MCMP 422 Tony Hazbun RHPH 406D Hazbun@pharmacy.purdue.edu Include MCMP 422 in the subject line

MCMP422 Immunology Immunology is important personally and professionally! Learn the language - use the glossary and index RNR - Reading, Note taking, Reviewing All materials in Chapters 1-5 are examinable (with exceptions) plus extra material from class

What and why? Immunology: Science of how the body responds to foreign agents Immune system: the organs, cells and molecules that defend and respond to pathogens/allergens Organ transplantation, cancer, immunodeficiency diseases, infectious diseases

Pharmacy and Therapy Perspective Immunology How do we recognize foreign structures? How do we recognize self vs non-self? How do we stop and remove invading agents? Pharmacy and Therapy Perspective How can we use the immune system as a therapeutic agent? How do drugs affect the immune system?

Chapter 1 Concepts What components make up the immune system? Cells, organs, cytokines and other molecules involved in the immune system What is the goal of the immune system? To clear pathogens in our body How do we classify immune responses? Innate and adaptive immune responses What are the side effects of the immune system? Autoimmune diseases, Allergies, Transplantation Rejection

Machinery of the Immune system 1. Tissues/organs bone marrow, thymus, spleen, lymph nodes 2. Cells lymphocytes, dendritic cells, macrophages, natural killer cells, granulocytes (neutrophils, basophils, eosinophils), mast cells Blood-borne proteins complement and mannose-binding proteins

Origin of Immunology - individuals who survived a disease seemed to be untouched upon re-exposure Vaccination/Immunization - procedure where disease is prevented by deliberate exposure to infectious agent that cannot cause disease. Vaccinia - mild disease caused by cowpox Edward Jenner - first demonstration of vaccination

Pathogen - any organism that can cause disease Figure 1-2

Diversity of Pathogens Four Classes Bacteria Fungi Viruses Parasites Opportunistic pathogens e.g. Pneumocystis carinii Pathogen-Host relationship

How Clean are You? Part of body Bacteria Head (scalp) 1,000,000 /cm2 Surface of skin 1000 /cm2 Saliva 100,000,000 /g Nose mucus 10,000,000 /g Faeces over 100,000,000 /g

Defenses against Pathogens Physical Defenses 1. Skin - Tough water-proof Barrier - Pathogen Penetration is difficult - Breached by wounds/mosquito 2. Mucosal surfaces - line body cavities - epithelial cells covered with mucus - mucus thick fluid layer containing glycoproteins, proteoglycans and enzymes - e.g. mucus in lungs traps pathogens Immune Defenses 1. Innate - physical defenses are part of innate immunity 2. Adaptive

Physical Barriers Lungs: Mucus, cilia trap and move pathogens Nose: Mucus traps pathogens which are then swallowed or blown out Mouth: Friendly bacteria, Saliva Eyes: lysozyme Stomach: acid neutralization Intestine: Friendly bacteria Urogenital tract: Slightly acid conditions

Immunity: Three Basic Parts Pathogen Recognition (Binding event) (Foreign) Signal Effector mechanisms (Self) Immune disorders Effector Cells Complement Two types of Immunity - Innate or Adaptive

Innate Immunity Ancient system - present in invertebrates naïve, immediate, everyday immunity Molecules recognize common features of pathogens Lectin Phagocytes, large lymphocytes (NK cells) Complement

Adaptive Immunity specialized, late, immunity Newer system - present in fish, birds, human specialized, late, immunity Molecules recognize specific features of pathogens Antibodies B and T cells - small lymphocytes Immunological memory

Figure 1-5 part 1 of 2 Example of Innate Immunity Complement - blood borne (serum) proteins that tag pathogens or attack them directly Effector cell - engulf bacteria, kill virus infected cells, attack pathogens Endocytosis - process by which extracellular material is taken up

