IMMUNOTOXICOLOGY

BAG / SMF. Ilmu Kesehatan Kulit

Dan Kelamin
 Immunotoxicology
Study of the effects of physical and
chemical agents on the immune system
Immune system is highly sensitive to
toxicants
Consists of many cells undergoing rapid
proliferation and differentiation;
(eg. Bone marrow)
Immunocompetence requires adequate
balance (cooperation) and functioning
between different cellular components
Functions of the Immune System

Defense

Homeostasis

Surveillance
 Anatomical Organization
External Secretory Systems
Respiratory, GI, urogenital

Internal Secretory Systems

Lymph system
Primary lymphatic organs (thymus, fetal liver,
bone marrow)
Secondary lymphatic organs (spleen, lymph
nodes, peritoneal cavity, lymph ducts)
Reticuloendothelial system (mononuclear
phagocytes; macrophages, monocytes)
 INNATE IMMUNITY

PHYSICAL CELLS CHEMICAL

BARRIERS granulocytes, BARRIERS
monocytes, pH, lipids, enzymes
Skin, mucous
macrophages
membrane

ADAPTIVE IMMUNITY

HUMORAL CELL MEDIATED

B cells MP T cells
antibodies lymphokines
 Cellular Components of the
Immune System
Neutrophils and Macrophages
Nonspecific (innate) immunity
Secrete biological mediators
Process and present antigen (MP)
Pattern recognition receptors; Fc receptors for
antibody coated antigens (MP)
Lymphocytes
Specific (acquired) immunity
Specific antigen receptors
B cells (humoral immunity)
T cells (cell mediated immunity)
 Cellular Interactions in the
Immune System
MP Activated
MP (+)

PA Sensitized Antigen
(+) Th
Th destruction
MP
(-) (+)
AG
Y
(90%) Tcyt
(10%) (+)
(-) Treg (-)
B
Sensitized
plasma
B Bmem
Hematopoietic Bone Marrow Stem Cells

Stem cells
can differentiate
into many
different cell types

How does it
decide what
to become?
Control of Stem Cell Differentiation

Two major influences:

1. ENVIRONMENT in which it develops: which primary

lymphoid organ (ie bone marrow vs thymus)

2. CYTOKINES AND GROWTH FACTORS stem cells are

exposed to during this process
 Origin and Development of
Immune Cells
BONE MARROW BLOOD TISSUE
noncommitted Cell Mediated
Stem dividing
T cell
thymus 90% Immunity
cells 30%
Lymphoid
bursa B cell B
10% Humoral
equivalent Immunity
Progenitor
cells Monocyte
3-5% MP
committed Myeloid
dividing PMN (70%)
eosinophil (<3%)
Granulocyte
basophil (<2%) mast
 Innate Immunity

Universal and evolutionarily conserved mechanism of host

defense against infection; first line of defense;
Predates adaptive immune response
Found in all multicellular organisms
(adaptive only in vertebrates)
Uses receptors and effectors that are ancient in their lineage
Provides protection against a wide variety of pathogens
(pattern recognition receptors)
Distinguishes self from non-self perfectly
Defects in innate immunity are very rare and almost always
lethal
Elie Metchnikoff: Father of Macrophages
1908 Nobel prize
Metchnikoff watched the reaction to a splinter inserted into a
starfish. Hemocytes (amoeba-like cells) arrived and tried to
ingest the foreign body; if they could not, they walled it off.
Mammals do exactly the same thing to foreign bodies.

Phagocytosis- to devour
 Oyster hemocyte Mouse macrophage

The similarity between an invertebrate and mammalian phagocyte is

striking; they also use many of the same mechanisms, including the
production of reactive oxygen species

http://www.mdsg.umd.edu/oysters/oysblood.htm http://itgmv1.fzk.de/www/itg/diabate/images.html#fig5
 Foreign Body Reactions in Humans
chromic catgut
The suture material is not digestible by
macrophages (MP), so it has been walled
off by fibroblasts.
Atlas of Granulomatous Diseases Yale Rosen, M.D.
silicone droplets
The clear (unstained) globules of silicone have
been released from a ruptured breast implant.
They cannot be ingested; at this early stage,
MP accumulate to wall off the material; note MP
that has become a multinucleated giant cell
showing the asteroid bodies characteristic of
the foreign body reaction.
 Innate Immunity: Functions

