Immunotoxicity:Contribution of Immunotoxicity:Contribution of Chemicals to Allergic DiseaseChemicals to Allergic Disease

Assesment, Examples, & MechanismsAssesment, Examples, & Mechanisms

MaryJane SelgradeNational Health & Environmental Effects Research Laboratory, U.S, EPA, Research

Triangle Pk, NCSelgrade.MaryJane@EPA.Gov

November 2, 2007

Lecture OutlineLecture Outline

• Mechanisms of immune mediated injury• Chemicals which elicit immune responses

– Contact sensitivity– Respiratory sensitivity– Food Allergy

• Chemicals which modulate responses to other antigens

HypersensitivityHypersensitivity

Definition: Excessive humoral or cellular response to an antigen which

can lead to tissue damage

Hypersensitivity: ClassificationHypersensitivity: 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 StagesTwo Stages(Distinguishes from irritation)(Distinguishes from irritation)

InductionSensitization(1st exposure)

ElicitationChallenge(subsequent exposure)

Type I (Immediate)Type I (Immediate)(Atopy)(Atopy)

Example:Bee Sting

SensitizationSensitization

Mediators (as such Histamine)

Bronchoconstriction

ElicitationElicitation

Mast CellDegranulation

Type 2 (Cytotoxic)Type 2 (Cytotoxic)SensitizationSensitization Ag Ab (or autoantibody)

Ag binds to cell surface

ElicitationElicitation

Cell lysis

Ab bind to cell bound antigen

Examples:Goodpastures syndromeHemolytic anemia

Type 3 (Arthus)Type 3 (Arthus)

SensitizationSensitizationAg Ab

ElicitationElicitationFormation ofAg/Ab complexes;Deposition in tissues

Activation of macrophagesAnd Complement

Influx of PMN’s, Eosinophils,Lymphocytes (Inflammation) Polymorphonuclear

leukocyte

Activatedmacrophage

Examples: Late onsetasthmatic response, fibrosis, serum sickness

Type 4 (Cell Mediated)Type 4 (Cell Mediated)

Inflammatory activity

Cytokines

Activatedmacrophage

ElicitationElicitation

Antigen

Ag presenting cell

Activated T cell

SensitizationSensitization

Antigen

Ag presenting cell

Clonal expansion

Example:Contact

Sensitivity

Class I MHC

peptide generally derived from protein produced within the cell

peptide generally derived from protein taken up by the cell

Class II MHC

cell membrane

Recognised by TCR of CD4 T cells

Recognised by TCR of CD8 T cells

cytoplasm

with bound peptide with bound peptide

Antigen Presented to T Cells Via MHC

IL-4 IL-10IL-5 IL-13 IL-6

TH1TH1

IL-2 TGFß IFN

Suppresses TH2

Suppresses TH1IgG2a, CF Antibody

DTH, CytotoxicityIgE, IgG2b, IgG1

Mast Cells + Eosinophils

TH2TH2

Defense Against Intracellular Pathogens

Inflammatory Diseases

Defense Against Parasitic Infection

Immediate-type Hypersensitivity

Type IV Responses Type IV Responses Immune cell

Th1 Th2 CD8 (CTL) Tc

antigen Soluble (MHC II)

Soluble(MHC II)

Cell-associatedMHC I)

Effector mechanism

Macrophage activation

Eosinophil activation

cytotoxicity

Examples Tuberculin RxContact dermatitis

Chronic asthma/allergy

Contact dermatitis

Potential Effects of Chemicals Potential Effects of Chemicals on Allergic Diseaseon 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 SensitizersPotential Contact Sensitizers• Cosmetics and Fragrances• Dyes• Preservatives (formaldehyde• Metals (Ni, Co, Be, Cr)• Pesticides

IL-1, IL-6, IL-12

ALLERGEN

LANGERHANS CELL (LC)

EDEMA AND ERYTHEMA

INDUCTION PHASE ELICITATION PHASE

LC AND LYMPHOCYTE INTERACTION

“PRIMED” LYMPHOCYTES

CELLULAR INFLUX

NONSPECIFIC INFLAMMATORY

MEDIATORS

SPECIFIC RESPONSE

Migration to Local Lymph Node

LYMPHOCYTE PROLIFERATION

Contact HypersensitivityContact HypersensitivityAllergic Contact Dermatitis (ACDAllergic Contact Dermatitis (ACD