One type of effector cell is the phagocyte

Figure 1-6 Innate Immunity Cytokines = signaling molecules --> inflammation/adaptive immunity Phagocytosis = “phagos” means to eat Inflammation is sometimes an unwanted by-product! Inflammatory cells = WBC’s contributing to inflammation

Inflammation Inflammation - local accumulation of fluid and cells involved in the immune response What happens when inflammation is induced Blood capillary dilation => heat (calor) & redness (rubor) - Local dilation of blood capillaries = increase of blood to the area (DOES NOT increase blood flow) Vascular dilation (vasodilation) => swelling (tumor) & pain (dolor) Extravasation - movement of cells/fluid into connective tissue. A) change in adhesiveness of the endothelial tissue allowing immune cells to attach and migrate into the connective tissue B) vascular dilation - gaps in endothelial cells

Example of inflammation gone bad: Sepsis Systemic inflammatory response syndrome (SIRS) Results from the body's systemic over-response to infection Treatment: broad-spectrum antibiotics and supportive therapy Disturbance of innate immunity during sepsis and multiorgan dysfunction syndrome (MODS) probably linked to uncontrolled activation of the complement system Future Drug therapies could be used that modulate pro-inflammatory and anti-inflammatory factors

Innate and Adaptive responses Pathogen independent Immediate (hours) Neutrophils Macrophages Mast cells Eosinophils Basophils NK cells “Large Lymphocytes” = NK cells Adaptive Pathogen-dependent Slower (days) Dendritic cells B cells T cells (CD4 or CD8) “Small Lymphocytes” = B & T cells Both systems “talk” to each other to modulate response Both systems use leukocytes = white blood cells

What if Innate Immunity is not Enough? Innate immunity keeps us healthy most of the time Some pathogens escape the innate immune process Need a specific system to adapt to a specific pathogen - Hence vertebrates evolved the Adaptive immune response

Principles of Adaptive Immunity Lymphocytes each with different specificity generated by gene rearrangements Small fraction of total pool of lymphocytes can recognize the pathogen Pathogen recognizing lymphocyte is amplified - Clonal amplification Pathogen recognizing lymphocyte can persist providing long-term immunological memory Primary vs Secondary immune response eg. Influenza/Measles/Vaccination

Figure 1-7 Characteristics of Innate vs Adaptive Immunity INNATE = genes are constant = genes are rearranged Leukocytes - white blood cells that increase the immune response to ongoing infection

Innate vs Adaptive Molecular Recognition Most important difference: Receptors used to recognize pathogens Innate immunity: Receptors recognize conserved structures present in many pathogens (usually a repetitive pattern) Pathogen-associated Molecular Patterns (PAMPs): LPS, peptidoglycan, lipids, mannose, bacterial DNA and viral RNA e.g. Mannose-binding Lectin (MBL) Adaptive immunity: Receptors recognize a specific structure unique to that pathogen e.g. Antibodies

Figure 1-11 part 1 of 2 Flowchart of Hematopoiesis Pluripotent stem cell Self-renewal

Flowchart of Hematopoiesis Figure 1-11 Leukocytes

Myeloid Lineage

Figure 1-9 part 3 of 6 Granulocytes (Myeloid progenitor) Polymorphonuclear leukocytes (PMLs) Figure 1-9 part 3 of 6 Neutrophils: Most abundant Phagocyte Effector cells of Innate Immunity Short-lived - Pus Eosinophils: Worms/intestinal parasites Amplify inflammation Bind IgE Very Toxic - Pathogen and host Chronic asthma Basophils: Rare Unknown function Bind to IgE

Figure 1-9 part 5 of 6 Circulate in blood Bigger than PMLs Look similar Immature form of macrophage Scavengers Phagocytose pathogens, cells, debris Secrete cytokines

Figure 1-13

Macrophages respond by two mechanisms - use 2 different receptors. Phagocytosis - Phagosome fuses with lysosome - toxic small molecules and hydrolytic enzymes kill/degrade the bacteria Signaling - bacterial component binds receptor - initiates transcription - inflammatory cytokines synthesized and secreted

