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Immunotoxicity:Contribution of
Chemicals to Allergic Disease
Assesment, Examples, & Mechanisms
MaryJane Selgrade
National Health & Environmental Effects
Research Laboratory, U.S, EPA, Research
Triangle Pk, NC
[email protected]
November 2, 2007
Lecture Outline
• Mechanisms of immune mediated injury
• Chemicals which elicit immune responses
– Contact sensitivity
– Respiratory sensitivity
– Food Allergy
• Chemicals which modulate responses to
other antigens
Hypersensitivity
Definition: Excessive humoral or
cellular response to an antigen which
can lead to tissue damage
Hypersensitivity: Classification
• Type 1: IgE mediated (Immediate type)
• Type 2: IgM, IgG, cytolysis of cells
• Type 3: IgM, IgG Immune complex
mediated
• Type 4: T-cell mediated (delayed-type)
(Gel & Coombs classification)
Two Stages
(Distinguishes from irritation)
Induction
Sensitization
(1st exposure)
Elicitation
Challenge
(subsequent exposure)
Type I (Immediate)
(Atopy)
Sensitization
Example:
Bee Sting
Elicitation
Mediators (as such Histamine)
Bronchoconstriction
Mast Cell
Degranulation
Type 2 (Cytotoxic)
Sensitization
Ag
Ab (or autoantibody)
Ag binds to cell surface
Elicitation
Ab bind to cell bound antigen
Cell lysis
Examples:
Goodpastures syndrome
Hemolytic anemia
Type 3 (Arthus)
Sensitization
Ag
Ab
Elicitation
Formation of
Ag/Ab complexes;
Deposition in tissues
Examples: Late onset
asthmatic response,
fibrosis, serum sickness
Activation of macrophages
And Complement
Activated
macrophage
Influx of PMN’s, Eosinophils,
Lymphocytes (Inflammation)
Polymorphonuclear
leukocyte
Type 4 (Cell Mediated)
Sensitization
Elicitation
Antigen
Antigen
Ag presenting cell
Ag presenting cell
Activated T cell
Clonal expansion
Example:
Contact
Sensitivity
Cytokines
Activated
macrophage
Inflammatory activity
Antigen Presented to T Cells Via MHC
Recognised by
TCR of CD8 T cells
Recognised by
TCR of CD4 T cells
peptide generally derived from
peptide generally derived
protein produced within the cell from protein taken up by the cell
  Class I MHC
with bound peptide


cell membrane
cytoplasm




Class II MHC
with bound peptide
TH1
IL-2 TGFß
TH2
IFN
IL-5 IL-13 IL-6 IL-4 IL-10
Suppresses TH2
Suppresses TH1
IgG2a, CF Antibody
DTH, Cytotoxicity
IgE, IgG2b, IgG1
Mast Cells + Eosinophils
Defense Against
Intracellular Pathogens
Defense Against Parasitic
Infection
Inflammatory Diseases
Immediate-type Hypersensitivity
Type IV Responses
Immune
cell
antigen
Th1
Soluble
(MHC II)
Effector
Macrophage
mechanism activation
Examples Tuberculin Rx
Contact
dermatitis
Th2
CD8 (CTL) Tc
Soluble
(MHC II)
Eosinophil
activation
Chronic
asthma/allergy
Cell-associated
MHC I)
cytotoxicity
Contact
dermatitis
Potential Effects of Chemicals
on Allergic Disease
• Can themselves act as antigens
– Haptens
• Contact sensitizers
• Respiratory sensitizers
• Systemic hypersensitivity (drugs)
– Proteins
• Respiratory allergens
• Food Allergens
• Systemic hypersensitivity (drugs)
• Can enhance development or expression of
allergic reactions
Potential Contact Sensitizers
•
•
•
•
•
Cosmetics and Fragrances
Dyes
Preservatives (formaldehyde
Metals (Ni, Co, Be, Cr)
Pesticides
Contact Hypersensitivity
Allergic Contact Dermatitis (ACD
INDUCTION PHASE
ELICITATION PHASE
EDEMA AND ERYTHEMA
ALLERGEN
LANGERHANS
CELL (LC)
IL-1B, IL-6, IL-12
NONSPECIFIC
INFLAMMATORY
MEDIATORS
Migration to Local
Lymph Node
CELLULAR
INFLUX
“PRIMED”
LYMPHOCYTES
LC AND
LYMPHOCYTE LYMPHOCYTE
INTERACTION PROLIFERATION
SPECIFIC RESPONSE
GUINEA PIG MODELS
HRIPT
Guinea Pig
Maximization
Test
Buehler
Assay
20 animals/ group
ID injection w/ and
without FCA plus topical
application:Days 5-8
Induction
Day 20-22 topical
challenge
Challenge
Read: 48,72 h after
challenge
HUMAN
Topical application closed patch:
Days 0, 6-8, 13-15
Endpoint
Human Repeat
Insult Patch
Test
Topical Application
Closed patch, 24 hr
3x/wk, 3wk
Day 27-28 topical
challenge untreated
flank, 6 h
Rest 2 wk, apply
patch 24 hr
Read: 21, 24, 48 h after
removing patch
Read 48 hr
erythema
>30% positive
Criteria
> 15% positive
Any positive
Mouse Ear Swelling Test
Sensitization
Application of
Agent or Vehicle
to Back (local)
or Abdomen
(systemic)
Induction Period
Challenge
Measure Ears: 24-48 hours post challenge
Application of
