FIS: Immunology Christopher F. Cuff, Ph.D. Rm. 2070 293-4622 CCUFF@HSC.WVU.EDU

FIS: Immunology

Christopher F. Cuff, Ph.D.Rm. 2070293-4622

CCUFF@HSC.WVU.EDU

Goals

Enable biomedical scientists to understand the role of microbes in health and disease at a level sufficient to integrate new knowledge in the field as it applies to his/her discipline.

Enable students to understand the nature and use of research tools developed from the study of microbiology and immunology (such as genetic manipulation and antibodies)

Microbiology and Immunology

A study of infectious organisms to understand the mechanisms of pathogenesis

A study of the host’s immune system to understand the mechanisms involved in the clearance of infection

Microbes as Infectious Agents

Multicellular - parasites, fungi

Unicellular - bacteria, fungi, protozoan parasites

Subcellular - viruses, prions

Infectious Agents: -bind to the host, replicate, in some cases produce toxins, -cause damage to the hosts’ tissues, -elicit an immune response from the host.

Distinguish from ‘normal flora’ that contributes to health of the host.

Red Blood Cell

Bacteria

Viruses

Bacteria

bacilli

streptococcistaphylococci

spirochetes

Viruses

Ebola HIV

HSV-1 Hepatitis B

Fungi

unicellular

multi-cellular

Parasites (unicellular and multi-cellular)

unicellular

multi-cellular

Robert Koch (1843-1910)

‘Koch’s Postulates’

The same organism must be found in all cases of a given disease.

The organism must be isolated and grown in pure culture.

The organisms from pure culture must reproduce the disease when inoculated into a healthy, susceptible animal.

The organism must then be again isolated from the experimentally-infected animal.

First to demonstrate that microorganisms cause a human disease (anthrax)

The Immune System:

System to maintain the integrity of the hosts’internal environment during insult from pathogens and toxins in the external environment.

Immunity is Mediated by:

Innate Factors (non-specific)&

Adaptive Factors (specific)

Innate Immunity

Anatomic Barriers

Intact epithelium(keratinized and non-keratinized)

Mucous

Cilia

Smooth muscle contraction

Physiologic (Chemical) BarrierstemperaturepH

enzymes (i.e. lysozyme, & peroxidase)

complement and acute phase reactants

Innate Immunity

Anatomic Barriers

Endocytic/Phagocytic Barriers

Phagocytic cells - internalize particles

Granulocytes (neutrophils, eosinophils)

Macrophages (monocytes)

Endocytosis: internalization of macromolecules(phagocytosis: specialized internalization of particlesSuch as whole bacteria)

Pinocytosis: Non-specific membrane invagination

Receptor Mediated Endocytosis: Endocytosis following binding of macromoleculesto surface receptors

Primary Lysozomes fromGolgi complex

Extracellular

Intracellular

Macromolecules binding to receptors on cell surface

Extracellular

Intracellular

Macromolecules binding to receptors on cell surface

Primary Lysozomes fromGolgi complex

EndosomeLysosome Fusion

Primary Lysozomes fromGolgi complex

Extracellular

Intracellular

Macromolecules binding to receptors on cell surface

Primary Lysozomes fromGolgi complex

ExtracellularMacromolecules binding to receptors on cell surface

Degradation of Macromolecules

Antimicrobial and cytotoxic activities of phagocytes

(in lysozomes)Oxygen-dependent

Oxygen-independent

reactive oxygen intermediatese.g. superoxide anions (O2

-) and hydrogen peroxide (H202)reactive nitrogen intermediates

e.g. nitric oxide (NO)

antibacterial proteins lysozyme and defensins

proteolytic enzymes elastase and cathepsin B, cathepsin L, and cathepsin G

Inflammation:

Non-specific reaction that occurs as a result of local tissue damage and the presence of foreign substances.

