Immunotoxicity:Contribution of Chemicals to Allergic Disease Assesment, Examples, & Mechanisms

1. Immunotoxicity:Contribution of Chemicals to Allergic DiseaseAssesment, Examples, & Mechanisms MaryJane Selgrade National Health & Environmental Effects Research Laboratory, U.S, EPA, Research Triangle Pk, NC Selgrade.MaryJane@EPA.Gov November 2, 2007

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

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

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

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

10. Antigen Presented to T Cells Via MHC

11. TH1 TH2 IL-2 TGFßIFN 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 Inflammatory Diseases Defense Against Parasitic Infection Immediate-type Hypersensitivity

12. Type IV Responses

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 • Systemic hypersensitivity (drugs) • 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 NONSPECIFIC INFLAMMATORY MEDIATORS IL-1B, IL-6, IL-12 LANGERHANS CELL (LC) Migration to Local Lymph Node CELLULAR INFLUX “PRIMED” LYMPHOCYTES SPECIFIC RESPONSE LC AND LYMPHOCYTE INTERACTION LYMPHOCYTE PROLIFERATION

16. Guinea Pig Maximization Test Buehler Assay Induction Challenge GUINEA PIG MODELS HUMAN HRIPT Human Repeat Insult Patch Test 20 animals/ group ID injection w/ and without FCA plus topical application:Days 5-8 Day 20-22 topical challenge Read: 48,72 h after challenge >30% positive Topical application - closed patch: Days 0, 6-8, 13-15 Day 27-28 topical challenge untreated flank, 6 h Read: 21, 24, 48 h after removing patch > 15% positive Topical Application Closed patch, 24 hr 3x/wk, 3wk Rest 2 wk, apply patch 24 hr Read 48 hr Any positive 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: 24-48 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 Endpoint expressed as dpm

19. Contact Hypersensitivity Models and Endpoint Mechanisms PHASE Induction LLNA proliferation 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. Skin penetration/ metabolism Protein reactivity Metabolic Transform Immune reactivity Using SAR to Predict ACD Predict ACD (Allergic Contact Dermatitis) Chemical See attached Chem characteristic pdf

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

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

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

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 TNFßIFN 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 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. Impact Goal –Relate Potency of GMO Protein to Other Food Proteins(picture is hypothetical)