Provides a barrier to prevent the spread of

infection
Mechanical (tight junctions, movement)
Chemical (fatty acids, enzymes, pH, antimicrobial
peptides)
Microbiological (normal flora)

Mucosal surfaces
Respiratory, GI, Reproductive

Skin (epithelial cells)

Wounds, burns, insect bites
 Innate Immunity: Functions

Identifies and eliminates pathogens

Non-adaptive recognition systems
Activates molecules that target the microbe and aid
in its identification
These factors may be surface expressed, released
from immune cells or present within circulatory
system
 Innate Immunity: Functions

Initiates an inflammatory response

Reaction to injury or infection
Trauma to tissues or cells
Presence of foreign material (non-self)
Infectious agents (viruses, bacteria, fungi)
Delivers effector molecules and immune cells to the
site of injury/infection
Components
Granulocytes, MP, secreted factors
Blood vessels (endothelium)
Plasma proteins
 Innate Immunity: Functions

Provides signals to alert the adaptive

immune system to activate an effective
specific immune response
Upregulation of co-stimulatory molecules
MHC class II, CD80/86
Induction of cytokine/chemokine
response : IL-4, IL-12
 Innate Immunity:
Cellular Components
Granulocytes
Polymorphonuclear leukocytes fight
pathogens
(PMN, neutrophils) inflammation
Eosinophils
Basophils (blood) allergic and
hypersensitivity reactions
Mast Cells (tissues)
Mononuclear Phagocytes (RES)
Monocytes (blood) fight pathogens
inflammation
Macrophages (tissue) cytotoxicity
immune regulation
Innate Immune Cells
 Origin and Development of
Macrophages and Granulocytes
Bone Marrow Blood Tissue
monoblast promonocyte monocyte

macrophage

basophilic basophil
Phagocytic promyelocyte
precursors

eosinophilic mast
eosinophil
promyelocyte cell
myeloblast
neutrophilic Neutrophil
promyelocyte (PMN)
Neutrophils
(PMN)

Present in blood (60-70% of WBC)

Not normally present in tissues
Short lifespan - 12 hours
Functions:
First cell at the site of infection/injury
Ingest
and kill microbes after bactericidal
mechanisms activated (binding to pathogen)
Mononuclear
Phagocytes

Blood - monocytes (1-6% WBC)

Tissues - macrophages
mature form of monocytes
found in tissues (ex., gastrointestinal tract, lung, liver,
brain, skin, spleen); reticuloendothelial system (RES)
Functions:
Phagocytize and kill after bactericidal mechanisms
activated
Produce cytokines/chemokines (initiate inflammation)
Antigen presentation (activate adaptive immunity)
Tumor surveillance and cytotoxicity
 Reticuloendothelial System

Consists of fixed and wandering

macrophages located throughout the body
(tissues, sinusoids, lymph system)
Major locations: Liver and Lung
Functions: antibacterial resistance, tumor
resistance, defense against shock, antigen
processing, lipid metabolism, protein
turnover, iron metabolism
 Origin and Development of
Macrophages and Granulocytes
Bone Marrow Blood Tissue
monoblast promonocyte monocyte

macrophage

basophilic basophil
Phagocytic promyelocyte
precursors

eosinophilic mast
eosinophil
promyelocyte cell
myeloblast
neutrophilic Neutrophil
promyelocyte (PMN)
 Functions of Phagocytic Cells

Macrophages and Neutrophils

Localization and removal of foreign substances
(ie., pathogens) (MP and PMN)
Inflammation- response to infection or injury
(MP and PMN)
Initiate wound healing (MP)
Secretory functions (MP)
Immunologic functions: process and present
antigen (MP)
Tumor cell surveillance and cytotoxicity (MP)
Functions of Phagocytic Leukocytes
(mono, MP and PMN)
Localization and Removal of Foreign
Substances (Inflammation and wound healing)
-Chemotaxis: directed migration to site of injury;
chemical mediators (chemotactic factors)
-Phagocytosis: ingestion of foreign substances
-Metabolic destruction: digestion and killing
Oxygen-dependent (MPO, ROI, RNI)
Oxygen-independent (cationic proteins,
lysozyme, TNF, porphorins
 Secretory Functions of
Macrophages
Second most potent secretory cell in
body

