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  1. 1. SOPHOMORE MEDICAL MICROBIOLOGY SECTION: IMMUNOLOGY OVERVIEW OF THE IMMUNE RESPONSE Lecturer: Louis B. Justement, Ph.D. 934-1429 Objectives: To review the functional relationships between the innate and acquired immune responses. This lecture will: 1) provide a more detailed description of the components of the innate immune response and their function; 2) describe the transition from the innate immune response to the adaptive immune response; and 3) provide contextual information regarding the anatomic regions of the body in which the innate and adaptive responses occur. Reading: Kuby Immunology – The topics covered are dealt with throughout the text. Thus, there is no specific reading assignment. THE INNATE IMMUNE RESPONSE – The First Line of Defense The Innate immune system serves three important functions: 1) Innate immunity provides the first response to microbes thereby preventing infection and effectively eliminating the invading microbes in many instances. 2) The effector mechanisms of innate immunity are often used to eliminate microbes even during an adaptive immune response. 3) Innate immunity to microbes stimulates adaptive immune responses and can influence the nature of the adaptive immune response to make them optimally effective against different types of microbes. The Skin – Physical Barrier and Immune Organ: 1) Epithelial barriers – • The epidermis comprises a physical barrier that microbes can not pass through. 2) Chemical barriers – • Defensins are produced by neutrophils. These are small polypeptides that exhibit antibacterial activity. • Additionally, the pH of the skin and oils or fatty acids produced by the epidermis and
  2. 2. dermis, respectively, inhibit microbial growth. 3) Immunological barriers – Epidermis: • Keratinocytes and melanocytes constitute the largest percentage of cells in the epidermis. Keratinocytes produce several cytokines that promote the innate immune response and mediate local inflammatory responses. • The epidermis also contains Langerhan’s cells. - These constitute only 1% of epidermal cells but because of their long extensions they are able to cover 25% of the epidermal surface. - These cells are immature dendritic cells, which are able to ingest antigens and present them on their surface in the context of MHC class II. - Langerhan’s cells that pick up antigen will retract their extensions, leave the epidermis and migrate to the dermis. In the dermis they will home to lymphatic capillaries, enter the lymphatic system and travel to regional lymphnodes as veiled cells. In the lymphnode, they will act as antigen presenting cells (APC) to present antigens to T cells thereby initiating the adaptive immune response. • Intraepidermal T lymphocytes (IEL) also reside in the epidermis. - IEL constitute 2% of skin-associated lymphocytes (the rest being located in the dermis). - These cells express a restricted set of antigen receptors on their surface that recognize microbial-specific antigens including polysaccharides and carbohydrates. - The antigen receptor on many IEL is comprised of γ and δ chains, which distinguishes these cells from the more common T cell population that expresses an α/β antigen receptor. Thus, the γ/δ positive IEL may be uniquely committed to recognizing antigens encountered at epithelial barriers. - These cells can “see” antigen that is presented in the context of the non- polymorphic CD1 molecule and therefore are not MHC restricted. Dermis: • The dermis is physically connected with the circulation and peripheral secondary lymphoid organs through postcapillary venules and lymphatic vessels, respectively. - The postcapillary venules provide a means of introducing immune cells and products of the immune system into the local microenvironment. - The lymphatic vessels drain lymph from the local microenvironment, which contains immune cells and antigens from the tissue. The lymph drains into regional lymphnodes where antigens are presented to T and B lymphocytes to
  3. 3. initiate the adaptive immune response. • The dermis also contains perivascular lymphocytes and macrophages. - The macrophages phagocytose particulate matter and present antigens in the context of MHC class II. - The T cells are α/β TCR positive and can be either CD4+ or CD8+. These T cells usually express markers typical of activated or memory cells. Features of Innate Immune Recognition of Microbes: The innate immune system has a unique specificity for the products of microbes that differs from the specificity of the adaptive immune system. 1) The cells and products of the innate immune system recognize and respond to structures that are characteristic of microbial pathogens and are not present on mammalian cells. 2) The microbial products recognized by the innate immune system are often essential for survival of the microbe. 3) The receptors of the innate immune system are encoded in the germline.
