Rzeszow University of Technology Unmethylated CpG DNA Chemokines N-formylmethionyl peptides Lipid mediators Microbe specific molecular pattern Chemokine receptors Toll-like receptors Integrin Expression Cytoskeletal reorganization Migration into tissue Opsonized microbes mannose , fucose LDL Generation of oxygen and nitrogen active metabolites Gene expression Phagocytic receptors phagocytosis microbes into phagolysosome Killing microbes Killing microbes Receptors and functional response of phagocytes E. Walajtys-Rode, Rzeszow University of Technology Characteristics of the innate immunity receptors 1. Specificity of the receptors is inherited in the genome 2. Receptors are expressed by all cells of particular type 3. Broad classes of pathogen are recognized 4. Receptors interacts with a range of molecular structures of given type 5. Receptor activation triggers immediate response E. Walajtys-Rode, Rzeszow University of Technology Natural killer cells - subset NK lymphocytes that kill intracellular microbe infected and cancer cells and secrete cytokines NK cells do not express specific lymphocyte lineage cells B or T antigen receptors NK cells are activated by specific set of receptors recognizing: • Antibody coated cells by FcRIIIa low affinity receptor for Fc region of IgG antybody • Cells infected with viruses and other intracellular pathogens possibly by recognizing adhesion molecules (integrins) • Cells lacking class MHC I molecules by complex mechanism including activating (lack of MHC I molecules) and inhibitory ( presence of MHC I E. Walajtys-Rode, Rzeszow University molecules ) receptors of Technology