IMMUNOLOGY Mary Joyce Saborrido-Teoxon, RMT, MD Dept. of Micro & Para FEU-NRMF Institute of Medicine

IMMUNOLOGY

Study of body’s protective and defensive mechanisms against foreign substances

Discriminate self vs. non self

Eliminate non-self (infectious agents)

Immune System

Collection of organs, tissues, cells and soluble factors that allow individuals to defend against harmful agents such as viruses, bacteria, fungi, parasitic organisms, and tumor cells

Two (2) Important Roles of the Immune System

1. Provides defense mechanism.

2. Identification and destruction of abnormal cells.

Innate vs. Adaptive

Innate Adaptive

Non-specific Natural 1st/ 2nd line Memory- NO Rxn time- RAPID CELLULAR:

◦ Phagocytes, mø, monocytes, NK cells, Mast cells

HUMORAL: ◦ Complement (Alternate)

◦ Cytokines

Specific Acquired 3rd line Memory- YES Rxn time- SLOW CELLULAR:

◦ Specific B (plasma cells) & T cells

◦ APCs HUMORAL:

◦ Abs ◦ Complement (Classic) ◦ Cytokines

First Line of Defense: Skin and Mucous Membranes

Mechanical Defenses

1.) Skin

A. Epidermis: Thin outer layer of epithelial tissue.

Contains Langerhans cells, dead cells, and keratin (waterproof).

B. Dermis: Thick inner layer of connective tissue.

Infections are rare in intact skin. Exceptions:

Hookworms can penetrate intact skin

Dermatophytes: “Skin loving” fungi

First Line of Defense: Skin and Mucous Membranes

• Mechanical Defenses

2.) Mucous Membranes:

Lines gastrointestinal, genitourinary, and respiratory tracts.

Two layers:

◦ Outer epithelial

◦ inner connective layer.

◦ Epithelial layer secretes mucus which maintains moist surfaces.

◦ Although they inhibit microbial entry, they offer less protection than skin.

◦ Several microorganisms are capable of penetrating mucous membranes:

Papillomavirus

Treponema pallidum

Enteroinvasive E. coli Entamoeba histolytica

First Line of Defense: Skin and Mucous Membranes

I. Mechanical Defenses

3. Lacrimal apparatus: Continual washing and blinking prevents microbes from settling on the eye surface.

4. Saliva: Washes microbes from teeth and mouth mucous membranes.

5. Mucus: Thick secretion that traps many microbes.

6. Nose Hair: Coated with mucus filter dust, pollen, and microbes.

7. Ciliary Escalator: Cilia on mucous membranes of lower respiratory tract move upwards towards throat at 1-3 cm/hour.

First Line of Defense: Skin and Mucous Membranes

I. Mechanical Defenses

8. Coughing and sneezing: Expel foreign objects.

9. Epiglottis: Covers larynx during swallowing.

10. Urination: Cleanses urethra.

11. Vaginal Secretions: Remove microbes from genital tract.

B. Chemical Defenses:

◦ Sebum: Oily substance produced by sebaceous glands that forms a protective layer over skin. Contains unsaturated fatty acids which inhibit growth of certain pathogenic bacteria and fungi.

◦ pH: Low, skin pH usually between 3 and 5. Caused by lactic acid and fatty acids.

◦ Perspiration: Produced by sweat glands. Contains lysozyme and acids.

◦ Lysozyme: Enzyme that breaks down gram-positive cell walls. Found in nasal secretions, saliva, and tears.

B. Chemical Defenses

◦ Gastric Juice: Mixture of hydrochloric acid, enzymes, and mucus. pH between 1.2 to 3 kills many microbes and destroys most toxins. Many enteric bacteria are protected by food particles. Helicobacter pylori neutralizes stomach acid and can grow in the stomach, causing gastritis and ulcers.

◦ Transferrins: Iron-binding proteins in blood which inhibit bacterial growth by reducing available iron.

II. Second Line of Defense

1. Phagocytosis:

◦ Derived from the Greek words “Eat and cell”.

