1. 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
September 18,2006

2. 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

3. Hypersensitivity
Definition: Excessive humoral or cellular response to an antigen which can lead to tissue damage

4. 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)

5. Two Stages (Distinguishes from irritation)
Induction
Sensitization
(1st exposure)
Elicitation
Challenge
(subsequent exposure)

6. Mediators (as such Histamine)
Type I (Immediate)
(Atopy)
Sensitization
Example:
Bee Sting
Mediators (as such Histamine)
Bronchoconstriction
Elicitation
Mast Cell
Degranulation

7. Type 2 (Cytotoxic) Cell lysis Ag Ab (or autoantibody) Sensitization
Ag binds to cell surface
Sensitization
Elicitation
Cell lysis
Ab bind to cell bound antigen
Examples:
Goodpastures syndrome
Hemolytic anemia

8. Type 3 (Arthus) Sensitization Ag Ab Elicitation Formation of
Ag/Ab complexes;
Deposition in tissues
Activation of macrophages
And Complement
Influx of PMN’s, Eosinophils,
Lymphocytes (Inflammation)
Polymorphonuclear
leukocyte
Activated
macrophage
Sensitization
Ag Ab
Examples: Late onset
asthmatic response,
fibrosis, serum sickness

9. Type 4 (Cell Mediated) Example: Contact Sensitivity Activated T cell
Sensitization
Antigen
Ag presenting cell
Clonal expansion
Inflammatory activity
Cytokines
Activated
macrophage
Elicitation
Antigen
Ag presenting cell
Activated T cell
Example:
Contact
Sensitivity

10. Antigen Presented to T Cells Via MHC

11. TH1 TH2 IL-2 TGFß IFN IL-5 IL-13 IL-6 IL-4 IL-10 Suppresses TH2
IgG2a, CF Antibody
DTH, Cytotoxicity
IgE, IgG2b, IgG1
Mast Cells + Eosinophils
Defense Against
Intracellular Pathogens
Inflammatory Diseases
Defense Against Parasitic
Infection
Immediate-type Hypersensitivity

12. Type IV Responses Immune cell Th1 Th2 CD8 (CTL) Tc antigen
Soluble (MHC II)
Soluble
(MHC II)
Cell-associated
MHC I)
Effector mechanism
Macrophage activation
Eosinophil activation
cytotoxicity
Examples
Tuberculin Rx
Contact dermatitis
Chronic asthma/allergy

13. 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
Can enhance development or expression of allergic reactions

14. Potential Contact Sensitizers
Cosmetics and Fragrances
Dyes
Preservatives (formaldehyde
Metals (Ni, Co, Be, Cr)
Pesticides

15. Contact Hypersensitivity Allergic Contact Dermatitis (ACD
INDUCTION PHASE
ELICITATION PHASE
EDEMA AND ERYTHEMA
ALLERGEN
IL-1B, IL-6, IL-12
NONSPECIFIC INFLAMMATORY MEDIATORS
LANGERHANS CELL (LC)
Migration to Local Lymph Node
CELLULAR INFLUX
“PRIMED” LYMPHOCYTES
SPECIFIC RESPONSE
LC AND LYMPHOCYTE INTERACTION
LYMPHOCYTE PROLIFERATION

16. GUINEA PIG MODELS Guinea Pig Maximization Test Buehler Assay Induction
20 animals/ group
ID injection w/ and without FCA plus topical application:
Days 5-8
Day topical challenge
Read: 48,72 h after challenge
>30% positive
Topical application - closed patch:
Days 0, 6-8, and 13-15
Day topical challenge
of the untreated flank for 6 h
Read: 21, 24, 48 h after removing patch
> 15% positive
Induction
To Summarize:
Point out: 1) endpoint
2) animals are challenged --- experience swelling and redness
3) 20 animals/ group
4) takes about 1 month
Challenge
Endpoint erythema
Criteria