  4. 4. The Role of Complement Activation in Innate Immunity: The complement (C’) system consists of several plasma proteins that serve to link recognition of microbes to effector functions and to the development of inflammation. 1) Complement activation aids in the destruction of microbes through the deposition of C’ components (e.g. C3b) on the surface of microbes and directly destroys microbes via the formation of the membrane attack complex (MAC) on their surface. 2) C’ activation leads to the production of chemotactic factors such as C5a and C3a that recruit phagocytic cells to the site of infection. 3) C’ recognition of microbes and its subsequent activation can occur via several mechanisms including the classical pathway, the alternative pathway and the lectin pathway. All of these lead to the same terminal event, which is formation of the membrane attack complex. Classical C’Pathway in Innate Immunity: In the classical pathway antigen:antibody complexes bind the first C’ component C1q leading to activation of the classical pathway. In innate immunity, the classical pathway can be triggered in the absence of antibody due to binding of plasma proteins to microbes. A microbial insult to the skin will lead to the generation of an inflammatory response. Inflammation is associated with increased vascular permeability, which causes an influx of plasma proteins into the site of infection. Many of these proteins are involved in the innate immune response including mannose binding lectin (MBL) and C reactive protein (CRP). MBL: • MBL is a plasma protein that has the ability to selectively bind to microbes due to its specificity for terminal fucose or mannose residues associated with microbial cell surface glycoproteins or glycolipids. • Because host cells do not express surface glycoproteins or glycolipids that contain terminal fucose or mannose residues, MBL binds only to microbes. • MBL is a hexamer that is structurally similar to C1q, this initial component of the classical C’ cascade. • Microbes coated with MBL can be bound by macrophages that express C1q receptors. This promotes phagocytosis and destruction of the microbe (i.e. the microbe is opsonized). • MBL can bind to C1s and C1r, which leads to activation of the C’ cascade via the lectin pathway. This will result in deposition of the MAC on the surface of the microbe leading to its destruction. Additionally, C3a and C5a will be produced leading to leukocyte recruitment. CRP: • CRP is a plasma protein so named because of its ability to bind to capsule of
  5. 5. pneumococcal bacteria. • It is a member of the pentraxin family and contains five identical globular subunits. • CRP typically binds to bacterial phospholipids such as phosphorylcholine thereby opsonizing the bacteria due to binding to the C1q receptor on macrophages. • CRP can also bind to C1q, which leads to activation of the classical C’ pathway. Alternative C’ Activation Pathway: The cell walls of gram-negative bacteria contain LPS, which will bind C3b that is produced by fluid phase proteolysis of C3. Binding of C3b to microbial surfaces promotes biding and cleavage of Factor B leading to the formation of the alternative C3 convertase, which promotes activation of the C’ cascade via the alternative pathway. Local and Systemic Effects of the Innate Immune Response: The early local reaction of innate immunity is the inflammatory response, in which leukocytes are recruited to the site of infection and activated to eradicate invading microbes. Local Effects: 1) Microbial products and tissue damage will initiate release of inflammatory mediators that promote increased vasodilation. 2) Increased vasodilation leads to significant increases in the concentration of acute-phase proteins that enter the site of inflammation. 3) Increased concentrations of microbial products, inflammatory mediators and acute-phase proteins will promote the production of cytokines and chemokines by local immune cells (e.g. macrophages) as well as endothelial cells and keratinocytes. 4) The elaboration of TNF, IL-1 and IL-6 leads to enhanced inflammation, recruitment and activation of leukocytes to sites of infection. TNF: • TNF causes vascular endothelial cells to express new surface receptors called adhesion molecules that promote adherence of leukocytes circulating in the blood. - Endothelial cells increase their expression E-selectin within 1-2 hours. E-selectin promotes rolling of neutrophils and increased diapedesis. - Subsequently, ICAM-1 and VCAM-1 are expressed by endothelial cells between 12-24 hours and 30-36 hours, respectively. These receptors bind to LFA-1 and Mac-1 on macrophages and VLA-4 on T cells, respectively to promote subsequent waves of leukocyte recruitment into infected tissue. • TNF stimulates endothelial cells and macrophages to produce chemokines that promote luekocyte chemotaxis from the blood into infected tissues. IL-1: • IL-1 is similar to TNF in that it will promote local inflammation and act on