◦ Phagocytosis is carried out by white blood cells: macrophages, neutrophils, and occasionally eosinophils.

◦ Neutrophils predominate early in infection.

◦ Wandering macrophages: Originate from monocytes that leave blood and enter infected tissue, and develop into phagocytic cells.

◦ Fixed Macrophages (Histiocytes): Located in liver, nervous system, lungs, lymph nodes, bone marrow, and several other tissues.

Phagocytic Cells: Macrophages (Monocytes), Neutrophils, and Eosinophils

Professional Phagocytic cells

These cells have enzymatic constituents in their granules to oxidize, kill, digest, and destroy particulate material that they ingest.

1.) Mononuclear phagocytes (formerly RES)

A. Monocytes (in the blood) B. Tissue Macrophages

A. Liver Kupffer cells B. Lungs Alveolar macrophages/ Dust cells C. Kidney Mesangial macrophages D. CNS Microglial cells E. Lymph nodes Dendritic cells F. Skin Langerhan’s cells G. Spleen Spleenic macrophages H. Connective tissue Histiocytes I. Bone Osteoclast J. Peyer’s patches K. Tonsils

Functions of MØ

Phagocytosis

Antigen Presentation

Cytokine Production

2. Polymorphonuclear leukocytes (PMNs)

A. Neutrophils (most aggressive phagocyte)

B. Eosinophils (antiparasitic phagocyte)

C. Basophils (secretory cells)

Stages of Phagocytosis

1. Chemotaxis: Phagocytes are chemically attracted to site of infection.

2. Adherence: Phagocyte plasma membrane attaches to surface of pathogen or foreign material.

Adherence can be inhibited by capsules (S.

pneumoniae) or M protein (S. pyogenes).

Opsonization: Coating process with opsonins that

facilitates attachment.

◦ Opsonins include antibodies and complement

proteins

Stages of Phagocytosis (Continued)

3. Ingestion: Plasma membrane of phagocytes extends projections (pseudopods) which engulf the microbe. Microbe is enclosed in a sac called phagosome.

4. Digestion: Inside the cell, phagosome fuses with lysosome to form a phagolysosome.

Lysosomal enzymes kill most bacteria within 30 minutes and include: Lysozyme: Destroys cell wall peptidoglycan Lipases and Proteases RNAses and DNAses

After digestion, residual body with undigestable material is discharged.

Stages of Phagocytosis

2. Inflammation

Triggered by tissue damage due to infection, heat, wound, etc.

Four Major Symptoms of Inflammation:

1. Redness

2. Pain

3. Heat

4. Swelling

May also observe:

5. Loss of function

Functions of Inflammation

1. Destroy and remove pathogens

2. If destruction is not possible, to limit

effects by confining the pathogen and its

products.

3. Repair and replace tissue damaged by

pathogen and its products.

Antimicrobial Substances:

I. Complement System: Large group of serum proteins that

participate in the lysis of foreign cells, inflammation, and

phagocytosis.

Three mechanisms of complement activation:

1. Classical Pathway: Initiated by an immune reaction of

antibodies.

2. Alternative Pathway: Initiated by direct interaction of

complement proteins with microbial polysaccharides.

Both pathways cleave a complement protein called C3,

which triggers a series of events.

3. Lectin pathway

II. INTERFERONS

Antiviral proteins that interfere with viral multiplication.

◦ Small proteins (15,000 to 30,000 kDa) ◦ Heat stable and resistant to low pH ◦ Important in acute and short term infections. ◦ Have no effect on infected cells. ◦ Host specific, but not virus specific. Interferon alpha and beta: Produced by virus

infected cells and diffuse to neighboring cells. Cause uninfected cells to produce antiviral proteins (AVPs).

Interferon gamma: Produced by lymphocytes. Causes neutrophils to kill bacteria.

