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 Selgrade.MaryJane@EPA.Gov September 18,2006

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)

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

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

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

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

Antigen Presented to T Cells Via MHC

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

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

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

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 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

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 20-22 topical challenge Read: 48,72 h after challenge >30% positive Topical application - closed patch: Days 0, 6-8, and 13-15 Day 27-28 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

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

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

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

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 Protein reactivity Metabolic Transform Skin penetration/ metabolism Immune Predict ACD (Allergic Contact Dermatitis) Chemical See attached

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

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)

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% Temperature transmitter Ag exhaust air oscillograph Differential Pressure transducer Measures pulmonary response

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 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

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 Impact