  6. 6. endothelial cells to upregulate adhesion molecule expression. Systemic Effects: 1) Production of TNF, IL-1 and IL-6 at moderate to high levels will result in systemic effects on the host associated with the acute-phase response or the systemic inflammatory response syndrome (SIRS). 2) The SIRS leads to neutrophilia, fever and increased levels of acute-phase reactants in the plasma. • TNF promotes neutrophilia • TNF and IL-1 act on the hypothalamus to induce fever (endogenous pyrogens) • TNF, IL1 and IL-6 act on the liver to induce the production of acute-phase proteins that circulate in the blood and enter sites of inflammation due to increased vasodilation. Cells of the Innate Immune System: The cell types that are most important in innate immunity are neutrophils, macrophages and natural killer cells (NK cells). Neutrophils and Macrophages: Response to microbes and inflammation. 1) Neutrophils and macrophages detect microbial products or mediators produced during infection through seven-transmembrane α-helical receptors that recognize 3 types of molecules – N-FMLP, chemokines and lipid mediators. • These receptors span the membrane 7 times and mediate signals that stimulate migration into sites of inflammation due to increased integrin avidity and cytoskeletal changes (chemotaxis) and production of microbicidal substances by activation of the respiratory burst. • These receptors recognize polypeptides that contain N-FMLP, which are produced by bacteria but not mammalian cells • These receptors also bind to a number of chemokines including IL-8 and C5a, a product of the complement activation cascade. • Lipid products that are recognized include platelet activating factor, prostaglandin E and leukotriene B4. 2) Macrophages express receptors on their surface that recognize lipopolysaccharide (LPS, endotoxin or exogenous pyrogen) a product of gram-negative bacteria, which stimulates microbicidal activity and the secretion of cytokines (TNF, IL-1, endogenous pyrogens). • The LPS-sensing system of macrophages consists of three components: a plasma protein called LPS-binding protein (LBP), a cell surface receptor called CD14 and a signal transducing subunit called mammalian Toll-like receptor 4. • Circulating LPS is picked up by LBP and delivered to CD14 after which LBP is
  7. 7. released. The Toll-like 4 subunit then transduces a signal leading to macrophage activation. 3) Neutrophils and Macrophages express receptors on their surface that recognize microbial products and opsonins , which promote binding and phagocytosis of microbes. • Three classes of receptors directly recognize and bind to microbes to promote phagocytosis. - Mannose receptor- a lectin that binds terminal mannose and fucose residues on glycoproteins and glycolipids - Scavenger receptors- were originally defined as molecules that bind oxidized or acetylated LDL. These receptors bind a variety of microbes. - Macrophage integrins (Mac-1)-also bind microbes to promote phagocytosis. • Neutrophils and macrophages express receptors for the Fc portion of IgG antibodies especially IgG1 and IgG3. These receptors are FcγRII and FcγRIII. - Microbes coated with IgG are readily and efficiently phagocytized by these cells due to opsonization by the antibody molecule. • Macrophages and neutrophils express receptors for other nonspecific opsonins that bind to microbes. These receptors recognize: - The complement system component C3b, which binds to CR1. - Fibrinogen-coated particles, which bind to Mac-1 and αvβ3 integrins. - Bacteria coated with either C1q (a component of the complement cascade) or mannose binding lectin (MBL), which bind to the C1q receptor on macrophages. Destruction of Microbes by Neutrophils and Macrophages. 1) Neutrophils and macrophages ingest microbes into vesicles where the microbes are destroyed. • Phagocytes use surface receptors to bind microbes and to extend cup-shaped extensions around the organism. • The extensions form a complete ring around the microbe after which an inside-out vesicle is pinched off. This vesicle is called a phagosome. • Cell surface receptors serve three important functions in this process: - The receptors physically attach to the microbe to be ingested. - The receptors deliver signals to the cell that are involved in remodeling the phagocyte membrane to promote membrane extension, closure and phagosome budding. - The receptors send signals that stimulate the microbicidal activity of the phagocytic cell.
  8. 8. 2) The principle function of Neutrophils and macrophages is to kill microbes by fusion of the phagosome with lysosomes and via the production of microbicidal molecules. • Fusion of phagosomes with lysosomes creates a phagolysosome in which proteolytic enzymes digest the microbe. Neutrophil lysosomes contain a particularly potent serine protease called elastase that has broad specificity. • Macrophage and neutrophils are also able to convert molecular oxygen to oxyhalide free radicals, which are highly reactive oxidizing agents that destroy microbes. - Phagocyte oxidase reduces molecular oxygen into reactive oxygen intermediates (ROIs) via a process called the respiratory burst. - Macrophages have a second free radical generating system called inducible nitric oxide synthase (iNOS). INOS catalyzes the conversion of arginine to citruline and freely diffusible nitric oxide gas is released. - Nitric oxide can combine with hydrogen peroxide or superoxide produced by the phagocyte oxidase enzyme to create highly reactive peroxynitrite radicals. 3) Macrophages Mediate Additional Effector Functions in Response to Activation. • Macrophages secrete cytokines, chemokines and lipid mediators. - IL-1 and TNF play an important role in promoting leukocyte recruitment and induction of the inflammatory response. These cytokines also have systemic effects on the host. - IL-12 and IL-15 are important for stimulation of NK cell function, including the production of IFNγ. - Chemokines such as IL-8, MCP 1-4, etc. are produced, which promote leukocyte migration into sites of inflammation - Lipid mediators such as PGE, LTB4 etc. are produced which also act as chemoattractants leading to leukocyte migration and activation. • Macrophages are activators of coagulation, produce mediators of chronic tissue remodeling, produce factors that promote fibroblast proliferation, and produce factors that regulate connective tissue synthesis. • Macrophages release proteases that belong to the matrix metalloproteinase family, which participate in tissue remodeling by degrading extracellular matrix proteins. Role of Natural Killer Cells in the Innate Immune Response.. 1) Natural killer (NK) cells play an important role in killing virally infected cells and cells that harbor other intracellular microbes. These cells exhibit the ability to kill target cells without the need to be activated – thus they are called natural killer cells. • NK cells constitute 5% to 20% of the mononuclear cells in blood and spleen and are