Enzymes capable of degrading

extracellular matrix proteins (collagenase,
elastase)
Products involved in host defense (ROI,
RNI, eicosanoids)
Regulatory proteins (IL-1, TNFa, pGE2)
Macrophage-derived Mediators
Binding proteins (transferrin, fibronectin)
Complement components
Proteolytic enzymes (lysozyme, neutral protease, acid
hydrolyases)
Enzyme inhibitors (a2-macroglobulin plasmin inhibitor)
Endogenous pyrogen (IL-1)
Reactive oxygen intermediates (superoxide, hydrogen
peroxide, hydroxyl radical)
Reactive nitrogen intermediates (nitric oxide,
peroxynitrite)
Bioactive lipids (PAF, PG, LT, TBX)
Chemotactic factors
Growth factors/cytokines (IL-1, TNF, IL-6, FGF, CSF)
Macrophage Mediators Can also
Damage Host Tissues

Bioactive ROI
Lipids
Activated
Macrophage

TNF-a Proteolytic
IL-1 Enzymes
chemokines
RNI
Reactive Oxygen Intermediates

H2O2
O2-
OH-

Lipid Peroxidation
Membrane, Protein and DNA
Damage
Reactive Nitrogen Intermediates

Nitric oxide and peroxynitrite

Nitric oxide- formed from l-arginine by the
enzyme nitric oxide synthase (NOS)
Macrophages: (NOSII) induced by
inflammatory cytokines (IFNg, TNFa) and
bacterially-derived products (LPS)
Highly labile; oxidizes nucleic acids,
membranes, proteins
Nitric oxide reacts with superoxide anion
forming peroxynitrite
Antitumor/antibacterial activity
Proinflammatory Cytokines

Tumor necrosis factor-a

Interleukin-1
Interleukin-6
Interleukin-18
Chemokines
Interferon-g
Tumor Necrosis Factor-a

Proinflammatory
Primes phagocytes to produce
ROI and RNI
Cytotoxic
Induces apoptosis and necrosis
Immune Functions of Macrophages

Tumor Surveillance

Tumor Cytotoxicity

Antigen processing and

presentation
Immune Functions of Macrophages

Antigenprocessing and
presentation

Tumor Cytotoxicity

Tumor Surveillance
 Macrophage Processing of
Antigens
Macrophages function as accessory cells or
antigen processing cells
Macrophage associated antigen is 1000x more
immunogenic
Processing of antigens involves change so that it
binds MHC II (Ia) proteins; may involve
unfolding, partial degradation, selection for
epitope with high affinity for MHC II
Required for T-helper cell recognition of antigens
Other APC: B cells, epithelial cells
 Antigen Processing

Phagocytosis of antigen
Partial degradation or unfolding
Binding to MHC II (Ia) proteins
Re-expression of processed antigen
on cell surface
Presentation to T-helper cells
Antigen Processing and
Presentation

II

Antigen
presentation
 Macrophage (APC)

MHC
Class II CD4
Processed
Antigen

T cell receptor Cytokines

CD4 Th Cell
 Adaptive Immunity:
Humoral Immunity

Mediated by B lymphocytes which produce

antibodies or immunoglobulins (Ig) in
response to antigen challenge
Antibodies: glycoproteins; selective, highly
specific; found in g-globulin fraction of
serum (humoral=blood)
Five Classes: physical, chemical and
antigenic differences
 Classes of Antibodies
IgM: primary immune response (7%); Type III
hypersensitivity reaction; immune complexes;
B cell receptor
IgG: secondary immune response, B memory
cells (70%)
IgA: external secretions, produced locally
against bacteria and viruses (15%)
IgE: Type I hypersensitivity reactions, minute
amounts
IgD: umbilical cord blood, primitive recognition
or regulation; B cell receptor
 Antibody Structure
N-terminus Variable region
Fab Fab
antigen binding site
ss ss
Light chain
papain (2 Fab fragments)
Heavy chain -ss-
pepsin ( 1 Fab fragment)
C-terminus
Fc Cellular binding site

