IMMUNOLOGY Mary Joyce Saborrido-Teoxon, RMT, MD Dept. of Micro & Para FEU-NRMF Institute of Medicine
May 31, 2015
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)