Agent
The Local Lymph Node Assay
Agent applied to ears
Days 1,2,3
Proliferation measure:
isotope incorporation
scintillation counting
IV 3H-thymidine
5 hour after IV injection
injection Day 6
Endpoint expressed as dpm
Contact Hypersensitivity Models and
Endpoint Mechanisms
PHASE
Induction
Elicitation
LLNA
GPMT and BA
MEST
Structure Activity Relationship (SAR) Models
• Computer modeling using chemical structure to
predict ability to induce CHS
• Based on following concepts:
– Biologic mechanism of chemical effect is related to
structure
– Chemicals with related structures have similar
mechanisms & hence effect
• How do SAR & QSAR models work
– Chemical structure added into program
– Structure compared to structures in data base of
know contact sensitizers
– Structures associated with sensitizers are flagged
Using SAR to Predict ACD
Chemical
Skin
penetration/
metabolism
Protein
reactivity
Metabolic
Transform
Predict
ACD
(Allergic Contact
Dermatitis)
Immune
reactivity
See attached Chem characteristic pdf
Prevalence of Asthma, United States
80
Rate (per thousand)
70
60
50
0- 4 yr
5-14
15-34
35-64
>65
Total
40
30
20
10
1980
19811983
19841986
19871989
19901992
19931994
Characteristics of Allergic
Asthma
• Immediate Response
• Late phase
– Bronchoconstriction
– Hyperresponsive to
non-specific stimuli
– IgE mediated (IL-4)
(methacholine)
– Eosinophilic
Inflammation
– Th2 mediated
(Il-5, IL-13)
Principal Asthma/Respiratory
Allergy Concerns for Toxicologist
• Proteins - e.g detergent enzymes, biotech
• Haptens (low molecular wt) - isocyanates &
anhydrides, platinum
• Adjuvants - air pollution
Method used to Assess for Potential
Pulmonary Hypersensitivity
Criteria for Positive Response
Respiratory rate 35%
Temp  0.6%
oscillograph
Differential
Pressure transducer
Measures pulmonary
response
exhaust
Ag
Temperature
transmitter
air
Protein allergens
•
•
•
•
•
Detergent Enzymes
Molds and spores
Latex
microbial pesticides
animal dander
*Not all proteins are equally allergenic; as yet no particular
amino acid sequences has been associated with allergenicity
Guinea Pig Intratracheal Test &
Detergent Enzymes Assessment
• IT dose response cytophilic Ab response to
unknown
• IT dose response to Alcalase (subtilisin B)
• Test protein/Reference protein = relative
potency
• Relative potency <1 set exposure level same as
alcalase
• Relative potency > 1 adjust by appropriate
factor
Low Molecular Wt (<3000 molecular
weight) Compounds
•
•
•
•
•
•
•
Toluene diisocyanate
Diphenylmethane diisocyanate
Phthalic anhydride
Trimellitic anhydride
Platinum salts
Reactive dyes
50 or so known allergens (asthmagens)
Assessing Low Molecular Wt (<3000) as
Potential Respiratory Allergens -Asthmagens
• Karol test - Inhalation exposure, respiratory
effects, Guinea pig
• Sarlo approach - Guinea Pigs
–
–
–
–
Structure activity
In vitro reactivity with protein
In vivo antibody response (IT exposure)
In vivo reactivity (Karol test)
Assessing Low Molecular Wt: Potential
Screening Hazard ID Approaches
• Mouse IgE test– exposure variation of LLNA
– assess total serum IgE
• Cytokine profiling
– exposure variation on LLNA
– assess cytokine profiles in draining lymph node,
look for differences in expression of Th1, Th2
profiles
• LLNA
Air Pollutants That Appear to Be
Adjuvants
• Rodent models
– Nitrogen dioxide (NO2)
– Residual oil fly ash (ROFA
• & Human
– Diesel exhaust
• & Rhesus Monkey
– Ozone
TH1
IL-2 TNFß
TH2
IFN
IL-5 IL-13 IL-6 IL-4 IL-10
Suppresses TH2
Suppresses TH1
IgG2a, CF Antibody
DTH, Cytotoxicity
IgE, IgG2b, IgG1
Mast Cells + Eosinophils
Defense Against
Intracellular Pathogens
Defense Against Parasitic
Infection
Inflammatory Diseases
Immediate-type Hypersensitivity
Conditions That Influence Th1/Th2
Polarization
• Nature or Dose of Antigen
• Local Micro Environment
– cytokines & other mediators produced by
macrophages & epithelial cells
• Genetics of Host
• Route of Exposure
Food allergens
• Around 5% of children and 2-3 % or adults
have food allergies
• Manifestations can range from life
threatening anaphylaxis, to respiratory or
gut irritation
• Peanut classic example
• Toxicologist involved because of GMOs
– acid resistant
– homology with known allergens
Goal –Relate Potency of GMO
Protein to Other Food Proteins
(picture is hypothetical)