NK cells

LGL / Null cells

Lack T cell receptor, CD3 proteins, and surface IgM & IgD

Thymus are not required for development

Activity not enhance by prior exposure

Associated w/ ADCC

CD56 & CD16

Functions of NK cells

Kill virus-infected/ Cancer cells

Killing ◦ Non-specific

◦ Not dependent on foreign antigen presentation by class I or II MHC proteins

◦ Activated by the failure of a cell to present self antigen

◦ Produce perforins & granzymes, w/c cause apoptosis of target cell

Adaptive Immunity

Adaptive Immunity

Antigen – Antibody reaction

Cells: ◦ B cells

◦ T cells

Antigens & Immunogens

Antigens molecules that react w/ Abs compound that does not

necessarily elicit an immune response Immunogens molecules that induce an

immune response

at least 2 antigenic determinant

Two properties of Antigens:

IMMUNOGENECITY ability to induce

specific immune response resulting to formation of antibodies or immune lymphocytes

ANTIGENECITY/ SPECIFICITY the

ability to react specifically with the antibody or cell that caused it to be produced.

Parts of Ag:

CARRIER PORTION ◦ The bigger part that is responsible for the MW of antigen

EPITOPE/ ANTIGENIC DETERMINANT ◦ Determines specificity of antigen, therefore, an antigen w/out epitope is said to be nonspecific.

Haptens

Molecule that is not immunogenic by itself but can react w/ specific antibody ◦ Incomplete Ag

◦ Small molecules (<10,000D)

◦ univalent

◦ HMW nucleic acids

◦ Drugs (e.g. Penicillin)

◦ Cathechol (plant oak)

CARRIER

A molecule that when coupled to a hapten, renders hapten immunogenic.

E.g: ◦ Albumin

◦ Globulin

◦ Synthetic polypeptides

Features of molecules that determine immunogenicity

Foreignness

Molecular size

Chemical-Structural Complexity

Antigenic Determinants (Epitopes)

Lymphoid System

Primary/ Central

Secondary/ Peripheral

Central/ Primary Lymphoid organs

Central/ Primary

are the sites for generation and early maturation of lymphocytes (B and T cells)

A. Bone Marrow (Bursa of Fabricus equivalent)

Hematopoeisis ◦ RBC

◦ Platelets

◦ Monocytes

◦ Granulocytes

Lymphopoeisis ◦ B cells

◦ T cell precursors NK cells

Dendritic cells

Mast cells

B. Thymus

Maturation & Differentiation of T cells

Secondary/ Peripheral

T & B cells Central L.T. Migrate Peripheral L.T. Respond to foreign

antigens

trap antigens

are the sites for initiation of most immune response

provide signals for recirculation of lymphocytes

A. Lymph nodes

Major antigen-trapping sites of the body

Filters foreign substances from the tissue fluids and lymph

Central organ for lymphocyte traffic and circulation

Lymph Nodes

PARTS: ◦ CORTEX (Germinal center) B cells

◦ PARACORTEX (Juxtamedullary) T cells

B. Spleen

Filters foreign substance from the blood

Critical line versus blood borne infections

Eliminates dead worn-out RBCs

Spleen

White pulp ◦ Marginal zone

◦ Germinal center

◦ PALS (mostly T cells)

◦ Primary follicles (mostly B cells)

Mucosa-associated Lymphoid Tissue (MALT)

GUT-associated lymphoid tissue (GALT)

Bronchus-associated lymphoid tissue (BALT)

BALT

L.T. beneath the respiratory mucosa and the aggregates of nodular lymphatic tissues called Tonsils.

Tonsils nodular aggregates of B cells &

diffuse areas that contain mostly of T cells

for airborne and alimentary

tract pathogens

Immune Cells

Embryonic development

Fetal liver

Yolk sac

Postnatal life

Bone Marrow

.. . … . . .. .

natural killer

(NK) cell

lymphocytes

HEMATOPOESIS

T CELLS ◦ Pre T cell Immature (thymocyte) Mature T cell Lymphokines

B CELLS ◦ Pro B cell Pre B cell Immature Mature B cell Plasma cells Abs

T cells

Responsible for foreign antigen recognition or cellular immune response, which include: ◦ rejection in organ transplantation

◦ regulation of antibody production

◦ secretion of soluble mediators

It has the ability to bind with sheep’s RBC forming rosette.