Five Cardinal Signs of Inflammation

1. Pain

2. Heat

3. Redness

4. Swelling

5. Loss of Function

Neutrophils RollingAlong Endothelium

SITE OF INFLAMMATION

Release of Vasoactive andChemotactic Substances

Margination: Neutrophil stops rolling and sticks to endothelium

SITE OF INFLAMMATION

Diapedesis: Neutrophil squeezes out ofblood vessel between endothelial cells

Chemotaxis: Neutrophil migrates toward site of tissue damage alongthe concentration gradient of chemotactic substances.

Chemotaxis: Neutrophil migrates toward site of tissue damage alongthe concentration gradient of chemotactic substances.

Margination: Neutrophil stops rolling and sticks to endothelium.

Diapedesis: Neutrophil squeezes out ofblood vessel between endothelial cells.

Interactions between endothelial cells and neutrophilsare mediated by molecules expressed on the surface ofboth the endothelial cells and the neutrophils.

The Complement System

Involved in inflammation and resistance to bacterial (and to a lesser extent fungal and viral) infections.

Complement deficiencies put patients in grave danger.

The Complement System

A system of about 20 proteins that are produced by the liver and found in blood serum.

Produced in an inactive form and are sequentially activatedto produce biologically active complexes.

Function of these complexes is 2-fold:direct killing of pathogens through formation of

the ‘membrane attack complex’ (MAC)enhance the inflammatory response by

opsonizing bacteria and increasing phagocyte chemotaxis

Complement components are sequentially activated

in a process often referred to as the ‘complement cascade’

1) the classical pathway2) the alternative pathway

3) lectin activation pathway

The complement cascade can be activated through 3 distinct pathways:

Complement components polymerize to form a Membrane Attack Complex (MAC) that inserts into the membrane and forms holes in the pathogen

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Intracellular

Extracellular

Complement enhances the activity of phagocytic cells byopsonizing bacteria

C3b

Chemotaxis: Neutrophil migrates toward site of tissue damage alongthe concentration gradient of chemotactic substances.

C5a

Alternative Pathway and Lectin Mediated Pathway of Complement Activation:

1) Collection of serum proteins that are sequentially activatedto enhance the inflammatory response.

2) Initiated by the spontaneous cleavage of C3 and recognition of bacterial mannose-containing surface molecules (mannans)

3) C5-C9 forms the MAC, C3b opsonizes the pathogen, thereby promoting phagocytosis, and C5a promotes chemotaxis of phagocytes to the site of inflammation.

Pathogen

Anatomic Barriers

Inflammatory Response (chemical and phagocytic barriers) -local release of vasoactive and chemotactic substances -neutrophil infiltration (margination, diapedesis, chemotaxis) -complement activation resulting in MAC, opsonization, and increased neutrophil migration

The Innate Arm of the Immune System Express Receptors for Components of Microbial Pathogens PAMPs - ‘pathogen-associated molecular patterns’

PRRs - ‘pattern recognition receptors’

ImmuneCell

PAMP

PRR

Soluble PRRs Mannan Binding Lectin (MBL)C-reactive Protein (CRP)Serum Amyloid Protein (SAP)LPS Binding Protein (LBP) Cell Bound PRRs  Toll-Like Receptors (TLRs) TLR2, TLR3, TLR4, TLR5, TLR9CD14

PRRs are Soluble or Expressed on the Cell Surface

Bind to various bacterial cell wall components andenhance killing

PAMPs  Bacterial lipopolysaccharide (LPS) – the outer coating of certain bacteria (Gram negative)

Bacterial cell wall peptidoglycan – the rigid outer layer of bacteria

Bacterial flagellin – the protein that makes flagella, which are used for motility

Mannan from yeast or bacteria

Unmethylated CpG DNA – nucleic acid bases

Double stranded RNA (dsRNA) from viruses

Binding of PAMPs by PRRs can stimulate cells of the innate arm of the immune system to become:

-more phagocytic,

-more effective and initiating adaptive immune responses.

