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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)