Subsets of T cells

1. T helper cell (CD4 marker) • Recognize Ag in association w/ MHC class II • Collaborate w/ B cells to produce Abs • Th1/Th2

2. T cytotoxic cell (CD8 marker) • Has killer function

3. T effector cell • Also called as TdTh cell • Responsible for delayed type of HPS

4. T suppressor cell (CD8 marker) ◦ Involved in presenting autoimmunity activated by

Ag

B cells

Have shorter life span (5-7 days)

Precursors, regulators, and effectors of immunity.

May transform or differentiate into plasma cell to produce immune antibodies.

CD19, CD20, CD21, CD22, CD35

Comparison of T & B cells

Feature T cells B cells

Antigen receptors Yes Yes

IgM on surface No Yes

CD3 proteins on surface Yes No

Clonal expansion after contact w/

specific antigen

Yes Yes

Immunoglobulin synthesis No Yes

Regulator of Antibody synthesis Yes No

IL-2, IL-4, IL-5 & Gamma interferon

synthesis

Yes No

Effector of CMI Yes No

Maturation in Thymus Yes No

Maturation in Bursa or its equivalent No Yes

CYTOKINES/ LYMPHOKINES

Soluble mediators that serves as the language for cell communication.

Either immune / non-immune

1. Interleukins

IL-1 T cell activation factor (MØ)

IL-2 Activates Tc cell

IL-3 Stimulates hematopoietic cells

IL-4 Activates B cell

IL-5 Activates eosinophils

IL-6 Activates B cell

IL-7 Differentiation and Maturation of T & B cell

IL-8 Activates Neutrophils

IL-9 Proliferation of T cells, thymocytes, mast cells

IL-10 Inhibition of cytokine synthesis

IL-11 Regulates hematopoiesis

IL-12 Activates NK cells

2. Interferons

INF α augments NK cell activity

INF β identical to IL-6

INF major mØ activator

antagonistic to IL-4

augments NK cell activity

3. TUMOR NECROSIS FACTOR

TNF α directly cytotoxic to tumor cells

TNF β lymphotoxin

CD MARKERS

CD1 - THYMOCYTES

CD2 – E ROSETTE RECEPTOR

CD3 – T CELLS (ALL) TCR

CD4 – T HELPER

CD8 – T SUPPRESSOR/CYTOTOXIC

CD19 – B CELL

CD56 – NK CELL

Immunoglobulins

Antibodies

Globulin proteins (immunoglobulins) that react specifically w/ the antigen that stimulated their production

20% of the protein in the blood plasma

Gamma globulins

Glycoproteins

Part of the adaptive immune response (humoral immunity)

Types:

1. Alloantibody

- produced after exposure to genetically different or non-self antigens of same species

2. Autoantibody

- produced in response to self antigen

Immunoglobulin Structure

Ig tx:

Pepsin ◦ One large F(ab)2 fragment

◦ LMW peptides

Papain ◦ Two (2) Fab fragments

◦ One (1) Fc

Major Ig Classes

5 classes/ Isotypes (constant heavy chain) ◦ IgG: gamma heavy chain

◦ IgA: alpha heavy chain

◦ IgM: mu heavy chain

◦ IgE: epsilon heavy chain

◦ IgD: Delta heavy chain

Property IgG IgA IgM IgE IgD

A. Physiologic

% of total Ig in Serum

75

15

9

0.004

0.2

Catabolic rate(1/2 life) 18-23 5-6.5 5-6 2.3 2.8

MW 150 170/ 400 900 190 180

Structure Monomer Mono/di Mono/penta Mono Mono

B. Biological

Agglutinating Capacity

+ +2 +4 - -

Complement fixing + - +4 - -

ADCC + - - - -

Mediation of allergic

Response

- - - +4 -

Placental transport + - - - -

Present in external

secretion

+ +4 ± +2 -

Receptor on B cell - - + - ?