Interchain disulfide bonds stabilize domain structure

which is tied to Ig function; differences in Ig
molecular weight due to differences in heavy chains
 Antibody Structure

IgM: pentamer

IgA: monomeric; becomes multimeric in

endothelium; acquires glycoprotein that protects
it against digestion
 Kinetics of Antibody
Production
Primaryimmune response: IgM
Secondary immune response: IgM and IgG
Shorter lag
Higher levels of specific IgG produced
Steady state level persists longer
IgG predominates
Quantitative difference between primary and
secondary immune response due to increase in
the number of potentially reactive B cells
Adjuvants: IgG produced after primary
response; secondary IgG response sustained
Kinetics of Antibody Production
 B Lymphocyte Differentiation
10%

Bursa Y Antigen
bone Pre-B equivalent B cell
marrow cell
90%

T cell-
Y processed
Sensitized B cell antigen
Y
Plasma cell Memory cell
MP

complement

Y Antigen AG Lysis
 Immune Response: B Cells
Antibody Clonal Maturation/
triggering Proliferation Differentiation

small Y Y B
B cell memory
Large lymphoblast
B cells B
B
Antigen B

U U U Y
B
Large lymphoblast
Small
B cell
B cells B U
B B
U memory
 Monoclonal Antibodies
Technique developed by Miller and Kohler (1980);
revolutionized immunology; involves development
of single antibody secreting immortalized cell

Inject mice with antigen (2 x)

After 2 weeks, collect spleen cells (B cells);
HGPRT+, Ig+, G-
Fuse with mouse myeloma (HGPRT-, Ig-, G+) in PEG
and HAT (hypoxanthine, aminopertin, thymidine) medium
Collect and clone hybridoma cells (HGPRT+, Ig+, G+)
Select for clones that produce specific antibody
 Monoclonal Antibodies
Monospecific: normally antibodies extremely
polymorphic even when directed against single
antigen; normal antisera heterogeneity, therefore no
two antisera identical
Reproducible: identical antibody more specific
and reliable
Unlimited quantities: permanent cell line, grows
indefinitely and produces very large amounts of
antibody.
Antigen need not be pure or characterized:
antibody reacts with only one determinant
 Monoclonal Antibodies

Potential Use
Reverse biological effects of certain agents
(digitalis, morphine, kepone, histamine)
Drug scavengers: low levels drugs and
chemicals (dioxin)
Drug targeting: cancer treatment
Tumor targeting
Identification and purification of proteins
 Antigens
Substance recognized as foreign
Hapten: foreign substance that binds antibody;
does not elicit immune response
Immunogen: foreign substance that binds
antigen and elicits immune response
Carrier: large protein
Hapten + Carrier = Immunogen
Epitope: part of antigen that is recognized by
antibody
Immunodominant site: epitopes that are more
highly charged or more accessible
 Types of Antigens
T-independent antigens
Complex carbohydrates
Do not require processing
Can directly interact with B cells
No memory
T-dependent antigens
Require macrophages or other APC
Require T-helper cells
Require major histocompatibility antigens
Mostly proteins
Antibody-Antigen Interactions

Binding of antigen to antibody

Occurs in variable region of antibody
molecule
Instantaneous
Exothermic
May form complexes
Cytotoxicity mediated by complement
(lysis)
 Cell Mediated Immunity

Mediated by T lymphocytes which release

soluble mediators (lymphokines)

Important in host defense against viruses,

certain bacteria, fungi, transplant rejection and
tumor surveillance; Type IV (delayed type)
hypersensitivity (DTH) reactions

T Cells: derived from precursor cells in bone

marrow; mature in thymus; become educated
 Types of T Cells
Regulatory T cells
T-helper cells: T4 (CD4) antigens (Th1, Th2)
T-regulatory (suppressor cells): surface
CD4+/CD25+; intracellular FoxP3 (naturally
occuring Treg)
Cytotoxic (killer) T cells: CD8 antigens
Identified by surface markers (CD4, CD8, CD25)
(monoclonal antibodies)
Work together to modulate immune response
Actions mediated by cytokines
 T-helper cells

TH1 cells: T helper inflammatory cells

Activate macrophages
Involved in cell-mediated allergies,
e.g. poison ivy
Promote rejection of transplanted tissue
Stimulated by cell-bound antigen, and secrete
lymphokines