17. Mouse Ear Swelling Test
Application of
Agent or Vehicle
to Back (local)
or Abdomen
(systemic)
Sensitization
Induction Period
Challenge
Application of
Agent
Measure Ears: hours post challenge

18. The Local Lymph Node Assay
Agent applied to ears Days 1,2,3
Proliferation measure:
isotope incorporation scintillation counting
5 hour after IV injection
IV 3H-thymidine injection Day 6
This slide demonstrates the basic procedures for a LLNA
The LLNA uses only the induction phase of contact hypersensitivity. As antigen is painted on the ears, langerhans cells undergo maturation to become effective antigen presenting cells while they transport the antigen to the draining auricular lymph node. When the antigen is presented to Tcells in the node, a proliferative response occurs. With the IV injection of triated thymidine or Iudr the proliferation can be objectively monitored by measuring it’s incorporation.
This assay, variations of this assay and validation studies have been published and on Sept 17 an ICCVAM Peer Review Panel of experts met to discuss this appropriateness of this procedure… which Lorraine Twerdock will speak to you about the perspective, conclusions that the Peer panel came to when evaluating this procedure and the recommendations that the PR panel had for ICCVAM on this assay.
Day 0,1,2: application of agent to ears
By day 3 mice rec IV 3H thymide
5hours later : nodes removed and excised for B-scintillations counting
(objective endpoitn; no need to elicit hypersensitivity reactions)
Endpoint expressed as dpm

19. Contact Hypersensitivity Models and Endpoint Mechanisms
PHASE
Induction
LLNA
proliferation
To summarize thus far-
CH can be broken down into phases.
The LLNA measures proliferation by incorporation of 3H-thymidine.
The GPMt or Buehler measure erythema by visual assessment.
Another murine test that I will just mention that does not have the magnitude of data that the LLNA has is the Mouse Ear Swelling Test- which is an objective measurement of edema.
GPMT and BA
Elicitation
MEST
erythema
edema

20. 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

21. Using SAR to Predict ACD
Protein
reactivity
Metabolic
Transform
Skin
penetration/
metabolism
Immune
Predict
ACD
(Allergic Contact
Dermatitis)
Chemical
See attached

22. Prevalence of Asthma, United States
0- 4 yr 80
5-14
15-34 70
35-64 60
>65
Total 50
Rate (per thousand) 40 30 20 10
1980
1981-
1984-
1987-
1990-
1993-
1983
1986
1989
1992
1994

23. Characteristics of Allergic Asthma
Immediate Response
Bronchoconstriction
IgE mediated (IL-4)
Late phase
Hyperresponsive to non-specific stimuli (methacholine)
Eosinophilic Inflammation
Th2 mediated (Il-5, IL-13)

24. Principal Asthma/Respiratory Allergy Concerns for Toxicologist
Proteins - e.g detergent enzymes, biotech
Haptens (low molecular wt) - isocyanates & anhydrides, platinum
Adjuvants - air pollution

25. Method used to Assess for Potential Pulmonary Hypersensitivity
Criteria for Positive Response
Respiratory rate 35%
Temp  0.6%
Temperature
transmitter Ag
exhaust
air
oscillograph
Differential
Pressure transducer
Measures pulmonary
response

26. 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

27. 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

28. 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)

29. 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)

30. 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

31. Air Pollutants That Appear to Be Adjuvants
Rodent models
Nitrogen dioxide (NO2)
Residual oil fly ash (ROFA
& Human
Diesel exhaust
& Rhesus Monkey
Ozone

32. TH1 TH2 IL-2 TGFß IFN IL-5 IL-13 IL-6 IL-4 IL-10 Suppresses TH2
IgG2a, CF Antibody
DTH, Cytotoxicity
IgE, IgG2b, IgG1
Mast Cells + Eosinophils
Defense Against
Intracellular Pathogens
Inflammatory Diseases
Defense Against Parasitic
Infection
Immediate-type Hypersensitivity

33. 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

34. 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

36. Goal –Relate Potency of GMO Protein to Other Food Proteins
Impact