Opsonization + - - - -

Polymeric form J chain - + + - -

Subclasses 4 2 - - -

1. IgG

Four subclasses: IgG1, IgG2, IgG3 & IgG4

Monomer

Highest concentration in plasma

Transported across the placenta

Activates complement

Opsonizes

Main Ab in the secondary immune response

Mediates Antibody-dependent cellular cytotoxicity (ADCC)

Comparison of IgG Subclasses

IgG1 IgG2 IgG3 IgG4

Serum

concentration ~840 ~240 ~70 ~50

Percent of Total

Serum IgG ~70 ~20 ~6 ~4

Half-life(days) ~23 ~23 ~7 ~23

Complement

binding ++ + +++ --

Placental

transfer +++ + +++ +++

2. IgA (Secretory)

Two subclasses: IgA1 & IgA2

Monomer/ Dimer

Main Ig in external secretions such as milk, tears, saliva & respiratory & intestinal mucus

Protects mucosal surfaces

Major protective factor in colostrum

It is present in the secretion as dimer w/ a J (joining) chain & secretory piece. J chain is made by B or plasma cell; Secretory piece made by epithelial cell

IgA

3. IgM

Produced in the primary response

Pentamer: serum (held by J chains)

Monomer: Ag receptor on B-cell surface

1st antibody that an infant makes

Most efficient at activating complement

Highest agglutinating capacity

IgM

4. IgE

Monomer

Mediates type I hypersensitivity

Main host defense against helminth infxn

5. IgD

Monomer

Uncertain

Present in the membrane of mature B cells

Antibody Variants

Isotypes

Allotypes

Idiotypes

Isotypes

Antigenic (amino acid) differences in their constant heavy regions

Heavy chain isotypes: 9

Total isotypes: 18

Allotypes

additional antigenic features of Ig that vary among individuals

Results from the substitution of only one or two amino acids in the constant regions (usually) of heavy or light chains

No biological significance

Idiotype

Antigenic determinants formed by the specific amino acids in the hypervariable region

Individual, unique differences between antibodies of different antigen binding specificities

Individually specific to each Ig molecule

Complement

Complement

Composed of several proteins found in human serum (other animal serum)

Synthesize in the liver (main)

Heat labile (inactivated by heating serum at 56 C for 30 mins)

3 Pathways:

Classic

Alternative

Lectin

CLASSIC LECTIN ALTERNATIVE

(+) Ag-Ab MBP Microbial surfaces

C1q,r,s C3 + B

MASP

C4 C2 D (protease)

C4b,2b/ C4b,2a C3 C3 C3b,Bb

(C3 CONVERTASE) (C3 CONVERTASE)

C4b,2b,3b/ C4b,2a,3b C3b,Bb,C3b

(C5 CONVERTASE) C5 (C5 CONVERTASE)

C5a + C5b

C5b,6,7

C5b,6,7,8,9

(MAC)

Lysis, Cytotoxicity

Functions of complement:

Anaphylatoxins – C3a, 4a, 5a

Chemoattractants- C5a, LTB4, IL-8, bacterial products

Opsonins – C3b, IgG

Bacterial cell lysis – C5b, 6, 7, 8, 9

Complement Regulatory Proteins

Protein Regulatory Functions

C1 INH Binds to C1, thereby preventing it from initiating

complement activation

Properdin Stabilizes the alternative pathway C convertase

C4bp Accelerates dissociation of C3 convertase (CP)

DAF Accelerates the dissociation of both C3 convertase

Factor I Cleaves and inactivates C3b and C4b

AI Proteolytically cleaves anaphylotoxins

MIRL/HRF/ S-

protein

Inhibits MAC formation

4 Phases of Ab response

Plateau

Log

Lag Decline

Primary

Lag (Longer)

Log

Plateau (Shorter)

Decline (Shorter)

Secondary (Anamnestic)

Lag (Shorter)

Log (Higher peak)

Plateau (Longer)

Decline (Gradual/Prolonged)