  9. 9. rare in other lymphoid organs. • NK cell activation is regulated by a balance between signals that are generated from activating receptors (CD16, CD2, CD28, Integrins) and inhibitory receptors (KIR, CD94/NKG2). • NK cells are induced to kill cells that are coated with antibody, infected by viruses or some intracellular bacteria, or cells that lack MHC class I. • NK cell expansion and function are stimulated by IL-12 and IL-15, which are produced by macrophages at the site of infection. 2) The second major effector function of NK cells is the production of Interferon gamma (IFNγ), which plays a critical role in activation of macrophages. Going From the Innate to the Adaptive Immune Response: 1) Neutrophils enter the site of inflammation, phagocytose microbes resulting in their destruction and subsequently die. They are short-lived participants in the innate response and therefore do not play an important role in generation of the adaptive immune response. 2) Cells that do play a role include Langerhan’s cells and macrophages. • Langerhan’s cells are immature dendritic cells that actively take up particulate microbial products, process them and present them on their surface in the context of MHC class II molecules. • In response to inflammatory cytokines, Langerhan’s cells retract their extensions and migrate to the dermis where they enter the lymphatic vessels as veiled cells. • Macrophages also take up microbes and their products, process them and present them on their surface in the context of MHC class II. Macrophages also migrate from the site of infection to local secondary lymphoid organs via the lymphatic system. 3) Due to increased vasodilation and influx of plasma at the site of inflammation, there is an increase in the amount of lymph that drains from the site of infection. This lymph contains Langerhan’s cells, macrophages and free microbial antigens. 4) The afferent lymphatic capillaries merge into lymphatic vessels that drain into the subcapsular region of local lymphnodes. • The lymphnodes are divided into three regions including the cortex, paracortex and medulla. These regions provide a physical separation of cells involved in the adaptive immune response. - The cortex is a B cell-rich area that contains both primary and secondary follicles. - The paracortex contains predominantly T cells - The medulla contains macrophages and plasma cells that are differentiated B
  10. 10. cells, which produce antibody. 5) The afferent lymphatics transport antigen presenting cells (APCs) and free antigen into the lympnode. Free antigen is picked up by antigen presenting cells in the lymphnode including macrophages and interdigitating dendritic cells. These antigens are processed and presented in the context of MHC class II to T cells in the paracortex. 6) Antigens are also picked up by B cells in the cortex via their antigen receptors and they are internalized and processed. B cells can present antigens on their surface in the context of MHC class II and act as APCs like macrophages and dendritic cells. 7) APCs entering the lympnode from sites of infection are activated and as a result upregulate the expression of key costimulatory molecules called B7.1 and B7.1. This enables these cells to provide two key signals to T cells that mediate their activation. • Signal 1 is delivered to the T cell via it antigen receptor complex, which recognizes antigenic peptides on the surface of APCs in the context of MHC class II. - The T cell antigen receptor (TCR) is a disulfide-linked heterodimer comprised of α and β chains. This structure recognizes peptide:MHC class II complexes. - The TCR is non-covalently associated with the CD3 complex and zeta chain homodimer. These structures actually transduce the signal in response to peptide:MHC class II binding to the TCR. • Signal 2 is delivered to the T cell when B7.1 or B7.2 bind to CD28 on the surface of the T cell. The combined effect of signal 1 and signal 2 leads to optimal T cell activation. 8) B cells that have bound antigen and presented it on their surface migrate from the cortex into the paracortex where they interact with activated T cells. • Signal 1 for the B cell is provided by antigen binding to the B cell antigen receptor (BCR) - BCR associated polypeptides called CD79a and b transduce a signal that leads to activation of the B cell and upregulation of B7.1 and B7.1. • A second signal is delivered to the B cell when it interacts with a T cells that has upregulated the expression of CD40 ligand which binds to CD40 on the B cell. This second signal promotes optimal activation of the B cell. 9) Activated B cells migrate to the cortex where they form secondary lymphoid follicles that contain germinal centers. It is here that B cells proliferate, undergo somatic mutation and differentiate into plasma cells. 10) Activated lymphocytes and their products (e.g. antibody molecules and cytokines) leave the lymphnode via the efferent lymphatic system. Ultimately the lymph drains back into the circulatory system via the thoracic duct.