immunotoxicity:contribution of chemicals to allergic disease assesment, examples, & mechanisms...
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Immunotoxicity:Contribution of Immunotoxicity:Contribution of Chemicals to Allergic DiseaseChemicals to Allergic Disease
Assesment, Examples, & MechanismsAssesment, Examples, & Mechanisms
MaryJane Selgrade
National Health & Environmental Effects Research Laboratory, U.S, EPA, Research
Triangle Pk, NC
November 2, 2007
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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
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HypersensitivityHypersensitivity
Definition: Excessive humoral or cellular response to an antigen which
can lead to tissue damage
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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)
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Two StagesTwo Stages(Distinguishes from irritation)(Distinguishes from irritation)
InductionSensitization(1st exposure)
ElicitationChallenge(subsequent exposure)
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Type I (Immediate)Type I (Immediate)(Atopy)(Atopy)
Example:Bee Sting
SensitizationSensitization
Mediators (as such Histamine)
Bronchoconstriction
ElicitationElicitation
Mast CellDegranulation
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Type 2 (Cytotoxic)Type 2 (Cytotoxic)
SensitizationSensitizationAg Ab (or autoantibody)
Ag binds to cell surface
ElicitationElicitation
Cell lysis
Ab bind to cell bound antigen
Examples:Goodpastures syndromeHemolytic anemia
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Type 3 (Arthus)Type 3 (Arthus)
SensitizationSensitization
Ag Ab
ElicitationElicitation
Formation 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
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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
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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
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IL-4 IL-10IL-5 IL-13 IL-6
TH1TH1
IL-2 TGFß IFN
Suppresses TH2
Suppresses TH1
IgG2a, CF Antibody DTH, Cytotoxicity
IgE, IgG2b, IgG1Mast Cells + Eosinophils
TH2TH2
Defense Against Intracellular Pathogens
Inflammatory Diseases
Defense Against Parasitic Infection
Immediate-type Hypersensitivity
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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
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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
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Potential Contact SensitizersPotential Contact Sensitizers
• Cosmetics and Fragrances
• Dyes
• Preservatives (formaldehyde
• Metals (Ni, Co, Be, Cr)
• Pesticides
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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
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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
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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
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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
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Contact Hypersensitivity Models and Contact Hypersensitivity Models and Endpoint MechanismsEndpoint Mechanisms
Elicitation
PHASE
LLNA
MEST
Induction
GPMT and BA
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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
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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
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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 t
hous
and)
0- 4 yr5-1415-3435-64>65Total
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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)
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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
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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%
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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
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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
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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)
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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)
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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
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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
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IL-4 IL-10IL-5 IL-13 IL-6
TH1TH1
IL-2 TNFß IFN
Suppresses TH2
Suppresses TH1
IgG2a, CF Antibody DTH, Cytotoxicity
IgE, IgG2b, IgG1Mast Cells + Eosinophils
TH2TH2
Defense Against Intracellular Pathogens
Inflammatory Diseases
Defense Against Parasitic Infection
Immediate-type Hypersensitivity
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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
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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
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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)