Star-shape In tissue Cellular messenger Cargo cell Connective tissue Unknown progenitor Granules Degranulation major contributor to inflammation and allergies

Lymphoid Lineage Cells Large lymphocytes NK cells Innate immunity Small lymphocytes B cells T cells Adaptive immunity

Figure 1-9 part 2 of 6 Lymp Large lymphocyte with granular cytoplasm Effector cell of innate immunity 1) kill viral infected cells 2) secrete cytokines that interfere with virus infections

Adaptive IR Small and immature Activated by pathogen Two types - B cell - T cell B cells have B cell receptors and secrete Ab T cells have T cell receptors

Erythroid Lineage

Figure 1-9 part 6 of 6 Giant nucleus Resident of bone marrow Fusion of precursor cells Fragments to make platelets Figure 1-9 part 6 of 6 Gas transport Infected by Plasmodium falciparum

Lymphoid Myeloid Erythroid Neulasta (Amgen): Granulocyte Colony-Stimulating Factor (G-CSF) Recovery from Neutropenia & protect against Bacterial disease Leukine (Schering-Plough): Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Recovery from Neutropenia and protection against Bacterial/fungal/parasitic disease

Centrifuged blood sample Plasma Red blood cells White blood cells

Figure 1-12

High WBC could be a sign of infection or leukemia Low WBC bone marrow diseases or HIV Polys = polymorphonucleocytes - mainly neutrophils High lymphocyte count indicates the bacterial or viral infection

Figure 1-15 The lymph system and sites of lymphoid tissue Primary (Red) and Secondary (yellow) GALT, BALT, MALT Thoracic Duct Lymphpatic vessels - fluid collection Lymph nodes - junctions of vessels

Recirculation Draining Lymph node Edema - is worse when patient is inactive Afferent (entry) Efferent (exit)

Figure 1-17 part 1 of 2 Communications Center Afferent vessels bring in the lymph from infected tissue Efferent vessels place of exit for non-activated lymphocytes

B-cell area (follicle)

Lymphocyte not activated T-cell area Afferent lymph Lymphocytes Efferent lymph artery Lymphocyte not activated T-cell area Afferent lymph Pathogen Dendritic cells Activated by dendritic cell T helper cell (lymph node) T helper cell (Infection site) Cytotoxic T cell (Infection site) Activate B cells Activate Macrophages Kills infected host cells Make Antibodies

Lymphocytes enter node through artery Tcells migrate to the T-cell area and if they meet a dendritic cell that is carrying pathogens from an infection site they get activated - to divide into functional effector cells. Some T-cells stay in the lymph node and become T-helper cells - secrete cytokines (soluble proteins) and have receptors that contact B-cells. This helps the B-cells differentiate into plasma cells. Plasma cells stay in the lymph or leave and pump out large amount of antibodies - a soluble form of their cell surface receptor A second type of activated T-cell is the T-helper cell that leaves the node to the infected area and interacts with macrophages and amplify inflammation Third type of T-cell is the cytotoxic T-Cell which kill cells infected with pathogen Remember 5 million lymphocytes are entering node every minute and only a few are activated in response to an infection.

Figure 1-19 Anatomy of immune function in the Spleen Blood filtering organ - remove old/damaged red cells (red pulp) Blood-borne pathogens e.g. malaria White pulp (Immune system) - similar to lymph node (except pathogens enter and leave by blood)

Activated lymphocytes Figure 1-20 M cells Activated lymphocytes M cells - specialized cells lining mucosal epithelium that deliver pathogen => activate lymphocytes

Adaptive Immunity Vertebrates only Specificity - recognition modules - BCR, Ab and TCR - gene rearrangement is the source of diversity - clonal selection Small lymphocytes - types and sub-types - functions