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

Endpoint erythema

Criteria

20 animals/ group

GUINEA PIG MODELSGUINEA PIG MODELS

Guinea Pig Guinea Pig Maximization Maximization

TestTest

Buehler Buehler AssayAssay

Induction

Challenge

HRIPTHRIPTHuman Repeat Human Repeat

Insult Patch Insult Patch TestTest

Topical ApplicationClosed patch, 24 hr3x/wk, 3wk

Rest 2 wk, apply patch 24 hr

Read 48 hr

Any positive

HUMANHUMAN

Mouse Ear Swelling TestMouse Ear Swelling Test

Application of Agent or Vehicle

to Back (local)or Abdomen(systemic)

Application of Agent

Measure Ears: 24-48 hours post challenge

Sensitization

Induction Period

Challenge

The Local Lymph Node AssayThe Local Lymph Node Assay

Agent applied to ears Days 1,2,3

IV 3H-thymidine injection Day 6

isotope incorporation scintillation counting

5 hour after IV injection

Proliferation measure:

Endpoint expressed as dpm

Contact Hypersensitivity Models and Contact Hypersensitivity Models and Endpoint MechanismsEndpoint Mechanisms

Elicitation

PHASE

LLNA

MEST

Induction

GPMT and BA

Structure Activity Relationship (SAR) ModelsStructure 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 ACDUsing SAR to Predict ACD

ChemicalChemical

PredictPredictACDACD

(AAllergic CContactDDermatitis)

See attached Chem characteristic pdfSee attached Chem characteristic pdf

ProteinProteinreactivityreactivity

Metabolic Metabolic TransformTransform

SkinSkinpenetration/penetration/metabolismmetabolism

ImmuneImmunereactivityreactivity

Prevalence of Asthma, United StatesPrevalence of Asthma, United States

10

20

30

40

50

60

70

80

1980 1981-1983

1984-1986

1987-1989

1990-1992

1993-1994

Rat

e (p

er th

ousa

nd)

0- 4 yr5-1415-3435-64>65Total

Characteristics of Allergic Characteristics of Allergic AsthmaAsthma

• Immediate ResponseImmediate Response– Bronchoconstriction– IgE mediated (IL-4)

• Late phaseLate phase– Hyperresponsive to

non-specific stimuli (methacholine)

– Eosinophilic Inflammation

– Th2 mediated (Il-5, IL-13)

Principal Asthma/Respiratory Principal Asthma/Respiratory Allergy Concerns for ToxicologistAllergy Concerns for Toxicologist

• Proteins - e.g detergent enzymes, biotech• Haptens (low molecular wt) - isocyanates &

anhydrides, platinum• Adjuvants - air pollution

Method used to Assess for PotentialMethod used to Assess for Potential

Pulmonary HypersensitivityPulmonary Hypersensitivity

Temperature transmitter

Ag

exhaust

air

oscillograph

DifferentialPressure transducerMeasures pulmonaryresponse

Criteria for Positive ResponseRespiratory rate 35%Temp 0.6%

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 & Guinea Pig Intratracheal Test & Detergent Enzymes AssessmentDetergent 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 Low Molecular Wt (<3000 molecular weight) Compoundsweight) 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 Assessing Low Molecular Wt (<3000) as Potential Respiratory Allergens -AsthmagensPotential 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 Assessing Low Molecular Wt: Potential Screening Hazard ID ApproachesScreening 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 Air Pollutants That Appear to Be AdjuvantsAdjuvants

• Rodent models– Nitrogen dioxide (NO2)– Residual oil fly ash (ROFA

• & Human– Diesel exhaust

• & Rhesus Monkey – Ozone

IL-4 IL-10IL-5 IL-13 IL-6

TH1TH1

IL-2 TNFß IFN

Suppresses TH2

Suppresses TH1IgG2a, CF Antibody

DTH, CytotoxicityIgE, IgG2b, IgG1

Mast Cells + Eosinophils

TH2TH2

Defense Against Intracellular Pathogens

Inflammatory Diseases

Defense Against Parasitic Infection

Immediate-type Hypersensitivity

Conditions That Influence Th1/Th2 Conditions That Influence Th1/Th2 PolarizationPolarization

• Nature or Dose of Antigen• Local Micro Environment

– cytokines & other mediators produced by macrophages & epithelial cells

• Genetics of Host• Route of Exposure

Food allergensFood 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 Goal –Relate Potency of GMO Protein to Other Food ProteinsProtein to Other Food Proteins

(picture is hypothetical)(picture is hypothetical)