TH2 cells: Stimulate B cells to produce

antibodies
 T Cell Lymphokines

Macrophage activity factor (IFNg)

Macrophage inhibitory factor (MIF)
Interferon (IFNa and IFNb)
Lymphotoxic factor
Interleukin 2 (IL-2,T cell growth
factor)
Interleukin 3 (IL-3, B cell growth
factor)
 T Lymphocyte Differentiation
MP
MP PA
Bone education Th
(+)
marrow (+)
Thymus (-) Sensitized
Th
Treg

(+)
(-)
Pre-T (-) B cell
(+)
Tcyt
Cytotoxic Y
cytokines lymphokines
 T Cell Education
Learning to distinguish between self and non-
self
Controlled by MHC (self marker) proteins
Selection of subpopulations of immature T cells
through their interactions with MHC proteins
displayed to them in thymus
Immature T cells recognize MHC alone; as they
mature, recognize MHC+antigen
Maturation dependent genetic alteration in T
cell receptor
Uneducated T cells are destroyed in thymus
Antigen Recognition by T Cells

Specific T cell receptor

T cells are MHC restricted; only recognize
antigen and MHC protein
T-helper cells recognize processed antigen
and MHC II (self)
Cytotoxic T cells recognize processed
antigen and MHC I (non-self or altered self)
Major Histocompatibility Complex
(MHC)
Large cluster of immune response genes; code for
proteins that direct T cell responses (mouse chrom 6;
human chrom 17); MHC I and MHC II
Allow immune system to distinguish self and non-self; to
destroy non-self or altered self (virally infected cells
and tumor cells)
Extreme polymorphism: millions of alleles or variants;
control individual responsiveness to antigen; inherit
haplotypes
Every individual unique; MHC markers genetically
determined; the closer two individual are genetically,
the closer their haplotype
 Major Histocompatibility
Complex

Class I MHC: expressed on all somatic

cells; classic transplantation antigens

Class II MHC: expressed on immune cells

(B,T, macrophages, thymus epithelium);
immune associated (IA) antigens; important in
immune regulation, cell-cell communication

Class III MHC: complement

 Activation of T Cells
altered
self
Macrophage
Antigen
processed MHC II
antigen
epitope
T cell
receptor CD4+
CD8+
T-helper
T-killer

Stimulatory cytokines
IL-3 gIFN IL-2
Cytotoxic lymphokines
B MP T
Potential Effects of Toxicants on
the Immune System

Immune Suppression
Decreased resistance to infection, impaired
surveillance; increased incidence cancer; delayed or
aberrant wound healing
General immune suppressants
Pharmaceuticals: corticosteroids (macrophage inhibitors;
lympholytic); methotrexate, cyclophosphamide (cytotoxic
agents; bone marrow suppressants)
Environmental/industrial chemicals (solvents, PAH, benzene,
pesticides, heavy metals, some air pollutants)
Agents of abuse (caabinoids, ethanol, tobacco)
Physical agents (UV, ionizing radiation)
Potential Effects of Toxicants on
the Immune System

Immune Enhancement
Autoimmunity: immune response against hosts own
tissues and cells; autoantibodies produced (ANA);
joints, connective tissue most sensitive; failure of
organism to recognize its own cells as self; leads to
inappropriate immune response against self antigens
Genetic factors
Chemicals/drugs
-lupus: genetic predisposition vs drug induced (hydralazine,
procainamide); increase Th or decreased Treg/s cells
-acute liver failure: alterations in self antigens; tienilic acid,
triglitizone
 Potential Effects of Toxicants on
the Immune System
Immune Enhancement
Hypersensitivity/allergic reactions: most common toxic
manifestations of chemicals; undesirable reactions produced by
normal immune system; require presensitization; most common in
lung (asthma-dust; pneumonitis- inorganic chem.; DTH-beryllium),
skin (contact dermatis) and GI track; drugs/chemicals can act as
haptens by conjugating to proteins or cells; become
immunogens; ex, penicillin allergy; poison ivy