Recognition concept Receptor or Antibody molecule Antigen - structure recognized by an Ab, BCR or TCR Epitope - particular sub-structure of the Ag that is bound Affinity - how much a molecule likes to bind to a structure

Small lymphocyte sub-types B-cells BCR is Immunoglobulin (Ig) Plasma cells - effector cells that secrete Ab T-cells Tc = cytotoxic (CD8+) TH = helper T-cells (CD4+) Th1 (inflammation) Th2 (help B-cells make AB)

Recognition modules of Adaptive immunity B-cell receptor (BCR) T cell receptor (TCR) B cells T cells Antibody is a secreted form of BCR TCR is membrane bound

Native vs Denatured Antigen processing Major Histocomp-atibility (MHC) B-cells T-cell

MHC APC - Antigen Presenting Cells Professional APC - macrophages - B cells Dendritic cells MHC I - all nucleated cells - intracelluar pathogens e.g. virus MHC II - immune cells - APC - extracellular Interact with cytotoxic T cells Interact with helper T cells

MHC class I communicates with cytotoxic T cells (Tc cells) Cellular ribosomes are subverted into making more virus proteins Some of those proteins are degraded in the cytoplasm and transported to ER MHC1 bind to these peptides and help to display them on the cell surface Cytotoxic T cells = Tc cells, Cytotoxic T-lymphocytes (CTLs)

MHC class II communicates with TH cells (TH1 or TH2) Also: Dendritic cells interact with naïve T-cells to initiate differentiation

Antibodies Produced by B-cells Humoral Immunity - Humor = “body fluids” Passive immunity - serum transferred to another individual can confer passive reistance due to transfer antibodies

Expel and/or destroy pathogen Antibodies Parasitic infection Parasite + Mast cell Neutralization Opsonization Inflammation Inflammation Mast cell activated Expel and/or destroy pathogen

Principles of Adaptive Immunity Diversity Specificity Memory Self-tolerance

Gene Rearrangement is the source of Diversity Germline configuration - the exact form of genes you inherit Somatic cells - all the cells of the body except germ cells Diversity Alternative combinations Imprecise joints Different types of chains B-cells - somatic hypermutation All this can happen in the absence of antigen

Clonal Selection Each cell = one receptor Millions of lymphocytes are generated Small subset will recognize a pathogen Proliferation and differentiation Acquired immunity - the adaptive immunity provided by immunological memory

Antibodies are usually very specific Figure 1-22

Concept Behind Vaccination Some memory lymphocytes Many lymphocytes Few specific lymphocytes

Pre-industrialization infants built immunity naturally Post-industrialization polio rate increased in adults hence a need for vaccination

Polio Vaccine - Inactive vs Oral “live” version VDPV - vaccine derived polio virus, cheap and easy to administer - mutations can lead to polio at extremely low rate - immunocompromized individuals can be carriers of VDPV

Principle of Self-tolerance B-cells with BCR that bind to self will undergo Apoptosis More complicated scheme of selection for T cells

Selection of T cells Thymocytes - immature T-cells Positive selection -Self MHC -cortex (epithelial cells) Negative selection Mechanism of Self-tolerance

Immunodeficiencies Inherited deficiencies e.g. Bubble boy disease Stress induced nutrition, emotional Pathogen caused deficiencies HIV - attacks CD4 T lymphocyte

Figure 1-32 Cells and molecules involved in Hypersensitivity Diseases IgE IgG CD4 TH1 CD8 CTL

Insulin-Dependent Diabetes Mellitus Beta cells of the islets of Langerhans in the pancreas are attacked Symptoms don’t show up for a long time Infection by a specific virus has been correlated with higher rate of IDDM Some of the activated CTL and Th1 cells will attack the healthy beta cells IDDM also has been correlated with certain polymorphisms (types) of the MHC molecule

Inflammatory Adaptive Immune Response Hygiene Hypothesis or Global Warming Hypothesis