ImmuneCellPAMP Danger, Danger !!!!

Innate Immunity

Anatomic/Physiologic barriers

Phagocytosis/Endocytosis by granulocytes and macrophages

When Innate Immunity Fails to Eliminate the Pathogen, Specific Immune Responses are Initiated

Properties of Specific Adaptive Immunity

1. Specificity (exquisite)

2. Memory

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Time (Days)

0 14 = 0 6 14

Pathogen A

1o anti-A

Pathogen A

Specificity and Memory

1o anti-B

2o anti-A

+ Pathogen B

Cells of the Immune System

Specific Immunity: Directed against foreign substances (termed antigens)following the failure of innate immunity

B-lymphocytes (Bursa-derived)

T-lymphocytes (Thymus-derived)

Antigen Presenting Cells (APC)

B-cells

B-cells produce and secrete proteins that bind to, and neutralize antigens (antibodies) .

B-cells that have contacted antigens furtherdifferentiate into either memory cells or plasma cells.

B-cells do not require APCs to become activated.

T-Lymphocytes

T-helper cells provide signals for the growth (proliferation) and differentiation of other immunologic effector cells. (Th)

T-cytotoxic cells can kill abnormal cells in the body - for example infected cells or tumor cells. (Tc)

T-regulatory cells can inhibit inflammatory responses. (Treg or Ts)

B-cells and T-cells are Antigen-Specific (Clonal)

Each T-cell recognizes a unique antigen on the surface of an APC.

Each B-cell recognizes a unique antigen, but does not require an APC.

Clonal Selection: Antigen Stimulates SpecificClones to Generate Antigen-Specific Responses

Expression of Differentiation Markers is used to Distinguish T-cells and B-cells

sIg

B-cell

TCRCD4

T-helper

TCRCD8

T-cytotoxic

Antigen-Presenting Cells (APC)

-dendritic cells, macrophages, and in some cases, B-cells.

-process antigens by endocytosis (or phagocytosis), degrading them into simplemolecular fragments, and re-expressingthese fragments on their cell surface.

Dendritic cell

dendritic cell macrophage B-cellAPCs

Primary Lymphoid Organs

Organs (Tissues) wherelymphocyte precursors mature to become lymphocytes

Bone marrow

Thymus

Fetal liverBursa of Fabricius (birds)

Pluripotent Hematopoietic Stem Cell

Lymphoid Stem Cell

earlypre-B

large pre-B

small pre-B

Virgin

Hematopoiesis

Plasma cell

MemoryB-cell

immaturethymocyte

T-helperT cytotoxic/suppressor

NK Cell

(blood cell formation)

RBC

EosinophilBasophil

Megakaryocyte

NeutrophilMonocyte

Macrophage

Cortex

Medulla

T-cell Development in the Thymus

90% ofthymocytes diein cortex

10% ofthymocytes mature to T-cells

Thymic InvolutionFollowing onset of puberty, the thymus decreases in size. This reduction in size results in decreased immune function that is

associated with old age.

Normal rat thymus

Secondary Lymphoid Tissues

Organs or tissues wherelymphocytes contact antigensand mediate their immune functions.

Spleen

Lymph Nodes

Lymphatic vessels

Splenic Blood Supply

Red Pulp

LFLF

LF

Paracortex (T-cells)Medulla

Cortex (B-cells)

PALS (T-cells)

Marginal Zone(B-cells)Afferent Lymphatics

Efferent LymphaticLymphaticBlood Supply

Lymph Node Spleen

Lymphatics drain tissueand direct dendritic cells with foreign substances or pathogens to the lymph nodes where they stimulate T-cells and B-cells

cortex

paracortexmedulla

Diagrams of a Lymph Node

Diagrams of Spleen

Summary

Innate and Specific (Adaptive)

Properties of the Immune Response

Cells and Tissues of the Immune System

Cells Involved in Innate and Specific Responses

Primary and Secondary LymphoidOrgans and Tissues