Chronic Inflammation: aberrant wound repair; foreign body

reaction; granuloma

General Immune Stimulation: nonspecific stimulation

macrophages and/or lymphocytes; ex, levamisole, copolymer,
estrogen, somatotropin
 Hypersensitivity Reactions
Type I: immediate (anaphylactic) reaction (within 15-30
min); can involve skin (urticaria, eczema), eyes
(conjunctivitis), nasopharynx (rhinitis), bronchopulmonary
tissue (asthma) or GI (gastroenteritis)

Primary exposure to antigen: binding of IgE to mast cells

Secondary exposure: cross bridging of antigen to IgE,
capping, Ca++ influx, release of vasoactive autacoids (ex.,
histamine, LT, PAF)

Response: Inhibition of vascular smooth muscle; bronchiole

constriction, hay fever, uticaria, acute anaphylaxis

Examples: latex, peanuts, isocyanates, cobalt, nickel

 Mast Cell Mediators

Histamine (increased vascular

permeability, constrict smooth muscles)
Serotonin (Increase vascular
permeability)
Eosinophil chemotactic factor
SRS-A (LTB) (constrict smooth muscles;
increased vascular permeability)
Platelet activating factor (platelet
aggregation)
 Hypersensitivity Reactions
Type II: Cytotoxic hypersensitivity; antibody
dependent (IgG and IgM)

Binding of antibody to antigen

Complement fixation
Activation of complement
Lysis of antigen/antibody complex by complement
Phagocytosis and degradation by macrophages
Examples: blood transfusions, organ transplants;
hemolytic anemia; Rh disease; Farmers lung-
biological dusts (hypersensitivity pneumonitis)
 Hypersensitivity Reactions
Type III: Intermediate reaction (6-24 hr)
Immune complex hypersensitivity; (Ab-Ag complexes)

Formation of immune complexes; mostly IgM; circulate in

blood; attach to vessel walls (esp. kidney); complement
fixation, phagocyte and platelet accumulation; increased
vessel permeability, edema, decreased blood flow, release
of hydrolytic enzymes and vasoactive substances; vessel
rupture, hemorrhage

Examples: arthus skin reaction-local; serum sickness-

systemic, protracted anaphylaxis, occupational diseases
 Hypersensitivity Reactions
Type IV: Delayed Type Hypersensitivity (DTH)
Reactions (48-72 hr)
Mediated by macrophages (MP) and cytotoxic T cells
(Tcyt)
Macrophage present processed antigen to T cells;
activation of Tcyt; MP and Tcyt release cytotoxic
mediators (CC, IL-2, IFNg, TNFa);
Examples: Tuberculin type allergy- other allergies of
infections; autoimmune diseases; allergic contact
dermatitis (acids, resins, preservatives, cosmetics,
beryllium, chromium, nickel)- agents act as small
MW haptens; form complete antigens by binding to
dermal proteins.
 Proceedures for Assessing
Immunotoxicology
Immunopathology
Good for assessing general immune alterations
-Hematology profile: leukocyte counts, differentials
-Clinical chemistry: Ig levels
-Histology: BM, nodes, spleen, thymus
-Organ weights: spleen, thymus
Host Resistance Assays
potentially useful as initial screen to measure
immunocompetence; rationale- immuno-
suppressed hosts are more susceptible to
infections or to cancer
Assessment of Phagocytic Cell
Functioning

Differentialcounts
Measurement of functional activity
(chemotaxis, phagocytosis,
cytotoxicity-bacteria, tumor cells)
Lysosomal enzyme activity
Clearance capcity RES (colloidal
carbon, 125-I-triolein)
Assessment of Humoral Immunity

Measurement of antibody titers (immunodiffusion,

hemagglutination, RIA, ELISA)
Measurement of antibody synthesis (plaque
forming assays)
Measurement of B cell surface receptors (EAC
rosettes)
Enumeration of B cells (Mab)
Measurement of B cell proliferation (polyclonal
activators-LPS, PWM)
 Assessment of Cell Mediated
Immunity
Enumeration of T cells (Mab)
Measurement of T cell proliferation in response
to mitogens (PHA, Con A)
Assessment of DTH response: measure skin
response to sensitizer (oxasalone, DCNB)
Graft vs Host reaction
T cell cytotoxicity- tumor cells
Lymphokine production
Mixed lymphocyte reaction